Regulation of vesicular trafficking by Parkinson's disease-associated genes

Directory of Open Access Journals (Sweden)

Tsuyoshi Inoshita

2015-10-01

Full Text Available The regulatory mechanisms that control intracellular vesicular trafficking play important roles in cellular function and viability. Neurons have specific vesicular trafficking systems for synaptic vesicle formation, release and recycling. Synaptic vesicular trafficking impairments induce neuronal dysfunction and physiological and behavioral disorders. Parkinson's disease (PD is an age-dependent neurodegenerative disorder characterized by dopamine depletion and loss of dopamine neurons in the midbrain. The molecular mechanism responsible for the neurodegeneration that occurs during PD is still not understood; however, recent functional analyses of familial PD causative genes suggest that a number of PD causative genes regulate intracellular vesicular trafficking, including synaptic vesicular dynamics. This review focuses on recent insights regarding the functions of PD causative genes, their relationship with vesicular trafficking and how mutations associated with PD affect vesicular dynamics and neuronal survival.

The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells

Directory of Open Access Journals (Sweden)

Giulia Chiaruttini

2016-03-01

Full Text Available Interleukin-12 (IL-12, produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells.

A vesicle trafficking protein αSNAP regulates Paneth cell differentiation in vivo.

Science.gov (United States)

Lechuga, Susana; Naydenov, Nayden G; Feygin, Alex; Jimenez, Antonio J; Ivanov, Andrei I

2017-05-13

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Astrocytic Vesicle Mobility in Health and Disease

Directory of Open Access Journals (Sweden)

Robert Zorec

2013-05-01

Full Text Available Astrocytes are no longer considered subservient to neurons, and are, instead, now understood to play an active role in brain signaling. The intercellular communication of astrocytes with neurons and other non-neuronal cells involves the exchange of molecules by exocytotic and endocytotic processes through the trafficking of intracellular vesicles. Recent studies of single vesicle mobility in astrocytes have prompted new views of how astrocytes contribute to information processing in nervous tissue. Here, we review the trafficking of several types of membrane-bound vesicles that are specifically involved in the processes of (i intercellular communication by gliotransmitters (glutamate, adenosine 5'-triphosphate, atrial natriuretic peptide, (ii plasma membrane exchange of transporters and receptors (EAAT2, MHC-II, and (iii the involvement of vesicle mobility carrying aquaporins (AQP4 in water homeostasis. The properties of vesicle traffic in astrocytes are discussed in respect to networking with neighboring cells in physiologic and pathologic conditions, such as amyotrophic lateral sclerosis, multiple sclerosis, and states in which astrocytes contribute to neuroinflammatory conditions.

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

Directory of Open Access Journals (Sweden)

Yves Briers

Full Text Available Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

Intracellular vesicles as reproduction elements in cell wall-deficient L-form bacteria.

Science.gov (United States)

Briers, Yves; Staubli, Titu; Schmid, Markus C; Wagner, Michael; Schuppler, Markus; Loessner, Martin J

2012-01-01

Cell wall-deficient bacteria, or L-forms, represent an extreme example of bacterial plasticity. Stable L-forms can multiply and propagate indefinitely in the absence of a cell wall. Data presented here are consistent with the model that intracellular vesicles in Listeria monocytogenes L-form cells represent the actual viable reproductive elements. First, small intracellular vesicles are formed along the mother cell cytoplasmic membrane, originating from local phospholipid accumulation. During growth, daughter vesicles incorporate a small volume of the cellular cytoplasm, and accumulate within volume-expanding mother cells. Confocal Raman microspectroscopy demonstrated the presence of nucleic acids and proteins in all intracellular vesicles, but only a fraction of which reveals metabolic activity. Following collapse of the mother cell and release of the daughter vesicles, they can establish their own membrane potential required for respiratory and metabolic processes. Premature depolarization of the surrounding membrane promotes activation of daughter cell metabolism prior to release. Based on genome resequencing of L-forms and comparison to the parental strain, we found no evidence for predisposing mutations that might be required for L-form transition. Further investigations revealed that propagation by intracellular budding not only occurs in Listeria species, but also in L-form cells generated from different Enterococcus species. From a more general viewpoint, this type of multiplication mechanism seems reminiscent of the physicochemical self-reproducing properties of abiotic lipid vesicles used to study the primordial reproduction pathways of putative prokaryotic precursor cells.

Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

International Nuclear Information System (INIS)

Verdin, Anthony; Lounes-Hadj Sahraoui, Anissa; Newsam, Ray; Robinson, Gary; Durand, Roger

2005-01-01

Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities

Polycyclic aromatic hydrocarbons storage by Fusarium solani in intracellular lipid vesicles

Energy Technology Data Exchange (ETDEWEB)

Verdin, Anthony [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France); Lounes-Hadj Sahraoui, Anissa [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)]. E-mail: lounes@univ-littoral.fr; Newsam, Ray [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Robinson, Gary [Department of Biosciences, University of Kent, Canterbury CT2 7NJ (United Kingdom); Durand, Roger [Laboratoire de Mycologie/Phytopathologie/Environnement, Universite du Littoral-Cote d' Opale, 17 avenue Bleriot, BP 699, 62228 Calais Cedex (France)

2005-01-01

Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi. - Fungi can store PAHs intracellularly in lipid vesicles independently of their PAH degradation abilities.

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

Science.gov (United States)

Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

2018-01-01

Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

Squalestatin alters the intracellular trafficking of a neurotoxic prion peptide

Directory of Open Access Journals (Sweden)

Williams Alun

2007-11-01

Full Text Available Abstract Background Neurotoxic peptides derived from the protease-resistant core of the prion protein are used to model the pathogenesis of prion diseases. The current study characterised the ingestion, internalization and intracellular trafficking of a neurotoxic peptide containing amino acids 105–132 of the murine prion protein (MoPrP105-132 in neuroblastoma cells and primary cortical neurons. Results Fluorescence microscopy and cell fractionation techniques showed that MoPrP105-132 co-localised with lipid raft markers (cholera toxin and caveolin-1 and trafficked intracellularly within lipid rafts. This trafficking followed a non-classical endosomal pathway delivering peptide to the Golgi and ER, avoiding classical endosomal trafficking via early endosomes to lysosomes. Fluorescence resonance energy transfer analysis demonstrated close interactions of MoPrP105-132 with cytoplasmic phospholipase A2 (cPLA2 and cyclo-oxygenase-1 (COX-1, enzymes implicated in the neurotoxicity of prions. Treatment with squalestatin reduced neuronal cholesterol levels and caused the redistribution of MoPrP105-132 out of lipid rafts. In squalestatin-treated cells, MoPrP105-132 was rerouted away from the Golgi/ER into degradative lysosomes. Squalestatin treatment also reduced the association between MoPrP105-132 and cPLA2/COX-1. Conclusion As the observed shift in peptide trafficking was accompanied by increased cell survival these studies suggest that the neurotoxicity of this PrP peptide is dependent on trafficking to specific organelles where it activates specific signal transduction pathways.

Rab proteins: The key regulators of intracellular vesicle transport

International Nuclear Information System (INIS)

Bhuin, Tanmay; Roy, Jagat Kumar

2014-01-01

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

Rab proteins: The key regulators of intracellular vesicle transport

Energy Technology Data Exchange (ETDEWEB)

Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

2014-10-15

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

Science.gov (United States)

Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

1994-08-01

The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. These results illustrate the internalization and intracellular

Galectin-3 guides intracellular trafficking of some human serotransferrin glycoforms

DEFF Research Database (Denmark)

Carlsson, Carl Michael; Bengtson, Per; Cucak, Helena

2013-01-01

these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3 bound glycoform increased in cancer, suggesting...

Bacterial pathogen manipulation of host membrane trafficking.

Science.gov (United States)

Asrat, Seblewongel; de Jesús, Dennise A; Hempstead, Andrew D; Ramabhadran, Vinay; Isberg, Ralph R

2014-01-01

Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.

Protein trafficking and maturation regulate intramembrane proteolysis.

Science.gov (United States)

Morohashi, Yuichi; Tomita, Taisuke

2013-12-01

Intramembrane-cleaving proteases (I-CLiPs) are membrane embedded proteolytic enzymes. All substrates identified so far are also membrane proteins, involving a number of critical cellular signaling as well as human diseases. After synthesis and assembly at the endoplasmic reticulum, membrane proteins are exported to the Golgi apparatus and transported to their sites of action. A number of studies have revealed the importance of the intracellular membrane trafficking in i-CLiP-mediated intramembrane proteolysis, not only for limiting the unnecessary encounter between i-CLiPs and their substrate but also for their cleavage site preference. In this review, we will discuss recent advances in our understanding of how each i-CLiP proteolysis is regulated by intracellular vesicle trafficking. This article is part of a Special Issue entitled: Intramembrane Proteases. Copyright © 2013 Elsevier B.V. All rights reserved.

Bovine lactoferrin and lactoferricin interfere with intracellular trafficking of Herpes simplex virus-1.

Science.gov (United States)

Marr, A K; Jenssen, H; Moniri, M Roshan; Hancock, R E W; Panté, N

2009-01-01

Although both lactoferrin (Lf), a component of the innate immune system of living organisms, and its N-terminal pepsin cleavage product lactoferricin (Lfcin) have anti-herpes activity, the precise mechanisms by which Lf and Lfcin bring about inhibition of herpes infections are not fully understood. In the present study, experiments were carried out to characterize the activity of bovine Lf and Lfcin (BLf and BLfcin) against the Herpes simplex virus-1 (HSV-1). HSV-1 cellular uptake and intracellular trafficking were studied by immunofluorescence microscopy. In comparison to the untreated infected control cells, both the BLf- and BLfcin-treated cells showed a significant reduction in HSV-1 cellular uptake. The few virus particles that were internalized appeared to have a delayed intracellular trafficking. Thus, in addition to their interference with the uptake of the virus into host cells, Lf and Lfcin also exert their antiviral effect intracellularly.

Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

Science.gov (United States)

Ojeda Naharros, Irene; Gesemann, Matthias; Mateos, José M; Barmettler, Gery; Forbes, Austin; Ziegler, Urs; Neuhauss, Stephan C F; Bachmann-Gagescu, Ruxandra

2017-12-01

Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

Highly specific detection of muscarinic M3 receptor, G protein interaction and intracellular trafficking in human detrusor using Proximity Ligation Assay (PLA).

Science.gov (United States)

Berndt-Paetz, Mandy; Herbst, Luise; Weimann, Annett; Gonsior, Andreas; Stolzenburg, Jens-Uwe; Neuhaus, Jochen

2018-05-01

-subtype coupling patterns. Detection of M3 interactions with endocytic trafficking proteins by PLA resulted in object sizes correlating with well-documented vesicle sizes of the endocytosis pathway. PLA enabled highly specific detection of M3 receptor expression, demonstration of M3/GP differential coupling and intracellular M3 trafficking in human detrusor smooth muscle cells. This new approach minimized background fluorescence and antibody cross-reactions resulting from single antibody application, and enhanced specificity due to the use of two primary antibodies. Use of subcellular markers allowed visualization of subcellular receptor location. PLA/CLSM allows analyses of muscarinic "receptor - G protein - promiscuity" and intracellular trafficking even in bladder paraffin sections and may give new insights into the etiology and pathology of BPS/IC. Copyright © 2018 Elsevier GmbH. All rights reserved.

Manipulation of Host Cholesterol by Obligate Intracellular Bacteria

Directory of Open Access Journals (Sweden)

Dhritiman Samanta

2017-05-01

Full Text Available Cholesterol is a multifunctional lipid that plays important metabolic and structural roles in the eukaryotic cell. Despite having diverse lifestyles, the obligate intracellular bacterial pathogens Chlamydia, Coxiella, Anaplasma, Ehrlichia, and Rickettsia all target cholesterol during host cell colonization as a potential source of membrane, as well as a means to manipulate host cell signaling and trafficking. To promote host cell entry, these pathogens utilize cholesterol-rich microdomains known as lipid rafts, which serve as organizational and functional platforms for host signaling pathways involved in phagocytosis. Once a pathogen gains entrance to the intracellular space, it can manipulate host cholesterol trafficking pathways to access nutrient-rich vesicles or acquire membrane components for the bacteria or bacteria-containing vacuole. To acquire cholesterol, these pathogens specifically target host cholesterol metabolism, uptake, efflux, and storage. In this review, we examine the strategies obligate intracellular bacterial pathogens employ to manipulate cholesterol during host cell colonization. Understanding how obligate intracellular pathogens target and use host cholesterol provides critical insight into the host-pathogen relationship.

The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery.

Science.gov (United States)

Jiang, Lijuan; Liang, Xin; Liu, Gan; Zhou, Yun; Ye, Xinyu; Chen, Xiuli; Miao, Qianwei; Gao, Li; Zhang, Xudong; Mei, Lin

2018-11-01

Protein nanocapsules have exhibited promising potential applications in the field of protein drug delivery. A major issue with various promising nano-sized biotherapeutics including protein nanocapsules is that owing to their particle size they are subject to cellular uptake via endocytosis, and become entrapped and then degraded within endolysosomes, which can significantly impair their therapeutic efficacy. In addition, many nano-sized biotherapeutics could be also sequestered by autophagosomes and degraded through the autolysosomal pathway. Thus, a limiting step in achieving an effective protein therapy is to facilitate the endosomal escape and auto-lysosomal escape to ensure cytosolic delivery of the protein drugs. Here, we prepared a protein nanocapsule based on BSA (nBSA) and the BSA nanocapsules modified with a bilayer of lauric acid (LA-nBSA) to investigate the escape effects from the endosome and autophagosome. The size distribution of nBSA and LA-nBSA analyzed using DLS presents a uniform diameter centered at 10 nm and 16 nm. The data also showed that FITC-labeled nBSA and LA-nBSA were taken up by the cells mainly through Arf-6-dependent endocytosis and Rab34-mediated macropinocytosis. In addition, LA-nBSA could efficiently escape from endosomal before the degradation in endo-lysosomes. Autophagy could also sequester the LA-nBSA through p62 autophagosome vesicles. These two types of nanocapsules underwent different intracellular destinies and lauric acid (LA) coating played a vital role in intracellular particle retention. In conclusion, the protein nanocapsules modified with LA could enhance the protein nanocapsules escape from intercellular trafficking vesicles, and protect the protein from degradation by the lysosomes.

Intracellular trafficking of the β-secretase and processing of amyloid precursor protein.

Science.gov (United States)

Zhi, Pei; Chia, Pei Zhi Cheryl; Chia, Cheryl; Gleeson, Paul A

2011-09-01

The main component of the amyloid plaques found in the brains of those with Alzheimer's disease (AD) is a polymerized form of the β-amyloid peptide (Aβ) and is considered to play a central role in the pathogenesis of this neurodegenerative disorder. Aβ is derived from the proteolytic processing of the amyloid precursor protein (APP). Beta site APP-cleaving enzyme, BACE1 (also known as β-secretase) is a membrane-bound aspartyl protease responsible for the initial step in the generation of Aβ peptide and is thus a prime target for therapeutic intervention. Substantive evidence now indicates that the processing of APP by BACE1 is regulated by the intracellular sorting of the enzyme and, moreover, perturbations in these intracellular trafficking pathways have been linked to late-onset AD. In this review, we highlight the recent advances in the understanding of the regulation of the intracellular sorting of BACE1 and APP and illustrate why the trafficking of these cargos represent a key issue for understanding the membrane-mediated events associated with the generation of the neurotoxic Aβ products in AD. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16.

Science.gov (United States)

Gottschalk, Elinor Y; Meneses, Patricio I

2015-09-01

The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study advances our knowledge of

Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

Science.gov (United States)

Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

2016-02-12

The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

Science.gov (United States)

Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

2008-05-20

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Intracellular Cholesterol Trafficking and Impact in Neurodegeneration

Directory of Open Access Journals (Sweden)

Fabian Arenas

2017-11-01

Full Text Available Cholesterol is a critical component of membrane bilayers where it plays key structural and functional roles by regulating the activity of diverse signaling platforms and pathways. Particularly enriched in brain, cholesterol homeostasis in this organ is singular with respect to other tissues and exhibits a heterogeneous regulation in distinct brain cell populations. Due to the key role of cholesterol in brain physiology and function, alterations in cholesterol homeostasis and levels have been linked to brain diseases and neurodegeneration. In the case of Alzheimer disease (AD, however, this association remains unclear with evidence indicating that either increased or decreased total brain cholesterol levels contribute to this major neurodegenerative disease. Here, rather than analyzing the role of total cholesterol levels in neurodegeneration, we focus on the contribution of intracellular cholesterol pools, particularly in endolysosomes and mitochondria through its trafficking via specialized membrane domains delineated by the contacts between endoplasmic reticulum and mitochondria, in the onset of prevalent neurodegenerative diseases such as AD, Parkinson disease, and Huntington disease as well as in lysosomal disorders like Niemann-Pick type C disease. We dissect molecular events associated with intracellular cholesterol accumulation, especially in mitochondria, an event that results in impaired mitochondrial antioxidant defense and function. A better understanding of the mechanisms involved in the distribution of cholesterol in intracellular compartments may shed light on the role of cholesterol homeostasis disruption in neurodegeneration and may pave the way for specific intervention opportunities.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles

Directory of Open Access Journals (Sweden)

Welsh Gavin I

2008-05-01

Full Text Available Abstract Background Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Results Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. Conclusion The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Sphingosine-1-phosphate receptors: Zooming in on ligand-induced intracellular trafficking and its functional implications

NARCIS (Netherlands)

Verzijl, Dennis; Peters, Stephan L. M.; Alewijnse, Astrid E.

2010-01-01

Regulatory processes including receptor phosphorylation and intracellular trafficking, also referred to as receptor internalization, are important processes to terminate G protein-coupled receptor (GPCR) signaling. Compelling evidence now indicates that internalization of a receptor is not

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

Science.gov (United States)

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

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

Directory of Open Access Journals (Sweden)

Maria Rödiger

2018-02-01

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

Internalization, Trafficking, Intracellular Processing and Actions of Antibody-Drug Conjugates.

Science.gov (United States)

Xu, Shi

2015-11-01

This review discusses the molecular mechanism involved in the targeting and delivery of antibody-drug conjugates (ADCs), the new class of biopharmaceuticals mainly designed for targeted cancer therapy. this review goes over major progress in preclinical and clinical studies of ADCs, in the past 5 years. The pharmacokinetics and pharmacodynamics of ADCs involve multiple mechanisms, including internalization of ADCs by target cells, intracellular trafficking, release of conjugated drugs, and payload. These mechanisms actually jointly determine the efficacy of ADCs. Therefore, the optimization of ADCs should take them as necessary rationales.

Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis.

Directory of Open Access Journals (Sweden)

Débora L Oliveira

2010-06-01

Full Text Available Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown.We characterized extracellular vesicle production in wild type (WT and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex or MVB functionality (vps23, late endosomal trafficking revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells.Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

Science.gov (United States)

Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

2017-07-01

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1 -/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

Directory of Open Access Journals (Sweden)

Dimitris Missirlis

Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

Trafficking of astrocytic vesicles in hippocampal slices

International Nuclear Information System (INIS)

Potokar, Maja; Kreft, Marko; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert

2009-01-01

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

Human cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells

DEFF Research Database (Denmark)

Merz, G S; Benedikz, Eirikur; Schwenk, V

1997-01-01

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C...... that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin......, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through...

Intracellular trafficking pathways of Cx43 gap junction channels.

Science.gov (United States)

Epifantseva, Irina; Shaw, Robin M

2018-01-01

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Cx43 protein has a short half-life, and rapid synthesis and timely delivery of those proteins to particular subdomains are crucial for the cellular organization of gap junctions and maintenance of intracellular coupling. Impairment in gap junction trafficking contributes to dangerous complications in diseased hearts such as the arrhythmias of sudden cardiac death. Of recent interest are the protein-protein interactions with the Cx43 carboxy-terminus. These interactions have significant impact on the full length Cx43 lifecycle and also contribute to trafficking of Cx43 as well as possibly other functions. We are learning that many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. This review highlights recent mechanisms identified in the trafficking of gap junction channels, involvement of other proteins contributing to the delivery of channels to the cell-cell border, and understanding of possible roles of the newly discovered alternatively translated isoforms in Cx43 biology. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional links between mucolipin-1 and Ca2+-dependent membrane trafficking in mucolipidosis IV

International Nuclear Information System (INIS)

LaPlante, Janice M.; Ye, C.P.; Quinn, Stephen J.; Goldin, Ehud; Brown, Edward M.; Slaugenhaupt, Susan A.; Vassilev, Peter M.

2004-01-01

Most of the membrane trafficking phenomena including those involving the interactions between endosomes and lysosomes are regulated by changes in intracellular Ca 2+ (Ca i ). These processes are disturbed in some types of mucolipidoses and other lysosomal storage disorders, such as mucolipidosis IV (MLIV), a neurological disorder that usually presents during the first year of life with blindness, cognitive impairment, and psychomotor delays. It is caused by mutations in MCOLN1, the gene encoding mucolipin-1 (MLN1), which we have recently established to represent a Ca 2+ -permeable cation channel that is transiently modulated by changes in Ca i . The cells of MLIV patients contain enlarged lysosomes that are likely associated with abnormal sorting and trafficking of these and related organelles. We studied fibroblasts from MLIV patients and found disturbed Ca 2+ signaling and large acidic organelles such as late endosomes and lysosomes (LEL) with altered cellular localization in these cells. The fusion between LEL vesicles in these cells was defective. This is a Ca 2+ -dependent process related to signaling pathways involved in regulation of Ca 2+ homeostasis and trafficking. The MLN1 channels could play a key role in Ca 2+ release from LEL vesicles, which triggers the fusion and trafficking of these organelles. The characterization of this MLN1-mediated Ca 2+ -dependent process should provide new insights into the pathophysiological mechanisms that lead to the development of MLIV and other mucolipidoses associated with similar disturbances in membrane trafficking

Quantification of the Force of Nanoparticle-Cell Membrane Interactions and Its Influence on Intracellular Trafficking of Nanoparticles

Science.gov (United States)

Vasir, Jaspreet K.; Labhasetwar, Vinod

2008-01-01

Understanding the interaction of nanoparticles (NPs) with the cell membrane and their trafficking through cells is imperative to fully explore the use of NPs for efficient intracellular delivery of therapeutics. Here, we report a novel method of measuring the force of NP-cell membrane interactions using atomic force microscopy (AFM). Poly(dl-lactide co-glycolide, PLGA) NPs functionalized with poly-l-lysine were used as a model system, to demonstrate that this force determines the adhesive interaction of NPs with the cell membrane and in turn the extent of cellular uptake of NPs, and hence that of the encapsulated therapeutic. Cellular uptake of NPs was monitored using AFM imaging, and the dynamics of their intracellular distribution was quantified using confocal microscopy. Results demonstrated that the functionalized NPs have a five-fold greater force of adhesion with the cell membrane and the time-lapse AFM images show their rapid internalization than unmodified NPs. The intracellular trafficking study showed that the functionalized NPs escape more rapidly and efficiently from late endosomes than unmodified NPs and result in 10-fold higher intracellular delivery of the encapsulated model protein. The findings described herein enhance our basic understanding of the NP-cell membrane interaction on the basis of physical phenomena that could have wider applications in developing efficient nanocarrier systems for intracellular delivery of therapeutics. PMID:18692238

Nanoparticle orientation to control RNA loading and ligand display on extracellular vesicles for cancer regression

Science.gov (United States)

Pi, Fengmei; Binzel, Daniel W.; Lee, Tae Jin; Li, Zhefeng; Sun, Meiyan; Rychahou, Piotr; Li, Hui; Haque, Farzin; Wang, Shaoying; Croce, Carlo M.; Guo, Bin; Evers, B. Mark; Guo, Peixuan

2018-01-01

Nanotechnology offers many benefits, and here we report an advantage of applying RNA nanotechnology for directional control. The orientation of arrow-shaped RNA was altered to control ligand display on extracellular vesicle membranes for specific cell targeting, or to regulate intracellular trafficking of small interfering RNA (siRNA) or microRNA (miRNA). Placing membrane-anchoring cholesterol at the tail of the arrow results in display of RNA aptamer or folate on the outer surface of the extracellular vesicle. In contrast, placing the cholesterol at the arrowhead results in partial loading of RNA nanoparticles into the extracellular vesicles. Taking advantage of the RNA ligand for specific targeting and extracellular vesicles for efficient membrane fusion, the resulting ligand-displaying extracellular vesicles were capable of specific delivery of siRNA to cells, and efficiently blocked tumour growth in three cancer models. Extracellular vesicles displaying an aptamer that binds to prostate-specific membrane antigen, and loaded with survivin siRNA, inhibited prostate cancer xenograft. The same extracellular vesicle instead displaying epidermal growth-factor receptor aptamer inhibited orthotopic breast cancer models. Likewise, survivin siRNA-loaded and folate-displaying extracellular vesicles inhibited patient-derived colorectal cancer xenograft.

Effect of serum proteins on polystyrene nanoparticle uptake and intracellular trafficking in endothelial cells

International Nuclear Information System (INIS)

Guarnieri, Daniela; Guaccio, Angela; Fusco, Sabato; Netti, Paolo A.

2011-01-01

The physico-chemical properties of nanoparticles (NPs), such as small dimensions, surface charge and surface functionalization, control their capability to interact with cells and, in particular, with sub-cellular components. This interaction can be also influenced by the adsorption of molecules present in biological fluids, like blood, on NP surface. Here, we analysed the effect of serum proteins on 49 and 100 nm red fluorescent polystyrene NP uptake in porcine aortic endothelial (PAE) cells, as a model for vascular transport. To this aim, NP uptake kinetic, endocytic pathway and intracellular trafficking were studied by monitoring NPs inside cells through confocal microscopy and multiple particle tracking (MPT). We demonstrated that NPs are rapidly internalized by cells in serum-free (SF) medium, according to a saturation kinetic. Conversely, in 10% foetal bovine serum-enriched (SE) medium, NP uptake rate results drastically reduced. Moreover, NP internalization depends on an active endocytic mechanism that does not involve clathrin- and caveolae-mediated vesicular transport, in both SE and SF media. Furthermore, MPT data indicate that NP intracellular trafficking is unaffected by protein presence. Indeed, approximately 50–60% of internalized NPs is characterized by a sub-diffusive behaviour, whereas the remaining fraction shows an active motion. These findings demonstrate that the unspecific protein adsorption on NP surface can affect cellular uptake in terms of internalization kinetics, but it is not effective in controlling active and cellular-mediated uptake mechanisms of NPs and their intracellular routes.

The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.

Science.gov (United States)

Carrion, Maria Dolores Perez; Marsicano, Silvia; Daniele, Federica; Marte, Antonella; Pischedda, Francesca; Di Cairano, Eliana; Piovesana, Ester; von Zweydorf, Felix; Kremmer, Elisabeth; Gloeckner, Christian Johannes; Onofri, Franco; Perego, Carla; Piccoli, Giovanni

2017-07-14

Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely β3-Cav2.1 at N-terminus part and β-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.

The conserved dileucine- and tyrosine-based motifs in MLV and MPMV envelope glycoproteins are both important to regulate a common Env intracellular trafficking

Directory of Open Access Journals (Sweden)

Lopez-Vergès Sandra

2006-09-01

Full Text Available Abstract Background Retrovirus particles emerge from the assembly of two structural protein components, Gag that is translated as a soluble protein in the cytoplasm of the host cells, and Env, a type I transmembrane protein. Because both components are translated in different intracellular compartments, elucidating the mechanisms of retrovirus assembly thus requires the study of their intracellular trafficking. Results We used a CD25 (Tac chimera-based approach to study the trafficking of Moloney murine leukemia virus and Mason-Pfizer monkey virus Env proteins. We found that the cytoplasmic tails (CTs of both Env conserved two major signals that control a complex intracellular trafficking. A dileucine-based motif controls the sorting of the chimeras from the trans-Golgi network (TGN toward endosomal compartments. Env proteins then follow a retrograde transport to the TGN due to the action of a tyrosine-based motif. Mutation of either motif induces the mis-localization of the chimeric proteins and both motifs are found to mediate interactions of the viral CTs with clathrin adaptors. Conclusion This data reveals the unexpected complexity of the intracellular trafficking of retrovirus Env proteins that cycle between the TGN and endosomes. Given that Gag proteins hijack endosomal host proteins, our work suggests that the endosomal pathway may be used by retroviruses to ensure proper encountering of viral structural Gag and Env proteins in cells, an essential step of virus assembly.

Evidence for small intracellular vesicles in human blood phagocytes containing cytochrome b558 and the adhesion molecule CD11b/CD18

NARCIS (Netherlands)

Calafat, J.; Kuijpers, T. W.; Janssen, H.; Borregaard, N.; Verhoeven, A. J.; Roos, D.

1993-01-01

Human neutrophils contain a rapidly mobilizable pool of so-called secretory vesicles distinct from the azurophil granules and specific granules. Using human albumin as a marker for these intracellular vesicles in immuno-electron microscopy, we found that part of the cytochrome b558 in non-purified

The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction [version 1; referees: 4 approved

Directory of Open Access Journals (Sweden)

Laura Picas

2016-03-01

Full Text Available Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction.

Rab7b at the intersection of intracellular trafficking and cell migration.

Science.gov (United States)

Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

2015-01-01

Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

OpenAIRE

Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

2008-01-01

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV cap...

Size-Dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-Based Drug-Delivery Systems.

Science.gov (United States)

Wang, Ting; Wang, Lu; Li, Xiaoming; Hu, Xingjie; Han, Yuping; Luo, Yao; Wang, Zejun; Li, Qian; Aldalbahi, Ali; Wang, Lihua; Song, Shiping; Fan, Chunhai; Zhao, Yun; Wang, Maolin; Chen, Nan

2017-06-07

Nanoparticles (NPs) have shown great promise as intracellular imaging probes or nanocarriers and are increasingly being used in biomedical applications. A detailed understanding of how NPs get "in and out" of cells is important for developing new nanomaterials with improved selectivity and less cytotoxicity. Both physical and chemical characteristics have been proven to regulate the cellular uptake of NPs. However, the exocytosis process and its regulation are less explored. Herein, we investigated the size-regulated endocytosis and exocytosis of carboxylated polystyrene (PS) NPs. PS NPs with a smaller size were endocytosed mainly through the clathrin-dependent pathway, whereas PS NPs with a larger size preferred caveolae-mediated endocytosis. Furthermore, our results revealed exocytosis of larger PS NPs and tracked the dynamic process at the single-particle level. These results indicate that particle size is a key factor for the regulation of intracellular trafficking of NPs and provide new insight into the development of more effective cellular nanocarriers.

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

DEFF Research Database (Denmark)

Jensen, Camilla Stampe; Misonou, Hiroaki

2015-01-01

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

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

Directory of Open Access Journals (Sweden)

Christopher R Bohl

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

6K2-induced vesicles can move cell to cell during turnip mosaic virus infection

Directory of Open Access Journals (Sweden)

Romain eGrangeon

2013-12-01

Full Text Available To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs. However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked towards the plasma membrane and were associated with plasmodesmata (PD. We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP to visualize how 6K2 move intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.

Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

International Nuclear Information System (INIS)

Lara, F.A.; Sant'Anna, C.; Lemos, D.; Laranja, G.A.T.; Coelho, M.G.P.; Reis Salles, I.; Michel, A.; Oliveira, P.L.; Cunha-e-Silva, N.; Salmon, D.; Paes, M.C.

2007-01-01

Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane

Trafficking and function of the tetraspanin CD63

Energy Technology Data Exchange (ETDEWEB)

Pols, Maaike S. [Cell Microscopy Center, Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht (Netherlands); Klumperman, Judith, E-mail: j.klumperman@umcutrecht.nl [Cell Microscopy Center, Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht (Netherlands)

2009-05-15

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.

Trafficking and function of the tetraspanin CD63

International Nuclear Information System (INIS)

Pols, Maaike S.; Klumperman, Judith

2009-01-01

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.

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

Directory of Open Access Journals (Sweden)

Lorena Brito de Souza

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

Self-Assembled Core-Shell-Type Lipid-Polymer Hybrid Nanoparticles: Intracellular Trafficking and Relevance for Oral Absorption.

