WorldWideScience

Sample records for activates junctional endocytosis

  1. 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.

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

    Science.gov (United States)

    Johnson, Kristen E.; Mitra, Shalini; Katoch, Parul; Kelsey, Linda S.; Johnson, Keith R.; Mehta, Parmender P.

    2013-01-01

    The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions. PMID:23363606

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

    Directory of Open Access Journals (Sweden)

    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.

    Science.gov (United States)

    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. VAMP4 Is an Essential Cargo Molecule for Activity-Dependent Bulk Endocytosis.

    Science.gov (United States)

    Nicholson-Fish, Jessica C; Kokotos, Alexandros C; Gillingwater, Thomas H; Smillie, Karen J; Cousin, Michael A

    2015-12-01

    The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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

    Science.gov (United States)

    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. Key physiological parameters dictate triggering of activity-dependent bulk endocytosis in hippocampal synapses.

    Directory of Open Access Journals (Sweden)

    Eva M Wenzel

    Full Text Available To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE. ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

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

    Science.gov (United States)

    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. Exocyst Sec10 protects renal tubule cells from injury by EGFR/MAPK activation and effects on endocytosis.

    Science.gov (United States)

    Fogelgren, Ben; Zuo, Xiaofeng; Buonato, Janine M; Vasilyev, Aleksandr; Baek, Jeong-In; Choi, Soo Young; Chacon-Heszele, Maria F; Palmyre, Aurélien; Polgar, Noemi; Drummond, Iain; Park, Kwon Moo; Lazzara, Matthew J; Lipschutz, Joshua H

    2014-12-15

    Acute kidney injury is common and has a high mortality rate, and no effective treatment exists other than supportive care. Using cell culture models, we previously demonstrated that exocyst Sec10 overexpression reduced damage to renal tubule cells and speeded recovery and that the protective effect was mediated by higher basal levels of mitogen-activated protein kinase (MAPK) signaling. The exocyst, a highly-conserved eight-protein complex, is known for regulating protein trafficking. Here we show that the exocyst biochemically interacts with the epidermal growth factor receptor (EGFR), which is upstream of MAPK, and Sec10-overexpressing cells express greater levels of phosphorylated (active) ERK, the final step in the MAPK pathway, in response to EGF stimulation. EGFR endocytosis, which has been linked to activation of the MAPK pathway, increases in Sec10-overexpressing cells, and gefitinib, a specific EGFR inhibitor, and Dynasore, a dynamin inhibitor, both reduce EGFR endocytosis. In turn, inhibition of the MAPK pathway reduces ligand-mediated EGFR endocytosis, suggesting a potential feedback of elevated ERK activity on EGFR endocytosis. Gefitinib also decreases MAPK signaling in Sec10-overexpressing cells to levels seen in control cells and, demonstrating a causal role for EGFR, reverses the protective effect of Sec10 overexpression following cell injury in vitro. Finally, using an in vivo zebrafish model of acute kidney injury, morpholino-induced knockdown of sec10 increases renal tubule cell susceptibility to injury. Taken together, these results suggest that the exocyst, acting through EGFR, endocytosis, and the MAPK pathway is a candidate therapeutic target for acute kidney injury.

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

    OpenAIRE

    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. Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling.

    Science.gov (United States)

    Vietti, Giulia; Ibouraadaten, Saloua; Palmai-Pallag, Mihaly; Yakoub, Yousof; Piret, Jean-Pascal; Marbaix, Etienne; Lison, Dominique; van den Brule, Sybille

    2016-01-01

    Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-β or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-β in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-β- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.

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

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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......On-state collector-emitter voltage (VCE) is a good indicator to determine the wear-out condition of power device modules. Further, it is a one of the Temperature Sensitive Electrical Parameters (TSEPs) and thus can be used for junction temperature estimation. In this paper, the junction temperature...

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  2. Endocytosis in filamentous fungi

    OpenAIRE

    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 ...

  3. Regulated RalBP1 binding to RalA and PSD-95 controls AMPA receptor endocytosis and LTD.

    Directory of Open Access Journals (Sweden)

    Kihoon Han

    2009-09-01

    Full Text Available Long-term depression (LTD is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR-dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.

  4. Thermally activated magnetization reversal in magnetic tunnel junctions

    Institute of Scientific and Technical Information of China (English)

    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.

  5. Active zone stability:insights from fly neuromuscular junction

    Institute of Scientific and Technical Information of China (English)

    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.

  6. Endocytosis of Receptor Tyrosine Kinases

    Science.gov (United States)

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  7. Delayed onset of positive feedback activation of Rab5 by Rabex-5 and Rabaptin-5 in endocytosis.

    Directory of Open Access Journals (Sweden)

    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

  8. Synaptic vesicle endocytosis.

    Science.gov (United States)

    Saheki, Yasunori; De Camilli, Pietro

    2012-09-01

    Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property relies on a highly efficient local endocytic recycling of synaptic vesicle membranes, which can be reused for hundreds, possibly thousands, of exo-endocytic cycles. Morphological, physiological, molecular, and genetic studies over the last four decades have provided insight into the membrane traffic reactions that govern this recycling and its regulation. These studies have shown that synaptic vesicle endocytosis capitalizes on fundamental and general endocytic mechanisms but also involves neuron-specific adaptations of such mechanisms. Thus, investigations of these processes have advanced not only the field of synaptic transmission but also, more generally, the field of endocytosis. This article summarizes current information on synaptic vesicle endocytosis with an emphasis on the underlying molecular mechanisms and with a special focus on clathrin-mediated endocytosis, the predominant pathway of synaptic vesicle protein internalization.

  9. Ankyrin-G Inhibits Endocytosis of Cadherin Dimers.

    Science.gov (United States)

    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

  10. Molecular Mechanisms of Endocytosis

    NARCIS (Netherlands)

    Riezman, H.; Woodman, P.G.; van Meer, G.; Marsh, M.

    1997-01-01

    Cells regulate their developmental and functional programs through their interaction with the external milieu, which requires communication across the plasma membrane. The plasma membrane is constantly being remodeled by endocytosis allowing cells to control how they respond to external stimuli. End

  11. The R-Ras/RIN2/Rab5 complex controls endothelial cell adhesion and morphogenesis via active integrin endocytosis and Rac signaling

    Institute of Scientific and Technical Information of China (English)

    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.

  12. Microwave resonant activation in hybrid single-gap/two-gap Josephson tunnel junctions

    Science.gov (United States)

    Carabello, Steven; Lambert, Joseph G.; Mlack, Jerome; Dai, Wenqing; Li, Qi; Chen, Ke; Cunnane, Daniel; Xi, X. X.; Ramos, Roberto C.

    2016-09-01

    Microwave resonant activation is a powerful, straightforward technique to study classical and quantum systems, experimentally realized in Josephson junction devices cooled to very low temperatures. These devices typically consist of two single-gap superconductors separated by a weak link. We report the results of the first resonant activation experiments on hybrid thin film Josephson junctions consisting of a multi-gap superconductor (MgB2) and a single-gap superconductor (Pb or Sn). We can interpret the plasma frequency in terms of theories both for conventional and hybrid junctions. Using these models, we determine the junction parameters including critical current, resistance, and capacitance and find moderately high quality factors of Q0˜ 100 for these junctions.

  13. Endocytosis of influenza viruses

    OpenAIRE

    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.

  14. Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

    OpenAIRE

    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...

  15. Drosophila cyfip regulates synaptic development and endocytosis by suppressing filamentous actin assembly.

    Science.gov (United States)

    Zhao, Lu; Wang, Dan; Wang, Qifu; Rodal, Avital A; Zhang, Yong Q

    2013-04-01

    The formation of synapses and the proper construction of neural circuits depend on signaling pathways that regulate cytoskeletal structure and dynamics. After the mutual recognition of a growing axon and its target, multiple signaling pathways are activated that regulate cytoskeletal dynamics to determine the morphology and strength of the connection. By analyzing Drosophila mutations in the cytoplasmic FMRP interacting protein Cyfip, we demonstrate that this component of the WAVE complex inhibits the assembly of filamentous actin (F-actin) and thereby regulates key aspects of synaptogenesis. Cyfip regulates the distribution of F-actin filaments in presynaptic neuromuscular junction (NMJ) terminals. At cyfip mutant NMJs, F-actin assembly was accelerated, resulting in shorter NMJs, more numerous satellite boutons, and reduced quantal content. Increased synaptic vesicle size and failure to maintain excitatory junctional potential amplitudes under high-frequency stimulation in cyfip mutants indicated an endocytic defect. cyfip mutants exhibited upregulated bone morphogenetic protein (BMP) signaling, a major growth-promoting pathway known to be attenuated by endocytosis at the Drosophila NMJ. We propose that Cyfip regulates synapse development and endocytosis by inhibiting actin assembly.

  16. The mechanochemistry of endocytosis.

    Directory of Open Access Journals (Sweden)

    Jian Liu

    2009-09-01

    Full Text Available Endocytic vesicle formation is a complex process that couples sequential protein recruitment and lipid modifications with dramatic shape transformations of the plasma membrane. Although individual molecular players have been studied intensively, how they all fit into a coherent picture of endocytosis remains unclear. That is, how the proper temporal and spatial coordination of endocytic events is achieved and what drives vesicle scission are not known. Drawing upon detailed knowledge from experiments in yeast, we develop the first integrated mechanochemical model that quantitatively recapitulates the temporal and spatial progression of endocytic events leading to vesicle scission. The central idea is that membrane curvature is coupled to the accompanying biochemical reactions. This coupling ensures that the process is robust and culminates in an interfacial force that pinches off the vesicle. Calculated phase diagrams reproduce endocytic mutant phenotypes observed in experiments and predict unique testable endocytic phenotypes in yeast and mammalian cells. The combination of experiments and theory in this work suggest a unified mechanism for endocytic vesicle formation across eukaryotes.

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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 ...

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

    DEFF Research Database (Denmark)

    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...

  20. 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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  2. Pharmacological attenuation of Paramecium fluid-phase endocytosis.

    Science.gov (United States)

    Wiejak, Jolanta; Surmacz, Liliana; Wyroba, Elzbieta

    2007-01-01

    Spectrophotometric quantification of fluid phase endocytosis in the presence of different pharmacological compounds was performed in the model unicellular eukaryote Paramecium. The kinetics of Lucifer Yellow Carbohydrazide (LY) uptake in cells exposed to forskolin and isoproterenol--known to stimulate phagocytosis in this cell--was analyzed. Reduction in both the rate of endocytosis and total accumulation of fluid phase marker was observed following the treatment. Forskolin diminished total LY accumulation by 11% and 21% after 5 min and 25 min of incubation, respectively, whereas the rate of uptake was lowered by 21% in comparison to control cells. The inhibitory effect ofisoproterenol was less pronounced than that of forskolin. The total accumulation of LY was decreased by 11% in 5 min as compared to the untreated cells and this effect was persistent upon further exposition to this reagent up to 25 min. To better understand these observations, the effect of inhibitors of PKA and cAMP phosphodiesterase on fluid phase uptake was tested. 3-isobutyl-1-methyl xanthine (IBMX) caused 12% decrease in LY accumulation after 5 min of incubation. In combination with isoproterenol or forskolin, IBMX enhanced their inhibitory effect on fluid endocytosis, which was lowered by 25% and 29%, respectively. The strongest inhibitory effect on fluid endocytosis was exerted by the 10 microM PKA inhibitor, which diminished endocytosis by 35% in 5 min. These results suggest that Paramecium fluid phase uptake may be regulated through activation of PKA, although the precise mechanism of this process has not yet been elucidated.

  3. Roles of Cortactin, an Actin Polymerization Mediator, in Cell Endocytosis

    Institute of Scientific and Technical Information of China (English)

    Li CHEN; Zhi-Wei WANG; Jian-wei ZHU; Xi ZHAN

    2006-01-01

    Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS 1 oncogene.Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents.Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 (SH3)-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation.Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Rocío eTalaverón

    2015-10-01

    Full Text Available The postnatal subventricular zone 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 subventricular zone is involved in NPC proliferation and in neuroblast migration towards the olfactory bulb. Subventricular zone 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 subventricular zone 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. 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 subventricular zone neurospheres. In addition, they demonstrate that subventricular zone-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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  9. 溶血磷脂酸对小胶质细胞活化和吞噬功能的影响%Effect of LPA on activation and endocytosis of microglia

    Institute of Scientific and Technical Information of China (English)

    付佩彩; 喻志源; 刘淼; 唐荣华; 王伟; 骆翔

    2012-01-01

    目的:观察溶血磷脂酸(LPA)对小胶质细胞活化及其吞噬功能的影响.方法:小胶质细胞系BV2细胞复苏后传代培养,取对数生长期细胞分为对照组和LPA组,LPA干预30 min后各组加入FITC标记的葡聚糖,在干预后1h、2h后收取细胞,用流式细胞仪测定各孔FITC-葡聚糖微粒吞噬率及荧光强度.结果:LPA干预1h时,吞噬率较对照组明显增加(P<0.05),2h时两组细胞吞噬率达到最高值.LPA干预后1h及2h时,吞噬荧光强度较对照组明显增加(P<0.01),而且吞噬率与荧光强度的乘积较对照组亦明显增加(P<0.01).结论:LPA可提高BV2细胞活化及吞噬功能,提示LPA在小胶质细胞活化及吞噬过程中发挥了重要作用.%Objectives To observe the effect of lysophosphatidic acid (LPA) on activation and endocytosis of microglia. Methods;BV2 cells in exponential phase were randomly divided into two groups: control group and LPA group. At time points (1 h and 2 h), cells were harvested for later use. FCM was used to detect the phagocytic rate of FITC-dextran and the fluorescence intensity. Results: As compared with control group, the phagocytic rate of FITC-dextran in LPA group was significantly increased at 1 h (P<0.05). At 2 h, the phagocytic rate of FITC-dextran reached the peak in both two groups. At time points (1 h and 2 h), as compared with control group, the fluorescence intensity in LPA group was increased (P<0. 01) and the product of phagocytic rate and fluorescence intensity in LPA group was significantly higher than that of control group (P<0. 01). Conclusion: LPA can promote activation and endocytosis of microglia in vitro, suggesting the roles of LPA in activation and endocytosis of microglia.

  10. Cell adhesion defines the topology of endocytosis and signaling.

    Science.gov (United States)

    Grossier, Jean-Philippe; Xouri, Georgia; Goud, Bruno; Schauer, Kristine

    2014-01-01

    Preferred sites of endocytosis have been observed in various cell types, but whether they occur randomly or are linked to cellular cues is debated. Here, we quantified the sites of endocytosis of transferrin (Tfn) and epidermal growth factor (EGF) in cells whose adhesion geometry was defined by micropatterns. 3D probabilistic density maps revealed that Tfn was enriched in adhesive sites during uptake, whereas EGF endocytosis was restricted to the dorsal cellular surface. This spatial separation was not due to distributions of corresponding receptors but was regulated by uptake mechanisms. Asymmetric uptake of Tfn resulted from the enrichment of clathrin and adaptor protein 2 at adhesive areas. Asymmetry in EGF uptake was strongly dependent on the actin cytoskeleton and led to asymmetry in EGF receptor activation. Mild alteration of actin dynamics abolished asymmetry in EGF uptake and decreased EGF-induced downstream signaling, suggesting that cellular adhesion cues influence signal propagation. We propose that restriction of endocytosis at distinct sites allows cells to sense their environment in an "outside-in" mechanism. PMID:24366944

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

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; Grimmer, S.; Iversen, T.G.;

    2000-01-01

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

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

    Science.gov (United States)

    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

  13. High-performance germanium n+/p junction by nickel-induced dopant activation of implanted phosphorus at low temperature

    Science.gov (United States)

    Wei, Huang; Chao, Lu; Jue, Yu; Jiang-Bin, Wei; Chao-Wen, Chen; Jian-Yuan, Wang; Jian-Fang, Xu; Chen, Wang; Cheng, Li; Song-Yan, Chen; Chun-Li, Liu; Hong-Kai, Lai

    2016-05-01

    High-performance Ge n+/p junctions were fabricated at a low formation temperature from 325 °C to 400 °C with a metal(nickel)-induced dopant activation technique. The obtained NiGe electroded Ge n+/p junction has a rectification ratio of 5.6× 104 and a forward current of 387 A/cm2 at -1 V bias. The Ni-based metal-induced dopant activation technique is expected to meet the requirement of the shallow junction of Ge MOSFET. Project supported by the National Natural Science Foundation of China (Grant Nos. 61176092 and 61474094), the National Basic Research Program of China (Grant Nos. 2012CB933503 and 2013CB632103), and the National Natural Science Foundation of China-National Research Foundation of Korea Joint Research Project (Grant No. 11311140251).

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    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

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    International Nuclear Information System (INIS)

    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

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

    OpenAIRE

    1992-01-01

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

  19. Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction.

    Science.gov (United States)

    Blunk, Aline D; Akbergenova, Yulia; Cho, Richard W; Lee, Jihye; Walldorf, Uwe; Xu, Ke; Zhong, Guisheng; Zhuang, Xiaowei; Littleton, J Troy

    2014-07-01

    Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, we carried out a chemical mutagenesis screen in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen we identified a mutation in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, act(E84K), harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous act(E84K) mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. act(E84K) mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in act(E84K) mutants. Genetic interactions between act(E84K) and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization.

  20. Exocytosis and endocytosis in juxtaglomerular cells

    DEFF Research Database (Denmark)

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

    2000-01-01

    ) 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....

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  3. Activity in right temporo-parietal junction is not selective for theory-of-mind.

    Science.gov (United States)

    Mitchell, Jason P

    2008-02-01

    Recent researchers have suggested that a region of right temporo-parietal junction (RTPJ) selectively subserves the attribution of beliefs to other people (Saxe R, Kanwisher N. 2003. People thinking about thinking people: fMRI investigations of theory of mind. NeuroImage. 19:1835-1842; Saxe R, Powell LJ. 2006. It's the thought that counts: specific brain regions for one component of theory of mind. Psychol Sci. 17:692-699; Saxe R, Wexler A. 2005. Making sense of another mind: the role of the right temporo-parietal junction. Neuropsychologia. 43:1391-1399). At the same time, a similar RTPJ region has been observed repeatedly in a variety of nonsocial tasks that require participants to redirect attention to task-relevant stimuli (e.g., Corbetta M, Shulman GL. 2002. Control of goal-directed and stimulus-driven attention in the brain. Nat Rev Neurosci. 3:201-215; Serences JT, Shomstein S, Leber AB, Golay X, Egeth HE, Yantis S. 2005. Coordination of voluntary and stimulus-driven attentional control in human cortex. Psychol Sci. 16:114-122). However, because these 2 sets of tasks have never been compared within the same participants, it remains unclear whether these observations refer to the exact same region of RTPJ or may instead involve neighboring regions with distinct functional profiles. To test the claim that there is a region of RTPJ selective for belief attribution, the current study used functional neuroimaging to examine the extent to which cortical loci identified by a "theory-of-mind localizer" also distinguish between trials on a target detection task that varied demands to reorient attention (i.e., a version of the "Posner cueing task"). Results were incompatible with claims of RTPJ selectivity for mental state attribution. Regardless of whether regions were defined from group analyses or were individually tailored for each participant, RTPJ activity was also modulated by the nonsocial attentional task. The overlap between theory-of-mind and attentional

  4. Right temporoparietal junction activation by a salient contextual cue facilitates target discrimination.

    Science.gov (United States)

    Geng, Joy J; Mangun, George R

    2011-01-01

    The right temporoparietal junction (R TPJ) is involved in stimulus-driven attentional control in response to the appearance of an unexpected target or a distractor that shares features with a task-relevant target. An unresolved question is whether these responses in R TPJ are due simply to the presence of a stimulus that is a potential target, or instead responds to any task-relevant information. Here, we addressed this issue by testing the sensitivity of R TPJ to a perceptually salient, non-target stimulus - a contextual cue. Although known to be a non-target, the contextual cue carried probabilistic information regarding the presence of a target in the opposite visual field. The contextual cue was therefore always of potential behavioral relevance, but only sometimes paired with a target. The appearance of the contextual cue alone increased activation in R TPJ, but more so when it appeared with a target. There was also greater connectivity between R TPJ and a network of attentional control and decision areas when the contextual cue was present. These results demonstrate that R TPJ is involved in the stimulus-driven representation of task-relevant information that can be used to engage an appropriate behavioral response.