Science.gov (United States)

Li, Qiuxia; Xia, Dengning; Tao, Jinsong; Shen, Aijun; He, Yuan; Gan, Yong; Wang, Chi

2017-10-01

Lipid-polymer hybrid nanoparticles (NPs) are advantageous for drug delivery. However, their intracellular trafficking mechanism and relevance for oral drug absorption are poorly understood. In this study, self-assembled core-shell lipid-polymer hybrid NPs made of poly(lactic-co-glycolic acid) (PLGA) and various lipids were developed to study their differing intracellular trafficking in intestinal epithelial cells and their relevance for oral absorption of a model drug saquinavir (SQV). Our results demonstrated that the endocytosis and exocytosis of hybrid NPs could be changed by varying the kind of lipid. A glyceride mixture (hybrid NPs-1) decreased endocytosis but increased exocytosis in Caco-2 cells, whereas the phospholipid (E200) (hybrid NPs-2) decreased endocytosis but exocytosis was unaffected as compared with PLGA nanoparticles. The transport of hybrid NPs-1 in cells involved various pathways, including caveolae/lipid raft-dependent endocytosis, and clathrin-mediated endocytosis and macropinocytosis, which was different from the other groups of NPs that involved only caveolae/lipid raft-dependent endocytosis. Compared with that of the reference formulation (nanoemulsion), the oral absorption of SQV-loaded hybrid NPs in rats was poor, probably due to the limited drug release and transcytosis of NPs across the intestinal epithelium. In conclusion, the intracellular processing of hybrid NPs in intestinal epithelia can be altered by adding lipids to the NP. However, it appears unfavorable to use PLGA-based NPs to improve oral absorption of SQV compared with nanoemulsion. Our findings will be essential in the development of polymer-based NPs for the oral delivery of drugs with the purpose of improving their oral absorption. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Tyrosine phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

Science.gov (United States)

Zhong, Li; Li, Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.; Weigel-Van Aken, Kirsten A.; Hobbs, Jacqueline A.; Zolotukhin, Sergei; Muzyczka, Nicholas; Srivastava, Arun

2008-01-01

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ~68% and ~74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which leads to ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy. PMID:18834608

Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

International Nuclear Information System (INIS)

Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

2008-01-01

We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

International Nuclear Information System (INIS)

Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

2010-01-01

The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 131 PRPR 134 NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

Endocytic pathway rapidly delivers internalized molecules to lysosomes: an analysis of vesicle trafficking, clustering and mass transfer.

Science.gov (United States)

Pangarkar, Chinmay; Dinh, Anh-Tuan; Mitragotri, Samir

2012-08-20

Lysosomes play a critical role in intracellular drug delivery. For enzyme-based therapies, they represent a potential target site whereas for nucleic acid or many protein drugs, they represent the potential degradation site. Either way, understanding the mechanisms and processes involved in routing of materials to lysosomes after cellular entry is of high interest to the field of drug delivery. Most therapeutic cargoes other than small hydrophobic molecules enter the cells through endocytosis. Endocytosed cargoes are routed to lysosomes via microtubule-based transport and are ultimately shared by various lysosomes via tethering and clustering of endocytic vesicles followed by exchange of their contents. Using a combined experimental and numerical approach, here we studied the rates of mass transfer into and among the endocytic vesicles in a model cell line, 3T3 fibroblasts. In order to understand the relationship of mass transfer with microtubular transport and vesicle clustering, we varied both properties through various pharmacological agents. At the same time, microtubular transport and vesicle clustering were modeled through diffusion-advection equations and the Smoluchowski equations, respectively. Our analysis revealed that the rate of mass transfer is optimally related to microtubular transport and clustering properties of vesicles. Further, the rate of mass transfer is highest in the innate state of the cell. Any perturbation to either microtubular transport or vesicle aggregation led to reduced mass transfer to lysosome. These results suggest that in the absence of an external intervention the endocytic pathway appears to maximize molecular delivery to lysosomes. Strategies are discussed to reduce mass transfer to lysosomes so as to extend the residence time of molecules in endosomes or late endosomes, thus potentially increasing the likelihood of their escape before disposition in the lysosomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid tissue regeneration induced by intracellular ATP delivery-A preliminary mechanistic study.

Directory of Open Access Journals (Sweden)

Harshini Sarojini

Full Text Available We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery-extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold

Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

International Nuclear Information System (INIS)

Chung, Le Thi Kim; Hosaka, Toshio; Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka; Teshigawara, Kiyoshi; Sakai, Tohru; Nakaya, Yutaka; Funaki, Makoto

2010-01-01

In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

Dysfunction of bovine endogenous retrovirus K2 envelope glycoprotein is related to unsuccessful intracellular trafficking.

Science.gov (United States)

Nakaya, Yuki; Miyazawa, Takayuki

2014-06-01

Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry

Cholera toxin subunit B-mediated intracellular trafficking of mesoporous silica nanoparticles toward the endoplasmic reticulum

Science.gov (United States)

Walker, William Andrew

In recent decades, pharmaceutical research has led to the development of numerous treatments for human disease. Nanoscale delivery systems have the potential to maximize therapeutic outcomes by enabling target specific delivery of these therapeutics. The intracellular localization of many of these materials however, is poorly controlled, leading to sequestration in degradative cellular pathways and limiting the efficacy of their payloads. Numerous proteins, particularly bacterial toxins, have evolved mechanisms to subvert the degradative mechanisms of the cell. Here, we have investigated a possible strategy for shunting intracellular delivery of encapsulated cargoes from these pathways by modifying mesoporous silica nanoparticles (MSNs) with the well-characterized bacterial toxin Cholera toxin subunit B (CTxB). Using established optical imaging methods we investigated the internalization, trafficking, and subcellular localization of our modified MSNs in an in vitro animal cell model. We then attempted to demonstrate the practical utility of this approach by using CTxB-modified mesoporous silica nanoparticles to deliver propidium iodide, a membrane-impermeant fluorophore.

Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

Science.gov (United States)

VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

2011-01-01

Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking.

Directory of Open Access Journals (Sweden)

Ruxandra Bachmann-Gagescu

2015-10-01

Full Text Available Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary

Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

International Nuclear Information System (INIS)

Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun

2006-01-01

We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

Exo- and endocytotic trafficking of SCAMP2.

Science.gov (United States)

Toyooka, Kiminori; Matsuoka, Ken

2009-12-01

Exo- and endocytotic membrane trafficking is an essential process for transport of secretory proteins, extracellular glycans, transporters and lipids in plant cells. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco BY-2 cells as a model system, we recently demonstrated that SCAMP2 positive structures containing secretory materials are transported from the Golgi apparatus to the plasma membrane (PM) and/or cell plate. This structure is consisted with clustered vesicles and was thus named the secretory vesicle cluster (SVC). Here, we have utilized the reversible photoswitching fluorescent protein Dronpa1 to trace the movement of SCAMP2 on the PM and cell plate. Activated SCAMP2-Dronpa fluorescence on the PM and cell plate moved into the BY-2 cells within several minutes, but did not spread around PM. This is consistent with recycling of SCAMP2 among endomembrane compartments such as the TGN, PM and cell plate. The relationship between SVC-mediated trafficking and exo- and endocytosis of plant cells is discussed taking into account this new data and knowledge provided by recent reports.

Intracellular trafficking of a pH-responsive drug metal complex.

Science.gov (United States)

Kheirolomoom, Azadeh; Ingham, Elizabeth S; Commisso, Joel; Abushaban, Neveen; Ferrara, Katherine W

2016-12-10

We previously developed a pH-responsive copper-doxorubicin (CuDox) cargo in lysolipid-based temperature-sensitive liposomes (LTSLs). The CuDox complex is released from the particle by elevated temperature; however, full release of doxorubicin from CuDox requires a reduced pH, such as that expected in lysosomes. The primary goal of this study is to evaluate the cellular uptake and intracellular trafficking of the drug-metal complex in comparison with intact liposomes and free drug. We found that the CuDox complex was efficiently internalized by mammary carcinoma cells after release from LTSLs. Intracellular doxorubicin and copper were 6-fold and 5-fold greater, respectively, after a 0.5h incubation with the released CuDox complex, as compared to incubation with intact liposomes containing the complex. Total cellular doxorubicin fluorescence was similar following CuDox and free doxorubicin incubation. Imaging and mass spectrometry assays indicated that the CuDox complex was initially internalized intact but breaks down over time within cells, with intracellular copper decreasing more rapidly than intracellular doxorubicin. Doxorubicin fluorescence was reduced when complexed with copper, and nuclear fluorescence was reduced when cells were incubated with the CuDox complex as compared with free doxorubicin. Therapeutic efficacy, which typically results from intercalation of doxorubicin with DNA, was equivalent for the CuDox complex and free doxorubicin and was superior to that of liposomal doxorubicin formulations. Taken together, the results suggest that quenched CuDox reaches the nucleus and remains efficacious. In order to design protocols for the use of these temperature-sensitive particles in cancer treatment, the timing of hyperthermia relative to drug administration must be examined. When cells were heated to 42°C prior to the addition of free doxorubicin, nuclear drug accumulation increased by 1.8-fold in cancer cells after 5h, and cytotoxicity increased 1

A versatile optical tool for studying synaptic GABAA receptor trafficking.

Science.gov (United States)

Lorenz-Guertin, Joshua M; Wilcox, Madeleine R; Zhang, Ming; Larsen, Mads B; Pilli, Jyotsna; Schmidt, Brigitte F; Bruchez, Marcel P; Johnson, Jon W; Waggoner, Alan S; Watkins, Simon C; Jacob, Tija C

2017-11-15

Live-cell imaging methods can provide critical real-time receptor trafficking measurements. Here, we describe an optical tool to study synaptic γ-aminobutyric acid (GABA) type A receptor (GABA A R) dynamics through adaptable fluorescent-tracking capabilities. A fluorogen-activating peptide (FAP) was genetically inserted into a GABA A R γ2 subunit tagged with pH-sensitive green fluorescent protein (γ2 pH FAP). The FAP selectively binds and activates Malachite Green (MG) dyes that are otherwise non-fluorescent in solution. γ2 pH FAP GABA A Rs are expressed at the cell surface in transfected cortical neurons, form synaptic clusters and do not perturb neuronal development. Electrophysiological studies show γ2 pH FAP GABA A Rs respond to GABA and exhibit positive modulation upon stimulation with the benzodiazepine diazepam. Imaging studies using γ2 pH FAP-transfected neurons and MG dyes show time-dependent receptor accumulation into intracellular vesicles, revealing constitutive endosomal and lysosomal trafficking. Simultaneous analysis of synaptic, surface and lysosomal receptors using the γ2 pH FAP-MG dye approach reveals enhanced GABA A R turnover following a bicucculine-induced seizure paradigm, a finding not detected by standard surface receptor measurements. To our knowledge, this is the first application of the FAP-MG dye system in neurons, demonstrating the versatility to study nearly all phases of GABA A R trafficking. © 2017. Published by The Company of Biologists Ltd.

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

DEFF Research Database (Denmark)

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

1992-01-01

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

Characterization of Leptin Intracellular Trafficking

Directory of Open Access Journals (Sweden)

E Walum

2009-12-01

Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

Science.gov (United States)

Matragkou, Ch; Papachristou, H; Karetsou, Z; Papadopoulos, G; Papamarcaki, T; Vizirianakis, I S; Tsiftsoglou, A S; Choli-Papadopoulou, T

2009-10-09

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its

Comparison of the intracellular trafficking itinerary of ctla-4 orthologues.

Directory of Open Access Journals (Sweden)

Satdip Kaur

Full Text Available CTLA-4 is an essential inhibitor of T cell immune responses. At steady state, most CTLA-4 resides in intracellular compartments due to constitutive internalisation mediated via a tyrosine based endocytic motif (YVKM within the cytoplasmic domain. This domain is highly conserved in mammals suggesting strong selective pressure. In contrast, the C-terminal domain varies considerably in non-mammals such as fish, xenopus and birds. We compared the ability of the C-terminus of these species to direct the trafficking of CTLA-4 with human CTLA-4. Using a chimeric approach, endocytosis was found to be conserved between human, xenopus and chicken CTLA-4 but was reduced substantially in trout CTLA-4, which lacks the conserved YXXM motif. Nevertheless, we identified an alternative YXXF motif in trout CTLA-4 that permitted limited endocytosis. Post-internalisation, CTLA-4 was either recycled or targeted for degradation. Human and chicken CTLA-4, which contain a YVKM motif, showed efficient recycling compared to xenopus CTLA-4 which contains a less efficient YEKM motif. Specific mutation of this motif in human CTLA-4 reduced receptor recycling. These findings suggest evolutionary development in the endocytic and recycling potential of CTLA-4, which may facilitate more refined functions of CTLA-4 within the mammalian immune system.

Platelet activating factor enhances synaptic vesicle exocytosis via PKC, elevated intracellular calcium, and modulation of synapsin 1 dynamics and phosphorylation

Directory of Open Access Journals (Sweden)

Jennetta W Hammond

2016-01-01

Full Text Available Platelet activating factor (PAF is an inflammatory phospholipid signaling molecule implicated in synaptic plasticity, learning and memory and neurotoxicity during neuroinflammation. However, little is known about the intracellular mechanisms mediating PAF’s physiological or pathological effects on synaptic facilitation. We show here that PAF receptors are localized at the synapse. Using fluorescent reporters of presynaptic activity we show that a non-hydrolysable analogue of PAF (cPAF enhances synaptic vesicle release from individual presynaptic boutons by increasing the size or release of the readily releasable pool and the exocytosis rate of the total recycling pool. cPAF also activates previously silent boutons resulting in vesicle release from a larger number of terminals. The underlying mechanism involves elevated calcium within presynaptic boutons and protein kinase C (PKC activation. Furthermore, cPAF increases synapsin I phosphorylation at sites 1 and 3, and increases dispersion of synapsin I from the presynaptic compartment during stimulation, freeing synaptic vesicles for subsequent release. These findings provide a conceptual framework for how PAF, regardless of its cellular origin, can modulate synapses during normal and pathologic synaptic activity.

Gliadin peptide P31-43 localises to endocytic vesicles and interferes with their maturation.

Directory of Open Access Journals (Sweden)

Maria Vittoria Barone

Full Text Available BACKGROUND: Celiac Disease (CD is both a frequent disease (1:100 and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs

[Development of a Novel Liposomal DDS by Manipulating Pharmacokinetics and Intracellular Trafficking for Drug Therapy and Nucleic Acid Medicine].

Science.gov (United States)

Hatakeyama, Hiroto

2018-01-01

　Nucleic acid therapy is expected to be a next generation medicine. We recently developed a multifunctional envelope-type nano device (MEND) for use as a novel delivery system. The modification of polyethylene glycol (PEG), i.e., PEGylation, is useful for achieving the delivery of MENDs to tumors via an enhanced permeability and retention (EPR) effect. However, PEGylation strongly inhibits the cellular uptake and endosomal escape of MEND, which results in significant loss of action, and therefore lost effectiveness, of the cargo therapeutic. For successful nucleic acid delivery in cancer treatment, the crucial problem associated with the use of PEG, known as the "PEG dilemma", must be solved. In this review, we describe the development and application of MEND in overcoming the PEG dilemma based on manipulating both the pharmacokinetics and intracellular trafficking of cellular uptake and endosomal release using a cleavable PEG lipid, a pH-sensitive fusogenic peptide, and a pH-sensitive cationic lipid. We also developed dual-ligand liposomes with a controlled diameter of around 300 nm, then modified these with a specific ligand and a cell penetrating peptide designed to target the neovasculature of tumors. Dual-ligand liposomes could induce an anti-tumor effect in drug resistant tumors by delivering drugs to tumor blood vessels, rather than to the cancer cells themselves. Here, we review our recent efforts to develop a novel liposomal drug delivery system (DDS) by manipulating pharmacokinetics and intracellular trafficking for drug therapy and nucleic acid medicine.

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis.

Science.gov (United States)

Ong, Edmund; Cahill, Catherine

2015-07-03

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

Science.gov (United States)

Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C; Fernandez-Garcia, Nieves

2017-01-01

Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

Pannexin2 oligomers localize into endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

Directory of Open Access Journals (Sweden)

Daniela eBoassa

2015-02-01

Full Text Available Pannexin2 (Panx2 is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS have been documented. Whereas Pannexin1 (Panx1 is fairly ubiquitous and Pannexin3 (Panx3 is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa and HEK293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the

The tinker, tailor, soldier in intracellular B12 trafficking.

Science.gov (United States)

Banerjee, Ruma; Gherasim, Carmen; Padovani, Dominique

2009-10-01

The recognition of eight discrete genetic complementation groups among patients with inherited cobalamin disorders provided early insights into the complexity of a cofactor-processing pathway that supports only two known B(12)-dependent enzymes in mammals. With the identification of all eight genes now completed, biochemical interrogations of their functions have started and are providing novel insights into a trafficking pathway involving porters that tinker with and tailor the active cofactor forms and editors that ensure the fidelity of the cofactor loading process. The principles of sequestration and escorted delivery of a rare and reactive organometallic cofactor that are emerging from studies on B(12) might be of general relevance to other cofactor trafficking pathways.

Role of LRRK2 in the regulation of dopamine receptor trafficking.

Directory of Open Access Journals (Sweden)

Mauro Rassu

Full Text Available Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD. Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1 and D2 (DRD2 trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

Eeyarestatin 1 interferes with both retrograde and anterograde intracellular trafficking pathways.

Directory of Open Access Journals (Sweden)

Mina-Olga Aletrari

Full Text Available The small molecule Eeyarestatin I (ESI inhibits the endoplasmic reticulum (ER-cytosol dislocation and subsequent degradation of ERAD (ER associated protein degradation substrates. Toxins such as ricin and Shiga/Shiga-like toxins (SLTx are endocytosed and trafficked to the ER. Their catalytic subunits are thought to utilise ERAD-like mechanisms to dislocate from the ER into the cytosol, where a proportion uncouples from the ERAD process, recovers a catalytic conformation and destroys their cellular targets. We therefore investigated ESI as a potential inhibitor of toxin dislocation.Using cytotoxicity measurements, we found no role for ES(I as an inhibitor of toxin dislocation from the ER, but instead found that for SLTx, ESI treatment of cells was protective by reducing the rate of toxin delivery to the ER. Microscopy of the trafficking of labelled SLTx and its B chain (lacking the toxic A chain showed a delay in its accumulation at a peri-nuclear location, confirmed to be the Golgi by examination of SLTx B chain metabolically labelled in the trans-Golgi cisternae. The drug also reduced the rate of endosomal trafficking of diphtheria toxin, which enters the cytosol from acidified endosomes, and delayed the Golgi-specific glycan modifications and eventual plasma membrane appearance of tsO45 VSV-G protein, a classical marker for anterograde trafficking.ESI acts on one or more components that function during vesicular transport, whilst at least one retrograde trafficking pathway, that of ricin, remains unperturbed.

Optogenetic acidification of synaptic vesicles and lysosomes.

Science.gov (United States)

Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

2015-12-01

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

Demonstration of differential quantitative requirements for NSF among multiple vesicle fusion pathways of GLUT4 using a dominant-negative ATPase-deficient NSF

International Nuclear Information System (INIS)

Chen Xiaoli; Matsumoto, Hideko; Hinck, Cynthia S.; Al-Hasani, Hadi; St-Denis, Jean-Francois; Whiteheart, Sidney W.; Cushman, Samuel W.

2005-01-01

In this study, we investigated the relative participation of N-ethylmaleimide-sensitive factor (NSF) in vivo in a complex multistep vesicle trafficking system, the translocation response of GLUT4 to insulin in rat adipose cells. Transfections of rat adipose cells demonstrate that over-expression of wild-type NSF has no effect on total, or basal and insulin-stimulated cell-surface expression of HA-tagged GLUT4. In contrast, a dominant-negative NSF (NSF-D1EQ) can be expressed at a low enough level that it has little effect on total HA-GLUT4, but does reduce both basal and insulin-stimulated cell-surface HA-GLUT4 by ∼50% without affecting the GLUT4 fold-translocation response to insulin. However, high expression levels of NSF-D1EQ decrease total HA-GLUT4. The inhibitory effect of NSF-D1EQ on cell-surface HA-GLUT4 is reversed when endocytosis is inhibited by co-expression of a dominant-negative dynamin (dynamin-K44A). Moreover, NSF-D1EQ does not affect cell-surface levels of constitutively recycling GLUT1 and TfR, suggesting a predominant effect of low-level NSF-D1EQ on the trafficking of GLUT4 from the endocytic recycling compared to the intracellular GLUT4-specific compartment. Thus, our data demonstrate that the multiple fusion steps in GLUT4 trafficking have differential quantitative requirements for NSF activity. This indicates that the rates of plasma and intracellular membrane fusion reactions vary, leading to differential needs for the turnover of the SNARE proteins

Cytoplasmic tail of coronavirus spike protein has intracellular

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

Structure-Function Correlation Analysis of Connexin50 Missense Mutations Causing Congenital Cataract: Electrostatic Potential Alteration Could Determine Intracellular Trafficking Fate of Mutants

Directory of Open Access Journals (Sweden)

Devroop Sarkar

2014-01-01

Full Text Available Connexin50 (Cx50 mutations are reported to cause congenital cataract probably through the disruption of intercellular transport in the lens. Cx50 mutants that undergo mistrafficking have generally been associated with failure to form functional gap junction channels; however, sometimes even properly trafficked mutants were found to undergo similar consequences. We hereby wanted to elucidate any structural bases of the varied functional consequences of Cx50 missense mutations through in silico approach. Computational studies have been done based on a Cx50 homology model to assess conservation, solvent accessibility, and 3-dimensional localization of mutated residues as well as mutation-induced changes in surface electrostatic potential, H-bonding, and steric clash. This was supplemented with meta-analysis of published literature on the functional properties of connexin missense mutations. Analyses revealed that the mutation-induced critical alterations of surface electrostatic potential in Cx50 mutants could determine their fate in intracellular trafficking. A similar pattern was observed in case of mutations involving corresponding conserved residues in other connexins also. Based on these results the trafficking fates of 10 uncharacterized Cx50 mutations have been predicted. Further experimental analyses are needed to validate the observed correlation.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

DEFF Research Database (Denmark)

Sezgin, Erdinc; Betul Can, Fatma; Schneider, Falk

2016-01-01

Cholesterol is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently...... for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase separated giant unilamellar vesicles (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C...... in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent cholesterol analogs in visualizing cellular cholesterol dynamics....

Endocytosis and Endosomal Trafficking in Plants.

Science.gov (United States)

Paez Valencia, Julio; Goodman, Kaija; Otegui, Marisa S

2016-04-29

Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

Science.gov (United States)

Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

2014-01-01

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

Directory of Open Access Journals (Sweden)

Irini Topalidou

2016-05-01

Full Text Available The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery.

Directory of Open Access Journals (Sweden)

Ming Wang

Full Text Available Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.

REGULATED VESICULAR TRAFFICKING OF SPECIFIC PCDH15 AND VLGR1 VARIANTS IN AUDITORY HAIR CELLS

Science.gov (United States)

Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T.; Cosgrove, Dominic

2012-01-01

Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal co-localization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular sub-pools. The apically trafficked pool co-localized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associates with membrane microdomains and SNAP25. Moreover, co-immunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions. PMID:23035094

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

International Nuclear Information System (INIS)

Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph; Bernatchez, Pascal

2011-01-01

Highlights: ► Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. ► Dysferlin interacts with key signaling proteins for transcytosis in EC. ► Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

Energy Technology Data Exchange (ETDEWEB)

Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph [UBC James Hogg Research Centre, Institute for Heart and Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia (Canada); University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia (Canada); Bernatchez, Pascal, E-mail: pbernatc@mail.ubc.ca [UBC James Hogg Research Centre, Institute for Heart and Lung Health, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia (Canada); University of Victoria - Genome BC Proteomics Centre, University of Victoria, Victoria, British Columbia (Canada)

2011-11-18

Highlights: Black-Right-Pointing-Pointer Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. Black-Right-Pointing-Pointer Dysferlin interacts with key signaling proteins for transcytosis in EC. Black-Right-Pointing-Pointer Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, ultimately, help bridging dysferlinopathies to aberrant membrane signaling.

The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells.

Science.gov (United States)

Shin, Jung Hoon; Park, Se-Ho

2014-05-01

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking.

Science.gov (United States)

Budayeva, Hanna G; Cristea, Ileana M

2016-10-01

previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ca2+-dependent mobility of vesicles capturing anti-VGLUT1 antibodies

International Nuclear Information System (INIS)

Stenovec, Matjaz; Kreft, Marko; Grilc, Sonja; Potokar, Maja; Kreft, Mateja Erdani; Pangrsic, Tina; Zorec, Robert

2007-01-01

Several aspects of secretory vesicle cycle have been studied in the past, but vesicle trafficking in relation to the fusion site is less well understood. In particular, the mobility of recaptured vesicles that traffic back toward the central cytoplasm is still poorly defined. We exposed astrocytes to antibodies against the vesicular glutamate transporter 1 (VGLUT1), a marker of glutamatergic vesicles, to fluorescently label vesicles undergoing Ca 2+ -dependent exocytosis and examined their number, fluorescence intensity, and mobility by confocal microscopy. In nonstimulated cells, immunolabeling revealed discrete fluorescent puncta, indicating that VGLUT1 vesicles, which are approximately 50 nm in diameter, cycle slowly between the plasma membrane and the cytoplasm. When the cytosolic Ca 2+ level was raised with ionomycin, the number and fluorescence intensity of the puncta increased, likely because the VGLUT1 epitopes were more accessible to the extracellularly applied antibodies following Ca 2+ -triggered exocytosis. In nonstimulated cells, the mobility of labeled vesicles was limited. In stimulated cells, many vesicles exhibited directional mobility that was abolished by cytoskeleton-disrupting agents, indicating dependence on intact cytoskeleton. Our findings show that postfusion vesicle mobility is regulated and may likely play a role in synaptic vesicle cycle, and also more generally in the genesis and removal of endocytic vesicles

PIST regulates the intracellular trafficking and plasma membrane expression of Cadherin 23

Directory of Open Access Journals (Sweden)

Oshima Kazuo

2010-10-01

Full Text Available Abstract Background The atypical cadherin protein cadherin 23 (CDH23 is crucial for proper function of retinal photoreceptors and inner ear hair cells. As we obtain more and more information about the specific roles of cadherin 23 in photoreceptors and hair cells, the regulatory mechanisms responsible for the transport of this protein to the plasma membrane are largely unknown. Results PIST, a Golgi-associated, PDZ domain-containing protein, interacted with cadherin 23 via the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI of cadherin 23. By binding to cadherin 23, PIST retained cadherin 23 in the trans-Golgi network of cultured cells. The retention was released when either of the two known cadherin 23-binding proteins MAGI-1 and harmonin was co-expressed. Similar to MAGI-1 and harmonin, PIST was detected in mouse inner ear sensory hair cells. Conclusions PIST binds cadherin 23 via its PDZ domain and retains cadherin 23 in trans-Golgi network. MAGI-1 and harmonin can compete with PIST for binding cadherin 23 and release cadherin 23 from PIST's retention. Our finding suggests that PIST, MAGI-1 and harmonin collaborate in intracellular trafficking of cadherin 23 and regulate the plasma membrane expression of cadherin 23.

Regulation of B cell differentiation by intracellular membrane associated proteins and microRNAs: role in the antibody response

Directory of Open Access Journals (Sweden)

Zheng eLou

2015-10-01

Full Text Available B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes and autophagosomes and protein factors specifically associated with these membranes, including Rab7, Atg5 and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, CSR/SHM, and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulate AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

Science.gov (United States)

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

Hierarchical unilamellar vesicles of controlled compositional heterogeneity.

Directory of Open Access Journals (Sweden)

Maik Hadorn

Full Text Available Eukaryotic life contains hierarchical vesicular architectures (i.e. organelles that are crucial for material production and trafficking, information storage and access, as well as energy production. In order to perform specific tasks, these compartments differ among each other in their membrane composition and their internal cargo and also differ from the cell membrane and the cytosol. Man-made structures that reproduce this nested architecture not only offer a deeper understanding of the functionalities and evolution of organelle-bearing eukaryotic life but also allow the engineering of novel biomimetic technologies. Here, we show the newly developed vesicle-in-water-in-oil emulsion transfer preparation technique to result in giant unilamellar vesicles internally compartmentalized by unilamellar vesicles of different membrane composition and internal cargo, i.e. hierarchical unilamellar vesicles of controlled compositional heterogeneity. The compartmentalized giant unilamellar vesicles were subsequently isolated by a separation step exploiting the heterogeneity of the membrane composition and the encapsulated cargo. Due to the controlled, efficient, and technically straightforward character of the new preparation technique, this study allows the hierarchical fabrication of compartmentalized giant unilamellar vesicles of controlled compositional heterogeneity and will ease the development of eukaryotic cell mimics that resemble their natural templates as well as the fabrication of novel multi-agent drug delivery systems for combination therapies and complex artificial microreactors.

Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

International Nuclear Information System (INIS)

Akbari, Vajihe; Abedi, Daryoush; Pardakhty, Abbas; Sadeghi-Aliabadi, Hojjat

2013-01-01

In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300–600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation

Energy Technology Data Exchange (ETDEWEB)

Akbari, Vajihe; Abedi, Daryoush [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of); Pardakhty, Abbas [Kerman University of Medical Sciences, Pharmaceutics Research Center (Iran, Islamic Republic of); Sadeghi-Aliabadi, Hojjat, E-mail: sadeghi@pharm.mui.ac.ir [Isfahan University of Medical Sciences, Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy (Iran, Islamic Republic of)

2013-04-15

In order to propose non-ionic surfactant vesicles (niosomes) for the treatment of intracellular infections, a remote loading method (active drug encapsulation) followed by sonication was used to prepare nano-niosome formulations containing ciprofloxacin (CPFX). Size analysis, size distribution and zeta potentials of niosomes were evaluated and then their antimicrobial activity, cellular uptake, cytotoxicity, intracellular distribution, and antibacterial activity against intracellular Staphylococcus aureus infection of murine macrophage-like, J774, cells were investigated in comparison to free drug. Our findings reveal that size and composition of the niosome formula can influence their in vitro biological properties. Vesicles in the 300-600 nm size range were phagocytosed to a greater degree by macrophages in comparison to other size vesicles. The minimum inhibitory concentrations (MICs) of CPFX-loaded niosomes were two to eightfold lower than MICs of free CPFX. In addition, niosome encapsulation of CPFX provided high intracellular antimicrobial activities while free CPFX is ineffective for eradicating intracellular forms of S. aureus. Encapsulation of CPFX in niosomes generally decreased its in vitro cytotoxicity. Our results show that niosomes are suitable drug delivery systems with good efficacy and safety properties to be proposed for drug targeting against intracellular infections.

Active elastohydrodynamics of vesicles in narrow blind constrictions

Science.gov (United States)

Fai, T. G.; Kusters, R.; Harting, J.; Rycroft, C. H.; Mahadevan, L.

2017-11-01

Fluid-resistance limited transport of vesicles through narrow constrictions is a recurring theme in many biological and engineering applications. Inspired by the motor-driven movement of soft membrane-bound vesicles into closed neuronal dendritic spines, here we study this problem using a combination of passive three-dimensional simulations and a simplified semianalytical theory for the active transport of vesicles forced through constrictions by molecular motors. We show that the motion of these objects is characterized by two dimensionless quantities related to the geometry and to the strength of forcing relative to the vesicle elasticity. We use numerical simulations to characterize the transit time for a vesicle forced by fluid pressure through a constriction in a channel and find that relative to an open channel, transport into a blind end leads to the formation of a smaller forward-flowing lubrication layer that strongly impedes motion. When the fluid pressure forcing is complemented by forces due to molecular motors that are responsible for vesicle trafficking into dendritic spines, we find that the competition between motor forcing and fluid drag results in multistable dynamics reminiscent of the real system. Our study highlights the role of nonlocal hydrodynamic effects in determining the kinetics of vesicular transport in constricted geometries.

ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

Science.gov (United States)

Shah, Abdul Haseeb; Banerjee, Atanu; Rawal, Manpreet Kaur; Saxena, Ajay Kumar; Mondal, Alok Kumar; Prasad, Rajendra

2015-08-01

The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Chlamydia trachomatis co-opts GBF1 and CERT to acquire host sphingomyelin for distinct roles during intracellular development.

Directory of Open Access Journals (Sweden)

Cherilyn A Elwell

2011-09-01

Full Text Available The strain designated Chlamydia trachomatis serovar that was used for experiments in this paper is Chlamydia muridarum, a species closely related to C. trachomatis (and formerly termed the Mouse Pneumonitis strain of C. trachomatis. [corrected]. The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM, a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM, for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic

Intracellular Environment-Responsive Stabilization of Polymer Vesicles Formed from Head-Tail Type Polycations Composed of a Polyamidoamine Dendron and Poly(L-lysine

Directory of Open Access Journals (Sweden)

Kenji Kono

2013-09-01

Full Text Available For the development of effective drug carriers, nanocapsules that respond to micro-environmental changes including a decrease in pH and a reductive environment were prepared by the stabilization of polymer vesicles formed from head-tail type polycations, composed of a polyamidoamine dendron head and a poly(L-lysine tail (PAMAM dendron-PLL, through the introduction of disulfide bonds between the PLL tails. Disulfide bonds were successfully introduced through the reaction of Lys residues in the PAMAM dendron-PLL polymer vesicles with 2-iminothiolane. The stabilization of PAMAM dendron-PLL polymer vesicles was confirmed by dynamic light scattering measurements. In acid-base titration experiments, nanocapsules cross-linked by disulfide bonds had a buffering effect during the cellular uptake process. The PAMAM dendron-PLL nanocapsules were used to incorporate the fluorescent dyes rhodamine 6G and fluorescein as a drug model. Cationic rhodamine 6G was generally not released from the nanocapsules because of the electrostatic barrier of the PLL membrane. However, the nanocapsules were destabilized at high glutathione concentrations corresponding to intracellular concentrations. Rhodamine 6G was immediately released from the nanocapsules because of destabilization upon the cleavage of disulfide bonds. This release of rhodamine 6G from the nanocapsules was also observed in HeLa cells by laser confocal microscopy.

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

International Nuclear Information System (INIS)

Endsley, Mark A.; Somasunderam, Anoma D.; Li, Guangyu; Oezguen, Numan; Thiviyanathan, Varatharasa; Murray, James L.; Rubin, Donald H.; Hodge, Thomas W.

2014-01-01

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4 + T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4 + T lymphocytes

Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

Energy Technology Data Exchange (ETDEWEB)

Endsley, Mark A., E-mail: maendsle@utmb.edu [Department Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555 (United States); Somasunderam, Anoma D., E-mail: asomasun@utmb.edu [Department Internal Medicine, Division of Infectious Diseases, University of Texas Medical Branch, 301 University Blvd, Galveston, TX 77555 (United States); Li, Guangyu, E-mail: LIG001@mail.etsu.edu [Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614 (United States); Oezguen, Numan, E-mail: numan.oezguen@bcm.edu [Department of Pathology and Immunology, Microbiome Center, Texas Children' s Hospital, Houston, TX 77030 (United States); Thiviyanathan, Varatharasa, E-mail: Varatharasa.Thiviyanathan@uth.tmc.edu [Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030 (United States); Murray, James L., E-mail: jmurray100@yahoo.com [GeneTAG Technology, Inc., 3155 Northwoods Place, Norcross, GA 30071 (United States); Rubin, Donald H., E-mail: don.h.rubin@vanderbilt.edu [Research Medicine, VA Tennessee Valley Healthcare System, 1310 24th Ave. South, Nashville, TN 37212 (United States); Departments of Medicine, Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 1161 21st Ave South, Nashville, TN 37232 (United States); Hodge, Thomas W., E-mail: twhodge3@gmail.com [Pre-clinical and Antiviral Research, Tamir Biotechnology, Inc., 12625 High Bluff Dr., Suite 113, San Diego, CA 92130 (United States); and others

2014-04-15

Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.

Stochastic models of intracellular transport

KAUST Repository

Bressloff, Paul C.

2013-01-09

The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures. © 2013 American Physical Society.

Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.

LENUS (Irish Health Repository)

Rogan, Mark P

2012-02-01

Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

Mechanisms of pH-Sensitivity and Cellular Internalization of PEOz-b-PLA Micelles with Varied Hydrophilic/Hydrophobic Ratios and Intracellular Trafficking Routes and Fate of the Copolymer.

Science.gov (United States)

Wang, Dishi; Zhou, Yanxia; Li, Xinru; Qu, Xiaoyou; Deng, Yunqiang; Wang, Ziqi; He, Chuyu; Zou, Yang; Jin, Yiguang; Liu, Yan

2017-03-01

pH-responsive polymeric micelles have shown promise for the targeted and intracellular delivery of antitumor agents. The present study aimed to elucidate the possible mechanisms of pH-sensitivity and cellular internalization of PEOz-b-PLA micelles in detail, further unravel the effect of hydrophilic/hydrophobic ratio of the micelles on their cellular internalization, and examine the intracellular trafficking routes and fate of PEOz-b-PLA after internalization of the micelles. The results of variations in the size and Zeta potential of PEOz-b-PLA micelles and cross-sectional area of PEOz-b-PLA molecules with pH values suggested that electrostatic repulsion between PEOz chains resulting from ionization of the tertiary amide groups along PEOz chain at pH lower than its pK a was responsible for pH-sensitivity of PEOz-b-PLA micelles. Furthermore, the studies on internalization of PEOz-b-PLA micelles by MCF-7 cells revealed that the uptake of PEOz-b-PLA micelles was strongly influenced by their structural features, and showed that PEOz-b-PLA micelles with hydrophilic/hydrophobic ratio of 1.7-2.0 exhibited optimal cellular uptake. No evident alteration in cellular uptake of PEOz-b-PLA micelles was detected by flow cytometry upon the existence of EIPA and chlorpromazine. However, the intracellular uptake of the micelles in the presence of MβCD and genistein was effectively inhibited. Hence, the internalization of such micelles by MCF-7 cells appeared to proceed mainly through caveolae/lipid raft-mediated endocytosis without being influenced by their hydrophilic/hydrophobic ratio. Confocal micrographs revealed that late endosomes, mitochondria and endoplasmic reticulum were all involved in the intracellular trafficking of PEOz-b-PLA copolymers following their internalization via endocytosis, and then part of them was excreted from tumor cells to extracellular medium. These findings provided valuable information for developing desired PEOz-b-PLA micelles to improve their

Focus on Extracellular Vesicles: Physiological Role and Signalling Properties of Extracellular Membrane Vesicles

Directory of Open Access Journals (Sweden)

Nunzio Iraci

2016-02-01

Full Text Available Extracellular vesicles (EVs are a heterogeneous population of secreted membrane vesicles, with distinct biogenesis routes, biophysical properties and different functions both in physiological conditions and in disease. The release of EVs is a widespread biological process, which is conserved across species. In recent years, numerous studies have demonstrated that several bioactive molecules are trafficked with(in EVs, such as microRNAs, mRNAs, proteins and lipids. The understanding of their final impact on the biology of specific target cells remains matter of intense debate in the field. Also, EVs have attracted great interest as potential novel cell-free therapeutics. Here we describe the proposed physiological and pathological functions of EVs, with a particular focus on their molecular content. Also, we discuss the advances in the knowledge of the mechanisms regulating the secretion of EV-associated molecules and the specific pathways activated upon interaction with the target cell, highlighting the role of EVs in the context of the immune system and as mediators of the intercellular signalling in the brain.

Calmodulin stimulation of calcium transport in carrot microsomal vesicles

International Nuclear Information System (INIS)

Pierce, W.S.; Sze, H.

1987-01-01

ATP-dependent 45 Ca 2+ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca 2+ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca 2+ uptake (at 1.17 g/cc) was not affected by oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca 2+ . These results are consistent with the ER being an important site of intracellular Ca 2+ regulation

Epithelial trafficking of Sonic hedgehog by megalin.

Science.gov (United States)

Morales, Carlos R; Zeng, Jibin; El Alfy, Mohamed; Barth, Jeremy L; Chintalapudi, Mastan Rao; McCarthy, Robert A; Incardona, John P; Argraves, W Scott

2006-10-01

We present here evidence of in vivo epithelial endocytosis and trafficking of non-lipid-modified Sonic hedgehog (ShhN) when infused into rat efferent ducts via microinjection. Initially, exogenous ShhN is detected in endocytic vesicles and early endosomes located near the apical plasma membrane of non-ciliated cells. Within 30-60 min following infusion, ShhN can be detected in lysosomes and at basolateral regions of non-ciliated cells. Basolaterally, ShhN was observed along the extracellular surfaces of interdigitated plasma membranes of adjacent cells and in the extracellular compartment underlying the efferent duct epithelium. Uptake and subcellular trafficking of infused ShhN by non-ciliated cells could be blocked by either anti-megalin IgG or the megalin antagonist, RAP. Ciliated cells, which do not express megalin, displayed little if any apical internalization of ShhN even though they were found to express Patched-1. However, ShhN was found in coated pits of lateral plasma membranes of ciliated cells as well as in underlying endocytic vesicles. We conclude that megalin-mediated endocytosis of ShhN can occur in megalin-expressing epithelia in vivo, and that the internalized ShhN can be targeted to the lysosome or transcytosed in the plane of the epithelium or across the epithelium. These findings highlight the multiple mechanisms by which megalin may influence Shh morphogen gradients in vivo.

Intracellular vesicle acidification promotes maturation of infectious poliovirus particles.

Directory of Open Access Journals (Sweden)

Alexsia L Richards

Full Text Available The autophagic pathway acts as part of the immune response against a variety of pathogens. However, several pathogens subvert autophagic signaling to promote their own replication. In many cases it has been demonstrated that these pathogens inhibit or delay the degradative aspect of autophagy. Here, using poliovirus as a model virus, we report for the first time bona fide autophagic degradation occurring during infection with a virus whose replication is promoted by autophagy. We found that this degradation is not required to promote poliovirus replication. However, vesicular acidification, which in the case of autophagy precedes delivery of cargo to lysosomes, is required for normal levels of virus production. We show that blocking autophagosome formation inhibits viral RNA synthesis and subsequent steps in the virus cycle, while inhibiting vesicle acidification only inhibits the final maturation cleavage of virus particles. We suggest that particle assembly, genome encapsidation, and virion maturation may occur in a cellular compartment, and we propose the acidic mature autophagosome as a candidate vesicle. We discuss the implications of our findings in understanding the late stages of poliovirus replication, including the formation and maturation of virions and egress of infectious virus from cells.

Endocytic vesicle rupture is a conserved mechanism of cellular invasion by amyloid proteins.

Science.gov (United States)

Flavin, William P; Bousset, Luc; Green, Zachary C; Chu, Yaping; Skarpathiotis, Stratos; Chaney, Michael J; Kordower, Jeffrey H; Melki, Ronald; Campbell, Edward M

2017-10-01

Numerous pathological amyloid proteins spread from cell to cell during neurodegenerative disease, facilitating the propagation of cellular pathology and disease progression. Understanding the mechanism by which disease-associated amyloid protein assemblies enter target cells and induce cellular dysfunction is, therefore, key to understanding the progressive nature of such neurodegenerative diseases. In this study, we utilized an imaging-based assay to monitor the ability of disease-associated amyloid assemblies to rupture intracellular vesicles following endocytosis. We observe that the ability to induce vesicle rupture is a common feature of α-synuclein (α-syn) assemblies, as assemblies derived from WT or familial disease-associated mutant α-syn all exhibited the ability to induce vesicle rupture. Similarly, different conformational strains of WT α-syn assemblies, but not monomeric or oligomeric forms, efficiently induced vesicle rupture following endocytosis. The ability to induce vesicle rupture was not specific to α-syn, as amyloid assemblies of tau and huntingtin Exon1 with pathologic polyglutamine repeats also exhibited the ability to induce vesicle rupture. We also observe that vesicles ruptured by α-syn are positive for the autophagic marker LC3 and can accumulate and fuse into large, intracellular structures resembling Lewy bodies in vitro. Finally, we show that the same markers of vesicle rupture surround Lewy bodies in brain sections from PD patients. These data underscore the importance of this conserved endocytic vesicle rupture event as a damaging mechanism of cellular invasion by amyloid assemblies of multiple neurodegenerative disease-associated proteins, and suggest that proteinaceous inclusions such as Lewy bodies form as a consequence of continued fusion of autophagic vesicles in cells unable to degrade ruptured vesicles and their amyloid contents.

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

DEFF Research Database (Denmark)

Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing

2008-01-01

fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...... exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1 mediated cellular sterol uptake....

Myeloid extracellular vesicles: messengers from the demented brain

Directory of Open Access Journals (Sweden)

Annamaria eNigro

2016-01-01

Full Text Available Blood-borne monocyte derived cells play a pivotal, initially unrecognized, role in most central nervous system disorders, including diseases initially classified as purely neurodegenerative (i.e. AD, PD, and ALS. Their trafficking to the brain and spinal cord has been extensively studied in classical neuroinflammatory disorders such as multiple sclerosis. Central nervous system resident myeloid cells, namely microglia and perivascular macrophages, also are in the spotlight of investigations on neurological disorders. Myeloid cells, such as infiltrating macrophages and microglia, have been described as having both protective and destructive features in neurological disorders, thus identification of their functional phenotype during disease evolution would be of paramount importance. Extracellular vesicles, namely exosomes and shed vesicles, are released by virtually any cell type and can be detected and identified in terms of cell origin in biological fluids. They therefore constitute an ideal tool to access information on cells residing in an inaccessible site such as the brain. We will review here available information on extracellular vesicles detection in neurological disorders with special emphasis on neurodegenerative diseases.

Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

Science.gov (United States)

Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

2015-08-01

ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods. Copyright © 2015 Elsevier B.V. All rights reserved.

The prenyl-binding protein PrBP/δ: a chaperone participating in intracellular trafficking.

Science.gov (United States)

Zhang, Houbin; Constantine, Ryan; Frederick, Jeanne M; Baehr, Wolfgang

2012-12-15

Expressed ubiquitously, PrBP/δ functions as chaperone/co-factor in the transport of a subset of prenylated proteins. PrBP/δ features an immunoglobulin-like β-sandwich fold for lipid binding, and interacts with diverse partners. PrBP/δ binds both C-terminal C15 and C20 prenyl side chains of phototransduction polypeptides and small GTP-binding (G) proteins of the Ras superfamily. PrBP/δ also interacts with the small GTPases, ARL2 and ARL3, which act as release factors (GDFs) for prenylated cargo. Targeted deletion of the mouse Pde6d gene encoding PrBP/δ resulted in impeded trafficking to the outer segments of GRK1 and cone PDE6 which are predicted to be farnesylated and geranylgeranylated, respectively. Rod and cone transducin trafficking was largely unaffected. These trafficking defects produce progressive cone-rod dystrophy in the Pde6d(-/-) mouse. Copyright © 2012 Elsevier Ltd. All rights reserved.

ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D2 receptors mediated by GRK and PKC in transfected cells

Science.gov (United States)

Cho, DI; Zheng, M; Min, C; Kwon, KJ; Shin, CY; Choi, HK; Kim, KM

2013-01-01

Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC-mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D2 receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D2 receptors internalized in a PKC-dependent manner. Conclusions and Implications GRK- and PKC-mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC. PMID:23082996

Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

Science.gov (United States)

Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

2014-02-12

Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

GLUT4 trafficking in a test tube.

Science.gov (United States)

Ramm, Georg; James, David E

2005-09-01

Insulin regulates glucose transport in muscle and fat cells by stimulating the translocation of GLUT4 from intracellular vesicles to the plasma membrane. In this issue of Cell Metabolism, Holman and colleagues reconstitute this process in vitro, providing a system that promises new breakthroughs in our understanding of this important metabolic process.

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

Science.gov (United States)

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

Directory of Open Access Journals (Sweden)

Yasuko Kitagishi

2015-01-01

Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Cell wall assembly and intracellular trafficking in plant cells are directly affected by changes in the magnitude of gravitational acceleration.

Directory of Open Access Journals (Sweden)

Youssef Chebli

Full Text Available Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions.

Overall energy conversion efficiency of a photosynthetic vesicle

Energy Technology Data Exchange (ETDEWEB)

Sener, Melih [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Strumpfer, Johan [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States; Singharoy, Abhishek [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Hunter, C. Neil [Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom; Schulten, Klaus [Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, United States; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States

2016-08-26

The chromatophore of purple bacteria is an intracellular spherical vesicle that exists in numerous copies in the cell and that efficiently converts sunlight into ATP synthesis, operating typically under low light conditions. Building on an atomic-level structural model of a low-light-adapted chromatophore vesicle from Rhodobacter sphaeroides, we investigate the cooperation between more than a hundred protein complexes in the vesicle. The steady-state ATP production rate as a function of incident light intensity is determined after identifying quinol turnover at the cytochrome bc1 complex (cytbc1) as rate limiting and assuming that the quinone/quinol pool of about 900 molecules acts in a quasi-stationary state. For an illumination condition equivalent to 1% of full sunlight, the vesicle exhibits an ATP production rate of 82 ATP molecules/s. The energy conversion efficiency of ATP synthesis at illuminations corresponding to 1%–5% of full sunlight is calculated to be 0.12-0.04, respectively. The vesicle stoichiometry, evolutionarily adapted to the low light intensities in the habitat of purple bacteria, is suboptimal for steady-state ATP turnover for the benefit of protection against over-illumination.

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

Science.gov (United States)

Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

2017-03-27

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Microinjecting FM4-64 validates it as a marker of the endocytic pathway in plants

NARCIS (Netherlands)

Gisbergen, van P.A.C.; Esseling-Ozdoba, A.; Vos, J.W.

2008-01-01

The amphiphilic dye FM4-64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We

Orchestrating cytoskeleton and intracellular vesicle traffic to build functional immunological synapses.

Science.gov (United States)

Soares, Helena; Lasserre, Rémi; Alcover, Andrés

2013-11-01

Immunological synapses are specialized cell-cell contacts formed between T lymphocytes and antigen-presenting cells. They are induced upon antigen recognition and are crucial for T-cell activation and effector functions. The generation and function of immunological synapses depend on an active T-cell polarization process, which results from a finely orchestrated crosstalk between the antigen receptor signal transduction machinery, the actin and microtubule cytoskeletons, and controlled vesicle traffic. Although we understand how some of these particular events are regulated, we still lack knowledge on how these multiple cellular elements are harmonized to ensure appropriate T-cell responses. We discuss here our view on how T-cell receptor signal transduction initially commands cytoskeletal and vesicle traffic polarization, which in turn sets the immunological synapse molecular design that regulates T-cell activation. We also discuss how the human immunodeficiency virus (HIV-1) hijacks some of these processes impairing immunological synapse generation and function. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

Directory of Open Access Journals (Sweden)

Lukas Aeberhard

2015-06-01

Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

International Nuclear Information System (INIS)

Ding Yun; Zhang Li; Goodwin, J. Shawn; Wang Ziqing; Liu Bingdong; Zhang Jingwu; Fan Guohuang

2008-01-01

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles.

Science.gov (United States)

Mandela, Prashant; Chandley, Michelle; Xu, Yao-Yu; Zhu, Meng-Yang; Ordway, Gregory A

2010-01-01

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors. Copyright 2010 Elsevier Ltd. All rights reserved.

Adenosine receptor desensitization and trafficking.

Science.gov (United States)

Mundell, Stuart; Kelly, Eamonn

2011-05-01

As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

Cellular trafficking of quantum dot-ligand bioconjugates and their induction of changes in normal routing of unconjugated ligands

DEFF Research Database (Denmark)

Tekle, Christina; van Deurs, Bo; Sandvig, Kirsten

2008-01-01

Can quantum dots (Qdots) act as relevant intracellular probes to investigate routing of ligands in live cells? The intracellular trafficking of Qdots that were coupled to the plant toxin ricin, Shiga toxin, or the ligand transferrin (Tf) was studied by confocal fluorescence microscopy. The Tf...

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed During Neuronal Development

Directory of Open Access Journals (Sweden)

Jenna M. Ramaker

2016-11-01

Full Text Available Proteolytic processing of the Amyloid Precursor Protein (APP produces beta-amyloid (Aβ peptide fragments that accumulate in Alzheimer’s Disease (AD, but APP may also regulate multiple aspects of neuronal development, albeit via mechanisms that are not well understood. APP is a member of a family of transmembrane glycoproteins expressed by all higher organisms, including two mammalian orthologs (APLP1 and APLP2 that have complicated investigations into the specific activities of APP. By comparison, insects express only a single APP-related protein (APP-Like, or APPL that contains the same protein interaction domains identified in APP. However, unlike its mammalian orthologs, APPL is only expressed by neurons, greatly simplifying an analysis of its functions in vivo. Like APP, APPL is processed by secretases to generate a similar array of extracellular and intracellular cleavage fragments, as well as an Aβ-like fragment that can induce neurotoxic responses in the brain. Exploiting the complementary advantages of two insect models (Drosophila melanogaster and Manduca sexta, we have investigated the regulation of APPL trafficking and processing with respect to different aspects of neuronal development. By comparing the behavior of endogenously expressed APPL with fluorescently tagged versions of APPL and APP, we have shown that some full-length protein is consistently trafficked into the most motile regions of developing neurons both in vitro and in vivo. Concurrently, much of the holoprotein is rapidly processed into N- and C-terminal fragments that undergo bi-directional transport within distinct vesicle populations. Unexpectedly, we also discovered that APPL can be transiently sequestered into an amphisome-like compartment in developing neurons, while manipulations targeting APPL cleavage altered their motile behavior in cultured embryos. These data suggest that multiple mechanisms restrict the bioavailability of the holoprotein to regulate

Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

Directory of Open Access Journals (Sweden)

Bartosz Różycki

Full Text Available The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

Polarized trafficking: the palmitoylation cycle distributes cytoplasmic proteins to distinct neuronal compartments.

Science.gov (United States)

Tortosa, Elena; Hoogenraad, Casper C

2018-02-01

In neurons, polarized cargo distribution occurs mainly between the soma and axonal and dendritic compartments, and requires coordinated regulation of cytoskeletal remodeling and membrane trafficking. The Golgi complex plays a critical role during neuronal polarization and secretory trafficking has been shown to differentially transport proteins to both axons and dendrites. Besides the Golgi protein sorting, recent data revealed that palmitoylation cycles are an efficient mechanism to localize cytoplasmic, non-transmembrane proteins to particular neuronal compartments, such as the newly formed axon. Palmitoylation allows substrate proteins to bind to and ride with Golgi-derived secretory vesicles to all neuronal compartments. By allowing cytoplasmic proteins to 'hitchhike' on transport carriers in a non-polarized fashion, compartmentalized depalmitoylation may act as a selective retention mechanism. Copyright © 2018 Elsevier Ltd. All rights reserved.

A fluorescent glycolipid-binding peptide probe traces cholesterol dependent microdomain-derived trafficking pathways.

Directory of Open Access Journals (Sweden)

Steffen Steinert

Full Text Available BACKGROUND: The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Abeta, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB. Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol

Multiple cues on the physiochemical, mesenchymal, and intracellular trafficking interactions with nanocarriers to maximize tumor target efficiency

Directory of Open Access Journals (Sweden)

Kim SW

2015-06-01

Full Text Available Sang-Woo Kim, Dongwoo Khang Nanomedicine Laboratory, Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, South Korea Abstract: Over the past 60 years, numerous medical strategies have been employed to overcome neoplasms. In fact, with the exception of lung, bronchial, and pancreatic cancers, the 5-year survival rate of most cancers currently exceeds 70%. However, the quality of life of patients during chemotherapy remains unsatisfactory despite the increase in survival rate. The side effects of current chemotherapies stem from poor target efficiency at tumor sites due to the uncontrolled biodistribution of anticancer agents (ie, conventional or current approved nanodrugs. This review discusses the effective physiochemical factors for determining biodistribution of nanocarriers and, ultimately, increasing tumor-targeting probability by avoiding the reticuloendothelial system. Second, stem cell-conjugated nanotherapeutics was addressed to maximize the tumor searching ability and to inhibit tumor growth. Lastly, physicochemical material properties of anticancer nanodrugs were discussed for targeting cellular organelles with modulation of drug-release time. A better understanding of suggested topics will increase the tumor-targeting ability of anticancer drugs and, ultimately, promote the quality of life of cancer patients during chemotherapy. Keywords: cancer, anticancer nanodrugs, mesenchymal stem cell, intracellular trafficking

Regulatory Multidimensionality of Gas Vesicle Biogenesis in Halobacterium salinarum NRC-1

Directory of Open Access Journals (Sweden)

Andrew I. Yao

2011-01-01

Full Text Available It is becoming clear that the regulation of gas vesicle biogenesis in Halobacterium salinarum NRC-1 is multifaceted and appears to integrate environmental and metabolic cues at both the transcriptional and posttranscriptional levels. The mechanistic details underlying this process, however, remain unclear. In this manuscript, we quantify the contribution of light scattering made by both intracellular and released gas vesicles isolated from Halobacterium salinarum NRC-1, demonstrating that each form can lead to distinct features in growth curves determined by optical density measured at 600 nm (OD600. In the course of the study, we also demonstrate the sensitivity of gas vesicle accumulation in Halobacterium salinarum NRC-1 on small differences in growth conditions and reevaluate published works in the context of our results to present a hypothesis regarding the roles of the general transcription factor tbpD and the TCA cycle enzyme aconitase on the regulation of gas vesicle biogenesis.

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

Energy Technology Data Exchange (ETDEWEB)

Sato, Mai [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Kitaguchi, Tetsuya [Cell Signaling Group, Waseda Bioscience Research Institute in Singapore (WABOIS), Waseda University, 11 Biopolis Way, 05-01/02 Helios, Singapore 138667 (Singapore); Numano, Rika [The Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tennpaku-cho, Toyohashi, Aichi 441-8580 (Japan); Ikematsu, Kazuya [Forensic Pathology and Science, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8523 (Japan); Kakeyama, Masaki [Laboratory of Environmental Health Sciences, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033 (Japan); Murata, Masayuki; Sato, Ken [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan); Tsuboi, Takashi, E-mail: takatsuboi@bio.c.u-tokyo.ac.jp [Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902 (Japan)

2012-04-06

Highlights: Black-Right-Pointing-Pointer Regulation of exocytosis by Rho GTPase Cdc42. Black-Right-Pointing-Pointer Cdc42 increases the number of fusion events from newly recruited vesicles. Black-Right-Pointing-Pointer Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott-Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

The small GTPase Cdc42 modulates the number of exocytosis-competent dense-core vesicles in PC12 cells

International Nuclear Information System (INIS)

Sato, Mai; Kitaguchi, Tetsuya; Numano, Rika; Ikematsu, Kazuya; Kakeyama, Masaki; Murata, Masayuki; Sato, Ken; Tsuboi, Takashi

2012-01-01

Highlights: ► Regulation of exocytosis by Rho GTPase Cdc42. ► Cdc42 increases the number of fusion events from newly recruited vesicles. ► Cdc42 increases the number of exocytosis-competent dense-core vesicles. -- Abstract: Although the small GTPase Rho family Cdc42 has been shown to facilitate exocytosis through increasing the amount of hormones released, the precise mechanisms regulating the quantity of hormones released on exocytosis are not well understood. Here we show by live cell imaging analysis under TIRF microscope and immunocytochemical analysis under confocal microscope that Cdc42 modulated the number of fusion events and the number of dense-core vesicles produced in the cells. Overexpression of a wild-type or constitutively-active form of Cdc42 strongly facilitated high-KCl-induced exocytosis from the newly recruited plasma membrane vesicles in PC12 cells. By contrast, a dominant-negative form of Cdc42 inhibited exocytosis from both the newly recruited and previously docked plasma membrane vesicles. The number of intracellular dense-core vesicles was increased by the overexpression of both a wild-type and constitutively-active form of Cdc42. Consistently, activation of Cdc42 by overexpression of Tuba, a Golgi-associated guanine nucleotide exchange factor for Cdc42 increased the number of intracellular dense-core vesicles, whereas inhibition of Cdc42 by overexpression of the Cdc42/Rac interactive binding domain of neuronal Wiskott–Aldrich syndrome protein decreased the number of them. These findings suggest that Cdc42 facilitates exocytosis by modulating both the number of exocytosis-competent dense-core vesicles and the production of dense-core vesicles in PC12 cells.

Ubiquilin 1 modulates amyloid precursor protein trafficking and Abeta secretion.

Science.gov (United States)

Hiltunen, Mikko; Lu, Alice; Thomas, Anne V; Romano, Donna M; Kim, Minji; Jones, Phill B; Xie, Zhongcong; Kounnas, Maria Z; Wagner, Steven L; Berezovska, Oksana; Hyman, Bradley T; Tesco, Giuseppina; Bertram, Lars; Tanzi, Rudolph E

2006-10-27

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with alpha-, beta-, or gamma-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (alpha- and beta-forms). Moreover, UBQLN1 knockdown increased levels of secreted Abeta40 and Abeta42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic "gatekeeper" that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Abeta.

Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles.

Science.gov (United States)

Digiacomo, Luca; Digman, Michelle A; Gratton, Enrico; Caracciolo, Giulio

2016-09-15

Fluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system's symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed flow motions, when compared to SpatioTemporal Image Correlation Spectroscopy (STICS). Although we present a specific example (lipoplexes in living cells), the emphasis is in the discussion of the method, its basic assumptions and its validation on numeric simulations. Recent advances in nanoparticle-based drug and gene delivery systems have pointed out the interactions at cellular and subcellular levels as key-factors for the efficiency of the adopted biomaterials. Such biochemical and biophysical interactions drive and affect the intracellular dynamics, that is commonly characterized by means of fluorescence microscopy and spectroscopy techniques. Here we present a novel Image Correlation Spectroscopy (ICS) method as a promising tool to capture the intracellular behavior of nanoparticles with high resolution and low background's sensitivity. This study overcomes some of the approximations

Periventricular Heterotopia: Shuttling of Proteins through Vesicles and Actin in Cortical Development and Disease

Directory of Open Access Journals (Sweden)

Volney L. Sheen

2012-01-01

Full Text Available During cortical development, proliferating neural progenitors exhibit polarized apical and basolateral membranes that are maintained by tightly controlled and membrane-specific vesicular trafficking pathways. Disruption of polarity through impaired delivery of proteins can alter cell fate decisions and consequent expansion of the progenitor pool, as well as impact the integrity of the neuroependymal lining. Loss of neuroependymal integrity disrupts radial glial scaffolding and alters initial neuronal migration from the ventricular zone. Vesicle trafficking is also required for maintenance of lipid and protein cycling within the leading and trailing edge of migratory neurons, as well as dendrites and synapses of mature neurons. Defects in this transport machinery disrupt neuronal identity, migration, and connectivity and give rise to a malformation of cortical development termed as periventricular heterotopia (PH. PH is characterized by a reduction in brain size, ectopic clusters of neurons localized along the lateral ventricle, and epilepsy and dyslexia. These anatomical anomalies correlate with developmental impairments in neural progenitor proliferation and specification, migration from loss of neuroependymal integrity and neuronal motility, and aberrant neuronal process extension. Genes causal for PH regulate vesicle-mediated endocytosis along an actin cytoskeletal network. This paper explores the role of these dynamic processes in cortical development and disease.

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

Science.gov (United States)

Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

2014-04-01

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Homology with vesicle fusion mediator syntaxin-1a predicts determinants ofepimorphin/syntaxin-2 function in mammary epithelial morphogenesis

Energy Technology Data Exchange (ETDEWEB)

Chen, Connie S.; Nelson, Celeste M.; Khauv, Davitte; Bennett, Simone; Radisky, Evette S.; Hirai, Yohei; Bissell, Mina J.; Radisky, Derek C.

2009-06-03

We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility.

Science.gov (United States)

Nojimoto, Fernanda D; Piffer, Renata C; Kiguti, Luiz Ricardo de A; Lameu, Claudiana; de Camargo, Antônio C M; Pereira, Oduvaldo C M; Pupo, André S

2009-09-15

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg(-1), ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca(2+) transients. Sibutramine 5 and 20 mg kg(-1) reduced ejaculation latency whereas 50 mg kg(-1) increased ejaculation latency. Sibutramine 3 to 30 microM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 microM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl(2). In HEK293 cells, sibutramine 10 to 100 microM inhibited intracellular Ca(2+) transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca(2+) may be involved in the delayed ejaculation observed with high doses of sibutramine.

Multiple effects of sibutramine on ejaculation and on vas deferens and seminal vesicle contractility

International Nuclear Information System (INIS)

Nojimoto, Fernanda D.; Piffer, Renata C.; Kiguti, Luiz Ricardo de A.; Lameu, Claudiana; Camargo, Antonio C.M. de; Pereira, Oduvaldo C.M.; Pupo, Andre S.