  5. Why does endocytosis in single cells care which side up?

    Science.gov (United States)

    Schauer, Kristine; Goud, Bruno

    2014-01-01

    Eukaryotic cells display an asymmetric distribution of cellular compartments relying on their adhesion and the underlying anisotropy of the actin and microtubule cytoskeleton. Studies using a minimal cell culture system based on confined adhesion on micropatterns have illustrated that trafficking compartments are well organized at the single cell level in response to the geometry of cellular adhesion cues. Expanding our analysis on cellular uptake processes, we have found that cellular adhesion additionally defines the topology of endocytosis and signaling. During endocytosis, transferrin (Tfn) and epidermal growth factor (EGF) concentrate at distinct cellular sites in micropatterned cells. Tfn is enriched in adhesive sites during uptake, whereas EGF endocytosis is restricted to the dorsal cellular surface. This unexpected dorsal/ventral asymmetry is regulated by uptake mechanisms and actin dynamics. Interestingly, restricted EGF uptake leads to asymmetry of EGF receptor activation that is required to sustain downstream signaling. Based on our results, we propose that differential sorting begins at the plasma membrane leading to spatially distinct intracellular trafficking routes that are well defined in space. We speculate that the intracellular positioning of trafficking compartments sustains an important coupling between the endocytic and signaling systems that allows cells to sense their environment. PMID:24717194

  6. Gap Junctions

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    Science.gov (United States)

    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. PMID:26871885

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

    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)

  12. Endocytosis-dependent coordination of multiple actin regulators is required for wound healing.

    Science.gov (United States)

    Matsubayashi, Yutaka; Coulson-Gilmer, Camilla; Millard, Tom H

    2015-08-01

    The ability to heal wounds efficiently is essential for life. After wounding of an epithelium, the cells bordering the wound form dynamic actin protrusions and/or a contractile actomyosin cable, and these actin structures drive wound closure. Despite their importance in wound healing, the molecular mechanisms that regulate the assembly of these actin structures at wound edges are not well understood. In this paper, using Drosophila melanogaster embryos, we demonstrate that Diaphanous, SCAR, and WASp play distinct but overlapping roles in regulating actin assembly during wound healing. Moreover, we show that endocytosis is essential for wound edge actin assembly and wound closure. We identify adherens junctions (AJs) as a key target of endocytosis during wound healing and propose that endocytic remodeling of AJs is required to form "signaling centers" along the wound edge that control actin assembly. We conclude that coordination of actin assembly, AJ remodeling, and membrane traffic is required for the construction of a motile leading edge during wound healing.

  13. Multiple Functions of Sterols in Yeast Endocytosis

    OpenAIRE

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zarubin, Dmitri P., E-mail: dmitri.zarubin@mtu-net.ru [Department of Physical and Collod Chemistry, Moscow State University of Technology and Management, 73 Zemlyanoi Val, Moscow 109803 (Russian Federation)

    2011-08-15

    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), {gamma}{sub i}, are actually complicated functions of mean ionic activity coefficients, {gamma}{sub {+-}}, and transport numbers of ions, t{sub i}. In the present paper specific functions {gamma}{sub i}({gamma}{sub {+-}}, t{sub i}) 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 {gamma}{sub i} quantities by definition of {gamma}{sub {+-}}. The functions are tested numerically on some of the reported {gamma}{sub i} 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

  19. EBIC investigations of junction activity and the role of oxygen in CdS/CuInSe/sub 2/ devices

    Energy Technology Data Exchange (ETDEWEB)

    Matson, R.J.; Noufi, R.; Ahrenkiel, R.K.; Powell, R.C.; Cahen, D.

    1986-01-15

    EBIC characterization of CdS/CuInSe/sub 2/ device cross sections has been used to investigate junction activity as a function of post-deposition oxidation treatments. It was determined that the deposition of CdS onto single-crystal p-CuInSe/sub 2/ results in type conversion and the formation of a CuInSe/sub 2/ homojunction, rather than the expected heterojunction. Homojunctions have also been observed in as-deposited CdS/CuInSe/sub 2/ thin film devices. Post-deposition oxygen treatments of thin film devices serve to move the device junction nearer, if not up to, the heteroface and to largely remove both large (mm) and small (..mu..m) scale spatial variations in the I/sub sc/, thereby contributing to device performance improvement. This appears to occur by type converting the unintended CuInSe/sub 2/ n-layer via the elimination of deep level donor states. These processes are reversible by chemical reduction. They are quite temperature sensitive and are active even at room temperatures. Both hydrazine and an electron beam act to remove ''loosely bound'' oxygen and thereby prepare the system for a more optimal and stable incorporation of oxygen. The CuInSe/sub 2/ film stoichiometry appears to determine the concentration and kinds of defects. The resulting defect chemistry, in concert with oxygen, determines the electrical properties of the material.

  20. Endocytosis of glycosylphosphatidylinositol-anchored proteins

    Directory of Open Access Journals (Sweden)

    Sabharanjak Shefali

    2009-10-01

    Full Text Available Abstract Glycosylphosphatidylinositol-anchored proteins (GPI-APs represent an interesting amalgamation of the three basic kinds of cellular macromolecules viz. proteins, carbohydrates and lipids. An unusually hybrid moiety, the GPI-anchor is expressed in a diverse range of organisms from parasites to mammalian cells and serves to anchor a large number of functionally diverse proteins and has been the center of attention in scientific debate for some time now. Membrane organization of GPI-APs into laterally-organized cholesterol-sphingolipid ordered membrane domains or "rafts" and endocytosis of GPI-APs has been intensely debated. Inclusion into or exclusion from these membrane domains seems to be the critical factor in determining the endocytic mechanisms and intracellular destinations of GPI-APs. The intracellular signaling as well as endocytic trafficking of GPI-APs is critically dependent upon the cell surface organization of GPI-APs, and the associations with these lipid rafts play a vital role during these processes. The mechanism of endocytosis for GPI-APs may differ from other cellular endocytic pathways, such as those mediated by clathrin-coated pits (caveolae, and is necessary for unique biological functions. Numerous intracellular factors are involved in and regulate the endocytosis of GPI-APs, and these may be variably dependent on cell-type. The central focus of this article is to describe the significance of the endocytosis of GPI-APs on a multitude of biological processes, ranging from nutrient-uptake to more complex immune responses. Ultimately, a thorough elucidation of GPI-AP mediated signaling pathways and their regulatory elements will enhance our understanding of essential biological processes and benefit as components of disease intervention strategies.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Interaction among Saccharomyces cerevisiae pheromone receptors during endocytosis

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Yeast Exocytic v-SNAREs Confer Endocytosis

    OpenAIRE

    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...

  11. Endocytosis of Integrin-Binding Human Picornaviruses

    Directory of Open Access Journals (Sweden)

    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.

  12. Endocytosis of integrin-binding human picornaviruses.

    Science.gov (United States)

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    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.

  13. Exocytosis and endocytosis in juxtaglomerular cells.

    Science.gov (United States)

    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

  14. Kinetics of virus entry by endocytosis

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2015-04-01

    Entry of virions into the host cells is either endocytotic or fusogenic. In both cases, it occurs via reversible formation of numerous relatively weak bonds resulting in wrapping of a virion by the host membrane with subsequent membrane rupture or scission. The corresponding kinetic models are customarily focused on the formation of bonds and do not pay attention to the energetics of the whole process, which is crucially dependent, especially in the case of endocytosis, on deformation of actin filaments forming the cytoskeleton of the host cell. The kinetic model of endocytosis, proposed by the author, takes this factor into account and shows that the whole process can be divided into a rapid initial transient stage and a long steady-state stage. The entry occurs during the latter stage and can be described as a first-order reaction. Depending on the details of the dependence of the grand canonical potential on the number of bonds, the entry can be limited either by the interplay of bond formation and membrane rupture (or scission) or by reaching a maximum of this potential.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    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...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  18. Negatively charged silver nanoparticles cause retinal vascular permeability by activating plasma contact system and disrupting adherens junction.

    Science.gov (United States)

    Long, Yan-Min; Zhao, Xing-Chen; Clermont, Allen C; Zhou, Qun-Fang; Liu, Qian; Feener, Edward P; Yan, Bing; Jiang, Gui-Bin

    2016-01-01

    Silver nanoparticles (AgNPs) have been extensively used as antibacterial component in numerous healthcare, biomedical and consumer products. Therefore, their adverse effects to biological systems have become a major concern. AgNPs have been shown to be absorbed into circulation and redistributed into various organs. It is thus of great importance to understand how these nanoparticles affect vascular permeability and uncover the underlying molecular mechanisms. A negatively charged mecaptoundeonic acid-capped silver nanoparticle (MUA@AgNP) was investigated in this work. Ex vivo experiments in mouse plasma revealed that MUA@AgNPs caused plasma prekallikrein cleavage, while positively charged or neutral AgNPs, as well as Ag ions had no effect. In vitro tests revealed that MUA@AgNPs activated the plasma kallikrein-kinin system (KKS) by triggering Hageman factor autoactivation. By using specific inhibitors aprotinin and HOE 140, we demonstrated that KKS activation caused the release of bradykinin, which activated B2 receptors and induced the shedding of adherens junction protein, VE-cadherin. These biological perturbations eventually resulted in endothelial paracellular permeability in mouse retina after intravitreal injection of MUA@AgNPs. The findings from this work provided key insights for toxicity modulation and biomedical applications of AgNPs. PMID:26399585

  19. Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis

    Science.gov (United States)

    Brosson, Sébastien; Fontaine, Frédéric; Vermeersch, Marjorie; Perez-Morga, David; Pays, Etienne; Bousbata, Sabrina; Salmon, Didier

    2016-01-01

    Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi. PMID:27685262

  20. Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells.

    Science.gov (United States)

    Faille, Dorothée; El-Assaad, Fatima; Mitchell, Andrew J; Alessi, Marie-Christine; Chimini, Giovanna; Fusai, Thierry; Grau, Georges E; Combes, Valéry

    2012-08-01

    Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca(2+) chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na(+)/H(+) exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Interleukin-6 modulation of intestinal epithelial tight junction permeability is mediated by JNK pathway activation of claudin-2 gene.

    Directory of Open Access Journals (Sweden)

    Rana Al-Sadi

    Full Text Available Defective intestinal epithelial tight junction (TJ barrier has been shown to be a pathogenic factor in the development of intestinal inflammation. Interleukin-6 (IL-6 is a pleiotropic, pro-inflammatory cytokine which plays an important role in promoting inflammatory response in the gut and in the systemic circulation. Despite its key role in mediating variety inflammatory response, the effect of IL-6 on intestinal epithelial barrier remains unclear. The purpose of this study was to investigate the effect of IL-6 on intestinal epithelial TJ barrier and to delineate the intracellular mechanisms involved using in-vitro (filter-grown Caco-2 monolayers and in-vivo model (mouse intestinal perfusion systems. Our results indicated that IL-6 causes a site-selective increase in Caco-2 intestinal epithelia TJ permeability, causing an increase in flux of small-sized molecules having molecular radius <4 Å. The size-selective increase in Caco-2 TJ permeability was regulated by protein-specific increase in claudin-2 expression. The IL-6 increase in TJ permeability required activation of JNK signaling cascade. The JNK pathway activation of AP-1 resulted in AP-1 binding to its binding sequence on the claudin-2 promoter region, leading to promoter activation and subsequent increase in claudin-2 gene transcription and protein synthesis and TJ permeability. Our in-vivo mouse perfusion showed that IL-6 modulation of mouse intestinal permeability was also mediated by AP-1 dependent increase in claudin-2 expression. In conclusion, our studies show for the first time that the IL-6 modulation of intestinal TJ permeability was regulated by JNK activation of AP-1 and AP-1 activation of claudin-2 gene.

  3. Clathrin-independent endocytosis: mechanisms and function

    DEFF Research Database (Denmark)

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

    2011-01-01

    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.......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...

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

    OpenAIRE

    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...

  5. Polycystin-2 activity is controlled by transcriptional coactivator with PDZ binding motif and PALS1-associated tight junction protein.

    Science.gov (United States)

    Duning, Kerstin; Rosenbusch, Deike; Schlüter, Marc A; Tian, Yuemin; Kunzelmann, Karl; Meyer, Nina; Schulze, Ulf; Markoff, Arseni; Pavenstädt, Hermann; Weide, Thomas

    2010-10-29

    Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology.

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

    DEFF Research Database (Denmark)

    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...

  7. Cdc42-dependent Modulation of Tight Junctions and Membrane Protein Traffic in Polarized Madin-Darby Canine Kidney Cells

    Science.gov (United States)

    Rojas, Raul; Ruiz, Wily G.; Leung, Som-Ming; Jou, Tzuu-Shuh; Apodaca, Gerard

    2001-01-01

    Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity. PMID:11514615

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

    OpenAIRE

    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 internali...

  9. Neuropeptides stimulate human osteoblast activity and promote gap junctional intercellular communication.

    Science.gov (United States)

    Ma, Wenhui; Zhang, Xuemin; Shi, Shushan; Zhang, Yingze

    2013-06-01

    Neuropeptides released from the skeletal nerve fibers have neurotransmitter and immunoregulatory roles; they exert paracrine biological effects on bone cells present close to the nerve endings expressing these signaling molecules. The aims of this study were a systematic investigation of the effects of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), Neuropeptide Y (NPY) and tyrosine hydroxylase (TH) on the cell viability and function of the human osteoblasts, and comparing their difference in the role of regulating bone formation. Cultures of normal human osteoblasts were treated with SP, CGRP, VIP, NPY or TH at three concentrations. We found that each of the five neuropeptides induced increases in cell viability of human osteoblasts. The stimulatory action of NPY was the highest, followed by VIP, SP and TH, while CGRP had the lowest stimulatory effect. The viability index of osteoblasts was inversely associated with the concentration of neuropeptides, and positively with the time of exposure. Moreover, the five neuropeptides increased the ALP activity and osteocalcin to different extents in a dose-dependent manner. The GJIC of osteoblasts was significantly promoted by neuropeptides. The results demonstrated that neuropeptides released from skeletal nerve endings after a stimulus appeared to be able to induce the proliferation and activity of osteoblasts via enhancing GJIC between cells, and further influence the bone formation. These findings may contribute toward a better understanding of the neural influence on bone remodeling and improving treatments related to bone diseases.

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

    LENUS (Irish Health Repository)

    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

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

    LENUS (Irish Health Repository)

    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

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

    LENUS (Irish Health Repository)

    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

  13. Binding and endocytosis of monoterbium transferrin by K562 cells

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Using isotopic labeling of human serum apotransferrin, the binding and the endocytosis of monoterbium transferrin (TbC-apotransferrin, TbC-apotransferrin- FeN) by K562 cells, a human leukemic cell line, have been investigated. There are about (8.58±2.41)×105 binding sites per cell surface at 0℃. The association constant for TbC-apo- transferrin binding is 4.1×107 mol-1@L, for TbC-apo- transferrin-FeN 2.7×107 mol-1@L at 0℃. At pH 7.4, upon warming cells to 37℃, endocytosis starts. The rate constants for the endocytosis are about 0.97 min-1 and 0.31 min-1 and the endocytosis ratio reaches 56% and 80% for TbC-apo- transferrin and TbC-apotransferrin-FeN, respectively.

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

    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......, careful process optimization is required. While macroscopic variations can easily be addressed using the proper spatial power compensation it is more difficult to completely eliminate the micro scale non-uniformity which is intimately linked to the laser beam profile, the amount of overlaps and the scan...... pitch. In this work, we will present micro scale sheet resistance uniformity measurements for shallow 0.5 keV B junctions and zoom in on the underlying effect of multiple subsequent laser scans. A variety of characterization techniques are used to extract the relevant junction parameters and the role...

  16. Probing endocytosis from the enterocyte brush border using fluorescent lipophilic dyes

    DEFF Research Database (Denmark)

    Danielsen, E Michael

    2015-01-01

    The small intestinal brush border is a specialized cell membrane that needs to withstand the solubilizing effect of bile salts during assimilation of dietary nutrients and to achieve detergent resistance; it is highly enriched in glycolipids organized in lipid raft microdomains. In the present work......-toluenesulfonate), and CellMask Orange plasma membrane stain were used to study endocytosis from the enterocyte brush border of organ-cultured porcine mucosal explants. All the dyes readily incorporated into the brush border but were not detectably endocytosed by 5 min, indicating a slow uptake compared with other cell types....... At later time points, FM 1-43 clearly appeared in distinct punctae in the terminal web region, previously shown to represent early endosomes (TWEEs). In contrast, the other dyes were relatively "endocytosis resistant" to varying degrees for periods up to 2 h, indicating an active sorting of lipids...

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

    Science.gov (United States)

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

    2016-09-15

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

  18. Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals.

    Science.gov (United States)

    Marland, Jamie Roslin Keynes; Hasel, Philip; Bonnycastle, Katherine; Cousin, Michael Alan

    2016-01-29

    Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. BiPO{sub 4}/BiOBr p–n junction photocatalysts: One-pot synthesis and dramatic visible light photocatalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhang Sheng, E-mail: lzsliu2008@hotmail.com [School of Material Science and Engineering, China University of Mining and Technology, Xuzhou 221116 (China); Wu, Bian Tao [School of Information and Electrical Engineering, China University of Mining and Technology, Xuzhou 221116 (China); Niu, Ji Nan; Feng, Pei Zhong; Zhu, Ya Bo [School of Material Science and Engineering, China University of Mining and Technology, Xuzhou 221116 (China)

    2015-03-15

    Highlights: • BiPO{sub 4}/BiOBr p–n junction photocatalysts were synthesized by a solvothermal method. • BiPO{sub 4}/BiOBr composites were constructed by inlaying BiPO{sub 4} particles into BiOBr microspheres. • BiPO{sub 4}/BiOBr photocatalysts exhibited the enhanced photocatalytic activity. • ·O{sub 2}{sup −} and h{sup +} were the main active species responsible for the degradation of RhB. - Abstract: BiPO{sub 4}/BiOBr p–n junction photocatalysts were successfully synthesized via a facile one-pot solvothermal method. The products were characterized by XRD, FE-SEM, HRTEM, XPS, DRS, PL and EIS. The obtained BiPO{sub 4}/BiOBr composites were constructed by inlaying BiPO{sub 4} nanoparticles into BiOBr hierarchical microspheres. Compared with BiPO{sub 4} and BiOBr, they exhibited significantly enhanced visible light photocatalytic activity towards the degradation of Rhodamine B (RhB). Among them, 10% BiPO{sub 4}/BiOBr showed the maximum value of the activity, whose degradation rate was about three times higher than that of pure BiOBr. The enhanced photocatalytic activity could be attributed to the formation of the BiPO{sub 4}/BiOBr p–n junction, which resulted in the effective separation and transfer of photogenerated electron–hole pairs. Moreover, the trapping experiments confirmed that ·O{sub 2}{sup −} and h{sup +} were the main active species responsible for the degradation of RhB.

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

    Directory of Open Access Journals (Sweden)

    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.

  2. LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.

    Science.gov (United States)

    Luz, Simão; Cihil, Kristine M; Brautigan, David L; Amaral, Margarida D; Farinha, Carlos M; Swiatecka-Urban, Agnieszka

    2014-05-23

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued ΔF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued ΔF508-CFTR in patients with cystic fibrosis.

  3. Substrate-induced ubiquitylation and endocytosis of yeast amino acid permeases.

    Science.gov (United States)

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

    2014-12-01

    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 catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

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

    2016-01-01

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

  7. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  8. 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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Systematic study of shallow junction formation on germanium substrates

    DEFF Research Database (Denmark)

    Hellings, Geert; Rosseel, Erik; Clarysse, Trudo;

    2011-01-01

    Published results on Ge junctions are benchmarked systematically using RS–XJ plots. The electrical activation level required to meet the ITRS targets is calculated. Additionally, new results are presented on shallow furnace-annealed B junctions and shallow laser-annealed As junctions. Co-implanti......Published results on Ge junctions are benchmarked systematically using RS–XJ plots. The electrical activation level required to meet the ITRS targets is calculated. Additionally, new results are presented on shallow furnace-annealed B junctions and shallow laser-annealed As junctions. Co...

  12. Prolonged adenosine A1 receptor activation in hypoxia and pial vessel disruption focal cortical ischemia facilitates clathrin-mediated AMPA receptor endocytosis and long-lasting synaptic inhibition in rat hippocampal CA3-CA1 synapses: differential regulation of GluA2 and GluA1 subunits by p38 MAPK and JNK.

    Science.gov (United States)

    Chen, Zhicheng; Xiong, Cherry; Pancyr, Cassandra; Stockwell, Jocelyn; Walz, Wolfgang; Cayabyab, Francisco S

    2014-07-16

    Activation of presynaptic adenosine A1 receptors (A1Rs) causes substantial synaptic depression during hypoxia/cerebral ischemia, but postsynaptic actions of A1Rs are less clear. We found that A1Rs and GluA2-containing AMPA receptors (AMPARs) form stable protein complexes from hippocampal brain homogenates and cultured hippocampal neurons from Sprague Dawley rats. In contrast, adenosine A2A receptors (A2ARs) did not coprecipitate or colocalize with GluA2-containing AMPARs. Prolonged stimulation of A1Rs with the agonist N(6)-cyclopentyladenosine (CPA) caused adenosine-induced persistent synaptic depression (APSD) in hippocampal brain slices, and APSD levels were blunted by inhibiting clathrin-mediated endocytosis of GluA2 subunits with the Tat-GluA2-3Y peptide. Using biotinylation and membrane fractionation assays, prolonged CPA incubation showed significant depletion of GluA2/GluA1 surface expression from hippocampal brain slices and cultured neurons. Tat-GluA2-3Y peptide or dynamin inhibitor Dynasore prevented CPA-induced GluA2/GluA1 internalization. Confocal imaging analysis confirmed that functional A1Rs, but not A2ARs, are required for clathrin-mediated AMPAR endocytosis in hippocampal neurons. Pharmacological inhibitors or shRNA knockdown of p38 MAPK and JNK prevented A1R-mediated internalization of GluA2 but not GluA1 subunits. Tat-GluA2-3Y peptide or A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine also prevented hypoxia-mediated GluA2/GluA1 internalization. Finally, in a pial vessel disruption cortical stroke model, a unilateral cortical lesion compared with sham surgery reduced hippocampal GluA2, GluA1, and A1R surface expression and also caused synaptic depression in hippocampal slices that was consistent with AMPAR downregulation and decreased probability of transmitter release. Together, these results indicate a previously unknown mechanism for A1R-induced persistent synaptic depression involving clathrin-mediated GluA2 and GluA1 internalization that

  13. Endocytosis of desmosomal plaques depends on intact actin filaments and leads to a nondegradative compartment

    DEFF Research Database (Denmark)

    Holm, Pernille K.; Hansen, Steen H.; Sandvig, Kirsten;

    1993-01-01

    Cellebiologi, human epithelial cell line, growth inhibition, desmosomes, clathrin-independent endocytosis, cytoskeleton, nondegradative compartment......Cellebiologi, human epithelial cell line, growth inhibition, desmosomes, clathrin-independent endocytosis, cytoskeleton, nondegradative compartment...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

    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

  17. Cluster B personality symptoms in persons at genetic risk for schizophrenia are associated with social competence and activation of the right temporo-parietal junction during emotion processing.

    Science.gov (United States)

    Goldschmidt, Micaela Giuliana; Villarreal, Mirta Fabiana; de Achával, Delfina; Drucaroff, Lucas Javier; Costanzo, Elsa Yolanda; Castro, Mariana Nair; Pahissa, Jaime; Camprodon, Joan; Nemeroff, Charles; Guinjoan, Salvador Martín

    2014-01-30

    Personality disorders are common in nonpsychotic siblings of patients with schizophrenia, and some personality traits in this group may be associated with an increased risk for full-blown psychosis. We sought to establish if faulty right-hemisphere activation induced by social cognitive tasks, as previously described in patients with schizophrenia, is associated with specific personality symptoms in their unaffected siblings. We observed that cluster B personality symptoms in this group were inversely related to activation in the right temporo parietal junction (rTPJ, a structure critical in social cognitive processing) in response to a basic emotion processing task and also to social competence, whereas in contrast to our initial hypothesis, cluster A traits were not associated with right hemisphere activation during emotion processing or with social competence. These findings suggest the existence of clinical traits in at-risk individuals which share a common neurobiological substrate with schizophrenia, in regards to social performance.

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

    Directory of Open Access Journals (Sweden)

    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. Cdk5 is essential for synaptic vesicle endocytosis

    DEFF Research Database (Denmark)

    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 dynamin...

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

    OpenAIRE

    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.

  1. High-density lipoprotein endocytosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Stefanie; Fruhwürth; Margit; Pavelka; Robert; Bittman; Werner; J; Kovacs; Katharina; M; Walter; Clemens; Rhrl; Herbert; Stangl

    2013-01-01

    AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

  2. Molecular electronic junction transport

    DEFF Research Database (Denmark)

    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 f...

  3. Alpha-synuclein promotes clathrin-mediated endocytosis of NMDA receptors in dopaminergic cells

    Institute of Scientific and Technical Information of China (English)

    Shun Yu; Furong Cheng; Xin Li; Yaohua Li; Tao Wang; Guangwei Liu; Andrius Baskys

    2012-01-01

    Loss of dopaminergic i a compensatory increase in nput to the striatum associated with Parkinson' s disease brings about glutamate release onto the dopaminergic cell bodies in the substantia nigra pars compacta (SNpc)[1] Glutamate over-activation of NMDA receptors on these cells can cause excitotoxicity and contribute to their further loss. NMDA receptor-mediated neuronal death is reduced by group I mGluR-mediated up-regulation of endocytosis protein RAB5B[2.3] Among proteins shown to interact with RAB5 proteins is a-synuclein

  4. Solitons in Josephson junctions

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Membrane Mechanics of Endocytosis in Cells with Turgor

    CERN Document Server

    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...

  11. Unconventional myosins, actin dynamics and endocytosis: a ménage à trois?

    Science.gov (United States)

    Soldati, Thierry

    2003-06-01

    Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation. PMID:12753645

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Architectural remodeling of the tonoplast during fluid-phase endocytosis.

    Science.gov (United States)

    Etxeberria, Ed; Gonzalez, Pedro; Pozueta-Romero, Javier

    2013-07-01

    During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration. PMID:23656870

  15. [6]-Gingerol Prevents Disassembly of Cell Junctions and Activities of MMPs in Invasive Human Pancreas Cancer Cells through ERK/NF-κB/Snail Signal Transduction Pathway

    Directory of Open Access Journals (Sweden)

    Sung Ok Kim

    2013-01-01

    Full Text Available To study the effects of [6]-gingerol, a ginger phytochemical, on tight junction (TJ molecules, we investigated TJ tightening and signal transduction pathways in human pancreatic duct cell-derived cancer cell line PANC-1. The following methods were utilized: MTT assay to determine cytotoxicity; zymography to examine matrix metalloproteinase (MMP activities; transepithelial electrical resistance (TER and paracellular flux for TJ measurement; RT-PCR and immunoblotting for proteins related to TJ and invasion; and EMSA for NF-κB activity in PANC-1 cells. Results revealed that TER significantly increased and claudin 4 and MMP-9 decreased compared to those of the control. TJ protein levels, including zonula occludens (ZO- 1, occludin, and E-cadherin, increased in [6]-gingerol-treated cells, which correlated with a decrease in paracellular flux and MMP activity. Furthermore, NF-κB/Snail nuclear translocation was suppressed via downregulation of the extracellular signal-regulated kinase (ERK pathway in response to [6]-gingerol treatment. Moreover, treatment with U0126, an ERK inhibitor, completely blocked NF-κB activity. In conclusion, these findings demonstrate that [6]-gingerol regulates TJ-related proteins and suppresses invasion and metastasis through NF-κB/Snail inhibition via inhibition of the ERK pathway. Therefore, [6]-gingerol may suppress the invasive activity of PANC-1 cells.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  17. Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry▿

    Science.gov (United States)

    Hernaez, Bruno; Alonso, Covadonga

    2010-01-01

    African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na+/H+ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed. PMID:19939916

  18. Ubiquitin-Mediated Regulation of Endocytosis by Proteins of the Arrestin Family

    Directory of Open Access Journals (Sweden)

    Michel Becuwe

    2012-01-01

    Full Text Available In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an “arrest” of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.

  19. TGF-β Signaling Is Associated with Endocytosis at the Pocket Region of the Primary Cilium

    DEFF Research Database (Denmark)

    Clement, Christian Alexandro; Ajbro, Katrine Dalsgaard; Koefoed, Karen;

    2013-01-01

    Transforming growth factor β (TGF-β) signaling is regulated by clathrin-dependent endocytosis (CDE) for the control of cellular processes during development and in tissue homeostasis. The primary cilium coordinates several signaling pathways, and the pocket surrounding the base and proximal part...... of the cilium is a site for CDE. We report here that TGF-β receptors localize to the ciliary tip and endocytic vesicles at the ciliary base in fibroblasts and that TGF-β stimulation increases receptor localization and activation of SMAD2/3 and ERK1/2 at the ciliary base. Inhibition of CDE reduced TGF......-β-mediated signaling at the cilium, and TGF-β signaling and CDE activity are reduced at stunted primary cilia in Tg737(orpk) fibroblasts. Similarly, TGF-β signaling during cardiomyogenesis correlated with accumulation of TGF-β receptors and activation of SMAD2/3 at the ciliary base. Our results indicate...

  20. Zipper and freeway shear zone junctions

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Aniek van der Vaart

    2015-12-01

    Full Text Available Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL, resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis.

  5. Trypanosoma cruzi Intracellular Amastigotes Isolated by Nitrogen Decompression Are Capable of Endocytosis and Cargo Storage in Reservosomes.

    Directory of Open Access Journals (Sweden)

    Cassiano Martin Batista

    Full Text Available Epimastigote forms of Trypanosoma cruzi (the etiologic agent of Chagas disease internalize and store extracellular macromolecules in lysosome-related organelles (LROs called reservosomes, which are positive for the cysteine protease cruzipain. Despite the importance of endocytosis for cell proliferation, macromolecule internalization remains poorly understood in the most clinically relevant proliferative form, the intracellular amastigotes found in mammalian hosts. The main obstacle was the lack of a simple method to isolate viable intracellular amastigotes from host cells. In this work we describe the fast and efficient isolation of viable intracellular amastigotes by nitrogen decompression (cavitation, which allowed the analysis of amastigote endocytosis, with direct visualization of internalized cargo inside the cells. The method routinely yielded 5x10(7 amastigotes--with typical shape and positive for the amastigote marker Ssp4--from 5x10(6 infected Vero cells (48 h post-infection. We could visualize the endocytosis of fluorescently-labeled transferrin and albumin by isolated intracellular amastigotes using immunofluorescence microscopy; however, only transferrin endocytosis was detected by flow cytometry (and was also analyzed by western blotting, suggesting that amastigotes internalized relatively low levels of albumin. Transferrin binding to the surface of amastigotes (at 4°C and its uptake (at 37°C were confirmed by binding dissociation assays using acetic acid. Importantly, both transferrin and albumin co-localized with cruzipain in amastigote LROs. Our data show that isolated T. cruzi intracellular amastigotes actively ingest macromolecules from the environment and store them in cruzipain-positive LROs functionally related to epimastigote reservosomes.

  6. DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans.

    Science.gov (United States)

    van der Vaart, Aniek; Rademakers, Suzanne; Jansen, Gert

    2015-12-01

    Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT) plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL), resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis.

  7. Magnetic tunnel junctions (MTJs)

    Institute of Scientific and Technical Information of China (English)

    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.

  8. Stacked Josephson Junctions

    DEFF Research Database (Denmark)

    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...

  9. An ultrasensitive sorting mechanism for EGF Receptor Endocytosis

    Directory of Open Access Journals (Sweden)

    Dikic Ivan

    2008-04-01

    Full Text Available Abstract Background The Epidermal Growth Factor (EGF receptor has been shown to internalize via clathrin-independent endocytosis (CIE in a ligand concentration dependent manner. From a modeling point of view, this resembles an ultrasensitive response, which is the ability of signaling networks to suppress a response for low input values and to increase to a pre-defined level for inputs exceeding a certain threshold. Several mechanisms to generate this behaviour have been described theoretically, the underlying assumptions of which, however, have not been experimentally demonstrated for the EGF receptor internalization network. Results Here, we present a mathematical model of receptor sorting into alternative pathways that explains the EGF-concentration dependent response of CIE. The described mechanism involves a saturation effect of the dominant clathrin-dependent endocytosis pathway and implies distinct steady-states into which the system is forced for low vs high EGF stimulations. The model is minimal since no experimentally unjustified reactions or parameter assumptions are imposed. We demonstrate the robustness of the sorting effect for large parameter variations and give an analytic derivation for alternative steady-states that are reached. Further, we describe extensibility of the model to more than two pathways which might play a role in contexts other than receptor internalization. Conclusion Our main result is that a scenario where different endocytosis routes consume the same form of receptor corroborates the observation of a clear-cut, stimulus dependent sorting. This is especially important since a receptor modification discriminating between the pathways has not been found experimentally. The model is not restricted to EGF receptor internalization and might account for ultrasensitivity in other cellular contexts.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Controlling Cellular Endocytosis at the Nanoscale

    Science.gov (United States)

    Battaglia, Giuseppe

    2011-03-01

    One of the most challenging aspects of drug delivery is the intra-cellular delivery of active agents. Several drugs and especially nucleic acids all need to be delivered within the cell interior to exert their therapeutic action. Small hydrophobic molecules can permeate cell membranes with relative ease, but hydrophilic molecules and especially large macromolecules such as proteins and nucleic acids require a vector to assist their transport across the cell membrane. This must be designed so as to ensure intracellular delivery without compromising cell viability. We have recently achieved this by using pH-sensitive poly(2-(methacryloyloxy)ethyl-phosphorylcholine)- co -poly(2-(diisopropylamino)ethyl methacrylate) (PMPC-PDPA) and poly(ethylene oxide)-co- poly(2-(diisopropylamino)ethyl methacrylate) (PEO-PDPA) diblock copolymers that self-assemble to form vesicles in aqueous solution. These vesicles combine a non-fouling PMPC or PEO block with a pH-sensitive PDPA block and have the ability to encapsulate both hydrophobic molecules within the vesicular membrane and hydrophilic molecules within their aqueous cores. The pH sensitive nature of the PDPA blocks make the diblock copolymers forming stable vesicles at physiological pH but that rapid dissociation of these vesicles occurs between pH 5 and pH 6 to form molecularly dissolved copolymer chains (unimers). We used these vesicles to encapsulate small and large macromolecules and these were successfully delivered intracellularly including nucleic acid, drugs, quantum dots, and antibodies. Dynamic light scattering, zeta potential measurements, and transmission electron microscopy were used to study and optimise the encapsulation processes. Confocal laser scanning microscopy, fluorescence flow cytometry and lysates analysis were used to quantify cellular uptake and to study the kinetics of this process in vitro and in vivo. We show the effective cytosolic delivery of nucleic acids, proteins, hydrophobic molecules

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Active tectonics and kinematic modeling at the triple junction between the East Anatolian Fault, the Dead Sea Fault and the Cyprus Arc

    Science.gov (United States)

    Cakir, Ziyadin; Meghraoui, Mustapha; Masson, Frédéric; Ferry, Matthieu; Ergintav, Semih; Inan, Sedat; Karabaçak, Volkan; Altunel, Erhan

    2010-05-01

    We investigate the kinematics and slip rates of active faults at the Maras-Antakya triple junction in southern Turkey (where the Anatolian, Arabian, African plates and Iskenderun microplate meet) using GPS measurements, active tectonics, paleoseismology and block modeling. Repeated GPS surveys between 1991 and 2004 allow us to determine horizontal velocities at 22 stations located across the East Anatolian Fault (EAF), the Dead Sea Fault (DSF) and the Cyprus Arc (CA). Field observations indicate that toward the southwest at Maras the EAF branches into the SW-NE trending Karatas - Osmaniye Fault segment (KOFS) and the SSW-NNE Karasu Fault (KF) that meets the DSF and the CA around Hatay to the south. The tectonic and geodetic field investigations indicate the existence of the Iskenderun block between the Anatolian and African plates. By using a simple kinematic model, we estimate that the relative left-lateral plate motion is 8.9±0.4 mm/yr across the EAF, 5.6±1.7 mm/yr across KOFS and 3.8±2 mm/yr across the Karasu fault. We also determine 1.8±1.1 mm/yr for the Karasu normal-component. However, modeling suggest that additional GPS benchmarks are required and in consequence we have recently installed 25 new GPS points (4 permanent and 21 campaign) at the triple junction. Geomorphological and paleoseismic studies yield left-lateral slip rates of 10.8±1 mm/yr and 6.0±0.3 mm/yr, respectively for the EAF and DSF, comparable with those obtained from GPS and deduced from block modeling. The kinematic modeling that combines GPS and tectonic results reveals the predominance of the westward movement of the Anatolian block with the Karasu Valley acting as a large pull-apart basin. Furthermore, we observe that the DSF transforms the Cyprus arc subduction into transpressive tectonics on the East Anatolian fault.

  14. 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

    Institute of Scientific and Technical Information of China (English)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2002-01-01

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

  17. Arrestin-mediated endocytosis of yeast plasma membrane transporters.

    Science.gov (United States)

    Nikko, Elina; Pelham, Hugh R B

    2009-12-01

    Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.

  18. Activation of epidermal toll-like receptor 2 enhances tight junction function – Implications for atopic dermatitis and skin barrier repair

    Science.gov (United States)

    Kuo, I-Hsin; Carpenter-Mendini, Amanda; Yoshida, Takeshi; McGirt, Laura Y.; Ivanov, Andrei I.; Barnes, Kathleen C.; Gallo, Richard L.; Borkowski, Andrew W.; Yamasaki, Kenshi; Leung, Donald Y.; Georas, Steve N.; De Benedetto, Anna; Beck, Lisa A.

    2012-01-01

    Atopic dermatitis (AD) is characterized by epidermal tight junction (TJ) defects and a propensity for Staphylococcus aureus (S. aureus) skin infections. S. aureus is sensed by many pattern recognition receptors including toll-like receptor (TLR) 2. We hypothesized that an effective innate immune response will include skin barrier repair and that this response is impaired in AD subjects. S. aureus-derived peptidoglycan (PGN) and synthetic TLR2 agonists enhanced TJ barrier and increased expression of TJ proteins, CLDN1, CLDN23, occludin and ZO-1 in primary human keratinocytes. A TLR2 agonist enhanced skin barrier recovery in human epidermis wounded by tape-stripping. Tlr2−/− mice had a delayed and incomplete barrier recovery following tape-stripping. AD subjects had reduced epidermal TLR2 expression as compared to nonatopic (NA) subjects, which inversely correlated (r= 0.654, P= 0.0004) with transepidermal water loss (TEWL). These observations indicate that TLR2 activation enhances skin barrier in murine and human skin and is an important part of a wound repair response. Reduced epidermal TLR2 expression observed in AD patients may play a role in their incompetent skin barrier. PMID:23223142

  19. Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network.

    Science.gov (United States)

    Springler, Alexandra; Hessenberger, Sabine; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    Deoxynivalenol (DON), produced by the plant pathogens Fusarium graminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways. PMID:27618100

  20. Early Activation of MAPK p44/42 Is Partially Involved in DON-Induced Disruption of the Intestinal Barrier Function and Tight Junction Network

    Science.gov (United States)

    Springler, Alexandra; Hessenberger, Sabine; Schatzmayr, Gerd; Mayer, Elisabeth

    2016-01-01

    Deoxynivalenol (DON), produced by the plant pathogens Fusarium graminearum and Fusarium culmorum, is one of the most common mycotoxins, contaminating cereal and cereal-derived products. Although worldwide contamination of food and feed poses health threats to humans and animals, pigs are particularly susceptible to this mycotoxin. DON derivatives, such as deepoxy-deoxynivalenol (DOM-1), are produced by bacterial transformation of certain intestinal bacteria, which are naturally occurring or applied as feed additives. Intestinal epithelial cells are the initial barrier against these food- and feed-borne toxins. The present study confirms DON-induced activation of MAPK p44/42 and inhibition of p44/42 by MAPK-inhibitor U0126 monoethanolate. Influence of DON and DOM-1 on transepithelial electrical resistance (TEER), viability and expression of seven tight junction proteins (TJ), as well as the potential of U0126 to counteract DON-induced effects, was assessed. While DOM-1 showed no effect, DON significantly reduced TEER of differentiated IPEC-J2 and decreased expression of claudin-1 and -3, while leaving claudin-4; ZO-1, -2, and -3 and occludin unaffected. Inhibition of p44/42 counteracted DON-induced TEER decrease and restored claudin-3, but not claudin-1 expression. Therefore, effects of DON on TEER and claudin-3 are at least partially p44/42 mediated, while effects on viability and claudin-1 are likely mediated via alternative pathways. PMID:27618100

  1. Hierarchical classification strategy for Phenotype extraction from epidermal growth factor receptor endocytosis screening

    NARCIS (Netherlands)

    L. Cao (Lu); M. Graauw (Marjo de); K. Yan (Kuan); L.C.J. Winkel (Leah C.J.); F.J. Verbeek (Fons)

    2016-01-01

    textabstractBackground: Endocytosis is regarded as a mechanism of attenuating the epidermal growth factor receptor (EGFR) signaling and of receptor degradation. There is increasing evidence becoming available showing that breast cancer progression is associated with a defect in EGFR endocytosis. In

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

    NARCIS (Netherlands)

    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.

  3. Flotillin-1 is essential for PKC-triggered endocytosis and membrane microdomain localization of DAT.

    Science.gov (United States)

    Cremona, M Laura; Matthies, Heinrich J G; Pau, Kelvin; Bowton, Erica; Speed, Nicole; Lute, Brandon J; Anderson, Monique; Sen, Namita; Robertson, Sabrina D; Vaughan, Roxanne A; Rothman, James E; Galli, Aurelio; Javitch, Jonathan A; Yamamoto, Ai

    2011-04-01

    Plasmalemmal neurotransmitter transporters (NTTs) regulate the level of neurotransmitters, such as dopamine (DA) and glutamate, after their release at brain synapses. Stimuli including protein kinase C (PKC) activation can lead to the internalization of some NTTs and a reduction in neurotransmitter clearance capacity. We found that the protein Flotillin-1 (Flot1), also known as Reggie-2, was required for PKC-regulated internalization of members of two different NTT families, the DA transporter (DAT) and the glial glutamate transporter EAAT2, and we identified a conserved serine residue in Flot1 that is essential for transporter internalization. Further analysis revealed that Flot1 was also required to localize DAT within plasma membrane microdomains in stable cell lines, and was essential for amphetamine-induced reverse transport of DA in neurons but not for DA uptake. In sum, our findings provide evidence for a critical role of Flot1-enriched membrane microdomains in PKC-triggered DAT endocytosis and the actions of amphetamine.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Al-Hussein, M., E-mail: m.alhussein@ju.edu.jo [Department of Physics, University of Jordan, Amman 11942 (Jordan); Hesse, H.C.; Weickert, J. [Ludwig-Maximilians-University Munich, Department of Physics and Center for NanoScience(CeNS), Amalienstr.54, 80799 Munich (Germany); Doessel, L.; Feng, X.; Muellen, K. [Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz (Germany); Schmidt-Mende, L. [Ludwig-Maximilians-University Munich, Department of Physics and Center for NanoScience(CeNS), Amalienstr.54, 80799 Munich (Germany)

    2011-10-31

    We investigate thin blend films of phenyl-substituted hexa-peri-hexabenzocoronenes (HBC) with various alkyl side chain lengths ((CH{sub 2})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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Absent activation in medial prefrontal cortex and temporoparietal junction but not superior temporal sulcus during the perception of biological motion in schizophrenia: a functional MRI study

    Directory of Open Access Journals (Sweden)

    Hashimoto N

    2014-11-01

    Full Text Available Naoki Hashimoto,1,2 Atsuhito Toyomaki,1 Masahiro Hirai,3 Tamaki Miyamoto,1 Hisashi Narita,1 Ryo Okubo,1 Ichiro Kusumi1 1Department of Psychiatry, Graduate School of Medicine, Hokkaido University, Sapporo, Japan; 2Child and Adolescent Psychiatry, Department of Psychiatry, University of California, San Francisco, CA, USA; 3Center for Development of Advanced Medical Technology, Jichi Medical University, Yakushiji, Shimotsuke, Tochigi, Japan Background: Patients with schizophrenia show disturbances in both visual perception and social cognition. Perception of biological motion (BM is a higher-level visual process, and is known to be associated with social cognition. BM induces activation in the “social brain network”, including the superior temporal sulcus (STS. Although deficits in the detection of BM and atypical activation in the STS have been reported in patients with schizophrenia, it remains unclear whether other nodes of the “social brain network” are also atypical in patients with schizophrenia.Purpose: We aimed to explore whether brain regions other than STS were involved during BM perception in patients with schizophrenia, using functional magnetic resonance imaging (fMRI.Methods and patients: Seventeen patients with schizophrenia, and 17 age- and sex- matched healthy controls, underwent fMRI scanning during a one-back visual task, containing three experimental conditions: (1 BM, (2 scrambled motion (SM, and (3 static condition. We used one-sample t-tests to examine neural responses selective to BM versus SM within each group, and two-sample t-tests to directly compare neural patterns to BM versus SM in schizophrenics versus controls.Results: We found significant activation in the STS region when BM was contrasted with SM in both groups, with no significant difference between groups. On the contrary, significant activation in the medial prefrontal cortex (MPFC and bilateral temporoparietal junction (TPJ was found only in the

  10. Equivalent Josephson junctions

    Science.gov (United States)

    Boyadjiev, T. L.; Semerdjieva, E. G.; Shukrinov, Yu. M.