2009-01-01

Sibutramine is an inhibitor of norepinephrine and 5-HT reuptake largely used in the management of obesity. Although a fairly safe drug, postmarketing adverse effects of sibutramine were reported including abnormal ejaculation in men. This study investigates the effects of sibutramine on ejaculation and vas deferens and seminal vesicle contractility. Adult male rats received sibutramine (5; 20; or 50 mg kg -1 , ip) and after 60 min were exposed to receptive females for determination of ejaculation parameters. The vasa deferentia and seminal vesicles of untreated rats were mounted in isolated organ baths for recording of isometric contractions and HEK293 cells loaded with fluorescent calcium indicator were used to measure intracellular Ca 2+ transients. Sibutramine 5 and 20 mg kg -1 reduced ejaculation latency whereas 50 mg kg -1 increased ejaculation latency. Sibutramine 3 to 30 μM greatly increased the sensitivity of the seminal vesicle and vas deferens to norepinephrine, but at concentrations higher than 10 μM there were striking depressions of maximal contractions induced by norepinephrine, carbachol and CaCl 2 . In HEK293 cells, sibutramine 10 to 100 μM inhibited intracellular Ca 2+ transients induced by carbachol. Depending on the doses, sibutramine either facilitates or inhibits ejaculation. Apart from its actions in the central nervous system, facilitation of ejaculation may result from augmented sensitivity of smooth muscles to norepinephrine while reductions of intracellular Ca 2+ may be involved in the delayed ejaculation observed with high doses of sibutramine.

Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

Science.gov (United States)

Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

2014-07-01

Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis.

Science.gov (United States)

Nakase, Ikuhiko; Noguchi, Kosuke; Fujii, Ikuo; Futaki, Shiroh

2016-10-17

Extracellular vesicles (EVs, exosomes) are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained.

Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

Science.gov (United States)

Prada, Ilaria; Meldolesi, Jacopo

2016-08-09

Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

Overcoming T. gondii infection and intracellular protein nanocapsules as biomaterials for ultrasonically controlled drug release.

Science.gov (United States)

Aw, M S; Paniwnyk, L

2017-09-26

One of the pivotal matters of concern in intracellular drug delivery is the preparation of biomaterials containing drugs that are compatible with the host target. Nanocapsules for oral delivery are found to be suitable candidates for targeting Toxoplasma gondii (T. gondii), a maneuvering and smart protozoic parasite found across Europe and America that causes a subtle but deadly infection. To overcome this disease, there is much potential of integrating protein-based cells into bioinspired nanocompartments such as via biodegradable cross-linked disulfide polyelectrolyte nanoparticles. The inner membrane vesicle system of these protein-drugs is not as simple as one might think. It is a complex transport network that includes sequential pathways, namely, endocytosis, exocytosis and autophagy. Unfortunately, the intracellular trafficking routes for nanoparticles in cells have not been extensively and intensively investigated. Hence, there lies the need to create robust protein nanocapsules for precise tracing and triggering of drug release to combat this protozoic disease. Protein nanocapsules have the advantage over other biomaterials due to their biocompatibility, use of natural ingredients, non-invasiveness, patient compliance, cost and time effectiveness. They also offer low maintenance, non-toxicity to healthy cells and a strictly defined route toward intracellular elimination through controlled drug delivery within the therapeutic window. This review covers the unprecedented opportunities that exist for constructing advanced nanocapsules to meet the growing needs arising from many therapeutic fields. Their versatile use includes therapeutic ultrasound for tumor imaging, recombinant DNA, ligand and functional group binding, the delivery of drugs and peptides via protein nanocapsules and polyelectrolytes, ultrasound-(US)-aided drug release through the gastrointestinal (GI) tract, and the recent progress in targeting tumor cells and a vast range of cancer therapies

Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

Science.gov (United States)

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2016-06-01

The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation. © 2016 The Authors.

Impact d'une perturbation mécanique ou photo-chimique sur le trafic intracellulaire

OpenAIRE

Aubertin , Kelly

2014-01-01

The cell cytoplasm is crowded with membrane-delimited compartments, permanently communicatingwith each other. Such communication is permitted by active transport, also called intracellulartrafficking, of vesicles along the cytoskeleton (actin filaments, microtubules), and mediated bymolecular motors. In order to perturbate this intracellular trafficking, two different ways to applycontrolled physical stresses have been performed. The first perturbation is a mechanical one : weused magnetic en...

Exocyst and autophagy-related membrane trafficking in plants.

Science.gov (United States)

Pecenková, Tamara; Markovic, Vedrana; Sabol, Peter; Kulich, Ivan; Žárský, Viktor

2017-12-18

Endomembrane traffic in eukaryotic cells functions partially as a means of communication; delivery of membrane in one direction has to be balanced with a reduction at the other end. This effect is typically the case during the defence against pathogens. To combat pathogens, cellular growth and differentiation are suppressed, while endomembrane traffic is poised towards limiting the pathogen attack. The octameric exocyst vesicle-tethering complex was originally discovered as a factor facilitating vesicle-targeting and vesicle-plasma membrane (PM) fusion during exocytosis prior to and possibly during SNARE complex formation. Interestingly, it was recently implicated both in animals and plants in autophagy membrane traffic. In animal cells, the exocyst is integrated into the mTOR-regulated energy metabolism stress/starvation pathway, participating in the formation and especially initiation of an autophagosome. In plants, the first functional link was to autophagy-related anthocyanin import to the vacuole and to starvation. In this concise review, we summarize the current knowledge of exocyst functions in autophagy and defence in plants that might involve unconventional secretion and compare it with animal conditions. Formation of different exocyst complexes during undisturbed cell growth, as opposed to periods of cellular stress reactions involving autophagy, might contribute to the coordination of endomembrane trafficking pathways. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

Science.gov (United States)

Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

Directory of Open Access Journals (Sweden)

Senthil Kumar A Natesan

2009-09-01

Full Text Available Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

International Nuclear Information System (INIS)

Kawaguchi, Takayuki; Tamori, Yoshikazu; Kanda, Hajime; Yoshikawa, Mari; Tateya, Sanshiro; Nishino, Naonobu; Kasuga, Masato

2010-01-01

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.

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

Directory of Open Access Journals (Sweden)

Ayako Kita

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

Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly.

Science.gov (United States)

Munson, M; Chen, X; Cocina, A E; Schultz, S M; Hughson, F M

2000-10-01

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia

Science.gov (United States)

Alves, Daiane S.; Thulin, Gunilla; Loffing, Johannes; Kashgarian, Michael

2015-01-01

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na+,K+-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na+,K+-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na+,K+-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na+,K+-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion–induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na+,K+-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na+,K+-ATPase to the energy state of renal epithelial cells. PMID:25788531

Akt Substrate of 160 kD Regulates Na+,K+-ATPase Trafficking in Response to Energy Depletion and Renal Ischemia.

Science.gov (United States)

Alves, Daiane S; Thulin, Gunilla; Loffing, Johannes; Kashgarian, Michael; Caplan, Michael J

2015-11-01

Renal ischemia and reperfusion injury causes loss of renal epithelial cell polarity and perturbations in tubular solute and fluid transport. Na(+),K(+)-ATPase, which is normally found at the basolateral plasma membrane of renal epithelial cells, is internalized and accumulates in intracellular compartments after renal ischemic injury. We previously reported that the subcellular distribution of Na(+),K(+)-ATPase is modulated by direct binding to Akt substrate of 160 kD (AS160), a Rab GTPase-activating protein that regulates the trafficking of glucose transporter 4 in response to insulin and muscle contraction. Here, we investigated the effect of AS160 on Na(+),K(+)-ATPase trafficking in response to energy depletion. We found that AS160 is required for the intracellular accumulation of Na(+),K(+)-ATPase that occurs in response to energy depletion in cultured epithelial cells. Energy depletion led to dephosphorylation of AS160 at S588, which was required for the energy depletion-induced accumulation of Na,K-ATPase in intracellular compartments. In AS160-knockout mice, the effects of renal ischemia on the distribution of Na(+),K(+)-ATPase were substantially reduced in the epithelial cells of distal segments of the renal tubules. These data demonstrate that AS160 has a direct role in linking the trafficking of Na(+),K(+)-ATPase to the energy state of renal epithelial cells. Copyright © 2015 by the American Society of Nephrology.

The cyclic nucleotide gated cation channel AtCNGC10 traffics from the ER via Golgi vesicles to the plasma membrane of Arabidopsis root and leaf cells

Directory of Open Access Journals (Sweden)

Andres Marilou A

2007-09-01

Full Text Available Abstract Background The cyclic nucleotide-gated ion channels (CNGCs maintain cation homeostasis essential for a wide range of physiological processes in plant cells. However, the precise subcellular locations and trafficking of these membrane proteins are poorly understood. This is further complicated by a general deficiency of information about targeting pathways of membrane proteins in plants. To investigate CNGC trafficking and localization, we have measured Atcngc5 and Atcngc10 expression in roots and leaves, analyzed AtCNGC10-GFP fusions transiently expressed in protoplasts, and conducted immunofluorescence labeling of protoplasts and immunoelectron microscopic analysis of high pressure frozen leaves and roots. Results AtCNGC10 mRNA and protein levels were 2.5-fold higher in roots than leaves, while AtCNGC5 mRNA and protein levels were nearly equal in these tissues. The AtCNGC10-EGFP fusion was targeted to the plasma membrane in leaf protoplasts, and lightly labeled several intracellular structures. Immunofluorescence microscopy with affinity purified CNGC-specific antisera indicated that AtCNGC5 and AtCNGC10 are present in the plasma membrane of protoplasts. Immunoelectron microscopy demonstrated that AtCNGC10 was associated with the plasma membrane of mesophyll, palisade parenchyma and epidermal cells of leaves, and the meristem, columella and cap cells of roots. AtCNCG10 was also observed in the endoplasmic reticulum and Golgi cisternae and vesicles of 50–150 nm in size. Patch clamp assays of an AtCNGC10-GFP fusion expressed in HEK293 cells measured significant cation currents. Conclusion AtCNGC5 and AtCNGC10 are plasma membrane proteins. We postulate that AtCNGC10 traffics from the endoplasmic reticulum via the Golgi apparatus and associated vesicles to the plasma membrane. The presence of the cation channel, AtCNGC10, in root cap meristem cells, cell plate, and gravity-sensing columella cells, combined with the previously reported

Regulation of vesicular traffic by a GTP-binding protein on the cytoplasmic surface of secretory vesicles in yeast

International Nuclear Information System (INIS)

Novick, P.J.; Goud, B.; Salminen, A.; Walworth, N.C.; Nair, J.; Potenza, M.

1988-01-01

Vesicular transport is an important mechanism for the intracellular traffic of proteins and lipids in eukaryotic cells. Vesicles mediate the passage of proteins between the various organelles of the secretory pathway and the exocytic release of these proteins into the extracellular environment. Vesicles also mediate the uptake of proteins and fluid from the external environment, delivering them to endosomes. Despite the generality of the vesicular transport mechanism, the process is not yet understood at a molecular level. The key questions that are addressed are (1) How are vesicles formed from the membrane of the donor organelle? (2) How are these vesicles transported? (3) How do the vesicles recognize the membrane of the target (acceptor) organelle? (4) How is membrane fusion accomplished? The genetic flexibility of yeast has been exploited to identify components of the cellular machinery required for vesicular transport

Trafficking through COPII stabilises cell polarity and drives secretion during Drosophila epidermal differentiation.

Directory of Open Access Journals (Sweden)

Michaela Norum

2010-05-01

Full Text Available The differentiation of an extracellular matrix (ECM at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation.We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus.Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes.

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

International Nuclear Information System (INIS)

Abu-Hamdah, Rania; Cho, Won Jin; Hoerber, J.K.H.; Jena, Bhanu P.

2006-01-01

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 μm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles

Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Abu-Hamdah, Rania [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Cho, Won Jin [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Hoerber, J.K.H. [Department of Physics, University of Bristol, Bristol BS8 1TD (United Kingdom); Jena, Bhanu P. [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States)]. E-mail: bjena@med.wayne.edu

2006-06-15

It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 {mu}m in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

The epithelial cell cytoskeleton and intracellular trafficking. I. Shiga toxin B-subunit system: retrograde transport, intracellular vectorization, and more.

Science.gov (United States)

Johannes, Ludger

2002-07-01

Many intracellular transport routes are still little explored. This is particularly true for retrograde transport between the plasma membrane and the endoplasmic reticulum. Shiga toxin B subunit has become a powerful tool to study this pathway, and recent advances on the molecular mechanisms of transport in the retrograde route and on its physiological function(s) are summarized. Furthermore, it is discussed how the study of retrograde transport of Shiga toxin B subunit allows one to design new methods for the intracellular delivery of therapeutic compounds.

Economics of human trafficking.

Science.gov (United States)

Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

2010-01-01

Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.

Imaging and controlling intracellular reactions: Lysosome transport as a function of diameter and the intracellular synthesis of conducting polymers

Science.gov (United States)

Payne, Christine

2014-03-01

Eukaryotic cells are the ultimate complex environment with intracellular chemical reactions regulated by the local cellular environment. For example, reactants are sequestered into specific organelles to control local concentration and pH, motor proteins transport reactants within the cell, and intracellular vesicles undergo fusion to bring reactants together. Current research in the Payne Lab in the School of Chemistry and Biochemistry at Georgia Tech is aimed at understanding and utilizing this complex environment to control intracellular chemical reactions. This will be illustrated using two examples, intracellular transport as a function of organelle diameter and the intracellular synthesis of conducting polymers. Using single particle tracking fluorescence microscopy, we measured the intracellular transport of lysosomes, membrane-bound organelles, as a function of diameter as they underwent transport in living cells. Both ATP-dependent active transport and diffusion were examined. As expected, diffusion scales with the diameter of the lysosome. However, active transport is unaffected suggesting that motor proteins are insensitive to cytosolic drag. In a second example, we utilize intracellular complexity, specifically the distinct micro-environments of different organelles, to carry out chemical reactions. We show that catalase, found in the peroxisomes of cells, can be used to catalyze the polymerization of the conducting polymer PEDOT:PSS. More importantly, we have found that a range of iron-containing biomolecules are suitable catalysts with different iron-containing biomolecules leading to different polymer properties. These experiments illustrate the advantage of intracellular complexity for the synthesis of novel materials.

Intracellular distribution of nontargeted quantum dots after natural uptake and microinjection

Science.gov (United States)

Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Valius, Mindaugas; Rotomskis, Ricardas

2013-01-01

Background: The purpose of this study was to elucidate the mechanism of natural uptake of nonfunctionalized quantum dots in comparison with microinjected quantum dots by focusing on their time-dependent accumulation and intracellular localization in different cell lines. Methods: The accumulation dynamics of nontargeted CdSe/ZnS carboxyl-coated quantum dots (emission peak 625 nm) was analyzed in NIH3T3, MCF-7, and HepG2 cells by applying the methods of confocal and steady-state fluorescence spectroscopy. Intracellular colocalization of the quantum dots was investigated by staining with Lysotracker®. Results: The uptake of quantum dots into cells was dramatically reduced at a low temperature (4°C), indicating that the process is energy-dependent. The uptake kinetics and imaging of intracellular localization of quantum dots revealed three accumulation stages of carboxyl-coated quantum dots at 37°C, ie, a plateau stage, growth stage, and a saturation stage, which comprised four morphological phases: adherence to the cell membrane; formation of granulated clusters spread throughout the cytoplasm; localization of granulated clusters in the perinuclear region; and formation of multivesicular body-like structures and their redistribution in the cytoplasm. Diverse quantum dots containing intracellular vesicles in the range of approximately 0.5–8 μm in diameter were observed in the cytoplasm, but none were found in the nucleus. Vesicles containing quantum dots formed multivesicular body-like structures in NIH3T3 cells after 24 hours of incubation, which were Lysotracker-negative in serum-free medium and Lysotracker-positive in complete medium. The microinjected quantum dots remained uniformly distributed in the cytosol for at least 24 hours. Conclusion: Natural uptake of quantum dots in cells occurs through three accumulation stages via a mechanism requiring energy. The sharp contrast of the intracellular distribution after microinjection of quantum dots in comparison

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

Directory of Open Access Journals (Sweden)

Leslie K Climer

2015-10-01

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

Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

Science.gov (United States)

Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

2015-11-01

The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

Science.gov (United States)

Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

2013-01-01

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

Osmotic Gradients Induce Bio-reminiscent Morphological Transformations in Giant Unilamellar Vesicles

Directory of Open Access Journals (Sweden)

Kamila eOglecka

2012-05-01

Full Text Available We report observations of large-scale, in-plane and out-of-plane membrane deformations in giant uni- and multilamellar vesicles composed of binary and ternary lipid mixtures in the presence of net transvesicular osmotic gradients. The lipid mixtures we examined consisted of binary mixtures of DOPC and DPPC lipids and ternary mixtures comprising POPC, sphingomyelin, and cholesterol over a range of compositions – both of which produce co-existing phases for selected ranges of compositions at room temperature under thermodynamic equilibrium. In the presence of net osmotic gradient, we find that the in-plane phase separation potential of these mixtures is non-trivially altered and a variety of out-of-plane morphological remodeling occurs. The repertoire of membrane deformations we observe display striking resemblance to their biological counterparts in live cells encompassing vesiculation, membrane fission and fusion, tubulation and pearling, as well as expulsion of entrapped vesicles from multicompartmental GUV architectures through large, self-healing transient pores. These observations suggest that the forces introduced by simple osmotic gradients across membrane boundaries could act as a trigger for shape-dependent membrane and vesicle trafficking activities. We speculate that such coupling of osmotic gradients with membrane properties might have provided lipid-mediated mechanisms during the early evolution of membrane compartmentalization in the absence of osmoregulatory protein machinery.

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles

Science.gov (United States)

Yasen, Aizezi; Herrera, Rossana; Rosbe, Kristina; Lien, Kathy; Tugizov, Sharof M.

2018-01-01

Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30–40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission. PMID:29277006

Synaptojanin 1 is required for endolysosomal trafficking of synaptic proteins in cone photoreceptor inner segments.

Directory of Open Access Journals (Sweden)

Ashley A George

Full Text Available Highly polarized cells such as photoreceptors require precise and efficient strategies for establishing and maintaining the proper subcellular distribution of proteins. The signals and molecular machinery that regulate trafficking and sorting of synaptic proteins within cone inner segments is mostly unknown. In this study, we show that the polyphosphoinositide phosphatase Synaptojanin 1 (SynJ1 is critical for this process. We used transgenic markers for trafficking pathways, electron microscopy, and immunocytochemistry to characterize trafficking defects in cones of the zebrafish mutant, nrc(a14 , which is deficient in phosphoinositide phosphatase, SynJ1. The outer segments and connecting cilia of nrc(a14 cone photoreceptors are normal, but RibeyeB and VAMP2/synaptobrevin, which normally localize to the synapse, accumulate in the nrc(a14 inner segment. The structure of the Endoplasmic Reticulum in nrc(a14 mutant cones is normal. Golgi develop normally, but later become disordered. Large vesicular structures accumulate within nrc(a14 cone photoreceptor inner segments, particularly after prolonged incubation in darkness. Cone inner segments of nrc (a14 mutants also have enlarged acidic vesicles, abnormal late endosomes, and a disruption in autophagy. This last pathway also appears exacerbated by darkness. Taken altogether, these findings show that SynJ1 is required in cones for normal endolysosomal trafficking of synaptic proteins.

HUMAN TRAFFICKING DRUG TRAFFICKING, AND THE DEATH PENALTY

Directory of Open Access Journals (Sweden)

Felicity Gerry

2016-12-01

Full Text Available Both Australia and Indonesia have made commitments to combatting human trafficking.  Through the experience of Mary Jane Veloso it can be seen that it is most often the vulnerable ‘mule’ that is apprehended by law enforcement and not the powerful leaders of crime syndicates. It is unacceptable that those vulnerable individuals may face execution for acts committed under threat of force, coercion, fraud, deception or abuse of power. For this reason it is vital that a system of victim identification is developed, including better training for law enforcement, legal representatives and members of the judiciary. This paper builds on submissions by authors for Australian Parliamentary Inquiry into Human Trafficking, and focusses on issues arising in the complex cross section of human trafficking, drug trafficking, and the death penalty with particular attention on identifying victims and effective reporting mechanisms in both Australia and Indonesia. It concludes that, in the context of human trafficking both countries could make three main improvements to law and policy, among others, 1 enactment of laws that create clear mandatory protection for human trafficking victims; 2 enactment of criminal laws that provides complete defence for victim of human trafficking; 3 enactment of corporate reporting mechanisms. Systemic protection and support is not sufficiently available without clear legislative protection as this paper suggests together with standardised referral mechanisms and effective financial reporting mechanisms. The implementation can be achieved through collaborative responses and inter-agency coordination with data collection and properly trained specialists.

Paromomycin affects translation and vesicle-mediated trafficking as revealed by proteomics of paromomycin -susceptible -resistant Leishmania donovani.

Directory of Open Access Journals (Sweden)

Bhavna Chawla

Full Text Available Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis (VL and is responsible for significant mortality and morbidity. Increasing resistance towards antimonial drugs poses a great challenge in chemotherapy of VL. Paromomycin is an aminoglycosidic antibiotic and is one of the drugs currently being used in the chemotherapy of cutaneous and visceral leishmaniasis. To understand the mode of action of this antibiotic at the molecular level, we have investigated the global proteome differences between the wild type AG83 strain and a paromomycin resistant (PRr strain of L. donovani. Stable isotope labeling of amino acids in cell culture (SILAC followed by quantitative mass spectrometry of the wild type AG83 strain and the paromomycin resistant (PRr strain identified a total of 226 proteins at ≥ 95% confidence. Data analysis revealed upregulation of 29 proteins and down-regulation of 21 proteins in the PRr strain. Comparative proteomic analysis of the wild type and the paromomycin resistant strains showed upregulation of the ribosomal proteins in the resistant strain indicating role in translation. Elevated levels of glycolytic enzymes and stress proteins were also observed in the PRr strain. Most importantly, we observed upregulation of proteins that may have a role in intracellular survival and vesicular trafficking in the PRr strain. Furthermore, ultra-structural analysis by electron microscopy demonstrated increased number of vesicular vacuoles in PRr strain when compared to the wild-type strain. Drug affinity pull-down assay followed by mass spectrometery identified proteins in L. donovani wild type strain that were specifically and covalently bound to paromomycin. These results provide the first comprehensive insight into the mode of action and underlying mechanism of resistance to paromomycin in Leishmania donovani.

Curcumin Mitigates the Intracellular Lipid Deposit Induced by Antipsychotics In Vitro.

Directory of Open Access Journals (Sweden)

Alberto Canfrán-Duque

Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, both inhibit cholesterol biosynthesis and impair the intracellular cholesterol trafficking, leading to lipid accumulation in the late endosome/lysosome compartment. In this study we examined if curcumin, a plant polyphenol that stimulates exosome release, can alleviate antipsychotic-induced intracellular lipid accumulation.HepG2 hepatocarcinoma cells were treated with antipsychotics or placebo and DiI-labelled LDL for 18 h and then exposed to curcumin for the last 2 h. Cells and media were collected separately and used for biochemical analyses, electron microscopy and immunocytochemistry. Exosomes were isolated from the incubation medium by ultracentrifugation.Curcumin treatment reduced the number of heterolysosomes and shifted their subcellular localization to the periphery, as revealed by electron microscopy, and stimulated the release of lysosomal β-hexosaminidase and exosome markers flotillin-2 and CD63 into the media. The presence of DiI in exosomes released by cells preloaded with DiI-LDL demonstrated the endolysosomal origin of the microvesicles. Furthermore, curcumin increased the secretion of cholesterol as well as LDL-derived DiI and [3H]-cholesterol, in association with a decrease of intracellular lipids. Thus, the disruption of lipid trafficking induced by FGAs or SGAs can be relieved by curcumin treatment. This polyphenol, however, did not mitigate the reduction of cholesterol esterification induced by antipsychotics.Curcumin stimulates exosome release to remove cholesterol (and presumably other lipids accumulated within the endolysosomal compartment, thereby normalizing intracellular lipid homeostasis. This action may help minimize the adverse metabolic effects of antipsychotic treatment, which should now be evaluated in clinical trials.

Atg9 is required for intraluminal vesicles in amphisomes and autolysosomes

Directory of Open Access Journals (Sweden)

C. A. Bader

2015-11-01

Full Text Available Autophagy is an intracellular recycling and degradation process, which is important for energy metabolism, lipid metabolism, physiological stress response and organism development. During Drosophila development, autophagy is up-regulated in fat body and midgut cells, to control metabolic function and to enable tissue remodelling. Atg9 is the only transmembrane protein involved in the core autophagy machinery and is thought to have a role in autophagosome formation. During Drosophila development, Atg9 co-located with Atg8 autophagosomes, Rab11 endosomes and Lamp1 endosomes-lysosomes. RNAi silencing of Atg9 reduced both the number and the size of autophagosomes during development and caused morphological changes to amphisomes/autolysosomes. In control cells there was compartmentalised acidification corresponding to intraluminal Rab11/Lamp-1 vesicles, but in Atg9 depleted cells there were no intraluminal vesicles and the acidification was not compartmentalised. We concluded that Atg9 is required to form intraluminal vesicles and for localised acidification within amphisomes/autolysosomes, and consequently when depleted, reduced the capacity to degrade and remodel gut tissue during development.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

Science.gov (United States)

Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

2018-03-01

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

Science.gov (United States)

Bao, Lan

2015-09-30

Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking.

Science.gov (United States)

Perkins, Lydia A; Yan, Qi; Schmidt, Brigitte F; Kolodieznyi, Dmytro; Saurabh, Saumya; Larsen, Mads Breum; Watkins, Simon C; Kremer, Laura; Bruchez, Marcel P

2018-02-06

Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.

From mucolipidosis type IV to Ebola: TRPML and two-pore channels at the crossroads of endo-lysosomal trafficking and disease.

Science.gov (United States)

Grimm, Christian; Butz, Elisabeth; Chen, Cheng-Chang; Wahl-Schott, Christian; Biel, Martin

2017-11-01

What do lysosomal storage disorders such as mucolipidosis type IV have in common with Ebola, cancer cell migration, or LDL-cholesterol trafficking? LDL-cholesterol, certain bacterial toxins and viruses, growth factors, receptors, integrins, macromolecules destined for degradation or secretion are all sorted and transported via the endolysosomal system (ES). There are several pathways known in the ES, e.g. the degradation, the recycling, or the retrograde trafficking pathway. The ES comprises early and late endosomes, lysosomes and recycling endosomes as well as autophagosomes and lysosome related organelles. Contact sites between the ES and the endoplasmic reticulum or the Golgi apparatus may also be considered part of it. Dysfunction of this complex intracellular machinery can cause or contribute to the development of a number of diseases ranging from neurodegenerative, infectious, or metabolic diseases to retinal and pigmentation disorders as well as cancer and autophagy-related diseases. Endolysosomal ion channels such as mucolipins (TRPMLs) and two-pore channels (TPCs) play an important role in intracellular cation/calcium signaling and homeostasis and appear to critically contribute to the proper function of the endolysosomal trafficking network. Copyright © 2017 Elsevier Ltd. All rights reserved.

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

Directory of Open Access Journals (Sweden)

Muriaux Delphine

2007-08-01

Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

Vesicles and vesicle fusion: coarse-grained simulations

DEFF Research Database (Denmark)

Shillcock, Julian C.

2010-01-01

of vesicles that is crucial for this transport is their ability to fuse to target membranes and release their contents to the distal side. In industry, some personal care products contain vesicles to help transport reagents across the skin, and research on drug formulation shows that packaging active......Biological cells are highly dynamic, and continually move material around their own volume and between their interior and exterior. Much of this transport encapsulates the material inside phospholipid vesicles that shuttle to and fro, fusing with, and budding from, other membranes. A feature...

Emergence and stability of intermediate open vesicles in disk-to-vesicle transitions.

Science.gov (United States)

Li, Jianfeng; Zhang, Hongdong; Qiu, Feng; Shi, An-Chang

2013-07-01

The transition between two basic structures, a disk and an enclosed vesicle, of a finite membrane is studied by examining the minimum energy path (MEP) connecting these two states. The MEP is constructed using the string method applied to continuum elastic membrane models. The results reveal that, besides the commonly observed disk and vesicle, open vesicles (bowl-shaped vesicles or vesicles with a pore) can become stable or metastable shapes. The emergence, stability, and probability distribution of these open vesicles are analyzed. It is demonstrated that open vesicles can be stabilized by higher-order elastic energies. The estimated probability distribution of the different structures is in good agreement with available experiments.

Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

OpenAIRE

Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian; Stürzl, Michael

2014-01-01

Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin a...

Gating characteristics control glutamate receptor distribution and trafficking in vivo.

Science.gov (United States)

Petzoldt, Astrid G; Lee, Yü-Hien; Khorramshahi, Omid; Reynolds, Eric; Plested, Andrew J R; Herzel, Hanspeter; Sigrist, Stephan J

2014-09-08

Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

Science.gov (United States)

Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

2015-11-05

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Abrin immunotoxin: targeted cytotoxicity and intracellular trafficking pathway.

Directory of Open Access Journals (Sweden)

Sudarshan Gadadhar

Full Text Available BACKGROUND: Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. METHODS: Protein synthesis assay using (3[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. RESULTS: We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. CONCLUSIONS: This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.

Rebooting Trafficking

Directory of Open Access Journals (Sweden)

Nicholas de Villiers

2016-09-01

Full Text Available While popular psychology and appeals to emotion have unfortunately dominated discussions of ‘sex trafficking’, this article suggests that feminist psychoanalytic film theory and theories of affect are still useful for making sense of the appeal of sensational exposés like Lifetime Television’s Human Trafficking (2005. The dynamic of identification with (and impersonation of a human trafficking ‘victim’ by the rescuing Immigration and Customs Enforcement agent (Mira Sorvino is particularly worthy of scrutiny. Film theory about the ‘rebooting’ of film franchises (iconic brands like Batman also helps explain the preponderance of similar programming—Sex Slaves (2005, Selling the Girl Next Door (2011, Trafficked (2016—and the way contemporary discourses of human trafficking have effectively rebranded the myth of ‘white slavery’.

Impact of lysosome status on extracellular vesicle content and release.

Science.gov (United States)

Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P

2016-12-01

Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. Copyright © 2016. Published by Elsevier B.V.

Elevated temperature inhibits recruitment of transferrin-positive vesicles and induces iron-deficiency genes expression in Aiptasia pulchella host-harbored Symbiodinium.

Science.gov (United States)

Song, Po-Ching; Wu, Tsung-Meng; Hong, Ming-Chang; Chen, Ming-Chyuan

2015-10-01

Coral bleaching is the consequence of disruption of the mutualistic Cnidaria-dinoflagellate association. Elevated seawater temperatures have been proposed as the most likely cause of coral bleaching whose severity is enhanced by a limitation in the bioavailability of iron. Iron is required by numerous organisms including the zooxanthellae residing inside the symbiosome of cnidarian cells. However, the knowledge of how symbiotic zooxanthellae obtain iron from the host cells and how elevated water temperature affects the association is very limited. Since cellular iron acquisition is known to be mediated through transferrin receptor-mediated endocytosis, a vesicular trafficking pathway specifically regulated by Rab4 and Rab5, we set out to examine the roles of these key proteins in the iron acquisition by the symbiotic Symbiodinium. Thus, we hypothesized that the iron recruitments into symbiotic zooxanthellae-housed symbiosomes may be dependent on rab4/rab5-mediated fusion with vesicles containing iron-bound transferrins and will be retarded under elevated temperature. In this study, we cloned a novel monolobal transferrin (ApTF) gene from the tropical sea anemone Aiptasia pulchella and confirmed that the association of ApTF with A. pulchella Rab4 (ApRab4) or A. pulchella Rab5 (ApRab5) vesicles is inhibited by elevated temperature through immunofluorescence analysis. We confirmed the iron-deficient phenomenon by demonstrating the induced overexpression of iron-deficiency-responsive genes, flavodoxin and high-affinity iron permease 1, and reduced intracellular iron concentration in zooxanthellae under desferrioxamine B (iron chelator) and high temperature treatment. In conclusion, our data are consistent with algal iron deficiency being a contributing factor for the thermal stress-induced bleaching of symbiotic cnidarians. Copyright © 2015 Elsevier Inc. All rights reserved.