    2008-01-01

    The magnetic field dependences of critical current are numerically constructed for a long Josephson junction with a shunt-or resistor-type microscopic inhomogeneities and compared to the critical curve of a junction with exponentially varying width. The numerical results show that it is adequate to replace the distributed inhomogeneity of a long Josephson junction by an inhomogeneity localized at one of its ends, which has certain technological advantages. It is also shown that the critical curves of junctions with exponentially varying width and inhomogeneities localized at the ends are unaffected by the mixed fluxon-antifluxon distributions of the magnetic flow. This fact may explain the improvement of the spectra of microwave radiation noted in the literature.

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

    Science.gov (United States)

    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

  12. Quantum Junction Solar Cells

    KAUST Repository

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  16. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

    Directory of Open Access Journals (Sweden)

    Kathleen Busman-Sahay

    Full Text Available Following antigen recognition, B cell receptor (BCR-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2 is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs. Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

  17. A feedback loop between dynamin and actin recruitment during clathrin-mediated endocytosis.

    Directory of Open Access Journals (Sweden)

    Marcus J Taylor

    Full Text Available Clathrin-mediated endocytosis proceeds by a sequential series of reactions catalyzed by discrete sets of protein machinery. The final reaction in clathrin-mediated endocytosis is membrane scission, which is mediated by the large guanosine triophosphate hydrolase (GTPase dynamin and which may involve the actin-dependent recruitment of N-terminal containing BIN/Amphiphysin/RVS domain containing (N-BAR proteins. Optical microscopy has revealed a detailed picture of when and where particular protein types are recruited in the ∼20-30 s preceding scission. Nevertheless, the regulatory mechanisms and functions that underpin protein recruitment are not well understood. Here we used an optical assay to investigate the coordination and interdependencies between the recruitment of dynamin, the actin cytoskeleton, and N-BAR proteins to individual clathrin-mediated endocytic scission events. These measurements revealed that a feedback loop exists between dynamin and actin at sites of membrane scission. The kinetics of dynamin, actin, and N-BAR protein recruitment were modulated by dynamin GTPase activity. Conversely, acute ablation of actin dynamics using latrunculin-B led to a ∼50% decrease in the incidence of scission, an ∼50% decrease in the amplitude of dynamin recruitment, and abolished actin and N-BAR recruitment to scission events. Collectively these data suggest that dynamin, actin, and N-BAR proteins work cooperatively to efficiently catalyze membrane scission. Dynamin controls its own recruitment to scission events by modulating the kinetics of actin and N-BAR recruitment to sites of scission. Conversely actin serves as a dynamic scaffold that concentrates dynamin and N-BAR proteins at sites of scission.

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

    OpenAIRE

    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...

  19. Chronic ethanol consumption in rats produces opioid antinociceptive tolerance through inhibition of mu opioid receptor endocytosis.

    Directory of Open Access Journals (Sweden)

    Li He

    Full Text Available It is well known that the mu-opioid receptor (MOR plays an important role in the rewarding properties of ethanol. However, it is less clear how chronic ethanol consumption affects MOR signaling. Here, we demonstrate that rats with prolonged voluntary ethanol consumption develop antinociceptive tolerance to opioids. Signaling through the MOR is controlled at many levels, including via the process of endocytosis. Importantly, agonists at the MOR that promote receptor endocytosis, such as the endogenous peptides enkephalin and β-endorphin, show a reduced propensity to promote antinociceptive tolerance than do agonists, like morphine, which do not promote receptor endocytosis. These observations led us to examine whether chronic ethanol consumption produced opioid tolerance by interfering with MOR endocytosis. Indeed, here we show that chronic ethanol consumption inhibits the endocytosis of MOR in response to opioid peptide. This loss of endocytosis was accompanied by a dramatic decrease in G protein coupled receptor kinase 2 (GRK2 protein levels after chronic drinking, suggesting that loss of this component of the trafficking machinery could be a mechanism by which endocytosis is lost. We also found that MOR coupling to G-protein was decreased in ethanol-drinking rats, providing a functional explanation for loss of opioid antinociception. Together, these results suggest that chronic ethanol drinking alters the ability of MOR to endocytose in response to opioid peptides, and consequently, promotes tolerance to the effects of opioids.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Endocytosis of VAMP is facilitated by a synaptic vesicle targeting signal

    Science.gov (United States)

    1996-01-01

    After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles. PMID:8647886

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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

    International Nuclear Information System (INIS)

    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. Junction-FET dosimeter

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 75 FR 76294 - Radio Broadcasting Services: Pacific Junction, IA

    Science.gov (United States)

    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...

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

    OpenAIRE

    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...

  10. Recording the dynamic endocytosis of single gold nanoparticles by AFM-based force tracing

    Science.gov (United States)

    Ding, Bohua; Tian, Yongmei; Pan, Yangang; Shan, Yuping; Cai, Mingjun; Xu, Haijiao; Sun, Yingchun; Wang, Hongda

    2015-04-01

    We utilized force tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NP invagination are accurately determined at the single-particle and microsecond level unprecedentedly.We utilized force tracing to directly record the endocytosis of single gold nanoparticles (Au NPs) with different sizes, revealing the size-dependent endocytosis dynamics and the crucial role of membrane cholesterol. The force, duration and velocity of Au NP invagination are accurately determined at the single-particle and microsecond level unprecedentedly. Electronic supplementary information (ESI) available: Details of the experimental procedures and the results of the control experiments. See DOI: 10.1039/c5nr01020a

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Bradykinin release avoids high molecular weight kininogen endocytosis.

    Directory of Open Access Journals (Sweden)

    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

  13. Actin-Based Feedback Circuits in Cell Migration and Endocytosis

    Science.gov (United States)

    Wang, Xinxin

    In this thesis, we study the switch and pulse functions of actin during two important cellular processes, cell migration and endocytosis. Actin is an abundant protein that can polymerize to form a dendritic network. The actin network can exert force to push or bend the cell membrane. During cell migration, the actin network behaves like a switch, assembling mostly at one end or at the other end. The end with the majority of the actin network is the leading edge, following which the cell can persistently move in the same direction. The other end, with the minority of the actin network, is the trailing edge, which is dragged by the cell as it moves forward. When subjected to large fluctuations or external stimuli, the leading edge and the trailing edge can interchange and change the direction of motion, like a motion switch. Our model of the actin network in a cell reveals that mechanical force is crucial for forming the motion switch. We find a transition from single state symmetric behavior to switch behavior, when tuning parameters such as the force. The model is studied by both stochastic simulations, and a set of rate equations that are consistent with the simulations. Endocytosis is a process by which cells engulf extracellular substances and recycle the cell membrane. In yeast cells, the actin network is transiently needed to overcome the pressure difference across the cell membrane caused by turgor pressure. The actin network behaves like a pulse, which assembles and then disassembles within about 30 seconds. Using a stochastic model, we reproduce the pulse behaviors of the actin network and one of its regulatory proteins, Las17. The model matches green fluorescence protein (GFP) experiments for wild-type cells. The model also predicts some phenotypes that modify or diminish the pulse behavior. The phenotypes are verified with both experiments performed at Washington University and with other groups' experiments. We find that several feedback mechanisms are

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  16. Screening of anti-hypoxia/reoxygenation agents by an in vitro method. Part 2: Inhibition of tyrosine kinase activation prevented hypoxia/reoxygenation-induced injury in endothelial gap junctional intercellular communication.

    Science.gov (United States)

    Zhang, Y W; Morita, I; Zhang, L; Shao, G; Yao, X S; Murota, S

    2000-03-01

    In this study, we demonstrated that hypoxia/reoxygenation (H/R) induced an injury in gap junctional intercellular communication (GJIC) after 2 h of reoxygenation in cultured HUVEC. Free radical scavenger (DMSO) and antioxidant (SOD) did not prevent this GJIC injury at all. Protein kinase C inhibitor (calphostin C) partly blocked this injury. However, the protein tyrosine kinase (PTK) inhibitor genistein completely inhibited this GJIC injury. Compounds 1 [laxogenin-3-O-alpha-L-arabinosyl-(1-->6)- beta-D-glucopyranoside], 2 (macrostemososide A), 3 [laxogenin-3-O-beta-D-xylopyranosyl-(1-->4)-alpha- L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside], 4 (chinenoside II), 5 (beta-sitosterol), 6 (daucosterine), 7 (ginsenoside-Rd), 29 (isocumarine), 52 (icariin), 53 (icariside), and 54 (icaritin), which showed obvious influence on H/R-induced PTK activation as stated in Part 1 (except 1), were explored for their effects on GJIC. The results showed that compounds 2-7 and 52-57 partly protected H/R-induced GJIC injury. Compounds 5 and 6 (especially 5), which showed the strongest inhibitory effects on PTK activation, completely blocked H/R-provoked GJIC injury. Compound 1, which did not influence PTK activation, failed to prevent this GJIC injury. In contrast, compound 29, which significantly promoted PTK activation, enhanced this H/R-induced GJIC injury further. Western blotting of connexin 43, an important gap junctional protein for modulating GJIC in HUVEC, revealed that interference with the gap junctional protein might be the most direct mechanism for compounds 2, 5, 29, and 53 to affect H/R-injured GJIC. PMID:10763583

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

    Directory of Open Access Journals (Sweden)

    Jiandong Sun

    2015-07-01

    Full Text Available 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 Ca2+ 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.

  18. Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor

    Institute of Scientific and Technical Information of China (English)

    Hua JIANG; Jie ZHANG; Bi-zhi SHI; Yu-hong XU; Zong-hai LI; Jian-ren GU

    2007-01-01

    Aim: To develop a simple method for monitoring protein localization of epidermal growth factor (EGF) in living cells. Methods: Enhanced green fluorescent protein (EGFP) was used as an autofluorescent tag to label EGF ligands. SDS-PAGE and Western blot analysis were used to detect the expression of the EGFP-tagged EGF (EGFP-EGF) protein. The cell-binding and internalization activity of EGFP-EGF were analyzed by fluorescence-activated cell sorting (FACS) and confocal micro-scopy. Results: EGFP-EGF protein was expressed in Escherichia coil and purified.A cell-binding assay demonstrated that the EGFP-EGF protein could bind effi-ciently to the cells expressing EGFR. The binding and intemalization of EGFP-EGF can be visualized even at a very low concentration under confocal microscopy.The FACS-based assay for internalization activity indicated the accumulation of internalized EGFP-EGF over time. Furthermore, the results of the competition assay indicated its EGFR binding specificity. Using such a method, it does not need to label EGF with chemicals and avoid light in the experimental process. Conclusion: The fusion protein EGFP-EGF has several characters including high sensitivity, stability and convenience for manipulation, and is a powerful tool for the study of EGF endocytosis.

  19. TGF-β Signaling Is Associated with Endocytosis at the Pocket Region of the Primary Cilium

    Directory of Open Access Journals (Sweden)

    Christian Alexandro Clement

    2013-06-01

    Full Text Available Transforming growth factor β (TGF-β signaling is regulated by clathrin-dependent endocytosis (CDE for the control of cellular processes during development and in tissue homeostasis. The primary cilium coordinates several signaling pathways, and the pocket surrounding the base and proximal part of the cilium is a site for CDE. We report here that TGF-β receptors localize to the ciliary tip and endocytic vesicles at the ciliary base in fibroblasts and that TGF-β stimulation increases receptor localization and activation of SMAD2/3 and ERK1/2 at the ciliary base. Inhibition of CDE reduced TGF-β-mediated signaling at the cilium, and TGF-β signaling and CDE activity are reduced at stunted primary cilia in Tg737orpk fibroblasts. Similarly, TGF-β signaling during cardiomyogenesis correlated with accumulation of TGF-β receptors and activation of SMAD2/3 at the ciliary base. Our results indicate that the primary cilium regulates TGF-β signaling and that the ciliary pocket is a compartment for CDE-dependent regulation of signal transduction.

  20. A yeast t-SNARE involved in endocytosis.

    Science.gov (United States)

    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

  1. Ouabain uptake by endocytosis in isolated guinea pig atria

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

    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

  3. Interaction between small GTPase Rab7 and PI3KC3 links autophagy and endocytosis: A new Rab7 effector protein sheds light on membrane trafficking pathways.

    Science.gov (United States)

    Lin, Mary Grace; Zhong, Qing

    2011-03-01

    Endocytosis and autophagy are both membrane trafficking pathways vital for cell survival. Endocytosis, the primary means by which cells internalize material such as cell-surface receptors and their protein ligands, is essential for proper cell growth and communication. Autophagy is a catabolic process that degrades cargo ranging from organelles to protein aggregates to bacteria, and it is important for maintaining cellular homeostasis. Defects in both endosome and autophagosome maturation lead to an array of human diseases, including cancer; however, the molecular mechanisms underlying endosome and autophagosome maturation are not well characterized. In the case of endocytosis, small GTPases, key players in membrane organization, are required for endosome maturation. Specifically, activation of the small GTPase Rab7 is required for the initiation of the early-to-late endosome transition, although how this is regulated is largely unknown. Now recent findings from our laboratory show that Rubicon, a component of the PI3KC3 complex, inhibits endosome maturation by preventing activation of Rab7. Not only do our results clarify the molecular link between PI3KC3 and Rab7 function in endosome maturation, they lead us to propose new models for PI3KC3 involvement in membrane trafficking, particularly at the convergence between the endosome and autophagosome pathways.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  9. Wireless Josephson Junction Arrays

    Science.gov (United States)

    Adams, Laura

    2015-03-01

    We report low temperature, microwave transmission measurements on a wireless two- dimensional network of Josephson junction arrays composed of superconductor-insulator -superconductor tunnel junctions. Unlike their biased counterparts, by removing all electrical contacts to the arrays and superfluous microwave components and interconnects in the transmission line, we observe new collective behavior in the transmission spectra. In particular we will show emergent behavior that systematically responds to changes in microwave power at fixed temperature. Likewise we will show the dynamic and collective response of the arrays while tuning the temperature at fixed microwave power. We discuss these spectra in terms of the Berezinskii-Kosterlitz-Thouless phase transition and Shapiro steps. We gratefully acknowledge the support Prof. Steven Anlage at the University of Maryland and Prof. Allen Goldman at the University of Minnesota. Physics and School of Engineering and Applied Sciences.

  10. Recruitment of endocytosis in sonopermeabilization-mediated drug delivery: a real-time study

    Science.gov (United States)

    Derieppe, Marc; Rojek, Katarzyna; Escoffre, Jean-Michel; de Senneville, Baudouin Denis; Moonen, Chrit; Bos, Clemens

    2015-07-01

    Microbubbles (MBs) in combination with ultrasound (US) can enhance cell membrane permeability, and have the potential to facilitate the cellular uptake of hydrophilic molecules. However, the exact mechanism behind US- and MB-mediated intracellular delivery still remains to be fully understood. Among the proposed mechanisms are formation of transient pores and endocytosis stimulation. In our study, we investigated whether endocytosis is involved in US- and MB-mediated delivery of small molecules. Dynamic fluorescence microscopy was used to investigate the effects of endocytosis inhibitors on the pharmacokinetic parameters of US- and MB-mediated uptake of SYTOX Green, a 600 Da hydrophilic model drug. C6 rat glioma cells, together with SonoVue® MBs, were exposed to 1.4 MHz US waves at 0.2 MPa peak-negative pressure. Collection of the signal intensity in each individual nucleus was monitored during and after US exposure by a fibered confocal fluorescence microscope designed for real-time imaging. Exposed to US waves, C6 cells pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, showed up to a 2.5-fold significant increase of the uptake time constant, and a 1.1-fold increase with genistein, an inhibitor of caveolae-mediated endocytosis. Both inhibitors slowed down the US-mediated uptake of SYTOX Green. With C6 cells and our experimental settings, these quantitative data indicate that endocytosis plays a role in sonopermeabilization-mediated delivery of small molecules with a more predominant contribution of clathrin-mediated endocytosis.

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

    Science.gov (United States)

    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

  12. 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.

    Science.gov (United States)

    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

  13. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Josephson junctions with ferromagnetic alloy interlayer

    International Nuclear Information System (INIS)

    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

    Energy Technology Data Exchange (ETDEWEB)

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

    Indian Academy of Sciences (India)

    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.

  17. Alpha-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis.

    Directory of Open Access Journals (Sweden)

    David Freeman

    Full Text Available α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

  18. Clathrin to Lipid Raft-Endocytosis via Controlled Surface Chemistry and Efficient Perinuclear Targeting of Nanoparticle.

    Science.gov (United States)

    Chakraborty, Atanu; Jana, Nikhil R

    2015-09-17

    Nanoparticle interacts with live cells depending on their surface chemistry, enters into cell via endocytosis, and is commonly trafficked to an endosome/lysozome that restricts subcellular targeting options. Here we show that nanoparticle surface chemistry can be tuned to alter their cell uptake mechanism and subcellular trafficking. Quantum dot based nanoprobes of 20-30 nm hydrodynamic diameters have been synthesized with tunable surface charge (between +15 mV to -25 mV) and lipophilicity to influence their cellular uptake processes and subcellular trafficking. It is observed that cationic nanoprobe electrostatically interacts with cell membrane and enters into cell via clathrin-mediated endocytosis. At lower surface charge (between +10 mV to -10 mV), the electrostatic interaction with cell membrane becomes weaker, and additional lipid raft endocytosis is initiated. If a lipophilic functional group is introduced on a weakly anionic nanoparticle surface, the uptake mechanism shifts to predominant lipid raft-mediated endocytosis. In particular, the zwitterionic-lipophilic nanoprobe has the unique advantage as it weakly interacts with anionic cell membrane, migrates toward lipid rafts for interaction through lipophilic functional group, and induces lipid raft-mediated endocytosis. While predominate or partial clathrin-mediated entry traffics most of the nanoprobes to lysozome, predominate lipid raft-mediated entry traffics them to perinuclear region, particularly to the Golgi apparatus. This finding would guide in designing appropriate nanoprobe for subcellular targeting and delivery.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-09-03

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

  20. An induced junction photovoltaic cell

    Science.gov (United States)

    Call, R. L.

    1974-01-01

    Silicon solar cells operating with induced junctions rather than diffused junctions have been fabricated and tested. Induced junctions were created by forming an inversion layer near the surface of the silicon by supplying a sheet of positive charge above the surface. Measurements of the response of the inversion layer cell to light of different wavelengths indicated it to be more sensitive to the shorter wavelengths of the sun's spectrum than conventional cells. The greater sensitivity occurs because of the shallow junction and the strong electric field at the surface.

  1. METAL-SEMICONDUCTOR JUNCTIONS

    OpenAIRE

    Flores, F.; Sánchez-Dehesa, J.; Guinea, F.

    1984-01-01

    In spite of many different models there seems to be a general lack of understanding about the mechanism of Schottky barrier formation. In order to elucidate this mechanism for covalent semiconductors, we present theoretical selfconsistent calculations for abrupt Si-Ag, C-Ag and Si-H-Ag junctions. Our results are in good agreement with the available experimental evidence, and show that the interface Fermi level is controlled by a density of states that appears in the semiconductor gap as a res...

  2. Roles of AP-2 in clathrin-mediated endocytosis.

    Directory of Open Access Journals (Sweden)

    Emmanuel Boucrot

    Full Text Available BACKGROUND: The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway. METHODOLOGY/PRINCIPAL FINDINGS: We report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37 degrees C. If cells are first incubated with LDL at 4 degrees C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37 degrees C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway. CONCLUSIONS/SIGNIFICANCE: These results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain

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

    International Nuclear Information System (INIS)

    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)

  4. Atomic Force Microscopy-based Cell Nanostructure for Ligand-conjugated Quantum Dot Endocytosis

    Institute of Scientific and Technical Information of China (English)

    Yun-Long PAN; Ji-Ye CAI; Li QIN; Hao WANG

    2006-01-01

    While it has been well demonstrated that quantum dots (QDs) play an important role in biological labeling both in vitro and in vivo,there is no report describing the cellular nanostructure basis of receptor-mediated endocytosis. Here, nanostructure evolution responses to the endocytosis of transferrin force microscopy (AFM). AFM-based nanostructure analysis demonstrated that the Tf-conjugated QDs were specifically and tightly bound to the cell receptors rrelated with the cell membrane receptor-mediated transduction.Consistently, confocal microscopic and flow cytometry results have demonstrated the specificity and the internalization of Tf-QD is linearly related to time. Moreover, while the nanoparticles on the cell membrane increased, the endocytosis was still nanoparticles did not interfere sterically with the binding and function of receptors. Therefore, ligand-conjugated QDs are potentially useful in biological labeling of cells at a nanometer scale.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; Torgersen, Maria Lyngaas; Raa, Hilde Andersen;

    2008-01-01

    that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has...... taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts...

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

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Faergeman, Nils J

    2008-01-01

    proximity to the cell membrane. Spatial surface intensity patterns of DHE as well as that of the lipid marker DiIC12 being assessed by statistical image analysis persisted over several minutes in cells having a constant overall curvature. Sites of sterol endocytosis appeared indistinguishable from other...... (CTL) combined with advanced image analysis were used to study spatiotemporal sterol distribution in living macrophages, adipocytes and fibroblasts. Sterol endocytosis was directly visualized by time-lapse imaging and noise-robust tracking revealing confined motion of DHE containing vesicles in close...

  8. 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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    NARCIS (Netherlands)

    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

  10. Study progress of cell endocytosis%细胞内吞作用的研究进展

    Institute of Scientific and Technical Information of China (English)

    Li Chen; Hui Li; Ren Zhao; jianwei Zhu

    2009-01-01

    Endocytosis is a process through which extracellular materials are transported into cell through membrane defor-mation. This process is not a simple step-by-step process in which a series of proteins function according to the chronological order, but rather a complex process comprising many members which are regulated precisely. The role of endocytosis is broadly divided into two categories, phagocytosis and pinocytosis, the latter is divided into four species in accordance with the size of endocytosis substances: dathrin dependent endocytosis, the diameter of dathrin-coated vesicle is 100-150 nm; caveolin dependent endocytosis, the diameter of caveolin protein-coated vesicle is 50-100 nm; macropinocytosis, the diam-eter of macropinocytosis is generally 0.5-2 μm, sometimes up to 5 pro; clathrin and caveolin independent endocytosis. Many proteins including endophilin A1, A2, A3, and endocytotic proteins B, Bla, and B1b as well as dynamin, acUn and Rab protein families are involved in endocytosis and play an important role in different stages. The abnormal endocytosis may be involved in the development of certain diseases.

  11. Josephson junction simulation of neurons

    OpenAIRE

    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...

  12. Fluid Flow at Branching Junctions

    OpenAIRE

    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.

  13. Angelman Syndrome Protein Ube3a Regulates Synaptic Growth and Endocytosis by Inhibiting BMP Signaling in Drosophila.

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Katia Cortese

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

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

    Directory of Open Access Journals (Sweden)

    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

  16. Herlitz junctional epidermolysis bullosa.

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

  18. Lipid rafts-mediated endocytosis and physiology-based cell membrane traffic models of doxorubicin liposomes.

    Science.gov (United States)

    Li, Yinghuan; Gao, Lei; Tan, Xi; Li, Feiyang; Zhao, Ming; Peng, Shiqi

    2016-08-01

    The clathrin-mediated endocytosis is likely a major mechanism of liposomes' internalization. A kinetic approach was used to assess the internalization mechanism of doxorubicin (Dox) loaded cationic liposomes and to establish physiology-based cell membrane traffic mathematic models. Lipid rafts-mediated endocytosis, including dynamin-dependent or -independent endocytosis of noncaveolar structure, was a dominant process. The mathematic models divided Dox loaded liposomes binding lipid rafts (B) into saturable binding (SB) and nonsaturable binding (NSB) followed by energy-driven endocytosis. The intracellular trafficking demonstrated early endosome-late endosome-lysosome or early/late endosome-cytoplasm-nucleus pathways. The three properties of liposome structures, i.e., cationic lipid, fusogenic lipid, and pegylation, were investigated to compare their contributions to cell membrane and intracellular traffic. The results revealed great contribution of cationic lipid DOTAP and fusogenic lipid DOPE to cell membrane binding and internalization. The valid Dox in the nuclei of HepG2 and A375 cells treated with cationic liposomes containing 40mol% of DOPE were 1.2-fold and 1.5-fold higher than that in the nuclei of HepG2 and A375 cells treated with liposomes containing 20mol% of DOPE, respectively, suggesting the dependence of cell type. This tendency was proportional to the increase of cell-associated total liposomal Dox. The mathematic models would be useful to predict intracellular trafficking of liposomal Dox.

  19. E-cadherin mediates adhesion and endocytosis of Aspergillus fumigatus blastospores in human epithelial cells

    Institute of Scientific and Technical Information of China (English)

    XU Xiao-yong; SHI Yi; ZHANG Peng-peng; ZHANG Feng; SHEN Yu-ying; SU Xin; ZHAO Bei-lei

    2012-01-01

    Background Aspergillus fumigatus (A.fumigatus) is a ubiquitous saprophytic fungus responsible for the majority of invasive mold infections in patients undergoing chemotherapy,organ transplantation or with persistent neutropenia.This study aimed to determine the role of E-cadherin for adhesion and endocytosis of A.fumigatus blastospores in the human epithelial cell line A549.Methods A.fumigatus blastospores were incubated with the total protein of A549 to investigate the binding of E-cadherin and blastospores followed by an affinity purification procedure.After establishing the adhesion model,the adhesion and endocytosis of A.fumigatus blastospores by A549 cells were evaluated by down-regulating E-cadherin of A549 cells using blocking antibody or small interfering RNA (siRNA).Results E-cadherin was adhered to the surface of A.fumigatus blastospore.Adhesion and endocytosis of the blastospores were reduced by blocking or down-regulating E-cadherin in A549 cells.Conclusions E-cadherin is a receptor for adhesion and endocytosis of A.fumigatus blastospores in epithelial cells.This may open a new approach to treat this fungal infection.

  20. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    Science.gov (United States)

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  1. Endocytosis of HERG is clathrin-independent and involves arf6.

    Directory of Open Access Journals (Sweden)

    Rucha Karnik

    Full Text Available The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal...

  3. Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.

    Science.gov (United States)

    Meng, Jianghui; Wang, Jiafu; Lawrence, Gary W; Dolly, J Oliver

    2013-08-01

    Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs. PMID:23640057

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  6. TRANSITIONAL FLOW IN CHANNEL JUNCTIONS

    Institute of Scientific and Technical Information of China (English)

    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.

  7. Thermal conductance of superlattice junctions

    Energy Technology Data Exchange (ETDEWEB)

    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.

  8. Thermal conductance of superlattice junctions

    Directory of Open Access Journals (Sweden)

    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.

  9. A simple method for fabricating p–n junction photocatalyst CuFe{sub 2}O{sub 4}/Bi{sub 4}Ti{sub 3}O{sub 12} and its photocatalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, W.; Jin, Y.; Gao, C.H.; Gu, W.; Jin, Z.M. [Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou 215123 (China); Lei, Y.L. [Center of Super-Diamond and Advanced Films (COSDAF) and Department of Physics and Materials Science, City University of Hong Kong, Hong Kong (China); Liao, L.S., E-mail: lsliao@suda.edu.cn [Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou 215123 (China)

    2014-02-14

    The synthesis of Bi{sub 4}Ti{sub 3}O{sub 12} and CuFe{sub 2}O{sub 4} powders was achieved using a conventional solid-state reaction and the Sol–Gel method, respectively. A novel p–n heterojunction photocatalyst CuFe{sub 2}O{sub 4}/Bi{sub 4}Ti{sub 3}O{sub 12} was subsequently prepared through ball milling. The structures, morphologies, and optical properties of the photocatalysts were comprehensively characterized. The transmission electron microscopy (TEM) images showed a clear interface between CuFe{sub 2}O{sub 4} and Bi{sub 4}Ti{sub 3}O{sub 12}, indicating that a heterojunction between CuFe{sub 2}O{sub 4} and Bi{sub 4}Ti{sub 3}O{sub 12} was formed during ball milling. In addition, the photocatalytic activity was evaluated based on the photocatalytic degradation of methyl orange (MO). The results indicated that the photocatalytic activity of the p–n heterojunction photocatalyst CuFe{sub 2}O{sub 4}/Bi{sub 4}Ti{sub 3}O{sub 12} was higher than that of Bi{sub 4}Ti{sub 3}O{sub 12} alone. The enhanced photocatalytic activity could be attributed to the formation of a heterojunction between CuFe{sub 2}O{sub 4} and Bi{sub 4}Ti{sub 3}O{sub 12}, which suppressed the recombination of photogenerated electron–hole pairs. We also investigated the effects of procedure time and dispersant (H{sub 2}O) during ball milling on the photocatalytic activity. The mechanisms underlying the observed photocatalytic activity were also described based on the semiconductor energy band theory and p–n junction principle. Moreover, the analysis of the radical scavengers confirmed that • O{sub 2}{sup −} and h{sup +} were the primary reactive species to cause the degradation of the MO. - Highlights: • A p–n heterojunction photocatalyst CuFe{sub 2}O{sub 4}/Bi{sub 4}Ti{sub 3}O{sub 12} was prepared by ball milling. • Water is added during the wet milling process to make the powders milled efficiently and dispersed highly. • The photocatalytical mechanism is discussed according to

  10. Neuromuscular junctional disorders.

    Science.gov (United States)

    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

  11. Josephson junctions with ferromagnetic interlayer

    International Nuclear Information System (INIS)

    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.

  12. Josephson junctions with ferromagnetic interlayer

    Energy Technology Data Exchange (ETDEWEB)

    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.

  13. Electronic thermometry in tunable tunnel junction

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. 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

    OpenAIRE

    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....

  15. Confocal Annular Josephson Tunnel Junctions

    Science.gov (United States)

    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.

  16. Confocal Annular Josephson Tunnel Junctions

    Science.gov (United States)

    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.

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

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

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

    Directory of Open Access Journals (Sweden)

    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.

  20. t-SNARE Phosphorylation Regulates Endocytosis in Yeast

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    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 ...

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

    NARCIS (Netherlands)

    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

  4. 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.

    Science.gov (United States)

    Nie, Yuxin; Deng, Ping; Zhao, Yayu; Wang, Penglei; Xing, Lili; Zhang, Yan; Xue, Xinyu

    2014-07-01

    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.

  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

    International Nuclear Information System (INIS)

    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. Endocytosis against the high turgor of guard cells

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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...

  11. Sortilin-Mediated Endocytosis Determines Levels of the Fronto-Temporal Dementia Protein, Progranulin

    DEFF Research Database (Denmark)

    Hu, Fenghua; Padukkavidana, Thihan; Vægter, Christian Bjerggaard;

    2010-01-01

    The most common inherited form of Fronto-Temporal Lobar Degeneration (FTLD) known stems from Progranulin (GRN) mutation, and exhibits TDP-43 plus ubiquitin protein aggregates in brain. Despite the causative role of GRN haploinsufficiency in FTLD-TDP, the neurobiology of this secreted glycoprotein......, and is fully normalized by Sort1 ablation. Sortilin-mediated PGRN endocytosis is likely to play a central role in FTLD-TDP pathophysiology...

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

    Science.gov (United States)

    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 ...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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 ...

  15. Endocytosis of fluorescent cyclodextrins by intestinal Caco-2 cells and its role in paclitaxel drug delivery.

    Science.gov (United States)

    Réti-Nagy, Katalin; Malanga, Milo; Fenyvesi, Éva; Szente, Lajos; Vámosi, György; Váradi, Judit; Bácskay, Ildikó; Fehér, Pálma; Ujhelyi, Zoltán; Róka, Eszter; Vecsernyés, Miklós; Balogh, György; Vasvári, Gábor; Fenyvesi, Ferenc

    2015-12-30

    Cyclodextrins are widely used excipients in pharmaceutical formulations. They are mainly utilized as solubilizers and absorption enhancers, but recent results revealed their effects on cell membranes and pharmacological barriers. In addition to the growing knowledge on their interaction with plasma membranes, it was confirmed that cyclodextrins are able to enter cells by endocytosis. The number of the tested cyclodextrins was limited, and the role of this mechanism in drug absorption and delivery is not known. Our aim was to examine the endocytosis of fluorescently labeled hydroxypropyl-β-cyclodextrin, random methyl-β-cyclodextrin and soluble β-cyclodextrin polymer, and the cellular uptake of the fluorescent paclitaxel derivative-random methyl-β-cyclodextrin complex. The studied cyclodextrin derivatives were able to enter Caco-2 intestinal cells and localized in vesicles in the cytoplasm, while their permeability was very limited through Caco-2 monolayers. We demonstrated for the first time that the fluorescent paclitaxel derivative and rhodamine-labeled random methyl-β-cyclodextrin were detected in the same intracellular vesicles after treating cells with their inclusion complex. These results indicate that the endocytosis of cyclodextrin complexes can contribute to drug absorption processes.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  18. AP-2-complex-mediated endocytosis of Drosophila Crumbs regulates polarity by antagonizing Stardust.

    Science.gov (United States)

    Lin, Ya-Huei; Currinn, Heather; Pocha, Shirin Meher; Rothnie, Alice; Wassmer, Thomas; Knust, Elisabeth

    2015-12-15

    Maintenance of epithelial polarity depends on the correct localization and levels of polarity determinants. The evolutionarily conserved transmembrane protein Crumbs is crucial for the size and identity of the apical membrane, yet little is known about the molecular mechanisms controlling the amount of Crumbs at the surface. Here, we show that Crumbs levels on the apical membrane depend on a well-balanced state of endocytosis and stabilization. The adaptor protein 2 (AP-2) complex binds to a motif in the cytoplasmic tail of Crumbs that overlaps with the binding site of Stardust, a protein known to stabilize Crumbs on the surface. Preventing endocytosis by mutation of AP-2 causes expansion of the Crumbs-positive plasma membrane domain and polarity defects, which can be partially rescued by removing one copy of crumbs. Strikingly, knocking down both AP-2 and Stardust leads to the retention of Crumbs on the membrane. This study provides evidence for a molecular mechanism, based on stabilization and endocytosis, to adjust surface levels of Crumbs, which are essential for maintaining epithelial polarity.

  19. A dynamin-actin interaction is required for vesicle scission during endocytosis in yeast.

    Science.gov (United States)

    Palmer, Sarah E; Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Mishra, Ritu; Johnson, Simeon; Goldberg, Martin W; Ayscough, Kathryn R

    2015-03-30

    Actin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that--like wild-type Vps1--it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.

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

    Science.gov (United States)

    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.

  1. 小鼠耳蜗内外毛细胞胞吞功能的实验研究%Different patterns of endocytosis identified in murine cochlear inner and outer hair cells

    Institute of Scientific and Technical Information of China (English)

    李四军; 柳柯; 唐嗣泉; 杨仕明

    2015-01-01

    目的:研究小鼠内外毛细胞胞吞功能的异同,探讨毛细胞胞吞功能与动物听功能之间的关系。方法选择出生后1月龄的正常C57BL/6J小鼠耳蜗基底膜在体外培养,以染料FM1-43为胞吞示踪剂,应用活细胞成像技术观察耳蜗内外毛细胞胞吞现象。结果内毛细胞的胞吞活动主要集中在细胞底部及核下区,而外毛细胞的胞吞活动则主要集中在核上区和外侧壁区。而且,在不同的观察时间点上,内毛细胞对FM1-43的摄入量均显著高于外毛细胞(P<0.05)。结论内毛细胞的胞吞活动集中出现在细胞底部及核下区,说明这种胞吞活动与内毛细胞带状突触的功能密切相关;外毛细胞的胞吞活动主要出现在核上区及细胞外侧壁,表明这种活动更多参与了外毛细胞纤毛及离子通道。内毛细胞比外毛细胞具有更强大的胞吞功能,表明内毛细胞在听功能的发育和维持中发挥着更为关键的作用。%Objective To report different patterns of endocytosis in murine cochlear inner and outer hair cells (IHCs&OHCs). Methods Normal C57BL/6J mice (1 month old) were used to observe different patterns of endocytosis in IHCs and OHCs in vitro using live cell imaging of FM1-43. Results Intense fluorescence den-sity of FM1-43 indicating massive endocytosis activities was identified at the base and blow the nucleus in IHCs. In contrast, major endocytosis activities in OHCs were found at or near the lateral cellular wall and above the nucleus. FM1-43 uptake in IHCs was higher than that in OHCs (P<0.05). Conclusion Active endocy-tosis at or near the basolateral area of the IHC suggests that endocytosis in IHCs may involve ribbon synapse plasticity. Endocytosis activities near the lateral wall and in the supranuclear area in OHC indicate that endocy-tosis in OHC involves tip-links or iron channels. Higher levels of FM1-43 uptake by IHCs may indicate IHC’s key roles in hearing

  2. Transport properties of molecular junctions

    CERN Document Server

    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 ...

  3. NbN tunnel junctions

    International Nuclear Information System (INIS)

    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

  4. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.

  5. Environmental Audit of the Grand Junction Projects Office

    Energy Technology Data Exchange (ETDEWEB)

    1991-08-01

    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. Environmental Audit of the Grand Junction Projects Office

    International Nuclear Information System (INIS)

    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

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

    KAUST Repository

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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?

    Science.gov (United States)

    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.

  10. Nano-Molecular Junctions on STM Tips

    Institute of Scientific and Technical Information of China (English)

    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.

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  13. Dynamics of pi-junction interferometer circuits

    DEFF Research Database (Denmark)

    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...

  14. Soliton excitations in Josephson tunnel junctions

    DEFF Research Database (Denmark)

    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...

  15. delta-biased Josephson tunnel junctions

    DEFF Research Database (Denmark)

    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...

  16. Soliton bunching in annular Josephson junctions

    DEFF Research Database (Denmark)

    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...

  17. GAP junctional communication in brain secondary organizers.

    Science.gov (United States)

    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

  18. Traumatic Tear of the Latissimus Dorsi Myotendinous Junction

    Science.gov (United States)

    Friedman, Michael V.; Stensby, J. Derek; Hillen, Travis J.; Demertzis, Jennifer L.; Keener, Jay D.

    2015-01-01

    A case of a latissimus dorsi myotendinous junction strain in an avid CrossFit athlete is presented. The patient developed acute onset right axillary burning and swelling and subsequent palpable pop with weakness while performing a “muscle up.” Magnetic resonance imaging examination demonstrated a high-grade tear of the right latissimus dorsi myotendinous junction approximately 9 cm proximal to its intact humeral insertion. There were no other injuries to the adjacent shoulder girdle structures. Isolated strain of the latissimus dorsi myotendinous junction is a very rare injury with a scarcity of information available regarding its imaging appearance and preferred treatment. This patient was treated conservatively and was able to resume active CrossFit training within 3 months. At 6 months postinjury, he had only a mild residual functional deficit compared with his preinjury level. PMID:26502450

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Fabrication of high quality ferromagnetic Josephson junctions

    International Nuclear Information System (INIS)

    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

  3. Fabrication of high quality ferromagnetic Josephson junctions

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. DJ-1 associates with lipid rafts by palmitoylation and regulates lipid rafts-dependent endocytosis in astrocytes.

    Science.gov (United States)

    Kim, Kwang Soo; Kim, Jin Soo; Park, Ji-Young; Suh, Young Ho; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2013-12-01

    Parkinson's disease (PD) is the second most common progressive neurodegenerative disease. Several genes have been associated with familial type PD, providing tremendous insights into the pathogenesis of PD. Gathering evidence supports the view that these gene products may operate through common molecular pathways. Recent reports suggest that many PD-associated gene products, such as α-synuclein, LRRK2, parkin and PINK1, associate with lipid rafts and lipid rafts may be associated with neurodegeneration. Here, we observed that DJ-1 protein also associated with lipid rafts. Palmitoylation of three cysteine residues (C46/53/106) and C-terminal region of DJ-1 were required for this association. Lipopolysaccharide (LPS) induced the localization of DJ-1 into lipid rafts in astrocytes. The LPS-TLR4 signaling was more augmented in DJ-1 knock-out astrocytes by the impairment of TLR4 endocytosis. Furthermore, lipid rafts-dependent endocytosis including the endocytosis of CD14, which play a major role in regulating TLR4 endocytosis was also impaired, but clathrin-dependent endocytosis was not. This study provides a novel function of DJ-1 in lipid rafts, which may contribute the pathogenesis of PD. Moreover, it also provides the possibility that many PD-related proteins may operate through common molecular pathways in lipid rafts.

  5. Duffy antigen receptor for chemokines mediates chemokine endocytosis through a macropinocytosis-like process in endothelial cells.

    Directory of Open Access Journals (Sweden)

    Yani Zhao

    Full Text Available The Duffy antigen receptor for chemokines (DARC shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated (125I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. (125I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression.(125I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.

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

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    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 lipid analysis revealed that fat absorption caused a marked increase in the microvillar membrane contents of free fatty acids. In conclusion, fat absorption rapidly induces a transient clathrin-dependent endocytosis via coated pits from the enterocyte brush border. The process selectively internalizes...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  12. Seebeck effect in molecular junctions

    International Nuclear Information System (INIS)

    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)

  13. Seebeck effect in molecular junctions

    Science.gov (United States)

    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.

  14. Imaging of cervicothoracic junction trauma

    OpenAIRE

    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...