Extracellular vesicles are present in mouse lymph and their level differs in atherosclerosis

Directory of Open Access Journals (Sweden)

Andreea Milasan

2016-09-01

Full Text Available The lymphatic system works in close collaboration with the cardiovascular system to preserve fluid balance throughout the body and is essential for the trafficking of antigen-presenting cells and lymphocytes to lymphoid organs. Recent findings have associated lymphatic dysfunction with the pathogenesis of cardiovascular-related diseases such as atherosclerosis, inflammation and obesity. Whether lymphatic dysfunction is a cause or a consequence of these diseases, as well as how, is under intensive investigation. Extracellular vesicles (EVs are submicron vesicles released by diverse cell types upon activation or apoptosis and are considered important biomarkers for several inflammatory diseases. Thus, it is critical to characterize the presence of EVs in various biological tissues and fluids to delineate their origins and, subsequently, their functions. In the past few years, new techniques allowing the quantitative and qualitative analysis of EVs have emerged, thus facilitating the onset of studies bridging these vesicles to the lymphatic system. Using several state-of-the-art approaches, this article reports the presence of diverse EVs inclusively derived from red blood cells and platelets in lymph of healthy animals. Our results suggest that lymph from atherosclerotic mice displays a higher concentration of EVs, bringing forward the concept that EVs contained in lymph could either be a biomarker for lymphatic dysfunction or, conversely, for inflammatory disease progression.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar CellsSummary

Directory of Open Access Journals (Sweden)

Scott W. Messenger

2015-11-01

Full Text Available Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI3P. PI(3,5P2 is necessary for maturation of early endosomes (EE to late endosomes (LE. Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1, and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8, bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular

Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

Directory of Open Access Journals (Sweden)

Katarzyna Jarmoszewicz

Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth.

Science.gov (United States)

Lyu, Junfang; Yang, Eun Ju; Head, Sarah A; Ai, Nana; Zhang, Baoyuan; Wu, Changjie; Li, Ruo-Jing; Liu, Yifan; Yang, Chen; Dang, Yongjun; Kwon, Ho Jeong; Ge, Wei; Liu, Jun O; Shim, Joong Sup

2017-11-28

Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents. Copyright © 2017 Elsevier B.V. All rights reserved.

Perturbed cholesterol and vesicular trafficking associated with dengue blocking in Wolbachia-infected Aedes aegypti cells.

Science.gov (United States)

Geoghegan, Vincent; Stainton, Kirsty; Rainey, Stephanie M; Ant, Thomas H; Dowle, Adam A; Larson, Tony; Hester, Svenja; Charles, Philip D; Thomas, Benjamin; Sinkins, Steven P

2017-09-13

Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia-infected Ae. aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia-carrying cells, suggesting dengue is inhibited in Wolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders. Wolbachia infection of mosquitoes can block dengue virus infection and is tested in field trials, but the mechanism of action is unclear. Using proteomics, Geoghegan et al. here identify effects of Wolbachia on cholesterol homeostasis and dengue virus replication in Aedes aegypti.

Exploring Anti-Bacterial Compounds against Intracellular Legionella

Science.gov (United States)

Harrison, Christopher F.; Kicka, Sébastien; Trofimov, Valentin; Berschl, Kathrin; Ouertatani-Sakouhi, Hajer; Ackermann, Nikolaus; Hedberg, Christian; Cosson, Pierre; Soldati, Thierry; Hilbi, Hubert

2013-01-01

Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an ‘accidental’ human pathogen and cause a severe pneumonia known as Legionnaires’ disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoeba castellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target. PMID:24058631

Exploring anti-bacterial compounds against intracellular Legionella.

Directory of Open Access Journals (Sweden)

Christopher F Harrison

Full Text Available Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the β-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.

The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

Science.gov (United States)

Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

2016-08-31

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Cystic Fibrosis, Cystic Fibrosis Transmembrane Conductance Regulator and Drugs: Insights from Cellular Trafficking.

Science.gov (United States)

Bridges, Robert J; Bradbury, Neil A

2018-01-01

The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein's absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

Science.gov (United States)

Fu, Yulong; Zhu, Jun-Yi; Zhang, Fujian; Richman, Adam; Zhao, Zhanzheng; Han, Zhe

2017-06-01

The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

Profiling extracellular vesicle release by the filarial nematode Brugia malayi reveals sex-specific differences in cargo and a sensitivity to ivermectin.

Directory of Open Access Journals (Sweden)

Hiruni Harischandra

2018-04-01

Full Text Available The filarial nematode Brugia malayi is an etiological agent of Lymphatic Filariasis. The capability of B. malayi and other parasitic nematodes to modulate host biology is recognized but the mechanisms by which such manipulation occurs are obscure. An emerging paradigm is the release of parasite-derived extracellular vesicles (EV containing bioactive proteins and small RNA species that allow secretion of parasite effector molecules and their potential trafficking to host tissues. We have previously described EV release from the infectious L3 stage B. malayi and here we profile vesicle release across all intra-mammalian life cycle stages (microfilariae, L3, L4, adult male and female worms. Nanoparticle Tracking Analysis was used to quantify and size EVs revealing discrete vesicle populations and indicating a secretory process that is conserved across the life cycle. Brugia EVs are internalized by murine macrophages with no preference for life stage suggesting a uniform mechanism for effector molecule trafficking. Further, the use of chemical uptake inhibitors suggests all life stage EVs are internalized by phagocytosis. Proteomic profiling of adult male and female EVs using nano-scale LC-MS/MS described quantitative and qualitative differences in the adult EV proteome, helping define the biogenesis of Brugia EVs and revealing sexual dimorphic characteristics in immunomodulatory cargo. Finally, ivermectin was found to rapidly inhibit EV release by all Brugia life stages. Further this drug effect was also observed in the related filarial nematode, the canine heartworm Dirofilaria immitis but not in an ivermectin-unresponsive field isolate of that parasite, highlighting a potential mechanism of action for this drug and suggesting new screening platforms for anti-filarial drug development.

Molecular features contributing to virus-independent intracellular localization and dynamic behavior of the herpesvirus transport protein US9.

Directory of Open Access Journals (Sweden)

Manuela Pedrazzi

Full Text Available Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at US9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of US9-related vesicles is the virus (or its parts, defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make US9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed US9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of US9. In these primary cells, transfected US9 mostly recapitulates the behavior of US9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y32Y33 and S34ES36 have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while US9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for US9 role in intracellular transport.

Readily releasable pool of synaptic vesicles measured at single synaptic contacts.

Science.gov (United States)

Trigo, Federico F; Sakaba, Takeshi; Ogden, David; Marty, Alain

2012-10-30

To distinguish between different models of vesicular release in brain synapses, it is necessary to know the number of vesicles of transmitter that can be released immediately at individual synapses by a high-calcium stimulus, the readily releasable pool (RRP). We used direct stimulation by calcium uncaging at identified, single-site inhibitory synapses to investigate the statistics of vesicular release and the size of the RRP. Vesicular release, detected as quantal responses in the postsynaptic neuron, showed an unexpected stochastic variation in the number of quanta from stimulus to stimulus at high intracellular calcium, with a mean of 1.9 per stimulus and a maximum of three or four. The results provide direct measurement of the RRP at single synaptic sites. They are consistent with models in which release proceeds from a small number of vesicle docking sites with an average occupancy around 0.7.

Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

Science.gov (United States)

DeVay, Rachel M; Delaria, Kathy; Zhu, Guoyun; Holz, Charles; Foletti, Davide; Sutton, Janette; Bolton, Gary; Dushin, Russell; Bee, Christine; Pons, Jaume; Rajpal, Arvind; Liang, Hong; Shelton, David; Liu, Shu-Hui; Strop, Pavel

2017-04-19

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

Vesicle electrohydrodynamics.

Science.gov (United States)

Schwalbe, Jonathan T; Vlahovska, Petia M; Miksis, Michael J

2011-04-01

A small amplitude perturbation analysis is developed to describe the effect of a uniform electric field on the dynamics of a lipid bilayer vesicle in a simple shear flow. All media are treated as leaky dielectrics and fluid motion is described by the Stokes equations. The instantaneous vesicle shape is obtained by balancing electric, hydrodynamic, bending, and tension stresses exerted on the membrane. We find that in the absence of ambient shear flow, it is possible that an applied stepwise uniform dc electric field could cause the vesicle shape to evolve from oblate to prolate over time if the encapsulated fluid is less conducting than the suspending fluid. For a vesicle in ambient shear flow, the electric field damps the tumbling motion, leading to a stable tank-treading state.

Quantitative modeling of the dynamics and intracellular trafficking of far-red light-activatable prodrugs: implications in stimuli-responsive drug delivery system.

Science.gov (United States)

Li, Mengjie; Thapa, Pritam; Rajaputra, Pallavi; Bio, Moses; Peer, Cody J; Figg, William D; You, Youngjae; Woo, Sukyung

2017-12-01

The combination of photodynamic therapy (PDT) with anti-tumor agents is a complimentary strategy to treat local cancers. We developed a unique photosensitizer (PS)-conjugated paclitaxel (PTX) prodrug in which a PS is excited by near-infrared wavelength light to site-specifically release PTX while generating singlet oxygen (SO) to effectively kill cancer cells with both PTX and SO. The aim of the present study was to identify the determinants influencing the combined efficacy of this light-activatable prodrug, especially the bystander killing effects from released PTX. Using PS-conjugated PTX as a model system, we developed a quantitative mathematical model describing the intracellular trafficking. Dynamics of the prodrug and the model predictions were verified with experimental data using human cancer cells in vitro. The sensitivity analysis suggested that parameters related to extracellular concentration of released PTX, prodrug uptake, target engagement, and target abundance are critical in determining the combined killing efficacy of the prodrug. We found that released PTX cytotoxicity was most sensitive to the retention time of the drug in extracellular space. Modulating drug internalization and conjugating the agents targeted to abundant receptors may provide a new strategy for maximizing the killing capacity of the far-red light-activatable prodrug system. These results provide guidance for the design of the PDT combination study in vivo and have implications for other stimuli-responsive drug delivery systems.

Vesicles and vesicle gels - structure and dynamics of formation

International Nuclear Information System (INIS)

Gradzielski, M

2003-01-01

Vesicles constitute an interesting morphology formed by self-aggregating amphiphilic molecules. They exhibit a rich structural variety and are of interest both from a fundamental point of view (for studying closed bilayer systems) and from a practical point of view (whenever one is interested in the encapsulation of active molecules). In many circumstances vesicular structures have to be formed by external forces, but of great interest are amphiphilic systems, where they form spontaneously. Here the question arises of whether this means that they are also thermodynamically stable structures, which at least in some systems appears to be the case. If such vesicles are well defined in size, it is possible to pack them densely and thereby form vesicle gels that possess highly elastic properties even for relatively low volume fractions of amphiphile. Conditions for the formation and the microstructure of such vesicle gels have been studied in some detail for the case of unilamellar vesicles. Another important and topical issue is the dynamics of vesicle formation/breakdown, as the understanding of the transition process will open the way to a deeper understanding of their stability and also allow controlling of the structures formed, by means of their formation processes. Significant progress in the study of the transformation processes has been achieved, in particular by means of time-resolved scattering experiments. (topical review)

Overproduction of individual gas vesicle proteins perturbs flotation, antibiotic production and cell division in the enterobacterium Serratia sp. ATCC 39006.

Science.gov (United States)

Monson, Rita E; Tashiro, Yosuke; Salmond, George P C

2016-09-01

Gas vesicles are intracellular proteinaceous organelles that facilitate bacterial colonization of static water columns. In the enterobacterium Serratia sp. ATCC 39006, gas vesicle formation requires the proteins GvpA1, GvpF1, GvpG, GvpA2, GvpK, GvpA3, GvpF2 and GvpF3 and the three gas vesicle regulatory proteins GvrA, GvrB and GvrC. Deletion of gvpC alters gas vesicle robustness and deletion of gvpN or gvpV results in small bicone vesicles. In this work, we assessed the impacts on gas vesicle formation when each of these 14 essential proteins was overexpressed. Overproduction of GvpF1, GvpF2, GvrA, GvrB or GvrC all resulted in significantly reduced gas vesicle synthesis. Perturbations in gas vesicle formation were also observed when GvpV and GvpA3 were in excess. In addition to impacts on gas vesicle formation, overproduction of GvrA or GvrB led to elevated biosynthesis of the tripyrrole pigment, prodigiosin, a secondary metabolite of increasing medical interest due to its antimalarial and anticancer properties. Finally, when GvpG was overexpressed, gas vesicles were still produced, but the cells exhibited a growth defect. Further analysis showed that induction of GvpG arrested cell growth and caused a drop in viable count, suggesting a possible physiological role for this protein linking gas vesicle biogenesis and binary fission. These combined results demonstrate that the stoichiometry of individual gas vesicle proteins is crucially important for controlled organelle morphogenesis and flotation and provides evidence for the first link between gas vesicle assembly and cell division, to our knowledge.

A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium

Science.gov (United States)

Ramsay, Joshua P.; Williamson, Neil R.; Spring, David R.; Salmond, George P. C.

2011-01-01

Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air–liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen. PMID:21873216

A quorum-sensing molecule acts as a morphogen controlling gas vesicle organelle biogenesis and adaptive flotation in an enterobacterium.

Science.gov (United States)

Ramsay, Joshua P; Williamson, Neil R; Spring, David R; Salmond, George P C

2011-09-06

Gas vesicles are hollow intracellular proteinaceous organelles produced by aquatic Eubacteria and Archaea, including cyanobacteria and halobacteria. Gas vesicles increase buoyancy and allow taxis toward air-liquid interfaces, enabling subsequent niche colonization. Here we report a unique example of gas vesicle-mediated flotation in an enterobacterium; Serratia sp. strain ATCC39006. This strain is a member of the Enterobacteriaceae previously studied for its production of prodigiosin and carbapenem antibiotics. Genes required for gas vesicle synthesis mapped to a 16.6-kb gene cluster encoding three distinct homologs of the main structural protein, GvpA. Heterologous expression of this locus in Escherichia coli induced copious vesicle production and efficient cell buoyancy. Gas vesicle morphogenesis in Serratia enabled formation of a pellicle-like layer of highly vacuolated cells, which was dependent on oxygen limitation and the expression of ntrB/C and cheY-like regulatory genes within the gas-vesicle gene cluster. Gas vesicle biogenesis was strictly controlled by intercellular chemical signaling, through an N-acyl homoserine lactone, indicating that in this system the quorum-sensing molecule acts as a morphogen initiating organelle development. Flagella-based motility and gas vesicle morphogenesis were also oppositely regulated by the small RNA-binding protein, RsmA, suggesting environmental adaptation through physiological control of the choice between motility and flotation as alternative taxis modes. We propose that gas vesicle biogenesis in this strain represents a distinct mechanism of mobility, regulated by oxygen availability, nutritional status, the RsmA global regulatory system, and the quorum-sensing morphogen.

Rapid granulation tissue regeneration by intracellular ATP delivery--a comparison with Regranex.

Directory of Open Access Journals (Sweden)

Jeffrey D Howard

Full Text Available This study tests a new intracellular ATP delivery technique for tissue regeneration and compares its efficacy with that of Regranex. Twenty-seven adult New Zealand white rabbits each underwent minimally invasive surgery to render one ear ischemic. Eight wounds were then created: four on the ischemic and four on the normal ear. Two wounds on one side of each ear were treated with Mg-ATP encapsulated lipid vesicles (ATP-vesicles while the two wounds on the other side were treated with Regranex. Wound healing time was shorter when ATP-vesicles were used. The most striking finding was that new tissue growth started to appear in less than 1 day when ATP-vesicles were used. The growth continued and covered the wound area within a few days, without the formation of a provisional matrix. Regranex-treated wounds did not have this growth pattern. In wounds treated by ATP-vesicles, histologic studies revealed extremely rich macrophage accumulation, along with active proliferating cell nuclear antigen (PCNA and positive BrdU staining, indicating in situ macrophage proliferation. Human macrophage culture suggested direct collagen production. These results support an entirely new healing process, which seems to have combined the conventional hemostasis, inflammation, and proliferation phases into a single one, thereby eliminating the lag time usually seen during healing process.

Lubricated immersed boundary method in two dimensions

Science.gov (United States)

Fai, Thomas G.; Rycroft, Chris H.

2018-03-01

Many biological examples of fluid-structure interaction, including the transit of red blood cells through the narrow slits in the spleen and the intracellular trafficking of vesicles into dendritic spines, involve the near-contact of elastic structures separated by thin layers of fluid. Motivated by such problems, we introduce an immersed boundary method that uses elements of lubrication theory to resolve thin fluid layers between immersed boundaries. We demonstrate 2nd-order accurate convergence for simple two-dimensional flows with known exact solutions to showcase the increased accuracy of this method compared to the standard immersed boundary method. Motivated by the phenomenon of wall-induced migration, we apply the lubricated immersed boundary method to simulate an elastic vesicle near a wall in shear flow. We also simulate the dynamics of a vesicle traveling through a narrow channel and observe the ability of the lubricated method to capture the vesicle motion on relatively coarse fluid grids.

Apolipoprotein L1 confers pH-switchable ion permeability to phospholipid vesicles.

Science.gov (United States)

Bruno, Jonathan; Pozzi, Nicola; Oliva, Jonathan; Edwards, John C

2017-11-03

Apolipoprotein L1 (ApoL1) is a human serum protein conferring resistance to African trypanosomes, and certain ApoL1 variants increase susceptibility to some progressive kidney diseases. ApoL1 has been hypothesized to function like a pore-forming colicin and has been reported to have permeability effects on both intracellular and plasma membranes. Here, to gain insight into how ApoL1 may function in vivo , we used vesicle-based ion permeability, direct membrane association, and intrinsic fluorescence to study the activities of purified recombinant ApoL1. We found that ApoL1 confers chloride-selective permeability to preformed phospholipid vesicles and that this selectivity is strongly pH-sensitive, with maximal activity at pH 5 and little activity above pH 7. When ApoL1 and lipid were allowed to interact at low pH and were then brought to neutral pH, chloride permeability was suppressed, and potassium permeability was activated. Both chloride and potassium permeability linearly correlated with the mass of ApoL1 in the reaction mixture, and both exhibited lipid selectivity, requiring the presence of negatively charged lipids for activity. Potassium, but not chloride, permease activity required the presence of calcium ions in both the association and activation steps. Direct assessment of ApoL1-lipid associations confirmed that ApoL1 stably associates with phospholipid vesicles, requiring low pH and the presence of negatively charged phospholipids for maximal binding. Intrinsic fluorescence of ApoL1 supported the presence of a significant structural transition when ApoL1 is mixed with lipids at low pH. This pH-switchable ion-selective permeability may explain the different effects of ApoL1 reported in intracellular and plasma membrane environments. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Intracellular siRNA delivery dynamics of integrin-targeted, PEGylated chitosan-poly(ethylene imine) hybrid nanoparticles

DEFF Research Database (Denmark)

Ragelle, Héloïse; Colombo, Stefano; Pourcelle, Vincent

2015-01-01

chitosan-poly(ethylene imine) hybrid nanoparticles. The amount of intracellular siRNA delivered by αvβ3-targeted versus non-targeted nanoparticles was quantified in the human non-small cell lung carcinoma cell line H1299 expressing enhanced green fluorescent protein (EGFP) using a stem-loop reverse...... that these nanoparticles might end up in late endosomes or lysosomes without releasing their cargo to the cell cytoplasm. Thus, the silencing efficiency of the chitosan-based nanoparticles is strongly dependent on the uptake and the intracellular trafficking in H1299 EGFP cells, which is critical information towards...

Astrocytic Pathological Calcium Homeostasis and Impaired Vesicle Trafficking in Neurodegeneration

Directory of Open Access Journals (Sweden)

Nina Vardjan

2017-02-01

Full Text Available Although the central nervous system (CNS consists of highly heterogeneous populations of neurones and glial cells, clustered into diverse anatomical regions with specific functions, there are some conditions, including alertness, awareness and attention that require simultaneous, coordinated and spatially homogeneous activity within a large area of the brain. During such events, the brain, representing only about two percent of body mass, but consuming one fifth of body glucose at rest, needs additional energy to be produced. How simultaneous energy procurement in a relatively extended area of the brain takes place is poorly understood. This mechanism is likely to be impaired in neurodegeneration, for example in Alzheimer’s disease, the hallmark of which is brain hypometabolism. Astrocytes, the main neural cell type producing and storing glycogen, a form of energy in the brain, also hold the key to metabolic and homeostatic support in the central nervous system and are impaired in neurodegeneration, contributing to the slow decline of excitation-energy coupling in the brain. Many mechanisms are affected, including cell-to-cell signalling. An important question is how changes in cellular signalling, a process taking place in a rather short time domain, contribute to the neurodegeneration that develops over decades. In this review we focus initially on the slow dynamics of Alzheimer’s disease, and on the activity of locus coeruleus, a brainstem nucleus involved in arousal. Subsequently, we overview much faster processes of vesicle traffic and cytosolic calcium dynamics, both of which shape the signalling landscape of astrocyte-neurone communication in health and neurodegeneration.

Internalization and Subcellular Trafficking of Poly-l-lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation.

Science.gov (United States)

Avaritt, Brittany R; Swaan, Peter W

2015-06-01

Internalization and intracellular trafficking of dendrimer-drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-l-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer-drug conjugates requires careful consideration to achieve successful drug delivery.

Sex for Sale: Globalization and Human Trafficking

OpenAIRE

Aiello, Annmarie

2009-01-01

The practice of trafficking has many different facets; drug trafficking, arms trafficking and human trafficking complete the top three illegal trafficking practices today. Human trafficking may be the third highest illegal trafficking practice, however there is inadequate mainstream information on the affects of the trade and horrifying issues that incorporate trafficking in human beings. This paper will discuss how the globalized world has been enabling trafficking in human beings with a con...

Human Trafficking

Science.gov (United States)

Wilson, David McKay

2011-01-01

The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

Science.gov (United States)

Lee, Byung Ha; Gauna, Adrienne E.; Perez, Geidys; Park, Yun-jong; Pauley, Kaleb M.; Kawai, Toshihisa; Cha, Seunghee

2013-01-01

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS. PMID:23382834

Key challenges in the combat of human trafficking : Evaluating the EU trafficking strategy and EU trafficking directive

NARCIS (Netherlands)

Bosma, Alice; Rijken, Conny

2016-01-01

The problem of trafficking in human beings (THB) is still omnipresent in Europe, despite the numerous preventive and retributive actions taken. This article evaluates the two most important EU-instruments to combat trafficking: the EU Directive and the EU Strategy. Based on secondary analysis of

Sinai Trafficking: Origin and Definition of a New Form of Human Trafficking

Directory of Open Access Journals (Sweden)

Mirjam van Reisen

2015-02-01

Full Text Available The phenomenon that is coined “Sinai Trafficking” started in 2009 in the Sinai desert. It involves the abduction, extortion, sale, torture, sexual violation and killing of men, women and children. Migrants, of whom the vast majority are from Eritrean descent, are abducted and brought to the Sinai desert, where they are sold and resold, extorted for very high ransoms collected by mobile phone, while being brutally and “functionally” tortured to support the extortion. Many of them die in Sinai. Over the last five years broadcasting stations, human rights organisations and academics have reported on the practices in the Sinai and some of these reports have resulted in some confusion on the modus operandi. Based on empirical research by the authors and the analysis of data gathered in more than 200 recorded interviews with Sinai hostages and survivors on the practices, this article provides a definition of Sinai Trafficking. It argues that the term Sinai Trafficking can be used to differentiate a particular new set of criminal practices that have first been reported in the Sinai Peninsula. The article further examines how the new phenomenon of Sinai Trafficking can be framed into the legal human trafficking definition. The interconnectedness of Sinai Trafficking with slavery, torture, ransom collection, extortion, sexual violence and other severe crimes is presented to substantiate the use of the trafficking framework. The plight of Sinai survivors in Israel and Egypt is explained to illustrate the cyclical process of the trafficking practices especially endured by Eritreans, introduced as the Human Trafficking Cycle. The article concludes by setting out areas for further research.

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

International Nuclear Information System (INIS)

Doller, Anke; Badawi, Amel; Schmid, Tobias; Brauß, Thilo; Pleli, Thomas; Meyer zu Heringdorf, Dagmar; Piiper, Albrecht; Pfeilschifter, Josef; Eberhardt, Wolfgang

2015-01-01

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D 1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E 2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on different Hu

Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

Science.gov (United States)

2012-01-01

Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR) and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application. PMID:22520625

Plasma membrane protein trafficking in plant-microbe interactions: a plant cell point of view

Directory of Open Access Journals (Sweden)

Nathalie eLeborgne-Castel

2014-12-01

Full Text Available In order to ensure their physiological and cellular functions, plasma membrane (PM proteins must be properly conveyed from their site of synthesis, i.e. the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters.

Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7

Directory of Open Access Journals (Sweden)

Vanessa M. D’Costa

2015-09-01

Full Text Available Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

Interference of the Histone Deacetylase Inhibits Pollen Germination and Pollen Tube Growth in Picea wilsonii Mast.

Directory of Open Access Journals (Sweden)

Yaning Cui

Full Text Available Histone deacetylase (HDAC is a crucial component in the regulation of gene expression in various cellular processes in animal and plant cells. HDAC has been reported to play a role in embryogenesis. However, the effect of HDAC on androgamete development remains unclear, especially in gymnosperms. In this study, we used the HDAC inhibitors trichostatin A (TSA and sodium butyrate (NaB to examine the role of HDAC in Picea wilsonii pollen germination and pollen tube elongation. Measurements of the tip-focused Ca2+ gradient revealed that TSA and NaB influenced this gradient. Immunofluorescence showed that actin filaments were disrupted into disorganized fragments. As a result, the vesicle trafficking was disturbed, as determined by FM4-64 labeling. Moreover, the distribution of pectins and callose in cell walls was significantly altered in response to TSA and NaB. Our results suggest that HDAC affects pollen germination and polarized pollen tube growth in Picea wilsonii by affecting the intracellular Ca2+ concentration gradient, actin organization patterns, vesicle trafficking, as well as the deposition and configuration of cell wall components.

Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

Science.gov (United States)

Chen, Mo; Qiu, Tao; Wu, Jiajie; Yang, Yang; Wright, Graham D; Wu, Min; Ge, Ruowen

2018-03-09

Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

Science.gov (United States)

Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

2013-03-06

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Regulation of AMPA Receptor Trafficking by Protein Ubiquitination

Directory of Open Access Journals (Sweden)

Jocelyn Widagdo

2017-10-01

Full Text Available The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer’s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer’s disease, chronic stress and epilepsy.

How to Use a Trafficked Woman. The Alliance between Political and Criminal Trafficking Organisations

Directory of Open Access Journals (Sweden)

John Davies

2011-03-01

Full Text Available The principal argument of this paper is that migrant women with secure mobility rights and supportive social networks can avoid or mitigate many trafficking harms. However the paper contends that some actors have conspired to prevent such circumstances so as to pursue diverse political agendas at the expense of migrant women. The paper’s analysis restructures the trafficking contest from organised criminals versus law enforcement agencies to principally a contest between migrant women and those political agents who benefit from the moral panic associated with trafficking. It is then argued that it is these more sophisticated political actors rather than organised criminals and the clients of sex workers are the most important stakeholders in sustaining or exploiting trafficking harm. Therefore, it is concluded that resolving many trafficking harms in the EEA could be achieved by subverting political traffickers through improving migration policy rather than fighting organised crime.

Consequences of SOS1 deficiency: Intracellular physiology and transcription

KAUST Repository

Ha, OhDong

2010-06-01

As much as there is known about the function of the sodium/proton antiporter SOS1 in plants, recent studies point towards a more general role for this protein. The crucial involvement in salt stress protection is clearly one of its functions –confined to the N-terminus, but the modular structure of the protein includes a segment with several domains that are functionally not studied but comprise more than half of the protein’s length. Additional functions of the protein appear to be an influence on vesicle trafficking, vacuolar pH and general ion homeostasis during salt stress. Eliminating SOS1 leads to the expression of genes that are not strictly salinity stress related. Functions that are regulated in sos1 mutants included pathogen responses, and effects on circadian rhythm.

The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

Directory of Open Access Journals (Sweden)

Cecilia Bucci

2014-10-01

Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

Science.gov (United States)

Bucci, Cecilia; Alifano, Pietro; Cogli, Laura

2014-01-01

Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways. PMID:25295627

FAT/CD36 is localized in sarcolemma and in vesicle-like structures in subsarcolemma regions but not in mitochondria

DEFF Research Database (Denmark)

Jeppesen, Jacob; Mogensen, Martin; Prats, Clara

2010-01-01

was performed on single muscle fibers dissected from soleus muscle of lean and obese Zucker rats and from the vastus lateralis muscle from humans. Co-staining against FAT/CD36 and MitoNEET clearly show that FAT/CD36 is highly present in sarcolemma and it also associates with some vesicle-like intracellular...

Intracellular Trafficking Modulation by Ginsenoside Rg3 Inhibits Brucella abortus Uptake and Intracellular Survival within RAW 264.7 Cells.

Science.gov (United States)

Huy, Tran Xuan Ngoc; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Arayan, Lauren Togonon; Min, WonGi; Lee, Hu Jang; Rhee, Man Hee; Chang, Hong Hee; Kim, Suk

2017-03-28

Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus -infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus -containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

Internalization and trafficking mechanisms of coxsackievirus B3 in HeLa cells

International Nuclear Information System (INIS)

Chung, Sun-Ku; Kim, Joo-Young; Kim, In-Beom; Park, Sang-Ick; Paek, Kyung-Hee; Nam, Jae-Hwan

2005-01-01

Coxsackievirus B3 (CVB3) is nonenveloped and has a single-stranded positive-sense RNA genome. CVB3 induces myocarditis and ultimately dilated cardiomyopathy. Although there are mounting evidences of an interaction between CVB3 particles and the cellular receptors, coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF), very little is known about the mechanisms of internalization and trafficking. In the present study, we used the CVB3 H3 strain, which is CAR-dependent but DAF-independent Woodruff variant and found that during entry, CVB3 particles were colocalized in clathrin, after interacting primarily with CAR, which was not recycled to the plasma membrane. We also found that CVB3 internalization was dependent on the function of dynamin, a large GTPase that has an essential role in endocytosis. Heat-shock cognate protein, Hsc70, which acts as a chaperone in the release of coat proteins from clathrin-coated vesicles (CCV), played a role in CVB3 trafficking processes. Moreover, endosomal acidification was crucial for CVB3 endocytosis. Finally, CVB3 was colocalized in early endosome autoantigen 1 (EEA1) molecules, which are involved in endosome-endosome tethering and fusion. In conclusion, these data together indicate that CVB3 uses clathrin-mediated endocytosis and is transcytosed to early endosomes

Measuring phospholipase D activity in insulin-secreting pancreatic beta-cells and insulin-responsive muscle cells and adipocytes.

Science.gov (United States)

Cazzolli, Rosanna; Huang, Ping; Teng, Shuzhi; Hughes, William E

2009-01-01

Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking processes. A role for PLD in regulating glucose homeostasis is emerging as the enzyme has recently been identified in events regulating exocytosis of insulin from pancreatic beta-cells and also in insulin-stimulated glucose uptake through controlling GLUT4 vesicle exocytosis in muscle and adipose tissue. We present methodologies for assessing cellular PLD activity in secretagogue-stimulated insulin-secreting pancreatic beta-cells and also insulin-stimulated adipocyte and muscle cells, two of the principal insulin-responsive cell types controlling blood glucose levels.

Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases.

Science.gov (United States)

Yamada, Kazuhiro; Gherasim, Carmen; Banerjee, Ruma; Koutmos, Markos

2015-12-04

In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+β fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Combating illicit trafficking

International Nuclear Information System (INIS)

Biro, L.L.; Grama, E.V.

2002-01-01

Full text: National Commission for Nuclear Activities Control (CNCAN) is the national authority, which is contact point for illicit trafficking and coordinates all measures and activities to combat and prevent illicit trafficking with nuclear material and radioactive sources. Legal framework regarding illicit trafficking has been improved due to new Physical Protection Regulations, Regulations on using the DBT, Regulations on requirements for qualification of guards and physical protection personnel, Design Basis Threat for each nuclear facility to avoid the unauthorized removal or theft of nuclear material or radioactive sources. New amendments of the Law for the safe deployment of nuclear activities, Law no. 111/1996, republished, in respect of illicit trafficking with nuclear material and radioactive sources are in the process to be approved by the Parliament. CNCAN is member of the Romanian Non-proliferation Group that is an interdepartmental mechanism of cooperation entered into force in August 1999. During the sessions of this group there are discussions focused on the preventing and combating illicit trafficking with nuclear material and radioactive sources. CNCAN is member of the Interministerial Council that controls import and export with strategic products including nuclear material, non nuclear material and equipment pertinent for proliferation of nuclear weapons. An Emergency Mobile Unit has been created in 2001 that contains instruments (gamma dose rate instruments portable and personal, contaminometers, mini MCA with CdZnTe detector, a CANBERRA Inspector with Nal, CdZnTe and HPGe detectors and 2 FiedSPEC, a mobile laboratory, 2 cars and individual equipment). CNCAN is cooperating with the Police through a National Plan to verify the authorization holders in order to prevent and combat illicit trafficking, and to find the orphan sources. CNCAN is the beneficiary of the PECO Project initiated by the European Commission in cooperation with the IAEA and

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

Energy Technology Data Exchange (ETDEWEB)

Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br [Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Rua José Lourenço Kelmer, Juiz de Fora, MG 36036-900 (Brazil); Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215 (United States); Weller, Peter F. [Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215 (United States)

2016-10-01

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

International Nuclear Information System (INIS)

Melo, Rossana C.N.; Weller, Peter F.