  15. Counting Statistics in Nanoscale Junctions

    OpenAIRE

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

    Science.gov (United States)

    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

  17. Thermoelectric efficiency of molecular junctions

    Science.gov (United States)

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

    2016-09-01

    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.

  18. Thermoelectric efficiency of molecular junctions.

    Science.gov (United States)

    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. Endocytosis of indium-tin-oxide nanoparticles by macrophages provokes pyroptosis requiring NLRP3-ASC-Caspase1 axis that can be prevented by mesenchymal stem cells.

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Regulation of neuronal axon specification by glia-neuron gap junctions in C. elegans

    Science.gov (United States)

    Meng, Lingfeng; Zhang, Albert; Jin, Yishi; Yan, Dong

    2016-01-01

    Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4. Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions. DOI: http://dx.doi.org/10.7554/eLife.19510.001 PMID:27767956

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Endocytosis and Trafficking of Natriuretic Peptide Receptor-A: Potential Role of Short Sequence Motifs

    Directory of Open Access Journals (Sweden)

    Kailash N. Pandey

    2015-07-01

    Full Text Available The targeted endocytosis and redistribution of transmembrane receptors among membrane-bound subcellular organelles are vital for their correct signaling and physiological functions. Membrane receptors committed for internalization and trafficking pathways are sorted into coated vesicles. Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP bind to guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA and elicit the generation of intracellular second messenger cyclic guanosine 3',5'-monophosphate (cGMP, which lowers blood pressure and incidence of heart failure. After ligand binding, the receptor is rapidly internalized, sequestrated, and redistributed into intracellular locations. Thus, NPRA is considered a dynamic cellular macromolecule that traverses different subcellular locations through its lifetime. The utilization of pharmacologic and molecular perturbants has helped in delineating the pathways of endocytosis, trafficking, down-regulation, and degradation of membrane receptors in intact cells. This review describes the investigation of the mechanisms of internalization, trafficking, and redistribution of NPRA compared with other cell surface receptors from the plasma membrane into the cell interior. The roles of different short-signal peptide sequence motifs in the internalization and trafficking of other membrane receptors have been briefly reviewed and their potential significance in the internalization and trafficking of NPRA is discussed.

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

    Science.gov (United States)

    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.

  8. The iTRAPs: Guardians of Synaptic Vesicle Cargo Retrieval During Endocytosis.

    Science.gov (United States)

    Gordon, Sarah L; Cousin, Michael A

    2016-01-01

    The reformation of synaptic vesicles (SVs) during endocytosis is essential for the maintenance of neurotransmission in central nerve terminals. Newly formed SVs must be generated with the correct protein cargo in the correct stoichiometry to be functional for exocytosis. Classical clathrin adaptor protein complexes play a key role in sorting and clustering synaptic vesicle cargo in this regard. However it is becoming increasingly apparent that additional "fail-safe" mechanisms exist to ensure the accurate retrieval of essential cargo molecules. For example, the monomeric adaptor proteins AP180/CALM and stonin-2 are required for the efficient retrieval of synaptobrevin II (sybII) and synaptotagmin-1 respectively. Furthermore, recent studies have revealed that sybII and synaptotagmin-1 interact with other SV cargoes to ensure a high fidelity of retrieval. These cargoes are synaptophysin (for sybII) and SV2A (for synaptotagmin-1). In this review, we summarize current knowledge regarding the retrieval mechanisms for both sybII and synaptotagmin-1 during endocytosis. We also define and set criteria for a new functional group of SV molecules that facilitate the retrieval of their interaction partners. We have termed these molecules intrinsic trafficking partners (iTRAPs) and we discuss how the function of this group impacts on presynaptic performance in both health and disease.

  9. The iTRAPs: guardians of synaptic vesicle cargo retrieval during endocytosis

    Directory of Open Access Journals (Sweden)

    Sarah Louise Gordon

    2016-02-01

    Full Text Available The reformation of synaptic vesicles during endocytosis is essential for the maintenance of neurotransmission in central nerve terminals. Newly formed synaptic vesicles must be generated with the correct protein cargo in the correct stoichiometry to be functional for exocytosis. Classical clathrin adaptor protein complexes play a key role in sorting and clustering synaptic vesicle cargo in this regard. However it is becoming increasingly apparent that additional fail-safe mechanisms exist to ensure the accurate retrieval of essential cargo molecules. For example, the monomeric adaptor proteins AP180/CALM and stonin-2 are required for the efficient retrieval of synaptobrevin II and synaptotagmin-1 respectively. Furthermore, recent studies have revealed that synaptobrevin II and synaptotagmin-1 interact with other synaptic vesicle cargoes to ensure a high fidelity of retrieval. These cargoes are synaptophysin (for synaptobrevin II and SV2A (for synaptotagmin-1. In this review we summarise current knowledge regarding the retrieval mechanisms for both synaptobrevin II and synaptotagmin-1 during endocytosis. We also define and set criteria for a new functional group of synaptic vesicle molecules that facilitate the retrieval of their interaction partners. We have termed these molecules intrinsic trafficking partners (iTRAPs and we discuss how the function of this group impacts on presynaptic performance in both health and disease.

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

    Science.gov (United States)

    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

  11. Chaos induced by coupling between Josephson junctions

    Science.gov (United States)

    Shukrinov, Yu. M.; Azemtsa-Donfack, H.; Botha, A. E.

    2015-02-01

    It is found that, in a stack of intrinsic Josephson junctions in layered high temperature superconductors under external electromagnetic radiation, the chaotic features are triggered by interjunction coupling, i.e., the coupling between different junctions in the stack. While the radiation is well known to produce chaotic effects in the single junction, the effect of interjunction coupling is fundamentally different and it can lead to the onset of chaos via a different route to that of the single junction. A precise numerical study of the phase dynamics of intrinsic Josephson junctions, as described by the CCJJ+DC model, is performed. We demonstrate the charging of superconducting layers, in a bias current interval corresponding to a Shapiro step subharmonic, due to the creation of a longitudinal plasma wave along the stack of junctions. With increase in radiation amplitude chaotic behavior sets in. The chaotic features of the coupled Josephson junctions are analyzed by calculations of the Lyapunov exponents. We compare results for a stack of junctions to the case of a single junction and prove that the observed chaos is induced by the coupling between the junctions. The use of Shapiro step subharmonics may allow longitudinal plasma waves to be excited at low radiation power.

  12. Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

    OpenAIRE

    Xia, Yi; Gong, Kai-zheng; Xu, Ming; Zhang, You-Yi; Guo, Ji-Hong; Song, Yao; Zhang, Ping

    2009-01-01

    Aim: β-adrenergic receptor (β-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of β-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function. Methods: The level of Cx43...

  13. Endocytosis of μ opioid receptors inhibits morphine tolerance%μ阿片受体的内吞抑制吗啡耐受的形成

    Institute of Scientific and Technical Information of China (English)

    吕庆琴; 陈霆隽; 洪炎国

    2012-01-01

    Opioids are the most effective analgesics. However, prolonged administration of morphine, the representative of opioids, results in tolerance, limiting the therapeutic utility of o-piate drugs. Studies have recently suggested that endocytosis of μ opioid receptors attenuates opioid tolerance. The ability of inducing endocytosis of opioid receptor is agonist-dependent. It has been shown that the endocytotic efficacy of opioids are negatively correlated with opioid tolerance. Receptor internalization reduced adaptive changes in signaling pathways that are involved in the development of opioid tolerance. Moreover, endocytosised μ-opioid receptors are rapidly recycled back to the cell membrane surface resuming their normal function. Therefore, tolerance does not occur. Thus, the study of receptor endocytosis and trafficking following the activation of the receptors can help the therapy for chronic pain.%阿片类药物是至今最有效的镇痛药,但是长期应用会产生药物耐受,大大限制了其临床应用.μ阿片受体和特定的激动剂结合后会出现内吞.研究发现,μ阿片受体是否内吞与耐受的发生有密切关系;加强μ阿片受体的内吞能够抑制受体耐受.不同的激动剂导致μ阿片受体内吞的能力是不同的;其导致耐受的能力和导致内吞的能力呈负相关.激动剂越容易引起μ受体内吞,就越不容易产生吗啡耐受.内吞的作用在于能抑制过度刺激μ受体而导致的环腺苷酸( cyclic adenosine monophosphate,cAMP)等兴奋性信号通路的激活,而内吞的μ受体也会很快回到细胞膜上,恢复和阿片类药物结合后激活抑制性GTP 结合蛋白的能力.因此,对受体的内吞和其后迁移过程展开研究,可能为慢性疼痛的治疗找到新的路径.

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

    Energy Technology Data Exchange (ETDEWEB)

    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).

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

    International Nuclear Information System (INIS)

    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)

  16. Algorithms for Junctions in Directed Acyclic Graphs

    CERN Document Server

    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.

  17. Fluxon dynamics in three stacked Josephson junctions

    DEFF Research Database (Denmark)

    Gorria, Carlos; Christiansen, Peter Leth; Gaididei, Yuri Borisovich;

    2002-01-01

    /sub -/, the coupling between junctions leads to a repulsion of the fluxons with the same polarity. Above this critical velocity a fluxon will induce radiation in the neighboring junctions, leading to a bunching of the fluxons in the stacked junctions. Using the Sakai-Bodin-Pedersen model, three coupled perturbed sine......The motion of fluxons of the same polarity in three vertically stacked Josephson junctions is studied. In this configuration the difference between exterior and interior junctions plays a more important role than in other configurations with several interior junctions. Below the Swihart velocity c......-Gordon equations are numerically studied for different values of coupling, damping, and bias parameters. In a narrow range of velocities bunching occurs. Outside this interval the fluxons split and new fluxons may be created. I-V characteristics are presented...

  18. String junction as a baryonic constituent

    CERN Document Server

    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.

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

    NARCIS (Netherlands)

    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

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

    Directory of Open Access Journals (Sweden)

    Soheil Aghamohammadzadeh

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

    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

  3. Molecular mechanism of synaptic vesicle endocytosis%突触囊泡内吞的分子机制

    Institute of Scientific and Technical Information of China (English)

    张妮; 王世伟; 苟兴春

    2014-01-01

    突触传递是神经系统实现其功能的最基本的方式。神经细胞进行着快速的突触囊泡信息传递而没有耗尽突触囊泡,这主要依赖于突触囊泡在神经末梢进行着精确而快速的内吞作用。本文将主要介绍四种突触囊泡的回收分子机制,网格蛋白介导的经典途径、Kiss-and-run、Bulk endocytosis以及2013年12月在Nature上报道的超速内吞机制。%Synaptic transmission is the most basic way for nervous system to realize its function. Neurons can sustain high rates of synaptic transmission without exhausting their supply of synaptic vesicles. This property re-lies on a highly efficient local endocytic recycling of synaptic vesicle membranes. This article summarizes four modes of synaptic vesicle endocytosis, which are clathrin-mediated endocytosis,kiss-and-run,bulk endocytosis and ultrafast endocytosis (reported in Nature in Dec. 2013), with an emphasis on the underlying molecular mechanisms.

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

    DEFF Research Database (Denmark)

    Beedholm, Rasmus; Grissom, Charles B.; Fedosov, Sergey N.;

    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 molecules...

  5. Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis

    NARCIS (Netherlands)

    Zuhorn, IS; Kalicharan, R; Hoekstra, D

    2002-01-01

    Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often

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

    Science.gov (United States)

    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

  7. Immunomodulatory effects of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells.

    Science.gov (United States)

    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

  8. Immunomodulatory effects of human umbilical cord Wharton's jelly-derived mesenchymal stem cells on differentiation, maturation and endocytosis of monocyte-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    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.

  9. The timing of cortical granule fusion, content dispersal, and endocytosis during fertilization of the hamster egg: an electrophysiological and histochemical study.

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Grand Junction Remedial Action Program

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

    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

  13. The Biochemical Properties and Functions of CALM and AP180 in Clathrin Mediated Endocytosis

    Directory of Open Access Journals (Sweden)

    Lia Moshkanbaryans

    2014-07-01

    Full Text Available Clathrin-mediated endocytosis (CME is a fundamental process for the regulated internalization of transmembrane cargo and ligands via the formation of vesicles using a clathrin coat. A vesicle coat is initially created at the plasma membrane by clathrin assembly into a lattice, while a specific cargo sorting process selects and concentrates proteins for inclusion in the new vesicle. Vesicles formed via CME traffic to different parts of the cell and fuse with target membranes to deliver cargo. Both clathrin assembly and cargo sorting functions are features of the two gene family consisting of assembly protein 180 kDa (AP180 and clathrin assembly lymphoid myeloid leukemia protein (CALM. In this review, we compare the primary structure and domain organization of CALM and AP180 and relate these properties to known functions and roles in CME and disease.

  14. Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis

    DEFF Research Database (Denmark)

    Confalonieri, S; Salcini, A E; Puri, C;

    2000-01-01

    for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function......Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required....... Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization...

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

    CERN Document Server

    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...

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

    CERN Document Server

    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...

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

    Directory of Open Access Journals (Sweden)

    Xavier eCharest-Morin

    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.

  18. Endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments.

    Directory of Open Access Journals (Sweden)

    Eric Bernard

    Full Text Available BACKGROUND: The replicative cycle of chikungunya virus (CHIKV, an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s hijacked by CHIKV to enter mammalian cells. METHODOLOGY/PRINCIPAL FINDINGS: Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl beta-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island. CONCLUSIONS: Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Multiplication in Silicon p-n Junctions

    DEFF Research Database (Denmark)

    Moll, John L.

    1965-01-01

    Multiplication values were measured in the collector junctions of silicon p-n-p and n-p-n transistors before and after bombardment by 1016 neutrons/cm2. Within experimental error there was no change either in junction fields, as deduced from capacitance measurements, or in multiplication values...

  1. Spin, Vibrations and Radiation in Superconducting Junctions

    NARCIS (Netherlands)

    Padurariu, C.

    2013-01-01

    This thesis presents the theoretical study of superconducting transport in several devices based on superconducting junctions. The important feature of these devices is that the transport properties of the junction are modified by the interaction with another physical system integrated in the super

  2. Hall effect in NS and SNS junctions

    OpenAIRE

    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.

  3. Gap junctions and connexin-interacting proteins

    NARCIS (Netherlands)

    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

  4. Genetics Home Reference: junctional epidermolysis bullosa

    Science.gov (United States)

    ... 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 ...

  5. Electrophysiological study in neuromuscular junction disorders

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  7. Josephson tunnel junction microwave attenuator

    DEFF Research Database (Denmark)

    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...... at different points in the current-voltage characteristic. Both numerical calculations based on the Tien-Gordon theory and 70-GHz microwave experiments have confirmed the wide dynamic range (more than 15-dB attenuation for one stage) and the low insertion loss in the ''open'' state. The performance of a fully...... integrated submillimeter receiver circuit which comprises a flux-flow oscillator (FFO) as local oscillator, a superconducting variable attenuator, and a microwave SIS detector with tuned-out capacitance is also reported....

  8. Coordinate transformation in the model of long Josephson junctions: geometrically equivalent Josephson junctions

    Science.gov (United States)

    Semerdzhieva, E. G.; Boyadzhiev, T. L.; Shukrinov, Yu. M.

    2005-10-01

    The transition from the model of a long Josephson junction of variable width to the model of a junction with a coordinate-dependent Josephson current amplitude is effected through a coordinate transformation. This establishes the correspondence between the classes of Josephson junctions of variable width and quasi-one-dimensional junctions with a variable thickness of the barrier layer. It is shown that for a junction of exponentially varying width the barrier layer of the equivalent quasi-one-dimensional junction has a distributed resistive inhomogeneity that acts as an attractor for magnetic flux vortices. The curve of the critical current versus magnetic field for a Josephson junction with a resistive microinhomogeneity is constructed with the aid of a numerical simulation, and a comparison is made with the critical curve of a junction of exponentially varying width. The possibility of replacing a distributed inhomogeneity in a Josephson junction by a local inhomogeneity at the end of the junction is thereby demonstrated; this can have certain advantages from a technological point of view.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  11. MQT observation in Bi2212 intrinsic Josephson junctions

    Science.gov (United States)

    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.

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

    CERN Document Server

    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...

  13. Graded junction termination extensions for electronic devices

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Axelsen, Lene Nygaard; Haugan, Ketil; Stahlhut, Martin; Kjølbye, Anne-Louise; Hennan, James K; Holstein-Rathlou, Niels-Henrik; Petersen, Jørgen Søberg; Nielsen, Morten Schak

    2007-03-01

    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 and by interfering with the gating of gap junctional channels.

  18. The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling

    DEFF Research Database (Denmark)

    Fraile-Ramos, A; Kledal, T N; Pelchen-Matthews, A;

    2001-01-01

    expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen...... mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands...... showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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 ...

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

    Institute of Scientific and Technical Information of China (English)

    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.

  3. Current trends in salivary gland tight junctions.

    Science.gov (United States)

    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

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

    International Nuclear Information System (INIS)

    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

  5. Role of endocytosis in sonoporation-mediated membrane permeabilization and uptake of small molecules: a electron microscopy study

    Science.gov (United States)

    Zeghimi, A.; Escoffre, J. M.; Bouakaz, A.

    2015-12-01

    Sonoporation is a physical method that has been successfully used to deliver drugs into living cells both in vitro and in vivo for experimental and therapeutic purposes. Despite numerous studies on this topic, often reporting successful outcomes, very little is known about the mechanisms involved in the hypothesized membrane permeabilization processes. In this study, electron microscopy was used to investigate the ultra-structural modifications of cell membranes, induced by sonoporation. Here, we demonstrate that sonoporation in the presence of microbubbles induces the formation of a significant number of transient and permeant structures at the membrane level. These structures were transient with a half-life of 10 min and had a heterogeneous size distribution ranging from a few nanometers to 150 nm. We demonstrated that the number and the size of these structures were positively correlated with the enhanced intracellular uptake of small molecules. In addition, we showed that these structures were associated with caveolae-dependent endocytosis for two thirds of the recorded events, with the remaining one third related to non-specific routes such as membrane disruptions as well as caveolae-independent endocytosis. In conclusion, our observations provide direct evidences of the involvement of caveolae-endocytosis in cell membrane permeabilization to small molecules after sonoporation.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  8. Bond resistances in molecular junctions

    Science.gov (United States)

    Painelli, Anna

    2006-03-01

    The description of molecular contacts is one of the hardest problems in modeling molecular junctions. In common approaches macroscopic leads ensure a finite potential drop and hence a driving force for the current. Recently, a different strategy is emerging where a steady-state DC current is forced in the molecule, by making resort to Lagrange multipliers, or by drawing a magnetic flux through the molecule. The strategy is promising, but two main problems remain to be solved: (1) the calculation of the potential drop needed to sustain the current, and (2) the definition of the potential profile along the molecule. Here the Joule law is used to evaluate the potential drop from the electrical power spent on the molecule, and continuity constraints for steady-state DC current are implemented to get information on the potential profile. Borrowing powerful concepts from the field of molecular spectroscopy, emphasis is put on the molecule, while clamping information about contacts in the molecular relaxation matrix. The molecule is described in a real-space approach, leading to a suggestive analogy between the molecule and an electrical circuit where resistances are associated with chemical bonds.

  9. Controllable spin transport in ferromagnetic graphene junctions

    OpenAIRE

    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...

  10. Heat dissipation in atomic-scale junctions

    OpenAIRE

    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...

  11. Junction Plasmon-Induced Molecular Reorientation

    Energy Technology Data Exchange (ETDEWEB)

    El-Khoury, Patrick Z.; Hu, Dehong; Hess, Wayne P.

    2013-10-17

    Time and frequency dependent intensity variations in sequences of Raman spectra recorded at plasmonic junctions can be assigned to molecular reorientation. This is revealed through Raman trajectories recorded at a nanojunction formed between a silver AFM tip and a corrugated silver surface coated with biphenyl-4,4’-dithiol. Molecular motion is not observed when the tip is retracted and only surface enhancement is operative. In effect, junction plasmon induced molecular reorientation is tracked.

  12. Pseudospin dynamics in multimode polaritonic Josephson junctions

    OpenAIRE

    Pavlovic, G.; Malpuech, G.; Shelykh, I. A.

    2012-01-01

    We analyzed multimode Josephson junctions with exciton-polaritons (polaritonic Josephson junctions) when several coupling mechanisms of fundamental and excited states are present. The applied method is based on Keldysh-Green function formalism and takes into account polariton pseudospin. We found that mean value of circular polarization degree in intrinsic Josephson oscillations and microscopic quantum self-trapping follow an oscillator behavior whose renormalizes due to intermode interaction...

  13. Binding and Endocytosis of Bovine Hololactoferrin by the Parasite Entamoeba histolytica

    Directory of Open Access Journals (Sweden)

    Guillermo Ortíz-Estrada

    2015-01-01

    Full Text Available Entamoeba histolytica is a human parasite that requires iron (Fe for its metabolic function and virulence. Bovine lactoferrin (B-Lf and its peptides can be found in the digestive tract after dairy products are ingested. The aim of this study was to compare virulent trophozoites recently isolated from hamster liver abscesses with nonvirulent trophozoites maintained for more than 30 years in cultures in vitro regarding their interaction with iron-charged B-Lf (B-holo-Lf. We performed growth kinetics analyses of trophozoites in B-holo-Lf and throughout several consecutive transfers. The virulent parasites showed higher growth and tolerance to iron than nonvirulent parasites. Both amoeba variants specifically bound B-holo-Lf with a similar Kd. However, averages of 9.45 × 105 and 6.65 × 106 binding sites/cell were found for B-holo-Lf in nonvirulent and virulent amoebae, respectively. Virulent amoebae bound more efficiently to human and bovine holo-Lf, human holo-transferrin, and human and bovine hemoglobin than nonvirulent amoebae. Virulent amoebae showed two types of B-holo-Lf binding proteins. Although both amoebae endocytosed this glycoprotein through clathrin-coated vesicles, the virulent amoebae also endocytosed B-holo-Lf through a cholesterol-dependent mechanism. Both amoeba variants secreted cysteine proteases cleaving B-holo-Lf. These data demonstrate that the B-Lf endocytosis is more efficient in virulent amoebae.

  14. The nuclear architectural protein HMGA1a triggers receptor-mediated endocytosis.

    Science.gov (United States)

    Wu, Wuwei; Wan, Wei; Li, Alexander D Q

    2009-11-01

    High mobility group proteins A (HMGA), nuclear architectural factors, locate in the cell nuclei and mostly execute gene-regulation function. However, our results reveal that a HMGA member (HMGA1a) has a unique plasma membrane receptor; this receptor specifically binds to HMGA-decorated species, effectively mediates endocytosis, and internalizes extracellular HMGA-functionalized cargoes. Indeed, dyes or nanoparticles labeled with HMGA1a protein readily enter Hela cells. Using a stratagem chemical cross-linker, we covalently bonded the HMGA receptor to the HMGA1a-GFP fusion protein, thus capturing the plasma membrane receptor. Subsequent Western blots and SDS-PAGE gel revealed that the HMGA receptor is a 26-kDa protein. Confocal live-cell microscopic imaging was used to monitor the whole endocytic process, in which the internalized HMGA1a-decorated species are transported by motor proteins on microtubules and eventually arrive at the late endosomes/lysosomes. Cell viability assays also suggested that extracellular HMGA1a protein directly influences the survival ability of Hela cells in a dose-dependent manner, implying versatility of HMGA1a protein and its potent role to suppress cancer cell survivability and to regulate growth. PMID:19739099

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

  18. Partial wrapping and spontaneous endocytosis of spherical nanoparticles by tensionless lipid membranes

    Science.gov (United States)

    Spangler, Eric J.; Upreti, Sudhir; Laradji, Mohamed

    2016-01-01

    Computer simulations of an implicit-solvent particle-based model are performed to investigate the interactions between small spherical nanoparticles and tensionless lipid bilayers. We found that nanoparticles are either unbound, wrapped by the bilayer, or endocytosed. The degree of wrapping increases with increasing the adhesion strength. The transition adhesion strength between the unbound and partially wrapped states decreases as the nanoparticle diameter is increased. We also observed that the transition adhesion strength between the wrapped states and endocytosis state decreases with increasing the nanoparticle diameter. The partial wrapping of the nanoparticles by the tensionless bilayer is explained by an elastic theory which accounts for the fact that the interaction between the nanoparticle and the bilayer extends beyond the contact region. The theory predicts that for small nanoparticles, the wrapping angle increases continuously with increasing the adhesion strength. However, for relatively large nanoparticles, the wrapping angle exhibits a discontinuity between weakly and strongly wrapped states. The size of the gap in the wrapping angle between the weakly wrapped and strongly wrapped states increases with decreasing the range of nanoparticle-bilayer interaction.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Alabi, AbdulRasheed A; Tsien, Richard W

    2013-01-01

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

  1. Protein Corona Modulates Uptake and Toxicity of Nanoceria via Clathrin-Mediated Endocytosis.

    Science.gov (United States)

    Mazzolini, Julie; Weber, Ralf J M; Chen, Hsueh-Shih; Khan, Abdullah; Guggenheim, Emily; Shaw, Robert K; Chipman, James K; Viant, Mark R; Rappoport, Joshua Z

    2016-08-01

    Particles present in diesel exhaust have been proposed as a significant contributor to the development of acute and chronic lung diseases, including respiratory infection and allergic asthma. Nanoceria (CeO2 nanoparticles) are used to increase fuel efficiency in internal combustion engines, are present in exhaust fumes, and could affect cells of the airway. Components from the environment such as biologically derived proteins, carbohydrates, and lipids can form a dynamic layer, commonly referred to as the "protein corona" which alters cellular nanoparticle interactions and internalization. Using confocal reflectance microscopy, we quantified nanoceria uptake by lung-derived cells in the presence and absence of a serum-derived protein corona. Employing mass spectrometry, we identified components of the protein corona, and demonstrated that the interaction between transferrin in the protein corona and the transferrin receptor is involved in mediating the cellular entry of nanoceria via clathrin-mediated endocytosis. Furthermore, under these conditions nanoceria does not affect cell growth, viability, or metabolism, even at high concentration. Alternatively, despite the antioxidant capacity of nanoceria, in serum-free conditions these nanoparticles induce plasma membrane disruption and cause changes in cellular metabolism. Thus, our results identify a specific receptor-mediated mechanism for nanoceria entry, and provide significant insight into the potential for nanoparticle-dependent toxicity.

  2. Toward complementary ionic circuits: the npn ion bipolar junction transistor.

    Science.gov (United States)

    Tybrandt, Klas; Gabrielsson, Erik O; Berggren, Magnus

    2011-07-01

    Many biomolecules are charged and may therefore be transported with ionic currents. As a step toward addressable ionic delivery circuits, we report on the development of a npn ion bipolar junction transistor (npn-IBJT) as an active control element of anionic currents in general, and specifically, demonstrate actively modulated delivery of the neurotransmitter glutamic acid. The functional materials of this transistor are ion exchange layers and conjugated polymers. The npn-IBJT shows stable transistor characteristics over extensive time of operation and ion current switch times below 10 s. Our results promise complementary chemical circuits similar to the electronic equivalence, which has proven invaluable in conventional electronic applications. PMID:21598973

  3. Photoinduced carrier annihilation in silicon pn junction

    Science.gov (United States)

    Sameshima, Toshiyuki; Motoki, Takayuki; Yasuda, Keisuke; Nakamura, Tomohiko; Hasumi, Masahiko; Mizuno, Toshihisa

    2015-08-01

    We report analysis of the photo-induced minority carrier effective lifetime (τeff) in a p+n junction formed on the top surfaces of a n-type silicon substrate by ion implantation of boron and phosphorus atoms at the top and bottom surfaces followed by activation by microwave heating. Bias voltages were applied to the p+ boron-doped surface with n+ phosphorus-doped surface kept at 0 V. The values of τeff were lower than 1 × 10-5 s under the reverse-bias condition. On the other hand, τeff markedly increased to 1.4 × 10-4 s as the forward-bias voltage increased to 0.7 V and then it leveled off when continuous-wave 635 nm light was illuminated at 0.74 mW/cm2 on the p+ surface. The carrier annihilation velocity S\\text{p + } at the p+ surface region was numerically estimated from the experimental τeff. S\\text{p + } ranged from 4000 to 7200 cm/s under the reverse-bias condition when the carrier annihilation velocity S\\text{n + } at the n+ surface region was assumed to be a constant value of 100 cm/s. S\\text{p + } markedly decreased to 265 cm/s as the forward-bias voltage increased to 0.7 V.

  4. Microfluidic mixing in a Y-junction open channel

    Directory of Open Access Journals (Sweden)

    Jue Nee Tan

    2012-09-01

    Full Text Available In the laminar regimes typical of microfluidic systems’, mixing is governed by molecular diffusion; however this process is slow in nature. Consequently, passive or active methods are usually sought for effective mixing. In this work, open fluidic channels will be investigated; these channels are bounded on all but one face by an air/fluid interface. Firstly, it will be shown that flow in open channels can merge at a Y-junction in a stable manner; hence two fluids can be brought into contact with each other. Secondly, the mixing of these two fluids will be studied. At high flow rates (>300 μl/min mixing occurs at the junction without need for additional intervention, this mixing is far swifter than can be expected from molecular diffusion. At lower flow rates, intervention is required. A major motivation for open fluidic channels is the ability to interact with the surrounding air environment; this feature is used to effect the desired mixing. It is shown that by blowing an air jet across the junction, shear stresses at the air/fluid interface causes a flow profile within the fluid inductive to rapid mixing of the fluids.

  5. Plasmon induced electric current in a molecular junction

    Science.gov (United States)

    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. Phase dynamics of low critical current density YBCO Josephson junctions

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Science.gov (United States)

    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

  10. Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

    OpenAIRE

    Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K; Hulley, Philippa A

    2014-01-01

    Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows qu...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    KAUST Repository

    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.

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

    DEFF Research Database (Denmark)

    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....

  15. Model Building to Facilitate Understanding of Holliday Junction and Heteroduplex Formation, and Holliday Junction Resolution

    Science.gov (United States)

    Selvarajah, Geeta; Selvarajah, Susila

    2016-01-01

    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…

  16. Josephson tunnel junctions with ferromagnetic interlayer

    International Nuclear Information System (INIS)

    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.)

  17. Josephson tunnel junctions with ferromagnetic interlayer

    Energy Technology Data Exchange (ETDEWEB)

    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.)

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

    Science.gov (United States)

    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. Evidences of endocytosis via caveolae following blood-brain barrier breakdown by Phoneutria nigriventer spider venom.

    Science.gov (United States)

    Soares, Edilene Siqueira; Mendonça, Monique Culturato Padilha; Irazusta, Silvia Pierre; Coope, Andressa; Stávale, Leila Miguel; da Cruz-Höfling, Maria Alice

    2014-09-17

    Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

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

    International Nuclear Information System (INIS)

    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)

  1. Particle detection with superconducting tunnel junctions

    International Nuclear Information System (INIS)

    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.)

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

    Science.gov (United States)

    Wu, Jie; Gao, Ming; Rice, Stephen G; Tsang, Candy; Beggs, John; Turner, Dharshaun; Li, Guohui; Yang, Bo; Xia, Kunkun; Gao, Fenfei; Qiu, Shenfeng; Liu, Qiang; Kerrigan, John F

    2016-06-01

    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.

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

    Directory of Open Access Journals (Sweden)

    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. Created-by-current states in long Josephson junctions

    Science.gov (United States)

    Boyadjiev, T. L.; Andreeva, O. Yu.; Semerdjieva, E. G.; Shukrinov, Yu. M.

    2008-08-01

    Critical curves "critical current-external magnetic field" of long Josephson junctions with inhomogeneity and variable width are studied. We demonstrate the existence of regions of magnetic field where some fluxon states are stable only if the external current through the junction is different from zero. Position and size of such regions depend on the length of the junction, its geometry, parameters of inhomogeneity and form of the junction. The noncentral (left and right) pure fluxon states appear in the inhomogeneous Josephson junction with the increase in the junction length. We demonstrate new bifurcation points with change in width of the inhomogeneity and amplitude of the Josephson current through the inhomogeneity.

  5. Switching current distributions in InAs nanowire Josephson junctions

    Science.gov (United States)

    Kim, Bum-Kyu; Doh, Yong-Joo

    2016-08-01

    We report on the switching current distributions in nano-hybrid Josephson junctions made of InAs semiconductor nanowires. The temperature dependence of the switching current distribution can be understood through the motion of Josephson phase particles escaping from a tilted washboard potential, and the data could be fitted well by using the macroscopic quantum tunneling, thermal activation or phase diffusion models, depending on temperature. Application of the gate voltage to tune the Josephson coupling strength enable us to adjust the effective temperature for the escape process, and holds promising for developing gate-tunable superconducting phase qubits.

  6. Functional assessment of gap junctions in monolayer and three-dimensional cultures of human tendon cells using fluorescence recovery after photobleaching

    Science.gov (United States)

    Kuzma-Kuzniarska, Maria; Yapp, Clarence; Pearson-Jones, Thomas W.; Jones, Andrew K.; Hulley, Philippa A.

    2014-01-01

    Abstract. Gap junction-mediated intercellular communication influences a variety of cellular activities. In tendons, gap junctions modulate collagen production, are involved in strain-induced cell death, and are involved in the response to mechanical stimulation. The aim of the present study was to investigate gap junction-mediated intercellular communication in healthy human tendon-derived cells using fluorescence recovery after photobleaching (FRAP). The FRAP is a noninvasive technique that allows quantitative measurement of gap junction function in living cells. It is based on diffusion-dependent redistribution of a gap junction-permeable fluorescent dye. Using FRAP, we showed that human tenocytes form functional gap junctions in monolayer and three-dimensional (3-D) collagen I culture. Fluorescently labeled tenocytes following photobleaching rapidly reacquired the fluorescent dye from neighboring cells, while HeLa cells, which do not communicate by gap junctions, remained bleached. Furthermore, both 18 β-glycyrrhetinic acid and carbenoxolone, standard inhibitors of gap junction activity, impaired fluorescence recovery in tendon cells. In both monolayer and 3-D cultures, intercellular communication in isolated cells was significantly decreased when compared with cells forming many cell-to-cell contacts. In this study, we used FRAP as a tool to quantify and experimentally manipulate the function of gap junctions in human tenocytes in both two-dimensional (2-D) and 3-D cultures. PMID:24390370

  7. 2,8-Diazaspiro[4.5]decan-8-yl)pyrimidin-4-amine potent CCR4 antagonists capable of inducing receptor endocytosis.

    Science.gov (United States)

    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

  8. Vortex structures in exponentially shaped Josephson junctions

    Science.gov (United States)

    Shukrinov, Yu. M.; Semerdjieva, E. G.; Boyadjiev, T. L.

    2005-04-01

    We report the numerical calculations of the static vortex structure and critical curves in exponentially shaped long Josephson junctions for in-line and overlap geometries. Stability of the static solutions is investigated by checking the sign of the smallest eigenvalue of the associated Sturm-Liouville problem. The change in the junction width leads to the renormalization of the magnetic flux in comparison with the case of a linear one-dimensional model. We study the influence of the model's parameters, and particularly, the shape parameter on the stability of the states of the magnetic flux. We compare the vortex structure and critical curves for the in-line and overlap geometries. Our numerically constructed critical curve of the Josephson junction matches well with the experimental one.

  9. Tunnel junction based memristors as artificial synapses

    Directory of Open Access Journals (Sweden)

    Andy eThomas

    2015-07-01

    Full Text Available We prepared magnesia, tantalum oxide and barium titanate based junction structures and investigated their memristive properties. The low amplitudes of the resistance change in these types of junctions are the major obstacle for their use. Here, we increased the amplitude of the resistance change from 10% up to 100%. Utilizing the memristive properties, we looked into the use of the junction structures as artificial synapses. We observed analogs of long-term potentiation, long-term depression and spike-time dependent plasticity in these simple two terminal devices. Finally, we suggest a possible pathway of these devices towards their integration in neuromorphic systems for storing analog synaptic weights and supporting the implementation of biologically plausible learning mechanisms.

  10. Holographic Josephson Junction from Massive Gravity

    CERN Document Server

    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.

  11. Synchronized switching in a josephson junction crystal.

    Science.gov (United States)

    Leib, Martin; Hartmann, Michael J

    2014-06-01

    We consider a superconducting coplanar waveguide resonator where the central conductor is interrupted by a series of uniformly spaced Josephson junctions. The device forms an extended medium that is optically nonlinear on the single photon level with normal modes that inherit the full nonlinearity of the junctions but are nonetheless accessible via the resonator ports. For specific plasma frequencies of the junctions, a set of normal modes clusters in a narrow band and eventually becomes entirely degenerate. Upon increasing the intensity of a red detuned drive on these modes, we observe a sharp and synchronized switching from low-occupation quantum states to high-occupation classical fields, accompanied by a pronounced jump from low to high output intensity. PMID:24949766

  12. Silicon fiber with p-n junction

    Energy Technology Data Exchange (ETDEWEB)

    Homa, D.; Cito, A.; Pickrell, G.; Hill, C.; Scott, B. [Department of Materials Science and Engineering, Virginia Polytechnic Institute and State University, 312 Holden Hall, Blacksburg, Virginia 24060 (United States)

    2014-09-22

    In this study, we fabricated a p-n junction in a fiber with a phosphorous doped silicon core and fused silica cladding. The fibers were fabricated via a hybrid process of the core-suction and melt-draw techniques and maintained overall diameters ranging from 200 to 900 μm and core diameters of 20–800 μm. The p-n junction was formed by doping the fiber with boron and confirmed via the current-voltage characteristic. The demonstration of a p-n junction in a melt-drawn silicon core fiber paves the way for the seamless integration of optical and electronic devices in fibers.

  13. Electronic Veselago lensing in graphene PN junctions

    Science.gov (United States)

    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.

  14. Esophagogastric junction distensibility in hiatus hernia.

    Science.gov (United States)

    Lottrup, C; McMahon, B P; Ejstrud, P; Ostapiuk, M A; Funch-Jensen, P; Drewes, A M

    2016-07-01

    Hiatus hernia is known to be an important risk factor for developing gastroesophageal reflux disease. We aimed to use the endoscopic functional lumen imaging probe (EndoFLIP) to evaluate the functional properties of the esophagogastric junction. EndoFLIP assessments were made in 30 patients with hiatus hernia and Barrett's esophagus, and in 14 healthy controls. The EndoFLIP was placed straddling the esophagogastric junction and the bag distended stepwise to 50 mL. Cross-sectional areas of the bag and intra-bag pressures were recorded continuously. Measurements were made in the separate sphincter components and hiatus hernia cavity. EndoFLIP measured functional aspects such as sphincter distensibility and pressure of all esophagogastric junction components and visualized all hiatus hernia present at endoscopy. The lower esophageal sphincter in hiatus hernia patients had a lower pressure (e.g. 47.7 ± 13.0 vs. 61.4 ± 19.2 mm Hg at 50-mL distension volume) and was more distensible (all P hiatus hernia patients, the crural diaphragm had a lower pressure (e.g. 29.6 ± 10.1 vs. 47.7 ± 13.0 mm Hg at 50-mL distension volume) and was more distensible (all P hiatus hernia and to measure the functional properties of the esophagogastric junction. Furthermore, EndoFLIP distinguished the separate esophagogastric junction components in hiatus hernia patients, and may help us understand the biomechanics of the esophagogastric junction and the mechanisms behind hiatal herniation. PMID:25789842

  15. Magnesium gating of cardiac gap junction channels.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  18. Rectangular-to-circular groove waveguide junction

    Institute of Scientific and Technical Information of China (English)

    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.

  19. Electron and Phonon Transport in Molecular Junctions

    DEFF Research Database (Denmark)

    Li, Qian

    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...... 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...

  20. Bursting behaviour in coupled Josephson junctions.

    Science.gov (United States)

    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

  1. Gap junctions-guards of excitability

    DEFF Research Database (Denmark)

    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....

  2. Microscopic tunneling theory of long Josephson junctions

    DEFF Research Database (Denmark)

    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 detai......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...

  3. The Geometric Field at a Josephson Junction

    CERN Document Server

    Atanasov, Victor

    2016-01-01

    A geometric potential from the kinetic term of a constrained to a curved hyper-plane of space-time quantum superconducting condensate is derived. An energy conservation relation involving the geometric field at every material point in the superconductor is demonstrated. At a Josephson junction the energy conservation relation implies the possibility to transform electric energy into geometric field energy, that is curvature of space-time. Experimental procedures to verify that the Josephson junction can act as a voltage-to-curvature converter are discussed.

  4. Common features of a vortex structure in long exponentially shaped Josephson junctions and Josephson junctions with inhomogeneities

    Science.gov (United States)

    Boyadjiev, T. L.; Semerdjieva, E. G.; Shukrinov, Yu. M.

    2007-09-01

    We study the vortex structure in three different models of the long Josephson junction: the exponentially shaped Josephson junction and the Josephson junctions with the resistor and the shunt inhomogeneities in the barrier layer. For these three models the critical curves “critical current-magnetic field” are numerically constructed. We develop the idea of the equivalence of the exponentially shaped Josephson junction and the rectangular junction with the distributed inhomogeneity and demonstrate that at some parameters of the shunt and the resistor inhomogeneities in the ends of the junction the corresponding critical curves are very close to the exponentially shaped one.

  5. Common features of a vortex structure in long exponentially shaped Josephson junctions and Josephson junctions with inhomogeneities

    Energy Technology Data Exchange (ETDEWEB)

    Boyadjiev, T.L. [Joint Institute for Nuclear Research, 141980 Dubna (Russian Federation); Semerdjieva, E.G. [Plovdiv University, 24 Tzar Asen Str., Plovdiv 4000 (Bulgaria); Shukrinov, Yu.M. [Joint Institute for Nuclear Research, 141980 Dubna (Russian Federation)], E-mail: shukrinv@theor.jinr.ru

    2007-09-01

    We study the vortex structure in three different models of the long Josephson junction: the exponentially shaped Josephson junction and the Josephson junctions with the resistor and the shunt inhomogeneities in the barrier layer. For these three models the critical curves 'critical current-magnetic field' are numerically constructed. We develop the idea of the equivalence of the exponentially shaped Josephson junction and the rectangular junction with the distributed inhomogeneity and demonstrate that at some parameters of the shunt and the resistor inhomogeneities in the ends of the junction the corresponding critical curves are very close to the exponentially shaped one.

  6. Preface: Charge transport in nanoscale junctions

    Science.gov (United States)

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

    OpenAIRE

    João Ramos Costa Andrade; Carla Cavalheiro da Silva

    1987-01-01

    Enteropathogenic E. coli (EPEC) infection of Hep-2 cells preoceeds through bacterial attachment to cell surface and internalization of adhered bacteria. EPEC attachment is a prerequisite for cell infection and is mediated by adhesins that recognize carbohydrate-containing receptors on cell membrane. Such endocytosis-inducer adhesins (EIA) also promote EPEC binding to infant enterocytes, suggesting that EIA may have an important role on EPEC gastroenteritis.A infecção de células Hep-2 por E. c...