2016-01-01

Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.

The SNARE protein vti1a functions in dense-core vesicle biogenesis

DEFF Research Database (Denmark)

Walter, Alexander M; Kurps, Julia; de Wit, Heidi

2014-01-01

overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion......The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially...... reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days...

Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

DEFF Research Database (Denmark)

Skjørringe, Tina; Pedersen, Per Amstrup; Thorborg, Sidsel Salling

2017-01-01

Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher...

The formation of endosymbiotic membrane compartments: membrane identity markers and the regulation of vesicle trafficking

NARCIS (Netherlands)

Ivanov, S.

2012-01-01

In symbiosis of plants and arbuscular mycorrhizal fungi as well as in rhizobium-legume symbiosis the microbes are hosted intracellularly, inside specialized membrane compartments of the host. These membrane compartments are morphologically different but similar in function, since they control

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

Science.gov (United States)

Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

2015-01-01

Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

Science.gov (United States)

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-01

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

South Africa – Safe Haven for Human Traffickers? Employing the Arsenal of Existing Law to Combat Human Trafficking

Directory of Open Access Journals (Sweden)

H Oosthuizen

2012-03-01

Full Text Available aving ratified the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, South Africa is obliged to adopt legislative measures that criminalise human trafficking and comply with other standards laid down in this international instrument. However, by mid-2011, South Africa had not enacted the required comprehensive counter-trafficking legislation. The question that now arises is if the absence of such anti-trafficking legislation poses an insurmountable obstacle to the prosecution of traffickers for trafficking-related activities. In asking this question the article examines the utilisation of existing crimes in order to prosecute and punish criminal activities committed during the human trafficking process. Firstly, a selection of existing common law and statutory crimes that may often be applicable to trafficking related activities is mapped out. Secondly, transitional trafficking provisions in the Children's Act 38 of 2005 and the Criminal Law (Sexual Offences and Related Matters Amendment Act 32 of 2007 are discussed. Finally, since the Prevention and Combating of Trafficking in Persons Bill B7 of 2010 will in all probability be enacted in the near future, the use of other criminal law provisions in human trafficking prosecutions, even after the passing of this bill into law, is reflected upon.

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

DEFF Research Database (Denmark)

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

2009-01-01

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

Human trafficking and the healthcare professional.

Science.gov (United States)

Barrows, Jeffrey; Finger, Reginald

2008-05-01

Despite the legislation passed in the 19th century outlawing human slavery, it is more widespread today than at the conclusion of the civil war. Modern human slavery, termed human trafficking, comes in several forms. The most common type of human trafficking is sex trafficking, the sale of women and children into prostitution. Labor trafficking is the sale of men, women, and children into hard labor for which they receive little or no compensation. Other forms of trafficking include child soldiering, war brides, and organ removal. Healthcare professionals play a critical role in both finding victims of human trafficking while they are still in captivity, as well as caring for their mental and physical needs upon release. Those working in the healthcare profession need to be educated regarding how a trafficking victim may present, as well as their unique healthcare needs.

P-glycoprotein trafficking as a therapeutic target to optimize CNS drug delivery.

Science.gov (United States)

Davis, Thomas P; Sanchez-Covarubias, Lucy; Tome, Margaret E

2014-01-01

The primary function of the blood-brain barrier (BBB)/neurovascular unit is to protect the central nervous system (CNS) from potentially harmful xenobiotic substances and maintain CNS homeostasis. Restricted access to the CNS is maintained via a combination of tight junction proteins as well as a variety of efflux and influx transporters that limits the transcellular and paracellular movement of solutes. Of the transporters identified at the BBB, P-glycoprotein (P-gp) has emerged as the transporter that is the greatest obstacle to effective CNS drug delivery. In this chapter, we provide data to support intracellular protein trafficking of P-gp within cerebral capillary microvessels as a potential target for improved drug delivery. We show that pain-induced changes in P-gp trafficking are associated with changes in P-gp's association with caveolin-1, a key scaffolding/trafficking protein that colocalizes with P-gp at the luminal membrane of brain microvessels. Changes in colocalization with the phosphorylated and nonphosphorylated forms of caveolin-1, by pain, are accompanied by dynamic changes in the distribution, relocalization, and activation of P-gp "pools" between microvascular endothelial cell subcellular compartments. Since redox-sensitive processes may be involved in signaling disassembly of higher-order structures of P-gp, we feel that manipulating redox signaling, via specific protein targeting at the BBB, may protect disulfide bond integrity of P-gp reservoirs and control trafficking to the membrane surface, providing improved CNS drug delivery. The advantage of therapeutic drug "relocalization" of a protein is that the physiological impact can be modified, temporarily or long term, despite pathology-induced changes in gene transcription. © 2014 Elsevier Inc. All rights reserved.

To discuss illicit nuclear trafficking

Energy Technology Data Exchange (ETDEWEB)

Balatsky, Galya I [Los Alamos National Laboratory; Severe, William R [Los Alamos National Laboratory; Wallace, Richard K [Los Alamos National Laboratory

2010-01-01

The Illicit nuclear trafficking panel was conducted at the 4th Annual INMM workshop on Reducing the Risk from Radioactive and Nuclear Materials on February 2-3, 2010 in Washington DC. While the workshop occurred prior to the Nuclear Security Summit, April 12-13 2010 in Washington DC, some of the summit issues were raised during the workshop. The Communique of the Washington Nuclear Security Summit stated that 'Nuclear terrorism is one of the most challenging threats to international security, and strong nuclear security measures are the most effective means to prevent terrorists, criminals, or other unauthorized actors from acquiring nuclear materials.' The Illicit Trafficking panel is one means to strengthen nuclear security and cooperation at bilateral, regional and multilateral levels. Such a panel promotes nuclear security culture through technology development, human resources development, education and training. It is a tool which stresses the importance of international cooperation and coordination of assistance to improve efforts to prevent and respond to incidents of illicit nuclear trafficking. Illicit trafficking panel included representatives from US government, an international organization (IAEA), private industry and a non-governmental organization to discuss illicit nuclear trafficking issues. The focus of discussions was on best practices and challenges for addressing illicit nuclear trafficking. Terrorism connection. Workshop discussions pointed out the identification of terrorist connections with several trafficking incidents. Several trafficking cases involved real buyers (as opposed to undercover law enforcement agents) and there have been reports identifying individuals associated with terrorist organizations as prospective plutonium buyers. Some specific groups have been identified that consistently search for materials to buy on the black market, but no criminal groups were identified that specialize in nuclear materials or isotope

Endosome-based protein trafficking and Ca2+ homeostasis in the heart

Directory of Open Access Journals (Sweden)

Jerry eCurran

2015-02-01

Full Text Available The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart and cardiovascular system. In the heart, these systems are essential for the regulation of cardiac calcium, both at the level of the plasma membrane, but also at local domains of the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, nucleus, and nuclear envelope. One intracellular pathway often overlooked in relation to cardiovascular calcium regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. The vast majority of what is known has been derived from heterologous cell systems. However, recently, more appropriate cell and animal models been developed that have allowed researchers to begin to understand how this system functions within the intact physiological environment. All excitable cells, including cardiomyocytes, depend on the proper expression and organization of multiple ion channels, pumps, exchangers, and transporters within the plasma membrane. As the endosomal system acts to regulate the expression and localization of membrane proteins, understanding the in vivo function of this system in the heart is important. This review will focus on endosome-based protein trafficking in the heart in both health and disease. Special emphasis will be given to the role played by the family of endocytic regulatory proteins, C-terminal Eps15 homology domain -containing proteins (EHDs, as recent data demonstrates that this family of proteins is essential for the proper trafficking and localization and of key proteins involved in excitation-contraction coupling.

Smuggled or trafficked?

Directory of Open Access Journals (Sweden)

Jacqueline Bhabha

2006-05-01

Full Text Available The UN Convention Against Transnational Organized Crime (TNC and its two Protocols on Trafficking and Smuggling, adopted in 2000, seek to distinguish between trafficking and smuggling. In reality these distinctions are often blurred. A more nuanced approach is needed to ensure protection for all those at risk.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

Science.gov (United States)

Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R.; Oker-Blom, Christian; Vattulainen, Ilpo; Vuento, Matti

2009-12-01

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter SCD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

International Nuclear Information System (INIS)

Pakkanen, Kirsi; Mäkelä, Anna R; Oker-Blom, Christian; Vuento, Matti; Salonen, Emppu; Vattulainen, Ilpo

2009-01-01

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter S CD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane

An Essential Role of Hrs/Vps27 in Endosomal Cholesterol Trafficking

Directory of Open Access Journals (Sweden)

Ximing Du

2012-01-01

Full Text Available The endosomal sorting complex required for transport (ESCRT plays a crucial role in the degradation of ubiquitinated endosomal membrane proteins. Here, we report that Hrs, a key protein of the ESCRT-0 complex, is required for the transport of low-density lipoprotein-derived cholesterol from endosomes to the endoplasmic reticulum. This function of Hrs in cholesterol transport is distinct from its previously defined role in lysosomal sorting and downregulation of membrane receptors via the ESCRT pathway. In line with this, knocking down other ESCRT proteins does not cause prominent endosomal cholesterol accumulation. Importantly, the localization and biochemical properties of key cholesterol-sorting proteins, NPC1 and NPC2, appear to be unchanged upon Hrs knockdown. Our data identify Hrs as a regulator of endosomal cholesterol trafficking and provide additional insights into the budding of intralumenal vesicles.

Formation of Oligovesicular Vesicles by Micromanipulation

Directory of Open Access Journals (Sweden)

Yukihisa Okumura

2011-09-01

Full Text Available Cell-sized lipid bilayer membrane vesicles (giant vesicles, GVs or semi-vesicles were formed from egg yolk phosphatidylcholine on a platinum electrode under applied electric voltage by electroformation. Micromanipulation of the semi-vesicle by first pressing its membrane with a glass microneedle and then withdrawing the needle left a GV in the interior of the vesicle. During the process, an aqueous solution of Ficoll that filled the needle was introduced into the newly formed inner vesicle and remained encapsulated. Approximately 50% of attempted micromanipulation resulted in the formation of an inner daughter vesicle, “microvesiculation”. By repeating the microvesiculation process, multiple inner GVs could be formed in a single parent semi-vesicle. A semi-vesicle with inner GVs could be detached from the electrode by scraping with a microneedle, yielding an oligovesicular vesicle (OVV with desired inner aqueous contents. Microvesiculation of a GV held on the tip of a glass micropipette was also possible, and this also produced an OVV. Breaking the membrane of the parent semi-vesicle by micromanipulation with a glass needle after microvesiculation, released the inner GVs. This protocol may be used for controlled formation of GVs with desired contents.

Combating Human Trafficking with Deep Multimodal Models

OpenAIRE

Tong, Edmund; Zadeh, Amir; Jones, Cara; Morency, Louis-Philippe

2017-01-01

Human trafficking is a global epidemic affecting millions of people across the planet. Sex trafficking, the dominant form of human trafficking, has seen a significant rise mostly due to the abundance of escort websites, where human traffickers can openly advertise among at-will escort advertisements. In this paper, we take a major step in the automatic detection of advertisements suspected to pertain to human trafficking. We present a novel dataset called Trafficking-10k, with more than 10,00...

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations

DEFF Research Database (Denmark)

Christensen, Henriette L; Păunescu, Teodor G; Matchkov, Vladimir

2017-01-01

fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na(+)-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7...

The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

Energy Technology Data Exchange (ETDEWEB)

Doller, Anke; Badawi, Amel [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Schmid, Tobias; Brauß, Thilo [Institut für Biochemie I (Pathobiochemie), Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pleli, Thomas [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Meyer zu Heringdorf, Dagmar [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Piiper, Albrecht [Medizinische Klinik 1, Schwerpunkt Gastroenterologie und Hepatologie, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Pfeilschifter, Josef [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany); Eberhardt, Wolfgang, E-mail: w.eberhardt@em.uni-frankfurt.de [Pharmazentrum Frankfurt/ZAFES, Klinikum der Goethe-Universität Frankfurt, Frankfurt/Main (Germany)

2015-01-01

The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

Vesicle-based rechargeable batteries

Energy Technology Data Exchange (ETDEWEB)

Stanish, I.; Singh, A. [Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Ave., S.W., Washington, DC 20375 (United States); Lowy, D.A. [Nova Research, Inc., 1900 Elkin St., Alexandria, VA 22308 (United States); Hung, C.W. [Department of Chemical Engineering, University of Maryland, College Park, MD 20742 (United States)

2005-05-02

Vesicle-based rechargeable batteries can be fabricated by mounting polymerized vesicles filled with ferrocyanide or ferricyanide to a conductive surface. The potential can be adjusted by changing the concentration ratio of hydroquinone and benzoquinone bound to the vesicle membranes. These batteries show promise as a means of supplying portable power for future autonomous nanosystems. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

Illicit Trafficking of Natural Radionuclides

Science.gov (United States)

Friedrich, Steinhäusler; Lyudmila, Zaitseva

2008-08-01

Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

Illicit Trafficking of Natural Radionuclides

International Nuclear Information System (INIS)

Friedrich, Steinhaeusler; Lyudmila, Zaitseva

2008-01-01

Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium ( 20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces

Human Trafficking and National Morality

Directory of Open Access Journals (Sweden)

William R. DI PIETRO

2015-12-01

Full Text Available The paper proposes that national morality is an important variable for explaining national anti-trafficking policy. It uses cross country regression analysis to see whether or not empirically national morality is a determinant of anti-trafficking policy. The findings of the paper are consistent with the notion that improved levels of national morality lead to better national anti-trafficking policy. National morality is found to be statistically relevant for national anti-trafficking policy when controlling for the extent of democracy, the share of the private sector in the economy, and the degree of globalization.

Probing intracellular motor protein activity using an inducible cargo trafficking assay.

Science.gov (United States)

Kapitein, Lukas C; Schlager, Max A; van der Zwan, Wouter A; Wulf, Phebe S; Keijzer, Nanda; Hoogenraad, Casper C

2010-10-06

Although purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living cells, we utilized the FKBP-rapalog-FRB heterodimerization system to develop an in vivo peroxisomal trafficking assay that allows inducible recruitment of exogenous and endogenous kinesin, dynein, and myosin motors to drive specific cargo transport. We demonstrate that cargo rapidly redistributes with distinct dynamics for each respective motor, and that combined (antagonistic) actions of more complex motor combinations can also be probed. Of importance, robust cargo redistribution is readily achieved by one type of motor protein and does not require the presence of opposite-polarity motors. Simultaneous live-cell imaging of microtubules and kinesin or dynein-propelled peroxisomes, combined with high-resolution particle tracking, revealed that peroxisomes frequently pause at microtubule intersections. Titration and washout experiments furthermore revealed that motor recruitment by rapalog-induced heterodimerization is dose-dependent but irreversible. Our assay directly demonstrates that robust cargo motility does not require the presence of opposite-polarity motors, and can therefore be used to characterize the motile properties of specific types of motor proteins. Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Human trafficking and health: a cross-sectional survey of NHS professionals’ contact with victims of human trafficking

Science.gov (United States)

Ross, Claire; Dimitrova, Stoyanka; Howard, Louise M; Dewey, Michael; Zimmerman, Cathy; Oram, Siân

2015-01-01

Objectives (1) To estimate the proportion of National Health Service (NHS) professionals who have come into contact with trafficked people and (2) to measure NHS professionals’ knowledge and confidence to respond to human trafficking. Design A cross-sectional survey. Setting Face-to-face mandatory child protection and/or vulnerable adults training sessions at 10 secondary healthcare provider organisations in England, and meetings of the UK College of Emergency Medicine. Participants 782/892 (84.4%) NHS professionals participated, including from emergency medicine, maternity, mental health, paediatrics and other clinical disciplines. Measures Self-completed questionnaire developed by an expert panel. Questionnaire asks about prior training and contact with potential victims of trafficking, perceived and actual human trafficking knowledge, confidence in responding to human trafficking, and interest in future human trafficking training. Results 13% participants reported previous contact with a patient they knew or suspected of having been trafficked; among maternity services professionals this was 20.4%. However, 86.8% (n=679) reported lacking knowledge of what questions to ask to identify potential victims and 78.3% (n=613) reported that they had insufficient training to assist trafficked people. 71% (n=556), 67.5% (n=528) and 53.4% (n=418) lacked confidence in making appropriate referrals for men, women and children, respectively, who had been trafficked. 95.3% (n=746) of respondents were unaware of the scale of human trafficking in the UK, and 76.5% (n=598) were unaware that calling the police could put patients in more danger. Psychometric analysis showed that subscales measuring perceived knowledge, actual knowledge and confidence to respond to human trafficking demonstrated good internal consistency (Cronbach's αs 0.93, 0.63 and 0.64, respectively) and internal correlations. Conclusions NHS professionals working in secondary care are in contact with potential

Understanding human trafficking in the United States.

Science.gov (United States)

Logan, T K; Walker, Robert; Hunt, Gretchen

2009-01-01

The topic of modern-day slavery or human trafficking has received increased media and national attention. However, to date there has been limited research on the nature and scope of human trafficking in the United States. This article describes and synthesizes nine reports that assess the U.S. service organizations' legal representative knowledge of, and experience with, human trafficking cases, as well as information from actual cases and media reports. This article has five main goals: (a) to define what human trafficking is, and is not; (b) to describe factors identified as contributing to vulnerability to being trafficked and keeping a person entrapped in the situation; (c) to examine how the crime of human trafficking differs from other kinds of crimes in the United States; (d) to explore how human trafficking victims are identified; and, (e) to provide recommendations to better address human trafficking in the United States.

Affinity between TBC1D4 (AS160 phosphotyrosine-binding domain and insulin-regulated aminopeptidase cytoplasmic domain measured by isothermal titration calorimetry

Directory of Open Access Journals (Sweden)

SangYoun Park*, Keon Young Kim, Sunmin Kim & Young Seok Yu

2012-06-01

Full Text Available Uptake of circulating glucose into the cells happens via the insulin-mediated signalling pathway, which translocates the glucosetransporter 4 (GLUT4 vesicles from the intracellular compartmentto the plasma membrane. RabㆍGTPases are involvedin this vesicle trafficking, where RabㆍGTPase-activatingproteins (RabGAP enhance the GTP to GDP hydrolysis.TBC1D4 (AS160 and TBC1D1 are functional RabGAPs in theadipocytes and the skeletonal myocytes, respectively. Theseproteins contain two phosphotyrosine-binding domains (PTBsat the amino-terminus of the catalytic RabGAP domain. Thesecond PTB has been shown to interact with the cytoplasmicregion of the insulin-regulated aminopeptidase (IRAP of theGLUT4 vesicle. In this study, we quantitatively measured the∼μM affinity (KD between TBC1D4 PTB and IRAP using isothermaltitration calorimetry, and further showed that IRAP residues1-49 are the major region mediating this interaction. Wealso demonstrated that the IRAP residues 1-15 are necessarybut not sufficient for the PTB interaction.

Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy

Directory of Open Access Journals (Sweden)

Thomas A. Ryan

2018-05-01

Full Text Available Optineurin is a multifunctional adaptor protein intimately involved in various vesicular trafficking pathways. Through interactions with an array of proteins, such as myosin VI, huntingtin, Rab8, and Tank-binding kinase 1, as well as via its oligomerisation, optineurin has the ability to act as an adaptor, scaffold, or signal regulator to coordinate many cellular processes associated with the trafficking of membrane-delivered cargo. Due to its diverse interactions and its distinct functions, optineurin is an essential component in a number of homeostatic pathways, such as protein trafficking and organelle maintenance. Through the binding of polyubiquitinated cargoes via its ubiquitin-binding domain, optineurin also serves as a selective autophagic receptor for the removal of a wide range of substrates. Alternatively, it can act in an ubiquitin-independent manner to mediate the clearance of protein aggregates. Regarding its disease associations, mutations in the optineurin gene are associated with glaucoma and have more recently been found to correlate with Paget’s disease of bone and amyotrophic lateral sclerosis (ALS. Indeed, ALS-associated mutations in optineurin result in defects in neuronal vesicular localisation, autophagosome–lysosome fusion, and secretory pathway function. More recent molecular and functional analysis has shown that it also plays a role in mitophagy, thus linking it to a number of other neurodegenerative conditions, such as Parkinson’s. Here, we review the role of optineurin in intracellular membrane trafficking, with a focus on autophagy, and describe how upstream signalling cascades are critical to its regulation. Current data and contradicting reports would suggest that optineurin is an important and selective autophagy receptor under specific conditions, whereby interplay, synergy, and functional redundancy with other receptors occurs. We will also discuss how dysfunction in optineurin-mediated pathways may lead

Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.

Science.gov (United States)

Chitcholtan, Kenny; Hampton, Mark B; Keenan, Jacqueline I

2008-12-01

Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.

The dynamin chemical inhibitor dynasore impairs cholesterol trafficking and sterol-sensitive genes transcription in human HeLa cells and macrophages.

Directory of Open Access Journals (Sweden)

Emmanuelle Girard

Full Text Available Intracellular transport of cholesterol contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we characterized the impact of dynasore, a recently described drug that specifically inhibits the enzymatic activity of dynamin, a GTPase regulating receptor endocytosis and cholesterol trafficking. Dynasore strongly inhibited the uptake of low-density lipoprotein (LDL in HeLa cells, and to a lower extent in human macrophages. In both cell types, dynasore treatment led to the abnormal accumulation of LDL and free cholesterol (FC within the endolysosomal network. The measure of cholesterol esters (CE further showed that the delivery of regulatory cholesterol to the endoplasmic reticulum (ER was deficient. This resulted in the inhibition of the transcriptional control of the three major sterol-sensitive genes, sterol-regulatory element binding protein 2 (SREBP-2, 3-hydroxy-3-methyl-coenzymeA reductase (HMGCoAR, and low-density lipoprotein receptor (LDLR. The sequestration of cholesterol in the endolysosomal compartment impaired both the active and passive cholesterol efflux in HMDM. Our data further illustrate the importance of membrane trafficking in cholesterol homeostasis and validate dynasore as a new pharmacological tool to study the intracellular transport of cholesterol.

Irradiation-induced fusion between giant vesicles and photoresponsive large unilamellar vesicles containing malachite green derivative.

Science.gov (United States)

Uda, Ryoko M; Yoshikawa, Yuki; Kitaba, Moe; Nishimoto, Noriko

2018-07-01

Light-initiated fusion between vesicles has attracted much attention in the research community. In particular, fusion between photoresponsive and non-photoresponsive vesicles has been of much interest in the development of systems for the delivery of therapeutic agents to cells. We have performed fusion between giant vesicles (GVs) and photoresponsive smaller vesicles containing malachite green (MG) derivative, which undergoes ionization to afford a positive charge on the molecule by irradiation. The fusion proceeds as the concentration of GV lipid increases toward equimolarity with the lipid of the smaller vesicle. It is also dependent on the molar percentage of photoionized MG in the lipid of the smaller vesicle. On the other hand, the fusion is hardly affected by the anionic component of the GV. The photoinduced fusion was characterized by two methods, involving the mixing of lipid membranes and of aqueous contents. Fluorescence microscopy revealed that irradiation triggered the fusion of a single GV with the smaller vesicles containing MG. Copyright © 2018 Elsevier B.V. All rights reserved.

Debate - Achievements of the Trafficking Protocol: Perspectives from the former UN Special Rapporteur on Trafficking in Persons

OpenAIRE

Joy N Ezeilo

2015-01-01

The United Nations (UN) Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, Supplementing the UN Convention against Transnational Organized Crime, 2000 (Trafficking Protocol), is a watershed in galvanising the global movement against human trafficking. Thanks to the Protocol, international and regional bodies, along with civil society groups, have become involved in researching the issue and supporting anti-trafficking efforts; and states have begun...

South Africa - safe haven for human traffickers? Employing the arsenal of existing law to combat human trafficking

OpenAIRE

Kruger, H B; Oosthuizen, H

2012-01-01

aving ratified the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, South Africa is obliged to adopt legislative measures that criminalise human trafficking and comply with other standards laid down in this international instrument. However, by mid-2011, South Africa had not enacted the required comprehensive counter-trafficking legislation. The question that now arises is if the absence of such anti-trafficking legislation poses an insurmountabl...

Does Legalized Prostitution Increase Human Trafficking?

OpenAIRE

Seo-Young Cho; Axel Dreher; Eric Neumayer

2011-01-01

This paper investigates the impact of legalized prostitution on human trafficking inflows. According to economic theory, there are two opposing effects of unknown magnitude. The scale effect of legalized prostitution leads to an expansion of the prostitution market, increasing human trafficking, while the substitution effect reduces demand for trafficked women as legal prostitutes are favored over trafficked ones. Our empirical analysis for a cross-section of up to 150 countries shows that th...

Security Implications of Human-Trafficking Networks

Science.gov (United States)

2007-06-15

to those security concerns. Background How is Human Trafficking Carried Out? While trafficking victims are often found in sweatshops , domestic...labor. This type of trafficking is often found in agricultural labor, the production of goods (typically called sweatshops ) and construction labor

Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice.

Science.gov (United States)

Kim, Suk; Kurokawa, Daisuke; Watanabe, Kenta; Makino, Sou-Ichi; Shirahata, Toshikazu; Watarai, Masahisa

2004-05-15

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and non-professional phagocytes, and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. Nicotinamidase/pyrazinamidase mutant (pncA mutant) of Brucella abortus failed to replicate in HeLa cells, and showed a lower rate of intracellular replication than that of wild-type strain in macrophages. Addition of nicotinic acid, but not nicotinamide, into medium supported intracellular replication of pncA mutant in HeLa cells and macrophages. The pncA mutant was not co-localizing with either late endosomes or lysosomes. The B. abortus virB4 mutant was completely cleared from the spleens of mice after 4 weeks, while the pncA mutant showed a 1.5-log reduction of the number of bacteria isolated from spleens after 10 weeks. Although pncA mutant showed reduced virulence in mice and defective intracellular replication, its ability to confer protection against the virulent B. abortus strain 544 was fully retained. These results suggest that PncA does not contribute to intracellular trafficking of B. abortus, but contributes to utilization of nutrients required for intracellular growth. Our results indicate that detailed characterizations of the pncA mutant may help the improvement of currently available live vaccines. Copyright 2004 Federation of European Microbiological Societies

Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

Science.gov (United States)

Abounit, Saïda; Bousset, Luc; Loria, Frida; Zhu, Seng; de Chaumont, Fabrice; Pieri, Laura; Olivo-Marin, Jean-Christophe; Melki, Ronald; Zurzolo, Chiara

2016-10-04

Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies. © 2016 The Authors.

Human trafficking and health: a cross-sectional survey of NHS professionals' contact with victims of human trafficking.

Science.gov (United States)

Ross, Claire; Dimitrova, Stoyanka; Howard, Louise M; Dewey, Michael; Zimmerman, Cathy; Oram, Siân

2015-08-20

(1) To estimate the proportion of National Health Service (NHS) professionals who have come into contact with trafficked people and (2) to measure NHS professionals' knowledge and confidence to respond to human trafficking. A cross-sectional survey. Face-to-face mandatory child protection and/or vulnerable adults training sessions at 10 secondary healthcare provider organisations in England, and meetings of the UK College of Emergency Medicine. 782/892 (84.4%) NHS professionals participated, including from emergency medicine, maternity, mental health, paediatrics and other clinical disciplines. Self-completed questionnaire developed by an expert panel. Questionnaire asks about prior training and contact with potential victims of trafficking, perceived and actual human trafficking knowledge, confidence in responding to human trafficking, and interest in future human trafficking training. 13% participants reported previous contact with a patient they knew or suspected of having been trafficked; among maternity services professionals this was 20.4%. However, 86.8% (n=679) reported lacking knowledge of what questions to ask to identify potential victims and 78.3% (n=613) reported that they had insufficient training to assist trafficked people. 71% (n=556), 67.5% (n=528) and 53.4% (n=418) lacked confidence in making appropriate referrals for men, women and children, respectively, who had been trafficked. 95.3% (n=746) of respondents were unaware of the scale of human trafficking in the UK, and 76.5% (n=598) were unaware that calling the police could put patients in more danger. Psychometric analysis showed that subscales measuring perceived knowledge, actual knowledge and confidence to respond to human trafficking demonstrated good internal consistency (Cronbach's αs 0.93, 0.63 and 0.64, respectively) and internal correlations. NHS professionals working in secondary care are in contact with potential victims of human trafficking, but lack knowledge and confidence in

Human trafficking and the dental professional.

Science.gov (United States)

O'Callaghan, Michael G

2012-05-01

"Human trafficking" is a term for a modern form of slavery. It is a criminal human rights violation and a significant health issue. Dental professionals can assist in recognizing victims of trafficking. The author conducted a PubMed search of the English-language literature through May 2011, which yielded no articles meeting the search criteria "dentistry" and "human trafficking prostitution." Given these results, the author reviewed articles published in medical journals, reports from both governmental and nongovernmental agencies and lay literature. The author examines the present state of human trafficking and provides information--including specific questions to ask--to help dentists identify victims. In addition, the author suggests means of notifying authorities and assisting trafficking victims. He also examines the health care needs of these patients. Human trafficking is a global problem, with thousands of victims in the United States, including many women and children. Dentists have a responsibility to act for the benefit of others, which includes detecting signs of abuse and neglect. Dental professionals are on the front lines with respect to encountering and identifying potential victims who seek dental treatment. Dentists can combat human trafficking by becoming informed and by maintaining vigilance in their practices.

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles

NARCIS (Netherlands)

Farsi, Z.; Preobraschenski, J.; Bogaart, G. van den; Riedel, D.; Jahn, R.; Woehler, A.

2016-01-01

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided

Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

Science.gov (United States)

Farr, Glen A.; Hull, Michael; Stoops, Emily H.; Bateson, Rosalie; Caplan, Michael J.

2015-01-01

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking. PMID:26424804

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

Science.gov (United States)

Farsi, Zohreh; Preobraschenski, Julia; van den Bogaart, Geert; Riedel, Dietmar; Jahn, Reinhard; Woehler, Andrew

2016-02-26

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters. Copyright © 2016, American Association for the Advancement of Science.

Following Intracellular Cholesterol Transport by Linear and Non-Linear Optical Microscopy of Intrinsically Fluorescent Sterols

DEFF Research Database (Denmark)

Wustner, D.

2012-01-01

Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow...... is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image...... analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented....

The Palermo Protocol: Trafficking Takes it All

Directory of Open Access Journals (Sweden)

Jónína Einarsdóttir

2014-12-01

Full Text Available The Palermo Protocol is the outcome of bargain and lobbying with global institutions, NGOs and government representatives embattling to enforce their interests. The outcome is the concept of trafficking that embraces the struggles against prostitution, slavery and child labour. This broad concept has allowed various local cultural practices and survival strategies of those who live under difficult conditions to become classified as trafficking. While such definition may facilitate fundraising there are adverse consequences to be considered. Firstly, hazardous conditions of children that obviously are not trafficking tend to become ignored. Second, the victims of “real” trafficking become invisible by the excessive number of children allegedly trafficked. Third, the broad definition of trafficking has contributed to criminalization of whole communities and consequent conflicts between NGOs engaged in anti-trafficking activities and the communities involved. Such a situation is not in the best interest of the children involved. Rather than spending huge amount of resources on the conventional anti-trafficking measures there is a need to address the root causes of whatsoever unacceptable condition a child is suffering from.

Light-induced dynamic structural color by intracellular 3D photonic crystals in brown algae.

Science.gov (United States)

Lopez-Garcia, Martin; Masters, Nathan; O'Brien, Heath E; Lennon, Joseph; Atkinson, George; Cryan, Martin J; Oulton, Ruth; Whitney, Heather M

2018-04-01

Natural photonic crystals are responsible for strong reflectance at selective wavelengths in different natural systems. We demonstrate that intracellular opal-like photonic crystals formed from lipids within photosynthetic cells produce vivid structural color in the alga Cystoseira tamariscifolia . The reflectance of the opaline vesicles is dynamically responsive to environmental illumination. The structural color is present in low light-adapted samples, whereas higher light levels produce a slow disappearance of the structural color such that it eventually vanishes completely. Once returned to low-light conditions, the color re-emerges. Our results suggest that these complex intracellular natural photonic crystals are responsive to environmental conditions, changing their packing structure reversibly, and have the potential to manipulate light for roles beyond visual signaling.

Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

DEFF Research Database (Denmark)

Bao, Jichen; Huang, Mingtao; Petranovic, Dina

2018-01-01

of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of alpha-amylase but did not induce production of reactive oxygen species. An expanded ER membrane......The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein...... recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-alpha-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast....

Fusion of Nonionic Vesicles

DEFF Research Database (Denmark)

Bulut, Sanja; Oskolkova, M. Z.; Schweins, R.

2010-01-01

We present an experimental study of vesicle fusion using light and neutron scattering to monitor fusion events. Vesicles are reproducibly formed with an extrusion procedure using an single amphiphile triethylene glycol mono-n-decyl ether in water. They show long-term stability for temperatures ar...... a barrier to fusion changing from 15 k(B)T at T = 26 degrees C to 10k(H) T at T = 35 degrees C. These results are compatible with the theoretical predictions using the stalk model of vesicle fusion....

Human Trafficking: How Nurses Can Make a Difference.

Science.gov (United States)

Scannell, Meredith; MacDonald, Andrea E; Berger, Amanda; Boyer, Nichole

Human trafficking is a human rights violation and a global health problem. Victims of human trafficking have medical and mental health sequelae requiring specific healthcare interventions. Healthcare professionals may be the initial contact that these victims make outside the world of trafficking. Healthcare professionals are key agents in the identification of human trafficking, which is essential in eliminating this public health problem. Unfortunately, healthcare professionals are not always able to detect signs of human trafficking. Failure to detect results in missed opportunities to assist victims. This is a case report of a victim of human trafficking who presented to an emergency department with medical and mental health issues. Despite numerous encounters with different healthcare professionals, signs and symptoms of human trafficking were not identified. Skilled assessment made by a forensic nurse alerted the healthcare team to clear features of human trafficking associated with this person. Through this case report we illustrate the key role the nurse played in identifying signs of human trafficking. Improvement of human trafficking educational programs is highlighted as a key adjunct to improving detection and facilitating the proper treatment of victims.

Role of Occupational Therapy in Combating Human Trafficking.

Science.gov (United States)

Gorman, Kathleen W; Hatkevich, Beth Ann

Human trafficking is a modern-day form of slavery that includes sex trafficking, labor trafficking, and trafficking of children. It is estimated that 35.8 million people are enslaved around the world. Because of the traumatic experiences that victims of human trafficking encounter, the needs of victims are extensive and require the services of several providers, including health care providers, for victims to transform into survivors and thrivers. Currently, the role of occupational therapy is minimal and unexplored. The profession of occupational therapy has the capacity of having a profound role in both providing client-centered care services to victims and survivors of human trafficking and partaking in preventive advocacy efforts to combat human trafficking. Further advocacy efforts are required to promote the profession of occupational therapy in combating human trafficking. Copyright © 2016 by the American Occupational Therapy Association, Inc.

RABA Members Act in Distinct Steps of Subcellular Trafficking of the FLAGELLIN SENSING2 Receptor[W

Science.gov (United States)

Choi, Seung-won; Tamaki, Takayuki; Ebine, Kazuo; Uemura, Tomohiro; Ueda, Takashi; Nakano, Akihiko

2013-01-01

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo–synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endo- and exocytosis. PMID:23532067

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

Science.gov (United States)

Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H G; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-02

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

High Glucose Represses hERG K+ Channel Expression through Trafficking Inhibition

Directory of Open Access Journals (Sweden)

Yuan-Qi Shi

2015-08-01

Full Text Available Background/Aims: Abnormal QT prolongation is the most prominent cardiac electrical disturbance in patients with diabetes mellitus (DM. It is well known that the human ether-ago-go-related gene (hERG controls the rapid delayed rectifier K+ current (IKr in cardiac cells. The expression of the hERG channel is severely down-regulated in diabetic hearts, and this down-regulation is a critical contributor to the slowing of repolarization and QT prolongation. However, the intracellular mechanisms underlying the diabetes-induced hERG deficiency remain unknown. Methods: The expression of the hERG channel was assessed via western blot analysis, and the hERG current was detected with a patch-clamp technique. Results: The results of our study revealed that the expression of the hERG protein and the hERG current were substantially decreased in high-glucose-treated hERG-HEK cells. Moreover, we demonstrated that the high-glucose-mediated damage to the hERG channel depended on the down-regulation of protein levels but not the alteration of channel kinetics. These discoveries indicated that high glucose likely disrupted hERG channel trafficking. From the western blot and immunoprecipitation analyses, we found that high glucose induced trafficking inhibition through an effect on the expression of Hsp90 and its interaction with hERG. Furthermore, the high-glucose-induced inhibition of hERG channel trafficking could activate the unfolded protein response (UPR by up-regulating the expression levels of activating transcription factor-6 (ATF-6 and the ER chaperone protein calnexin. In addition, we demonstrated that 100 nM insulin up-regulated the expression of the hERG channel and rescued the hERG channel repression caused by high glucose. Conclusion: The results of our study provide the first evidence of a high-glucose-induced hERG channel deficiency resulting from the inhibition of channel trafficking. Furthermore, insulin promotes the expression of the hERG channel

Biopolitical management, economic calculation and "trafficked women".

Science.gov (United States)

Berman, Jacqueline

2010-01-01

Narratives surrounding human trafficking, especially trafficking in women for sex work, employ gendered and racialized tropes that have among their effects, a shrouding of women's economic decision-making and state collusion in benefiting from their labour. This paper explores the operation of these narratives in order to understand the ways in which they mask the economics of trafficking by sensationalizing the sexual and criminal aspects of it, which in turn allows the state to pursue political projects under the guise of a benevolent concern for trafficked women and/or protection of its own citizens. This paper will explore one national example: Article 18 of Italian Law 40 (1998). I argue that its passage has led to an increase in cooperation with criminal prosecution of traffickers largely because it approaches trafficked women as capable of making decisions about how and what they themselves want to do. This paper will also consider a more global approach to trafficking embedded in the concept of "migration management", an International Organization for Migration (IOM) framework that is now shaping EU, US and other national immigration laws and policies that impact trafficking. It will also examine the inherent limitations of both the national and global approach as an occasion to unpack how Article 18 and Migration Management function as forms of biopolitical management that participate in the production of "trafficking victims" into a massified population to be managed, rather than engender a more engaged discussion of what constitutes trafficking and how to redress it.

The International Society for Extracellular Vesicles launches the first massive open online course on extracellular vesicles

OpenAIRE

L?sser, Cecilia; Th?ry, Clotilde; Buz?s, Edit I.; Mathivanan, Suresh; Zhao, Weian; Gho, Yong Song; L?tvall, Jan

2016-01-01

The International Society for Extracellular Vesicles (ISEV) has organised its first educational online course for students and beginners in the field of extracellular vesicles (EVs). This course, “Basics of Extracellular Vesicles,” uses recorded lectures from experts in the field and will be open for an unlimited number of participants. The course is divided into 5 modules and can be accessed at www.coursera.org/learn/extracellular-vesicles. The first module is an introduction to the field co...

Barriers to combating human trafficking in Colombia

OpenAIRE

Wilcox, Daniel Joseph

2015-01-01

Approved for public release; distribution is unlimited Despite international and domestic policies and programs intended to combat human trafficking, Colombia remains one of the countries with the highest instances of human trafficking in the Western Hemisphere. Factors contributing to human trafficking in Colombia, such as internal violence and displacement, drug trafficking, a weak central government, and widespread corruption, have overpowered what energies the government marshaled agai...

Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

Science.gov (United States)

Carbone, Marilena; Sabbatella, Gianfranco; Antonaroli, Simonetta; Orlando, Viviana; Biagioni, Stefano; Nucara, Alessandro

2016-09-01

A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions.

Trafficking as a Human Rights Violation: Is South Africa's Curriculum Stuck in a Traffick Jam?

Science.gov (United States)

du Preez, Petro; Simmonds, Shan

2013-01-01

Human trafficking is a form of modern day slavery and is often collectively referred to as a human rights violation. However, human trafficking is more complex than this suggests as this article attempts to demonstrate. It begins by describing the landscape of international trends in human trafficking, with particular attention to child…

Targeting eukaryotic Rab proteins: a smart strategy for chlamydial survival and replication.

Science.gov (United States)

Damiani, María Teresa; Gambarte Tudela, Julián; Capmany, Anahí

2014-09-01

Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy. © 2014 John Wiley & Sons Ltd.

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

Science.gov (United States)

Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

2018-03-01

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

Seminal vesicle cycts

International Nuclear Information System (INIS)

Alpern, M.B.; Dorfman, R.E.; Gross, B.H.; Gottlieb, C.A.; Sandler, M.A.

1990-01-01

PURPOSE: Adult polycystic kidney disease (APKCD), an autosomal dominant disorder, causes cyst formation in the kidney, liver, pancreas, esophagus, ovaries, uterus, and brain. This paper describes four APKCD patients with CT evidence of seminal vesicle cysts (SVCs). Four patients (aged 45-65 years) underwent abdominal/pelvic CT with oral and intravenous contrast material. Three were evaluated for possible renal transplantation and one for sepsis material. All seminal vesicles contained cystic masses with fluid that measured between 0 and 30 HU. Seminal vesicle thickness was 3-4 cm (normal, 1.5 cm). High-density walls separated the 3-12-mm diameter cysts. All patients demonstrated typical renal stigmata of APKCD. One patient had hepatic cysts, and none had cysts elsewhere. Postmortem examination in one patient confirmed the SVCs

Women trafficking: causes, concerns, care!

Science.gov (United States)

Khowaja, Shaneela Sadaruddin; Tharani, Ambreen Jawed; Agha, Ajmal; Karamaliani, Rozina Sherali

2012-08-01

Pakistan is both a country of origin and destination as far as women trafficking is concerned. Poverty, gender discrimination, lack of education, and ignorance about legal rights are some of the underlying causes. Available data suggest several areas of concern, like, for instance: direct health effects, maladaptive coping leading to the use of illicit drugs, and inaccessibility to healthcare facilities. Therefore, numerous interventions would be required at three levels: the prevention of trafficking, the protection of victims and the prosecution of the traffickers.

Health implications of human trafficking.

Science.gov (United States)

Richards, Tiffany A

2014-01-01

Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population. © 2014 AWHONN.

Dual-layered nanogel-coated hollow lipid/polypeptide conjugate assemblies for potential pH-triggered intracellular drug release.

Directory of Open Access Journals (Sweden)

Wen-Hsuan Chiang

Full Text Available To achieve effective intracellular anticancer drug delivery, the polymeric vesicles supplemented with the pH-responsive outlayered gels as a delivery system of doxorubicin (DOX were developed from self-assembly of the lipid/polypeptide adduct, distearin grafted poly(γ-glutamic acid (poly(γ-GA, followed by sequential deposition of chitosan and poly(γ-GA-co-γ-glutamyl oxysuccinimide-g-monomethoxy poly(ethylene glycol in combination with in situ covalent cross-linking on assembly surfaces. The resultant gel-caged polymeric vesicles (GCPVs showed superior performance in regulating drug release in response to the external pH change. Under typical physiological conditions (pH 7.4 and 37 °C at which the γ-GA/DOX ionic pairings remained mostly undisturbed, the dense outlayered gels of GCPVs significantly reduced the premature leakage of the uncomplexed payload. With the environmental pH being reduced from pH 7.4 to 4.7, the drug liberation was appreciably promoted by the massive disruption of the ionic γ-GA/DOX complexes along with the significant swelling of nanogel layers upon the increased protonation of chitosan chain segments. After being internalized by HeLa cells via endocytosis, GCPVs exhibited cytotoxic effect comparable to free DOX achieved by rapidly releasing the payload in intracellular acidic endosomes and lysosomes. This strongly implies the great promise of such unique GCPVs as an intracellular drug delivery carrier for potential anticancer treatment.

Impermeability effects in three-dimensional vesicles

International Nuclear Information System (INIS)

Biscari, P; Canevese, S M; Napoli, G

2004-01-01

We analyse the effects of the impermeability constraint on the equilibrium shapes of a three-dimensional vesicle hosting a rigid inclusion. A given alteration of the inclusion and/or vesicle parameters leads to shape modifications of different orders of magnitude, when applied to permeable or impermeable vesicles. Moreover, the enclosed-volume constraint wrecks the uniqueness of stationary equilibrium shapes, and gives rise to pear-shaped or stomatocyte-like vesicles

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

Directory of Open Access Journals (Sweden)

Lojk J

2015-02-01

Full Text Available Jasna Lojk,1 Vladimir B Bregar,1 Maruša Rajh,1 Katarina Miš,2 Mateja Erdani Kreft,3 Sergej Pirkmajer,2 Peter Veranič,3 Mojca Pavlin1 1Group for Nano and Biotechnological Applications, Faculty of Electrical Engineering, 2Institute of Pathophysiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Abstract: Magnetic nanoparticles (NPs are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA in three cell types: Chinese Hamster Ovary (CHO, mouse melanoma (B16 cell line, and primary human myoblasts (MYO. We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better

MR imaging of the seminal vesicles

International Nuclear Information System (INIS)

Edson, S.B.; Hricak, H.; Chun-Fang Chang, Y.

1987-01-01

The seminal vesicles of 56 healthy males and 23 males with pathologic conditions were studied with a .35-T magnet and spin-echo (SE) techniques (repetition time/echo time [msec] = 500/30 and 2,000/60). The authors analyzed (1) the size and relative signal intensity of seminal vesicles compared to surrounding fat, muscle, or urine; (2) the effect of aging on the size and signal intensity of the vesicles, and (3) the appearance of the seminal vesicles in different pathologic conditions. In the transverse plane, the normal seminal vesicle measures 31 +- 7 mm in length and 17 +- 4 mm in width. Its size or signal intensity did not change significantly with age. On SE = 500/30 images the seminal vesicles were isointense with muscle; on SE = 2,000/60 images they were isointense or slightly hypointense relative to fat. MR imaging was highly sensitive for displaying seminal vesicle pathology, based on asymmetry in size and changes in signal intensities. MR imaging provides unique information but its role in pathologic conditions needs to be further explored

Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

Energy Technology Data Exchange (ETDEWEB)

Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Wang, Zai [Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Li, Jian; Cai, Jian-Ping [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [School of Biomedical Sciences and Shenzhen Institute of Research and Innovation, The University of Hong Kong, Pokfulam (Hong Kong); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China); Zhang, Tie-Mei, E-mail: tmzhang126@126.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China)

2016-08-05

Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

International Nuclear Information System (INIS)

Cui, Ju; Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan; Wang, Zai; Li, Jian; Cai, Jian-Ping; Huang, Jian-Dong; Zhang, Tie-Mei

2016-01-01

Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

Directory of Open Access Journals (Sweden)

Claudia Orelio

Full Text Available Shwachman-Diamond Syndrome (SDS is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

Human liver cell trafficking mutants: characterization and whole exome sequencing.

Directory of Open Access Journals (Sweden)

Fei Yuan

Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

Spontaneous charged lipid transfer between lipid vesicles.

Science.gov (United States)

Richens, Joanna L; Tyler, Arwen I I; Barriga, Hanna M G; Bramble, Jonathan P; Law, Robert V; Brooks, Nicholas J; Seddon, John M; Ces, Oscar; O'Shea, Paul

2017-10-03

An assay to study the spontaneous charged lipid transfer between lipid vesicles is described. A donor/acceptor vesicle system is employed, where neutrally charged acceptor vesicles are fluorescently labelled with the electrostatic membrane probe Fluoresceinphosphatidylethanolamine (FPE). Upon addition of charged donor vesicles, transfer of negatively charged lipid occurs, resulting in a fluorescently detectable change in the membrane potential of the acceptor vesicles. Using this approach we have studied the transfer properties of a range of lipids, varying both the headgroup and the chain length. At the low vesicle concentrations chosen, the transfer follows a first-order process where lipid monomers are transferred presumably through the aqueous solution phase from donor to acceptor vesicle. The rate of transfer decreases with increasing chain length which is consistent with energy models previously reported for lipid monomer vesicle interactions. Our assay improves on existing methods allowing the study of a range of unmodified lipids, continuous monitoring of transfer and simplified experimental procedures.

Trafficking of α-L-fucosidase in lymphoid cells

International Nuclear Information System (INIS)

DiCioccio, R.A.; Brown, K.S.

1987-01-01

The quantity of α-L-fucosidase in human serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. To investigate this, lymphoid cell lines derived from individuals with either low, intermediate or high α-L-fucosidase in serum were established. Steady state levels of extracellular α-L-fucosidase protein and activity overlapped among the cell lines. Thus, in vivo serum phenotypes of α-L-fucosidase are not adequately expressed in this system. α-L-Fucosidase was also metabolically labelled with 35 S-methionine, immunoprecipitated, and examined by SDS-PAGE. Cells pulse-labelled from 0.25-2 h had a major intracellular form of enzyme (Mr = 58,000). Cells pulsed for 1.5 h and chased for 21 h with unlabeled methionine had an intracellular form of Mr = 60,000 and an extracellular form of Mr = 62,000. Cells treated with chloroquine had only the 58,000-form both intra- and extra-cellularly. Moreover, chloroquine did not effect the quantitative distribution of α-L-fucosidase between cells and medium. In fibroblasts, chloroquine enhanced the secretion of newly made lysosomal enzymes and blocked the processing of intercellular enzyme forms from a higher to a lower molecular mass. Thus, there are trafficking differences between α-L-fucosidase in lymphoid cells and lysosomal enzymes in fibroblasts. This suggests that alternative targeting mechanisms for lysosomal enzymes exist in these cells

STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma membrane, ER, and ERC

DEFF Research Database (Denmark)

Garbarino, J.; Pan, M. H.; Chin, H. F.

2012-01-01

small hairpin RNA knockdown technology to reduce STARD4 expression in HepG2 cells. In a cholesterol-poor environment, we found that a reduction in STARD4 expression leads to retention of cholesterol at the plasma membrane, reduction of endoplasmic reticulum-associated cholesterol, and decreased ACAT...... synthesized cholesteryl esters. Furthermore, D4 KD cells exhibited a reduced rate of sterol transport to the endocytic recycling compartment after cholesterol repletion. Although these cells displayed normal endocytic trafficking in cholesterol-poor and replete conditions, cell surface low density lipoprotein...... membrane and the endocytic recycling compartment to the endoplasmic reticulum and perhaps other intracellular compartments as well. -Garbarino, J., M. Pan, H.F. Chin, F.W. Lund, F.R. Maxfield, and J.L. Breslow. STARD4 knockdown in HepG2 cells disrupts cholesterol trafficking associated with the plasma...

Perdagangan Orang (Trafficking) sebagai Pelanggaran Hak Asasi Manusia

OpenAIRE

Munthe, Riswan

2015-01-01

Human trafficking is garbage of civilization which is hard to be fought. This sentence provide an invasion for all that human trafficking is a common enemy. Human trafficking is often done by agent who has national even international network, has power, strong physically and arrogance. Due to the victim of human trafficking is the group in the lower class of economy and education. Generally the victim of human trafficking is everyone without exception. Since Indonesian independence, it is con...

Child organ trafficking: global reality and inadequate international response.

Science.gov (United States)

Bagheri, Alireza

2016-06-01

In organ transplantation, the demand for human organs has grown far faster than the supply of organs. This has opened the door for illegal organ trade and trafficking including from children. Organized crime groups and individual organ brokers exploit the situation and, as a result, black markets are becoming more numerous and organized organ trafficking is expanding worldwide. While underprivileged and vulnerable men and women in developing countries are a major source of trafficked organs, and may themselves be trafficked for the purpose of illegal organ removal and trade, children are at especial risk of exploitation. With the confirmed cases of children being trafficked for their organs, child organ trafficking, which once called a "modern urban legend", is a sad reality in today's world. By presenting a global picture of child organ trafficking, this paper emphasizes that child organ trafficking is no longer a myth but a reality which has to be addressed. It argues that the international efforts against organ trafficking and trafficking in human beings for organ removal have failed to address child organ trafficking adequately. This chapter suggests that more orchestrated international collaboration as well as development of preventive measure and legally binding documents are needed to fight child organ trafficking and to support its victims.

Was Trafficking in Persons Really Criminalised?

Directory of Open Access Journals (Sweden)

Kristiina Kangaspunta

2015-04-01

Full Text Available This paper examines the successes and setbacks in the criminal justice response to trafficking in persons. While today, the majority of countries have passed specific legislation criminalising human trafficking in response to the United Nations Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, there are still very few convictions of trafficking. Using currently available knowledge, this paper discusses four possible reasons for low conviction rates. Further, the paper suggests that due to the heavy dependency on victim testimonies when prosecuting trafficking in persons crimes, members of criminal organisations that are easily identifiable by victims may face criminal charges more frequently than other members of the criminal group, particularly those in positions of greater responsibility who profit the most from the criminal activities. In this context, the exceptionally high number of women among convicted offenders is explored.

Ultrasound-responsive ultrathin multiblock copolyamide vesicles

Science.gov (United States)

Huang, Lei; Yu, Chunyang; Huang, Tong; Xu, Shuting; Bai, Yongping; Zhou, Yongfeng

2016-02-01

This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation.This study reports the self-assembly of novel polymer vesicles from an amphiphilic multiblock copolyamide, and the vesicles show a special structure with an ultrathin wall thickness of about 4.5 nm and a combined bilayer and monolayer packing model. Most interestingly, the vesicles are ultrasound-responsive and can release the encapsulated model drugs in response to ultrasonic irradiation. Electronic supplementary information (ESI) available: Details of experiments and characterization, and FT-IR, TEM, DPD, FL and micro-DSC results. See DOI: 10.1039/c5nr08596a

Gun Trafficking and the Southwest Border

Science.gov (United States)

2009-07-29

felons, drug traffickers, and juvenile gang members from acquiring firearms from gun traffickers. These criminals often acquire firearms from persons...renounced their U.S. citizenship; (8) persons restrained under a court-order from harassing, stalking , or threatening an intimate partner or child of... gang members from acquiring firearms from gun traffickers. These criminals often acquire firearms from a person who otherwise is not prohibited to

Responsive Polydiacetylene Vesicles for Biosensing Microorganisms

Directory of Open Access Journals (Sweden)

Estelle Lebègue

2018-02-01

Full Text Available Polydiacetylene (PDA inserted in films or in vesicles has received increasing attention due to its property to undergo a blue-to-red colorimetric transition along with a change from non-fluorescent to fluorescent upon application of various stimuli. In this review paper, the principle for the detection of various microorganisms (bacteria, directly detected or detected through the emitted toxins or through their DNA, and viruses and of antibacterial and antiviral peptides based on these responsive PDA vesicles are detailed. The analytical performances obtained, when vesicles are in suspension or immobilized, are given and compared to those of the responsive vesicles mainly based on the vesicle encapsulation method. Many future challenges are then discussed.

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

Directory of Open Access Journals (Sweden)

Julien Burlaud-Gaillard

Full Text Available The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy. CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Acyl-CoA binding proteins; structural and functional conservation over 2000 MYA

DEFF Research Database (Denmark)

Faergeman, Nils J; Wadum, Majken; Feddersen, Søren

2007-01-01

-CoA binding protein, ACBP, has been proposed to play a pivotal role in the intracellular trafficking and utilization of long-chain fatty acyl-CoA esters. Depletion of acyl-CoA binding protein in yeast results in aberrant organelle morphology incl. fragmented vacuoles, multi-layered plasma membranes...... and accumulation of vesicles of variable sizes. In contrast to synthesis and turn-over of glycerolipids, the levels of very-long-chain fatty acids, long-chain bases and ceramide are severely affected by Acb1p depletion, suggesting that Acb1p, rather than playing a general role, serves specific roles in cellular...

Assisting victims of human trafficking: strategies to facilitate identification, exit from trafficking, and the restoration of wellness.

Science.gov (United States)

Hodge, David R

2014-04-01

Human trafficking is a pressing social justice concern. Social work is uniquely situated to address this problem. However, despite the profession's commitment to social justice, the scholarship to equip social workers to address this issue has been largely absent from professional discourse. To address this gap, this article helps social work practitioners to assist victims of human trafficking. After orienting readers to the scope and process of human trafficking, the topics of victim identification, exit from trafficking, and the restoration of psychological wellness are discussed. By equipping themselves in these three areas, practitioners can advance social justice on behalf of some of the most exploited people in the world.

Trafficking in Persons for Ransom and the Need to Expand the Interpretation of Article 3 of the UN Trafficking Protocol

Directory of Open Access Journals (Sweden)

Mogos O Brhane

2015-04-01

Full Text Available As the nature of trafficking in persons continues to manifest itself in myriad ways all over the world, interpretation of the UN Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children (Trafficking Protocol, should be broadened to include newly emerging practices that are similar in nature to those it has already embraced under its definition. The Protocol appears to encompass other forms of trafficking which are unnamed or unforeseen by the definition provided under Article 3. It is time to expand its spectrum. Northeast Africa is plagued by a unique form of trafficking in persons—trafficking in persons for ransom. This involves a practice where people are smuggled, abducted, kidnapped and tortured to compel their relatives and families to pay ransom money. Victims are nationals of Eritrea, Ethiopia, Sudan and South Sudan. However, as Northeast Africa hosts particularly high numbers of Eritrean migrants and the largest Eritrean diaspora globally, Eritreans are very vulnerable to being targeted for trafficking for ransom. As trafficking for ransom is an emerging trend, legal ramifications have never been studied in full. Few reports try to address legal issues around the phenomenon, and those that do only give it a few paragraphs of attention. There is need for a closer look at this form of trafficking.

The political aspects of human trafficking

Directory of Open Access Journals (Sweden)

N. M. Lukach

2014-01-01

The negative international results of human trafficking are researched by the author. Namely, problems of governance organization that are created by powerful criminal groups of human traffickers and smugglers, mass stay of a significant number of non­citizens in the country; formation of the negative international image of the origin, destination or transit country as the state which is unable to counter effectively illegal migration and human trafficking.

Human Trafficking in the Emergency Department

OpenAIRE

Patel, Ronak B; Ahn, Roy; Burke, Thomas F

2010-01-01

Human trafficking continues to persist, affecting up to 200 million people worldwide. As clinicians in emergency departments commonly encounter victims of intimate partner violence, some of these encounters will be with trafficking victims. These encounters provide a rare opportunity for healthcare providers to intervene and help. This case report of a human trafficking patient from a teaching hospital illustrates the complexity in identifying these victims. Clinicians can better identify pot...

Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation

Energy Technology Data Exchange (ETDEWEB)

Acar, Handan [Institute; Department; Samaeekia, Ravand [Institute; Department; Schnorenberg, Mathew R. [Institute; Department; Medical; Sasmal, Dibyendu K. [Institute; Huang, Jun [Institute; Tirrell, Matthew V. [Institute; Institute; LaBelle, James L. [Department

2017-08-24

Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a resonance energy transfer (FRET)-based tracking system. Using this platform, components in real time using a Forster we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

Transcriptome of extracellular vesicles released by hepatocytes.

Directory of Open Access Journals (Sweden)

Felix Royo

Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

Depletion of intracellular zinc from neurons by use of an extracellular chelator in vivo and in vitro.

Science.gov (United States)

Frederickson, Christopher J; Suh, Sang W; Koh, Jae-Young; Cha, Yoo K; Thompson, Richard B; LaBuda, Christopher J; Balaji, Rengarajan V; Cuajungco, Math P

2002-12-01

The membrane-impermeable chelator CaEDTA was introduced extracellularly among neurons in vivo and in vitro for the purpose of chelating extracellular Zn(2+). Unexpectedly, this treatment caused histochemically reactive Zn(2+) in intracellular compartments to drop rapidly. The same general result was seen with intravesicular Zn(2+), which fell after CaEDTA infusion into the lateral ventricle of the brain, with perikaryal Zn(2+) in Purkinje neurons (in vivo) and with cortical neurons (in vitro). These findings suggest either that the volume of zinc ion efflux and reuptake is higher than previously suspected or that EDTA can enter cells and vesicles. Caution is therefore warranted in attempting to manipulate extracellular or intracellular Zn(2+) selectively.

Spontaneous Vesicle Recycling in the Synaptic Bouton

Directory of Open Access Journals (Sweden)

Sven eTruckenbrodt

2014-12-01

Full Text Available The trigger for synaptic vesicle exocytosis is Ca2+, which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca2+ levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca2+ sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca2+. The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs responding to Ca2+ fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles

International Nuclear Information System (INIS)

Brown, R.E.; Sugar, I.P.; Thompson, T.E.

1985-01-01

The transfer kinetics of the neutral glycosphingolipid gangliotetraosylceramide (asialo-GM1) were investigated by monitoring tritiated asialo-GM1 movement from donor to acceptor vesicles. Two different methods were employed to separate donor and acceptor vesicles at desired time intervals. In one method, a negative charge was imparted to dipalmitoylphosphatidylcholine donor vesicles by including 10 mol% dipalmitoylphosphatidic acid. Donors were separated from neutral dipalmitoylphosphatidylcholine acceptor vesicles by ion-exchange chromatography. In the other method, small, unilamellar donor vesicles and large, unilamellar acceptor vesicles were coincubated at 45 degrees C and then separated at desired time intervals by molecular sieve chromatography. The majority of asialo-GM1 transfer to acceptor vesicles occurred as a slow first-order process with a half-time of about 24 days assuming that the relative concentration of asialo-GM1 in the phospholipid matrix was identical in each half of the donor bilayer and that no glycolipid flip-flop occurred. Asialo-GM1 net transfer was calculated relative to that of [ 14 C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. A nearly identical transfer half-time was obtained when the phospholipid matrix was changed from dipalmitoylphosphatidylcholine to palmitoyloleoylphosphatidylcholine. Varying the acceptor vesicle concentration did not significantly alter the asialo-GM1 transfer half-time. This result is consistent with a transfer mechanism involving diffusion of glycolipid through the aqueous phase rather than movement of glycolipid following formation of collisional complexes between donor and acceptor vesicles. (Abstract Truncated)

Immunomodulatory role for membrane vesicles released by THP-1 macrophages and respiratory pathogens during macrophage infection.

Science.gov (United States)

Volgers, Charlotte; Benedikter, Birke J; Grauls, Gert E; Savelkoul, Paul H M; Stassen, Frank R M

2017-11-13

During infection, inflammation is partially driven by the release of mediators which facilitate intercellular communication. Amongst these mediators are small membrane vesicles (MVs) that can be released by both host cells and Gram-negative and -positive bacteria. Bacterial membrane vesicles are known to exert immuno-modulatory and -stimulatory actions. Moreover, it has been proposed that host cell-derived vesicles, released during infection, also have immunostimulatory properties. In this study, we assessed the release and activity of host cell-derived and bacterial MVs during the first hours following infection of THP-1 macrophages with the common respiratory pathogens non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa. Using a combination of flow cytometry, tunable resistive pulse sensing (TRPS)-based analysis and electron microscopy, we demonstrated that the release of MVs occurs by both host cells and bacteria during infection. MVs released during infection and bacterial culture were found to induce a strong pro-inflammatory response by naive THP-1 macrophages. Yet, these MVs were also found to induce tolerance of host cells to secondary immunogenic stimuli and to enhance bacterial adherence and the number of intracellular bacteria. Bacterial MVs may play a dual role during infection, as they can both trigger and dampen immune responses thereby contributing to immune defence and bacterial survival.

Intracellular proton conductance of the hepatitis C virus p7 protein and its contribution to infectious virus production.