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

    OpenAIRE

    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...

  9. Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity.

    Science.gov (United States)

    El Hage, Tatiana; Merlen, Clémence; Fabrega, Sylvie; Authier, François

    2007-05-01

    Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic

  10. Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity.

    Science.gov (United States)

    El Hage, Tatiana; Merlen, Clémence; Fabrega, Sylvie; Authier, François

    2007-05-01

    Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic

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

    Science.gov (United States)

    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.

  12. Desmosomal Molecules In and Out of Adhering Junctions: Normal and Diseased States of Epidermal, Cardiac and Mesenchymally Derived Cells

    Directory of Open Access Journals (Sweden)

    Sebastian Pieperhoff

    2010-01-01

    Full Text Available Current cell biology textbooks mention only two kinds of cell-to-cell adhering junctions coated with the cytoplasmic plaques: the desmosomes (maculae adhaerentes, anchoring intermediate-sized filaments (IFs, and the actin microfilament-anchoring adherens junctions (AJs, including both punctate (puncta adhaerentia and elongate (fasciae adhaerentes structures. In addition, however, a series of other junction types has been identified and characterized which contain desmosomal molecules but do not fit the definition of desmosomes. Of these special cell-cell junctions containing desmosomal glycoproteins or proteins we review the composite junctions (areae compositae connecting the cardiomyocytes of mature mammalian hearts and their importance in relation to human arrhythmogenic cardiomyopathies. We also emphasize the various plakophilin-2-positive plaques in AJs (coniunctiones adhaerentes connecting proliferatively active mesenchymally-derived cells, including interstitial cells of the heart and several soft tissue tumor cell types. Moreover, desmoplakin has also been recognized as a constituent of the plaques of the complexus adhaerentes connecting certain lymphatic endothelial cells. Finally, we emphasize the occurrence of the desmosomal transmembrane glycoprotein, desmoglein Dsg2, out of the context of any junction as dispersed cell surface molecules in certain types of melanoma cells and melanocytes. This broadening of our knowledge on the diversity of AJ structures indicates that it may still be too premature to close the textbook chapters on cell-cell junctions.

  13. Spin and valley transports in junctions of Dirac fermions

    OpenAIRE

    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 ...

  14. The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Delmas, B; Besnardeau, L;

    1998-01-01

    adsorption to the pAPN-MDCK cells. TGEV was also observed in endocytic pits and apical vesicles after 3 to 10 min of incubation at 38 degrees C. The number of pits and apical vesicles was increased by the TGEV incubation, indicating an increase in endocytosis. After 10 min of incubation, a distinct TGEV......-pAPN-containing population of large intracellular vesicles, morphologically compatible with endosomes, was found. A higher density of pAPN receptors was observed in the pits beneath the virus particles than in the surrounding plasma membrane, indicating that TGEV recruits pAPN receptors before endocytosis. Ammonium chloride...... and bafilomycin A1 markedly inhibited the TGEV infection as judged from virus production and protein biosynthesis analyses but did so only when added early in the course of the infection, i.e., about 1 h after the start of endocytosis. Together our results point to an acid intracellular compartment as the site...

  15. Quasiparticle dynamics in superconducting tunnel junctions

    NARCIS (Netherlands)

    Kozorezov, A.G.; Brammertz, G.; Hijmering, R.A.; Wigmore, J.K.; Peacock, A.; Martin, B.; Verhoeve, P.; Golubov, A.A.; Rogalla, H.

    2006-01-01

    Superconducting Tunnel Junctions (STJs) used as single photon detectors possess extreme sensitivity and excellent resolving power. However, like many other cryogenic detectors they operate under extremely non-equilibrium conditions. In order to understand the physics of the non-equilibrium, non-stat

  16. Polyphosphonium-based ion bipolar junction transistors.

    Science.gov (United States)

    Gabrielsson, Erik O; Tybrandt, Klas; Berggren, Magnus

    2014-11-01

    Advancements in the field of electronics during the past few decades have inspired the use of transistors in a diversity of research fields, including biology and medicine. However, signals in living organisms are not only carried by electrons but also through fluxes of ions and biomolecules. Thus, in order to implement the transistor functionality to control biological signals, devices that can modulate currents of ions and biomolecules, i.e., ionic transistors and diodes, are needed. One successful approach for modulation of ionic currents is to use oppositely charged ion-selective membranes to form so called ion bipolar junction transistors (IBJTs). Unfortunately, overall IBJT device performance has been hindered due to the typical low mobility of ions, large geometries of the ion bipolar junction materials, and the possibility of electric field enhanced (EFE) water dissociation in the junction. Here, we introduce a novel polyphosphonium-based anion-selective material into npn-type IBJTs. The new material does not show EFE water dissociation and therefore allows for a reduction of junction length down to 2 μm, which significantly improves the switching performance of the ion transistor to 2 s. The presented improvement in speed as well the simplified design will be useful for future development of advanced iontronic circuits employing IBJTs, for example, addressable drug-delivery devices. PMID:25553192

  17. Radiation comb generation with extended Josephson junctions

    Energy Technology Data Exchange (ETDEWEB)

    Solinas, P., E-mail: paolo.solinas@spin.cnr.it [SPIN-CNR, Via Dodecaneso 33, 16146 Genova (Italy); Bosisio, R., E-mail: riccardo.bosisio@nano.cnr.it [SPIN-CNR, Via Dodecaneso 33, 16146 Genova (Italy); NEST, Instituto Nanoscienze-CNR and Scuola Normale Superiore, I-56127 Pisa (Italy); Giazotto, F., E-mail: giazotto@sns.it [NEST, Instituto Nanoscienze-CNR and Scuola Normale Superiore, I-56127 Pisa (Italy)

    2015-09-21

    We propose the implementation of a Josephson radiation comb generator based on an extended Josephson junction subject to a time dependent magnetic field. The junction critical current shows known diffraction patterns and determines the position of the critical nodes when it vanishes. When the magnetic flux passes through one of such critical nodes, the superconducting phase must undergo a π-jump to minimize the Josephson energy. Correspondingly, a voltage pulse is generated at the extremes of the junction. Under periodic driving, this allows us to produce a comb-like voltage pulses sequence. In the frequency domain, it is possible to generate up to hundreds of harmonics of the fundamental driving frequency, thus mimicking the frequency comb used in optics and metrology. We discuss several implementations through a rectangular, cylindrical, and annular junction geometries, allowing us to generate different radiation spectra and to produce an output power up to 10 pW at 50 GHz for a driving frequency of 100 MHz.

  18. Graphene-based magnetic tunnel junctions

    Science.gov (United States)

    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.

  19. Intercellular junctions in nerve-free hydra

    DEFF Research Database (Denmark)

    McDowall, A W; Grimmelikhuijzen, C J

    1980-01-01

    with particles in an "enplaque conformation appearing as a raised plateau on the E-face or as a depression on the P-face; (ii) structures morphologically similar to gap junctions in rat liver, containing particles on the P-face and corresponding pits on the E-face, both having hexagonal packing with a lattice...

  20. Fluxon Dynamics in Elliptic Annular Josephson Junctions

    DEFF Research Database (Denmark)

    Monaco, Roberto; Mygind, Jesper

    2016-01-01

    We analyze the dynamics of a magnetic flux quantum (current vortex) trapped in a current-biased long planar elliptic annular Josephson tunnel junction. The system is modeled by a perturbed sine-Gordon equation that determines the spatial and temporal behavior of the phase difference across the tu...

  1. TOPICAL REVIEW: Intrinsic Josephson junctions: recent developments

    Science.gov (United States)

    Yurgens, A. A.

    2000-08-01

    Some recent developments in the fabrication of intrinsic Josephson junctions (IJJ) and their application for studying high-temperature superconductors are discussed. The major advantages of IJJ and unsolved problems are outlined. The feasibility of three-terminal devices based on the stacked IJJ is briefly evaluated.

  2. Electric Field Effect in Intrinsic Josephson Junctions

    Science.gov (United States)

    Koyama, T.

    The electric field effect in intrinsic Josephson junction stacks (IJJ's) is investigated on the basis of the capacitively-coupled IJJ model. We clarify the current-voltage characteristics of the IJJ's in the presence of an external electric field. It is predicted that the IJJ's show a dynamical transition to the voltage state as the external electric field is increased.

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

    Science.gov (United States)

    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

  4. Structure Stability of Ⅰ-Type Carbon Nanotube Junctions

    Institute of Scientific and Technical Information of China (English)

    夏丹; 袁喆; 李家明

    2002-01-01

    Carbon nanotubes with junctions may play an important role in future ‘nanoelectronics' and future ‘nano devices'.In particular, junctions constructed with metal and semiconducting nanotubes have potential applications. Basedon the orthogonal tight-binding molecular dynamics method, we present our study of the structure stability ofI-type carbon nanotube junctions.

  5. Shunted-Josephson-junction model. I. The autonomous case

    DEFF Research Database (Denmark)

    Belykh, V. N.; Pedersen, Niels Falsig; Sørensen, O. H.

    1977-01-01

    The shunted-Josephson-junction model: the parallel combination of a capacitance, a phase-dependent conductance, and an ideal junction element biased by a constant current, is discussed for arbitrary values of the junction parameters. The main objective is to provide a qualitative understanding of...

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.

    Directory of Open Access Journals (Sweden)

    Rosa Molfetta

    Full Text Available The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

  8. Endocytosis Pathways of the Folate Tethered Star-Shaped PEG-PCL Micelles in Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yu-Lun Li

    2014-03-01

    Full Text Available This study reports on the cellular uptake of folate tethered micelles using a branched skeleton of poly(ethylene glycol and poly(ε-caprolactone. The chemical structures of the copolymers were characterized by proton nuclear magnetic resonance spectroscopy, and Fourier transform infrared spectroscopy. Doxorubicin (DOX was utilized as an anticancer drug. The highest drug loading efficiencies of DOX in the folate decorated micelle (DMCF and folate-free micelle (DMC were found to be 88.5% and 88.2%, respectively, depending on the segment length of the poly(ε-caprolactone in the copolymers. A comparison of fluorescent microscopic images of the endocytosis pathway in two cell lines, human breast cancer cells (MCF-7 and human oral cavity carcinoma cells (KB, revealed that the micelles were engulfed by KB and MCF-7 cells following in vitro incubation for one hour. Flow cytometric analysis revealed that free folic acid can inhibit the uptake of DOX by 48%–57% and 26%–39% in KB cells and MCF-7 cells, respectively. These results prove that KB cells are relatively sensitive to folate-tethered micelles. Upon administering methyl-β-cyclodextrin, an inhibitor of the caveolae-mediated endocytosis pathway, the uptake of DOX by KB cells was reduced by 69% and that by MCF-7 cells was reduced by 56%. This finding suggests that DMCF enters cells via multiple pathways, thus implying that the folate receptor is not the only target of tumor therapeutics.

  9. Endocytosis-mediated HIV-1 entry and its significance in the elusive behavior of the virus in astrocytes.

    Science.gov (United States)

    Chauhan, Ashok; Mehla, Rajeev; Vijayakumar, Theophilus Sunder; Handy, Indhira

    2014-05-01

    Astrocytes protect neurons but also evoke a proinflammatory response to injury and viral infections including HIV. We investigated the mechanism of HIV-1 infection in primary astrocytes, which showed minimal but productive viral infection independent of CXCR4. As with ectopic-CD4-expressing astrocytes, lysosomotropic agents led to increased HIV-1 infection in wild-type but not Rabs 5, 7, and 11-ablated astrocytes. Instead, HIV-1 infection was decreased in Rab-depleted astrocytes, corroborating viral entry by endocytosis. HIV-1 produced persistent infection in astrocytes (160 days); no evidence of latent infection was seen. Notably, one caveat is that endosomal modifiers enhanced wild-type HIV-1 infection (M- and T-tropic) in astrocytes, suggesting endocytic entry of the virus. Impeding endocytosis by inhibition of Rab 5, 7 or 11 will inhibit HIV infection in astrocytes. Although the contribution of such low-level infection in astrocytes to neurological complications is unclear, it may serve as an elusive viral reservoir in the central nervous system.

  10. Lateral V/VOx/V Tunnel Junctions Formed by Anodic Oxidation

    Science.gov (United States)

    Kirkwood, David; West, Kevin; Lu, Jiwei; Wolf, Stuart

    2008-03-01

    Anodization has been found to be a simple and cost effective technique to produce oxide films of many transition metals. In this work, we have used anodic oxidation as a means of fabricating lateral V/VOx/V junctions. Vanadium wires grown by ion beam deposition were patterned by lithography and an active working window was defined on the wire. VOx was then grown under galvanostatic control in a two electrode electrochemical micro-cell. A droplet of oxygen rich saturated Boric acid was used as the electrolyte to electrically connect the Vandium working electrode to a Platinum wire counter electrode. A constant current of approximately 100 μA/cm^2 was maintained through the cell for various amounts of time. Electrical measurements of the resulting V/VOx/V junctions indicate a metal to insulator transition (MIT) near 340 ^oK that is similar to the structural phase transition and accompanied MIT of VO2 which occurs at this temperature. A 4-fold change in resistance is observed in the junctions. Below this transition temperature a typical junction behavior is observed with a dramatic change in resistance state from high to low with increasing applied current. This non-linear IV characteristic on the junction with a size of 5 μm by 15 μm suggests that the anodized VOx film behaves like a tunneling barrier.

  11. Modeling the power flow in normal conductor-insulator-superconductor junctions

    International Nuclear Information System (INIS)

    Normal conductor-insulator-superconductor (NIS) junctions promise to be interesting for x-ray and phonon sensing applications, in particular due to the expected self-cooling of the N electrode by the tunneling current. Such cooling would enable the operation of the active element of the sensor below the cryostat temperature and at a correspondingly higher sensitivity. It would also allow the use of NIS junctions as microcoolers. At present, this cooling has not been realized in large area junctions (suitable for a number of detector applications). In this article, we discuss a detailed modeling of the heat flow in such junctions; we show how the heat flow into the normal electrode by quasiparticle back-tunneling and phonon absorption from quasiparticle pair recombination can overcompensate the cooling power. This provides a microscopic explanation of the self-heating effects we observe in our large area NIS junctions. The model suggests a number of possible solutions. copyright 1998 American Institute of Physics

  12. Photocurrent spectroscopy of exciton and free particle optical transitions in suspended carbon nanotube pn-junctions

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Shun-Wen [Department of Physics and Astronomy, University of Southern California, Los Angeles, California 90089 (United States); Theiss, Jesse; Hazra, Jubin; Aykol, Mehmet; Kapadia, Rehan [Department of Electrical Engineering, University of Southern California, Los Angeles, California 90089 (United States); Cronin, Stephen B. [Department of Physics and Astronomy, University of Southern California, Los Angeles, California 90089 (United States); Department of Electrical Engineering, University of Southern California, Los Angeles, California 90089 (United States); Department of Chemistry, University of Southern California, Los Angeles, California 90089 (United States)

    2015-08-03

    We study photocurrent generation in individual, suspended carbon nanotube pn-junction diodes formed by electrostatic doping using two gate electrodes. Photocurrent spectra collected under various electrostatic doping concentrations reveal distinctive behaviors for free particle optical transitions and excitonic transitions. In particular, the photocurrent generated by excitonic transitions exhibits a strong gate doping dependence, while that of the free particle transitions is gate independent. Here, the built-in potential of the pn-junction is required to separate the strongly bound electron-hole pairs of the excitons, while free particle excitations do not require this field-assisted charge separation. We observe a sharp, well defined E{sub 11} free particle interband transition in contrast with previous photocurrent studies. Several steps are taken to ensure that the active charge separating region of these pn-junctions is suspended off the substrate in a suspended region that is substantially longer than the exciton diffusion length and, therefore, the photocurrent does not originate from a Schottky junction. We present a detailed model of the built-in fields in these pn-junctions, which, together with phonon-assistant exciton dissociation, predicts photocurrents on the same order of those observed experimentally.

  13. Salt-inducible kinase 1 regulates E-cadherin expression and intercellular junction stability

    OpenAIRE

    Eneling, Kristina; Brion, Laura; Pinto, Vanda; Pinho, Maria J.; Sznajder, Jacob I.; Mochizuki , Naoki; Emoto, Kazuo; Soares-da-Silva, Patricio; Bertorello, Alejandro M.

    2012-01-01

    The protein kinase liver kinase B1 (LKB1) regulates cell polarity and intercellular junction stability. Also, LKB1 controls the activity of salt-inducible kinase 1 (SIK1). The role and relevance of SIK1 and its downstream effectors in linking the LKB1 signals within these processes are partially understood. We hypothesize that SIK1 may link LKB1 signals to the maintenance of epithelial junction stability by regulating E-cadherin expression. Results from our studies using a mouse lung alveolar...

  14. Nanostructured thin films for multiband-gap silicon triple junction solar cells

    OpenAIRE

    R. E. I. Schropp; Li, H. B. T.; Franken, R.H.; Rath, J.K.; van der Werf, C.H.M.; Schuttauf, J.A.; Stolk, R.L.

    2008-01-01

    By implementing nanostructure in multiband-gap proto-Si/proto-SiGe/nc-Si:H triple junction n–i–p solar cells, a considerable improvement in performance has been achieved. The unalloyed active layers in the top and bottom cell of these triple junction cells are deposited by Hot-Wire CVD. A significant current enhancement is obtained by using textured Ag/ZnO back contacts instead of plain stainless steel. We studied the correlation between the integrated current density in the long-wavelength r...

  15. Two-dimensional Josephson junction arrays coupled through a high-Q cavity

    DEFF Research Database (Denmark)

    Filatrella, G.; Pedersen, Niels Falsig; Wiesenfeld, K.

    2001-01-01

    The problem of disordered two-dimensional arrays of underdamped Josephson junctions is addressed. Our simulations show that when coupled to a high-Q cavity, the array exhibits synchronized behavior, and the power emitted can be considerably increased once enough junctions are activated to pump the...... cavity. The highly resonant cavity induces synchronized behavior, which is qualitatively different than what is familiar from other studies on nonlinear oscillator arrays, for example the Kuramoto model. We also address the effects of disorder, as well as the role of detuning between the spontaneous...... emission frequency of the junctions and the cavity resonant frequency. We show with a simple argument that we can predict the scaling behavior of disorder with the size of the array. The consequences for the design of microwave oscillators in the Gigahertz region are discussed...

  16. Spin-polarized transport in a normal/ferromagnetic/normal zigzag graphene nanoribbon junction

    Institute of Scientific and Technical Information of China (English)

    Tian Hong-Yu; Wang Jun

    2012-01-01

    We investigate the spin-dependent electron transport in single and double normal/ferromagnetic/normal zigzag graphene nanoribbon (NG/FG/NG) junctions.The ferromagnetism in the FG region originates from the spontaneous magnetization of the zigzag graphene nanoribbon.It is shown that when the zigzag-chain number of the ribbon is even and only a single transverse mode is actived,the single NG/FG/NG junction can act as a spin polarizer and/or a spin analyzer because of the valley selection rule and the spin-exchange field in the FG,while the double NG/FG/NG/FG/NG junction exhibits a quantum switching effect,in which the on and the off states switch rapidly by varying the cross angle between two FG magnetizations.Our findings may shed light on the application of magnetized graphene nanoribbons to spintronics devices.

  17. Ultra Shallow Arsenic Junctions in Germanium Formed by Millisecond Laser Annealing

    DEFF Research Database (Denmark)

    Hellings, G.; Rosseel, E.; Simoen, E.;

    2011-01-01

    Millisecond laser annealing is used to fabricate ultra shallow arsenic junctions in preamorphized and crystalline germanium, with peak temperatures up to 900 degrees C. At this temperature, As indiffusion is observed while yielding an electrically active concentration up to 5.0 x 10(19) cm(-3...

  18. Laser Annealing of Self-Aligned As+ Implants in Contact Windows for Ultrashallow Junction Formation

    NARCIS (Netherlands)

    Biasotto, C.; Gonda, V.; Nanver, L.K.; Van der Cingel, J.; Jovanovic, V.

    2009-01-01

    In the past it has been shown that ultrashallow junctions with minimum lateral dimensions can be made by implanting self-aligned to the contact window and using one-shot excimer laser annealing (ELA) to activate the dopants. Besides the recrystallization of the implanted Si, the final structuring at

  19. Gap junctions suppress electrical but not [Ca(2+)] heterogeneity in resistance arteries

    DEFF Research Database (Denmark)

    Hald, Bjørn Olav; Welsh, Donald G; von Holstein-Rathlou, Niels-Henrik;

    2014-01-01

    arises, a virtual arteriole was developed that introduces variation in the activities of ion-transport proteins between cells. By varying the level of heterogeneity and subpopulations of gap junctions (GJs), the resulting simulations shows that GJs suppress electrical variation but can only reduce...

  20. Planar Josephson tunnel junctions in a transverse magnetic field

    DEFF Research Database (Denmark)

    Monacoa, R.; Aarøe, Morten; Mygind, Jesper;

    2007-01-01

    Traditionally, since the discovery of the Josephson effect in 1962, the magnetic diffraction pattern of planar Josephson tunnel junctions has been recorded with the field applied in the plane of the junction. Here we discuss the static junction properties in a transverse magnetic field where...... demagnetization effects imposed by the tunnel barrier and electrodes geometry are important. Measurements of the junction critical current versus magnetic field in planar Nb-based high-quality junctions with different geometry, size, and critical current density show that it is advantageous to use a transverse...... magnetic field rather than an in-plane field. The conditions under which this occurs are discussed....