Directory of Open Access Journals (Sweden)

Ann L Wozniak

2010-09-01

Full Text Available The hepatitis C virus (HCV p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H(+ conductance in vesicles and was able to rapidly equilibrate H(+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5 vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H(+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H(+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection

14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

Directory of Open Access Journals (Sweden)

Michael P Grant

Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

Granule protein processing and regulated secretion in neutrophils

Directory of Open Access Journals (Sweden)

Avinash eSheshechalam

2014-09-01

Full Text Available Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

Trafficking in Persons: The U.S. and International Response

Science.gov (United States)

2006-07-07

lucrative business of international trafficking; ! the high demand, worldwide, for trafficked women and children as sex workers, cheap sweatshop labor , and...raid brothels, women are often detained and punished, subjected to human rights abuses in jail, and swiftly deported. Few steps have been taken to...of trafficking. Many countries have no specific laws aimed at trafficking in humans . Traffickers and Their Victims Chinese, Asian, Mexican, Central

Human Trafficking and Commercialization of Surrogacy in India

Directory of Open Access Journals (Sweden)

Pyali Chatterjee

2014-10-01

Full Text Available The Supreme Court of India, In Baby Manji Yamada versus Union of India & Anr. [2008] INSC 1656, popularly known as Manji Case, declared that Commercial Surrogacy is legal in India. As we know that, India is a developing country and here, most of the peoples are very poor and illiterate. Recently, human trafficking was increase with an uncontrollable rate in the entire world. In addition, making Commercialization of Surrogacy legal had already give birth to a new form of trafficking. Where, illiterate women from poor section is trafficked to run the reproductive industry of the Surrogacy. As we know that the traffickers, they used to trafficked girls/women for prostitution but now after the legalization of Commercial Surrogacy, they will trafficked girl/women for the reproductive industry as a raw material. The Immoral Trafficking Prevention Act (ITPA, 1956 and Sections 366(A and 372 of the Indian Penal Code, 1860 are the existing laws of India, which deals with human trafficking. However, none of these provisions contains any solution, to deal with this new serious issue of trafficking of women/girls for the purpose of Commercial Surrogacy in reproductive industries. These existing laws as well as the pending draft bill of Assisted Reproductive Technologies (ART Regulation Bill, 2010 needs an amendment to check this crime against women once again to protect the rights and health of the women.

Detection and Physicochemical Characterization of Membrane Vesicles (MVs of Lactobacillus reuteri DSM 17938

Directory of Open Access Journals (Sweden)

Rossella Grande

2017-06-01

Full Text Available Membrane vesicles (MVs are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs, and planktonic (pMVs phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM and Dynamic Light Scattering (DLS. Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM analysis. eDNA (extracellular DNA was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA. DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems.

Rab11-family of interacting protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication.

Science.gov (United States)

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

2013-01-01

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion. © 2012 Blackwell Publishing Ltd.

Human trafficking law and social structures.

Science.gov (United States)

Wooditch, Alese

2012-08-01

Human trafficking has only recently emerged at the forefront of policy reform, even in developed nations. Yet, heightened awareness of the issue has not translated into effective policy as the majority of nations have ineffective antitrafficking practices; many countries have failed to criminalize human trafficking, whereas others do not actively enforce statutes in place. By applying Black's theory of law, this study offers a preliminary understanding into the variation of global prosecutorial efforts in human trafficking and adequacy of antitrafficking law. To isolate this relationship, the effects of trafficking markets are controlled. As with prior research, the study finds limited support for the theory. The article concludes with a discussion on the implications of the quantity of antitrafficking law and morphology association for policy development.

Organized crime-trafficking with human being

OpenAIRE

Jelenová, Jana

2011-01-01

Organized crime - Trafficking in human beings This thesis deals with the criminal offence of trafficking in human beings under Sec. 168 of the Czech Criminal Code. A trafficking in human being is not a frequent criminal offence but with its consequences belongs to the most dangerous crimes. After the Velvet revolution the relevance of this crime has raised subsequently and therefore the regulation of this crime requires particular attention. It is important to find new ways and improve curren...

Chloroform induces outstanding crystallization of poly(hydroxybutyrate) (PHB) vesicles within bacteria.

Science.gov (United States)

Rebois, Rolando; Onidas, Delphine; Marcott, Curtis; Noda, Isao; Dazzi, Alexandre

2017-03-01

Poly[(R)-3-hydroxyalkanoate]s or PHAs are aliphatic polyesters produced by numerous microorganisms. They are accumulated as energy and carbon reserve in the form of small intracellular vesicles. Poly[(R)-3-hydroxybutyrate] (PHB) is the most ubiquitous and simplest PHA. An atomic force microscope coupled with a tunable infrared laser (AFM-IR) was used to record highly spatially resolved infrared spectra of commercial purified PHB and native PHB within bacteria. For the first time, the crystallinity degree of native PHB within vesicle has been directly evaluated in situ without alteration due to the measure or extraction and purification steps of the polymer: native PHB is in crystalline state at 15% whereas crystallinity degree reaches 57% in commercial PHB. Chloroform addition on native PHB induces crystallization of the polymer within bacteria up to 60%. This possibility of probing and changing the physical state of polymer in situ could open alternative ways of production for PHB and others biopolymers. Graphical abstract An atomic force microscope coupled with a tunable infrared laser (AFM-IR) has been used to record local infrared spectra of biopolymer PHB within bacteria. Deconvolution of those spectra has allowed to determine in situ the crystallinity degree of native PHB.

a Study of Akachi Adimora-Ezeigbo's Trafficked

African Journals Online (AJOL)

Trafficking is frowned at in Nigeria, yet people are perpetually trafficked. In this research work, the researcher examines the novel in line with sociological approach so that the ills of human trafficking as it is a case in the contemporary society would be seen. The researcher believes that when the ills are exposed, there ...

Female sex trafficking: conceptual issues, current debates, and future directions.

Science.gov (United States)

Meshkovska, Biljana; Siegel, Melissa; Stutterheim, Sarah E; Bos, Arjan E R

2015-01-01

Female sex trafficking is a pressing concern. In this article, we provide a comprehensive overview of relevant issues regarding the concept of female sex trafficking and research in the field of human trafficking, drawing on a variety of disciplines, including economics, gender and sexuality studies, psychology, sociology, law, and social work. We discuss the debates surrounding the definition of human trafficking, compare and contrast it with human smuggling, and outline connections between female sex trafficking and the issue of sex work and prostitution. We further discuss the history and current estimations of female sex trafficking. We then outline the main actors in female sex trafficking, including trafficked persons, traffickers, clients, and service providers, and we overview the trafficking process from recruitment to identification, recovery, and (re)integration. Finally, we conclude with recommendations for future research that tie together the concepts of vulnerability, exploitation, and long-term recovery and (re)integration.

Amyloglucosidase enzymatic reactivity inside lipid vesicles

Directory of Open Access Journals (Sweden)

Kim Jin-Woo

2007-10-01

Full Text Available Abstract Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG (EC 3.2.1.3 from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC multilamellar vesicles (MLVs and large unilamellar vesicles (LUVs was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.

Expression and Trafficking of the γ Subunit of Na,K-ATPase in Hypertonically Challenged IMCD3 Cells

International Nuclear Information System (INIS)

Pihakaski-Maunsbach, Kaarina; Nonaka, Shoichi; Maunsbach, Arvid B.

2008-01-01

The γ subunit (FXYD2) of Na,K-ATPase is an important regulator of the sodium pump. In this investigation we have analysed the trafficking of γ to the plasma membrane in cultures of inner medullary collecting duct cells (IMCD3) following acute hypertonic challenge and brefeldin A (BFA) treatment. Following hypertonic challenging for 24 hr immunofluorescence labeling revealed initial co-localization of the γ subunit and 58K Golgi protein in the cytoplasm, but no co-localization of α1 and Golgi protein. Exposure of the challenged cells to BFA prevented the subsequent incorporation of γ into the basolateral plasma membrane. The γ subunit instead remained in cytoplasmic vesicles while cell proliferation and cell viability decreased simultaneously. Following removal of BFA from the hypertonic medium the IMCD3 cells recovered with distinct expression of γ in the basolateral membrane. The α1 subunit was only marginally influenced by BFA. The results demonstrate that the γ subunit trafficks to the plasma membrane via the Golgi apparatus, despite the absence of a signal sequence. The results also suggest that the γ and α subunits do not traffic together to the plasma membrane, and that the γ and α subunit have different turnover rates during these experimental conditions

Normative framework for combating trafficking in human organs

Directory of Open Access Journals (Sweden)

Banović Božidar

2017-01-01

Full Text Available Trafficking in human organs is a specific and complex criminal phenomenon that takes place in several phases, with the participation of a large number of actors, often covering several jurisdictions. To effectively countering are necessary conceptual and terminological demarcation of organ trafficking from other similar phenomena, fundamental scientifically research of its shape and dynamics, and the construction of an adequate legislative framework, both at the national and international level. This paper first analyses the conceptual - terminological framework in which research of organ trafficking is range, and then presents a review and analysis of the development of ethical and normative international standards aimed at prevention and suppression this phenomenon. In the end, we analysed the current solutions in the domestic legislation. Nowadays, there are two parallel legislative regime that regulate the matter of organ trafficking. The first legislative regime that organ trafficking treated exclusively as a modality of transnational crime of human trafficking, and a second, recent, that organ trafficking treats as forbidden (incriminating activity that violates a regulatory system of organ (tissue cells transplantation.

Trafficking in Persons Report

Science.gov (United States)

2009-06-01

people Burmese Lu kon ku de Trade in people French La traite des personnes The trade of people Japanese Jinshin bai bai The buying and selling of...commercial sex among young men. In 2008, the government partially funded an NGO to conduct an anti-trafficking awareness campaign in cinemas and in...A significant number of Japanese women and girls have also been reported as sex trafficking victims. During the last year, a number of Paraguayan

Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy

Directory of Open Access Journals (Sweden)

M. Malatesta

2012-04-01

Full Text Available Chitosan-based nanoparticles (NPs deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus.

The hotel industry's role in combatting sex trafficking

OpenAIRE

Winters, Brandon R.

2017-01-01

Approved for public release; distribution is unlimited Human trafficking is a global concern that victimizes countless individuals worldwide. The hotel industry, which traffickers often exploit, is in a unique position to assist in the prevention of sex trafficking; therefore, it plays a vital role in the overall fight against human trafficking. This thesis applies policy analysis and exploratory research to understand how and to what degree the U.S.-based hotel industry can affect efforts...

Human trafficking in Germany: strengthening victim's human rights

OpenAIRE

Follmar-Otto, Petra; Rabe, Heike

2009-01-01

The first study - "A human rights approach against human trafficking - International obligations and the status of implementation in Germany" - analyses how the prohibition of human trafficking and the resulting state obligations are anchored in human rights. The more recent specialised international agreements on human trafficking and law-making in the European Union are then presented. The emphasis is on the Council of Europe Convention, which professes to treat human trafficking in a human...

Human trafficking and exploitation: A global health concern.

Science.gov (United States)

Zimmerman, Cathy; Kiss, Ligia

2017-11-01

In this collection review, Cathy Zimmerman and colleague introduce the PLOS Medicine Collection on Human Trafficking, Exploitation and Health, laying out the magnitude of the global trafficking problem and offering a public health policy framework to guide responses to trafficking.

The cellular response to vascular endothelial growth factors requires co-ordinated signal transduction, trafficking and proteolysis.

Science.gov (United States)

Smith, Gina A; Fearnley, Gareth W; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

2015-08-18

VEGFs (vascular endothelial growth factors) are a family of conserved disulfide-linked soluble secretory glycoproteins found in higher eukaryotes. VEGFs mediate a wide range of responses in different tissues including metabolic homoeostasis, cell proliferation, migration and tubulogenesis. Such responses are initiated by VEGF binding to soluble and membrane-bound VEGFRs (VEGF receptor tyrosine kinases) and co-receptors. VEGF and receptor splice isoform diversity further enhances complexity of membrane protein assembly and function in signal transduction pathways that control multiple cellular responses. Different signal transduction pathways are simultaneously activated by VEGFR-VEGF complexes with membrane trafficking along the endosome-lysosome network further modulating signal output from multiple enzymatic events associated with such pathways. Balancing VEGFR-VEGF signal transduction with trafficking and proteolysis is essential in controlling the intensity and duration of different intracellular signalling events. Dysfunction in VEGF-regulated signal transduction is important in chronic disease states including cancer, atherosclerosis and blindness. This family of growth factors and receptors is an important model system for understanding human disease pathology and developing new therapeutics for treating such ailments. © 2015 Authors.

Report on combating of illicit trafficking. The Illicit Trafficking Combat Project Group. January 2000

Energy Technology Data Exchange (ETDEWEB)

NONE

2000-01-01

The objective of this study is to propose improvements of different means and methods for the prevention of illicit trafficking and the proliferation of nuclear weapons. An essential requirement imposed on authorities and operators through basic nuclear legislation is the establishment and implementation of: a) nuclear material accountancy and control; b) physical protection of nuclear material and facilities; and c) export/import control of nuclear material and, so called, dual-use items. The legislation would make it possible for the State nuclear authority to be informed continuously about the location and quantity of nuclear material in the country, and to monitor that it is under satisfactory protection and guard in accordance with the requirements. The introduction of requirements on licensing of practices and activities involving dual-use items, and other products used for nuclear weapons manufacturing, would also give the authority the possibility to prevent unauthorised transfers of such products. The nuclear legislation must clearly stipulate the distribution of responsibilities between nuclear authority, operator and employees. This would give a solid basis for attaining high safety culture, including positive motivation and active commitment among executives and other employees. It would also ensure that nuclear material would not be lost through negligence or internal criminal activities. A further step of improvements on the national level, would be the urgent and firm introduction of modern Quality Assurance and Internal Control Systems into all forms of nuclear activities. In modern, safety related quality control systems, the operator alone is responsible for all activities and operations, and he must establish an internal control that will ensure that the authority's rules and requirements are thoroughly implemented and adhered to. The operator's organisation, including competence and procedures, must be approved by the State authority. By means

Report on combating of illicit trafficking. The Illicit Trafficking Combat Project Group. January 2000

Energy Technology Data Exchange (ETDEWEB)

NONE

2000-01-01

The objective of this study is to propose improvements of different means and methods for the prevention of illicit trafficking and the proliferation of nuclear weapons. An essential requirement imposed on authorities and operators through basic nuclear legislation is the establishment and implementation of: a) nuclear material accountancy and control; b) physical protection of nuclear material and facilities; and c) export/import control of nuclear material and, so called, dual-use items. The legislation would make it possible for the State nuclear authority to be informed continuously about the location and quantity of nuclear material in the country, and to monitor that it is under satisfactory protection and guard in accordance with the requirements. The introduction of requirements on licensing of practices and activities involving dual-use items, and other products used for nuclear weapons manufacturing, would also give the authority the possibility to prevent unauthorised transfers of such products. The nuclear legislation must clearly stipulate the distribution of responsibilities between nuclear authority, operator and employees. This would give a solid basis for attaining high safety culture, including positive motivation and active commitment among executives and other employees. It would also ensure that nuclear material would not be lost through negligence or internal criminal activities. A further step of improvements on the national level, would be the urgent and firm introduction of modern Quality Assurance and Internal Control Systems into all forms of nuclear activities. In modern, safety related quality control systems, the operator alone is responsible for all activities and operations, and he must establish an internal control that will ensure that the authority's rules and requirements are thoroughly implemented and adhered to. The operator's organisation, including competence and procedures, must be approved by the State authority

Activation of Host IRE1α-Dependent Signaling Axis Contributes the Intracellular Parasitism of Brucella melitensis

Directory of Open Access Journals (Sweden)

Aseem Pandey

2018-04-01

Full Text Available Brucella spp. are intracellular vacuolar pathogens that causes brucellosis, a worldwide zoonosis of profound importance. We previously demonstrated that the activity of host unfolded protein response (UPR sensor IRE1α (inositol-requiring enzyme 1 and ER-associated autophagy confer susceptibility to Brucella melitensis and Brucella abortus intracellular replication. However, the mechanism by which host IRE1α regulates the pathogen intracellular lifestyle remains elusive. In this study, by employing a diverse array of molecular approaches, including biochemical analyses, fluorescence microscopy imaging, and infection assays using primary cells derived from Ern1 (encoding IRE1 conditional knockout mice, we address this gap in our understanding by demonstrating that a novel IRE1α to ULK1, an important component for autophagy initiation, signaling axis confers susceptibility to Brucella intracellular parasitism. Importantly, deletion or inactivation of key signaling components along this axis, including IRE1α, BAK/BAX, ASK1, and JNK as well as components of the host autophagy system ULK1, Atg9a, and Beclin 1, resulted in striking disruption of Brucella intracellular trafficking and replication. Host kinases in the IRE1α-ULK1 axis, including IRE1α, ASK1, JNK1, and/or AMPKα as well as ULK1, were also coordinately phosphorylated in an IRE1α-dependent fashion upon the pathogen infection. Taken together, our findings demonstrate that the IRE1α-ULK1 signaling axis is subverted by the bacterium to promote intracellular parasitism, and provide new insight into our understanding of the molecular mechanisms of intracellular lifestyle of Brucella.

Human trafficking and exploitation: A global health concern.

Directory of Open Access Journals (Sweden)

Cathy Zimmerman

2017-11-01

Full Text Available In this collection review, Cathy Zimmerman and colleague introduce the PLOS Medicine Collection on Human Trafficking, Exploitation and Health, laying out the magnitude of the global trafficking problem and offering a public health policy framework to guide responses to trafficking.

The concept of exploitation in international human trafficking law

OpenAIRE

von der Pütten, Tuija Kaarina

2017-01-01

Human trafficking is commonly known as a criminal practice that takes place in the framework of sex trade: women and children are trafficked within a state, or from one state to another, for the purpose of sexual exploitation. Similarly, the early 20th century international conventions aimed to tackle ‘white slave traffic’, trafficking of women and children for sexual exploitation. However, it is misleading to see trafficking only within this context. People are trafficked so that they can be...

Report on combating of illicit trafficking. The Illicit Trafficking Combat Project Group. January 2000

International Nuclear Information System (INIS)

2000-01-01

The objective of this study is to propose improvements of different means and methods for the prevention of illicit trafficking and the proliferation of nuclear weapons. An essential requirement imposed on authorities and operators through basic nuclear legislation is the establishment and implementation of: a) nuclear material accountancy and control; b) physical protection of nuclear material and facilities; and c) export/import control of nuclear material and, so called, dual-use items. The legislation would make it possible for the State nuclear authority to be informed continuously about the location and quantity of nuclear material in the country, and to monitor that it is under satisfactory protection and guard in accordance with the requirements. The introduction of requirements on licensing of practices and activities involving dual-use items, and other products used for nuclear weapons manufacturing, would also give the authority the possibility to prevent unauthorised transfers of such products. The nuclear legislation must clearly stipulate the distribution of responsibilities between nuclear authority, operator and employees. This would give a solid basis for attaining high safety culture, including positive motivation and active commitment among executives and other employees. It would also ensure that nuclear material would not be lost through negligence or internal criminal activities. A further step of improvements on the national level, would be the urgent and firm introduction of modern Quality Assurance and Internal Control Systems into all forms of nuclear activities. In modern, safety related quality control systems, the operator alone is responsible for all activities and operations, and he must establish an internal control that will ensure that the authority's rules and requirements are thoroughly implemented and adhered to. The operator's organisation, including competence and procedures, must be approved by the State authority. By means

PI3KC2{alpha}, a class II PI3K, is required for dynamin-independent internalization pathways

DEFF Research Database (Denmark)

Krag, Claudia; Malmberg, Emily Kim; Salcini, Anna Elisabetta

2010-01-01

as fluid-phase endocytosis. Our data suggest a general role for PI3KC2a in regulating physiologically relevant dynamin-independent internalization pathways by recruiting early endosome antigen 1 (EEA1) to vesicular compartments, a step required for the intracellular trafficking of vesicles generated...... screen using a cell line expressing a diphtheria toxin receptor (DTR, officially known as HBEGF) anchored to GPI (DTR-GPI), which internalizes diphtheria toxin (DT, officially known as DTX) in a dynamin-independent manner, identified PI3KC2a, a class II phosphoinositide 3-kinase (PI3K), as a specific...... regulator of dynamin-independent DT internalization. We found that the internalization of several proteins that enter the cell through dynamin-independent pathways led to a relocalization of PI3KC2a to cargo-positive vesicles. Furthermore, downregulation of PI3KC2a impaired internalization of CD59 as well...

RNA trafficking in parasitic plant systems

Science.gov (United States)

LeBlanc, Megan; Kim, Gunjune; Westwood, James H.

2012-01-01

RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs, and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host–parasite connections and the potential significance of host RNAs for the parasite. Additional research on host–parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking. PMID:22936942

RNA trafficking in parasitic plant systems

Directory of Open Access Journals (Sweden)

Megan L LeBlanc

2012-08-01

Full Text Available RNA trafficking in plants contributes to local and long-distance coordination of plant development and response to the environment. However, investigations of mobile RNA identity and function are hindered by the inherent difficulty of tracing a given molecule of RNA from its cell of origin to its destination. Several methods have been used to address this problem, but all are limited to some extent by constraints associated with accurately sampling phloem sap or detecting trafficked RNA. Certain parasitic plant species form symplastic connections to their hosts and thereby provide an additional system for studying RNA trafficking. The haustorial connections of Cuscuta and Phelipanche species are similar to graft junctions in that they are able to transmit mRNAs, viral RNAs, siRNAs and proteins from the host plants to the parasite. In contrast to other graft systems, these parasites form connections with host species that span a wide phylogenetic range, such that a high degree of nucleotide sequence divergence may exist between host and parasites and allow confident identification of most host RNAs in the parasite system. The ability to identify host RNAs in parasites, and vice versa, will facilitate genomics approaches to understanding RNA trafficking. This review discusses the nature of host parasite connections and the potential significance of host RNAs for the parasite. Additional research on host-parasite interactions is needed to interpret results of RNA trafficking studies, but parasitic plants may provide a fascinating new perspective on RNA trafficking.

Thermodynamics and kinetics of vesicles formation processes.

Science.gov (United States)

Guida, Vincenzo

2010-12-15

Vesicles are hollow aggregates, composed of bilayers of amphiphilic molecules, dispersed into and filled with a liquid solvent. These aggregates can be formed either as equilibrium or as out of equilibrium meta-stable structures and they exhibit a rich variety of different morphologies. The surprising richness of structures, the vast range of industrial applications and the presence of vesicles in a number of biological systems have attracted the interest of numerous researchers and scientists. In this article, we review both the thermodynamics and the kinetics aspects of the phenomena of formation of vesicles. We start presenting the thermodynamics of bilayer membranes formation and deformation, with the aim of deriving the conditions for the existence of equilibrium vesicles. Specifically, we use the results from continuum thermodynamics to discuss the possibility of formation of stable equilibrium vesicles, from both mixed amphiphiles and single component systems. We also link the bilayer membrane properties to the molecular structure of the starting amphiphiles. In the second part of this article, we focus on the dynamics and kinetics of vesiculation. We review the process of vesicles formation both from planar lamellar phase under shear and from isotropic micelles. In order to clarify the physical mechanisms of vesicles formation, we continuously draw a parallel between emulsification and vesiculation processes. Specifically, we compare the experimental results, the driving forces and the relative scaling laws identified for the two processes. Describing the dynamics of vesicles formation, we also discuss why non equilibrium vesicles can be formed by kinetics control and why they are meta-stable. Understanding how to control the properties, the stability and the formation process of vesicles is of fundamental importance for a vast number of industrial applications. Copyright © 2009. Published by Elsevier B.V.

NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus.

Directory of Open Access Journals (Sweden)

Onkar Sharma

2016-03-01

Full Text Available A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase. When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO, and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells.

Cargo Release from Polymeric Vesicles under Shear

Directory of Open Access Journals (Sweden)

Yingying Guo

2018-03-01

Full Text Available In this paper we study the release of cargo from polymeric nano-carriers under shear. Vesicles formed by two star block polymers— A 12 B 6 C 2 ( A B C and A 12 B 6 A 2 ( A B A —and one linear block copolymer— A 14 B 6 ( A B , are investigated using dissipative particle dynamics (DPD simulations. A - and C -blocks are solvophobic and B -block is solvophilic. The three polymers form vesicles of different structures. The vesicles are subjected to shear both in bulk and between solvophobic walls. In bulk shear, the mechanisms of cargo release are similar for all vesicles, with cargo travelling through vesicle membrane with no preferential release location. When sheared between walls, high cargo release rate is only observed with A B C vesicle after it touches the wall. For A B C vesicle, the critical condition for high cargo release rate is the formation of wall-polymersome interface after which the effect of shear rate in promoting cargo release is secondary. High release rate is achieved by the formation of solvophilic pathway allowing cargo to travel from the vesicle cavity to the vesicle exterior. The results in this paper show that well controlled target cargo release using polymersomes can be achieved with polymers of suitable design and can potentially be very useful for engineering applications. As an example, polymersomes can be used as carriers for surface active friction reducing additives which are only released at rubbing surfaces where the additives are needed most.

Human Trafficking, Mental Illness, and Addiction: Avoiding Diagnostic Overshadowing.

Science.gov (United States)

Stoklosa, Hanni; MacGibbon, Marti; Stoklosa, Joseph

2017-01-01

This article reviews an emergency department-based clinical vignette of a trafficked patient with co-occurring pregnancy-related, mental health, and substance use disorder issues. The authors, including a survivor of human trafficking, draw on their backgrounds in addiction care, human trafficking, emergency medicine, and psychiatry to review the literature on relevant general health and mental health consequences of trafficking and propose an approach to the clinical complexities this case presents. In their discussion, the authors explicate the deleterious role of implicit bias and diagnostic overshadowing in trafficked patients with co-occurring addiction and mental illness. Finally, the authors propose a trauma-informed, multidisciplinary response to potentially trafficked patients. © 2017 American Medical Association. All Rights Reserved.

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

Science.gov (United States)

Bahouth, Suleiman W; Nooh, Mohammed M

2017-08-01

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction

PICK1 regulates the trafficking of ASIC1a and acidotoxicity in a BAR domain lipid binding-dependent manner

Directory of Open Access Journals (Sweden)

Jin Wenying

2010-12-01

Full Text Available Abstract Background Acid-sensing ion channel 1a (ASIC1a is the major ASIC subunit determining acid-activated currents in brain neurons. Recent studies show that ASIC1a play critical roles in acid-induced cell toxicity. While these studies raise the importance of ASIC1a in diseases, mechanisms for ASIC1a trafficking are not well understood. Interestingly, ASIC1a interacts with PICK1 (protein interacting with C-kinase 1, an intracellular protein that regulates trafficking of several membrane proteins. However, whether PICK1 regulates ASIC1a surface expression remains unknown. Results Here, we show that PICK1 overexpression increases ASIC1a surface level. A BAR domain mutant of PICK1, which impairs its lipid binding capability, blocks this increase. Lipid binding of PICK1 is also required for PICK1-induced clustering of ASIC1a. Consistent with the effect on ASIC1a surface levels, PICK1 increases ASIC1a-mediated acidotoxicity and this effect requires both the PDZ and BAR domains of PICK1. Conclusions Taken together, our results indicate that PICK1 regulates trafficking and function of ASIC1a in a lipid binding-dependent manner.

Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

International Nuclear Information System (INIS)

Born, E J; Hartman, S V; Holstein, S A

2013-01-01

Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways

Update: What Nurses Need to Know about Human Trafficking.

Science.gov (United States)

Washburn, Joy

Nurses are key people who interact with victims of human trafficking in healthcare and other settings. This article provides a current overview of human trafficking, explains legal definitions, elements for protocols in healthcare settings when trafficking is suspected, nursing roles and responses, interview tools, resources, public health recommendations, and nursing education approaches to address human trafficking.

Getting out of the game: desistance from drug trafficking.

Science.gov (United States)

Campbell, Howard; Hansen, Tobin

2012-11-01

This ethnographic study was conducted along the U.S.-Mexico border, the centre of the western hemispheric illicit drugs trade. It examines factors that encouraged or discouraged drug traffickers to "get out of the game" (a common slang reference to leaving the drug business). In-depth, life history interviews were conducted of thirty ex-traffickers in the El Paso/Ciudad Juárez area. Participants discussed their experiences exiting drug trafficking and their retrospective, often conflicted, feelings about the trade. Although leaving drug trafficking is a complex and multi-faceted process, the principle factors for study participants were (1) punishment (by authorities or other traffickers), (2) self-image and identity, (3) social ties, (4) life course changes and (5) drug use/abuse. Traffickers often want to quit, but their divided self-identities make it difficult to relinquish the power and exhilaration they derive from the illicit drugs business. Harm reduction policies are needed that address the embeddedness of trafficker identities in dense webs of family, community, street gangs and transnational cartels, and the larger society, as well as the seductive appeal of Hollywood and pro-cartel narco-media. Traffickers need pathways that allow them to exit the illicit drugs business without surrendering their identity. Prison sentences are not enough to encourage traffickers to stop-also needed are culturally sensitive policies that help traffickers get out of the game and stay out. Copyright © 2012 Elsevier B.V. All rights reserved.

National Human Trafficking Initiatives: Dimensions of Policy Diffusion.

Science.gov (United States)

Yoo, Eun-Hye; Boyle, Elizabeth Heger

2015-01-01

The implementation of criminal law involves formal law enforcement, education and public outreach aimed at preventing criminal activity, and providing services for victims. Historically, quantitative research on global trends has tended to focus on a single policy dimension, potentially masking the unique factors that affect the diffusion of each policy dimension independently. Using an ordered-probit model to analyze new human trafficking policy data on national prosecution, prevention, and victim-protection efforts, we find that global ties and domestic interest groups matter more in areas where international law is less defined. While prosecution, officially mandated by the Trafficking Protocol, was relatively impervious to global ties and domestic interest groups, both trafficking prevention and victim protection were associated with these factors. Our findings also suggest that fear of repercussions is not a major driver of state actions to combat trafficking-neither ratification of the Trafficking Protocol nor levels of United States aid were associated with greater implementation of anti-trafficking measures.

Type IV Secretion System of Brucella spp. and its Effectors

Directory of Open Access Journals (Sweden)

Yuehua eKe

2015-10-01

Full Text Available Brucella spp. cause brucellosis in domestic and wild animals. They are intracellular bacterial pathogens and used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we will discuss roles of Brucella VirB T4SS and in more detail of all 15 identified effectors, which may be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells, suggesting that it plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. So, we listed some key molecular events in the intracellular life cycle of Brucella potentially targeted by the VirB T4SS effectors. Elucidating functions of the effectors secreted will be crucial to clarifying mechanism of T4SS during infection. Studying the effectors secreted by Brucella spp. might provide insights into the mechanisms by which the bacteria hijack the host signaling pathways, which help us to develop better vaccines and therapies against brucellosis.

Type IV secretion system of Brucella spp. and its effectors.

Science.gov (United States)

Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

2015-01-01

Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

Human Trafficking: A Review for Mental Health Professionals

Science.gov (United States)

Yakushko, Oksana

2009-01-01

This article provides a review of current research on human trafficking for mental health practitioners and scholars. In addition to an overview of definitions, causes and processes of trafficking, the article highlights mental health consequences of trafficking along with suggestions for treatment of survivors. Directions for counseling services,…

Entrapment and Enmeshment Schemes Used by Sex Traffickers.

Science.gov (United States)

Reid, Joan A

2016-09-01

Emerging research suggests that sex traffickers/pimps control the majority of trafficked girls in the United States. The youthfulness of these victims and their lack of psychosocial maturity severely diminish their ability to detect exploitative motives or withstand manipulation of traffickers. A review of 43 cases of sexually exploited girls involving non-relative traffickers and 10 semi-structured interviews with social service providers revealed numerous scripts and schemes used by sex traffickers to entrap and entangle victims including boyfriend/lover scripts, ruses involving debt bondage, friendship or faux-family scripts, threats of forced abortion or to take away children, and coerced co-offending. These findings inform potential prevention efforts and highlight the need for multi-systemic, victim-centered approaches to intervention. © The Author(s) 2014.

'Trafficking' or 'personal use': do people who regularly inject drugs understand Australian drug trafficking laws?

Science.gov (United States)

Hughes, Caitlin E; Ritter, Alison; Cowdery, Nicholas; Sindicich, Natasha

2014-11-01

Legal thresholds for drug trafficking, over which possession of an illicit drug is deemed 'trafficking' as opposed to 'personal use', are employed in all Australian states and territories excepting Queensland. In this paper, we explore the extent to which people who regularly inject drugs understand such laws. Participants from the seven affected states/territories in the 2012 Illicit Drug Reporting System (n = 823) were asked about their legal knowledge of trafficking thresholds: whether, if arrested, quantity possessed would affect legal action taken; and the quantities of heroin, methamphetamine, cocaine and cannabis that would constitute an offence of supply. Data were compared against the actual laws to identify the accuracy of knowledge by drug type and state, and sociodemographics, use and purchasing patterns related to knowledge. Most Illicit Drug Reporting System participants (77%) correctly said that quantity possessed would affect charge received. However, only 55.8% nominated any specific quantity that would constitute an offence of supply, and of those 22.6% nominated a wrong quantity, namely a quantity that was larger than the actual quantity for supply (this varied by state and drug). People who regularly inject drugs have significant gaps in knowledge about Australian legal thresholds for drug trafficking, particularly regarding the actual threshold quantities. This suggests that there may be a need to improve education for this population. Necessity for accurate knowledge would also be lessened by better design of Australian drug trafficking laws. © 2014 Australasian Professional Society on Alcohol and other Drugs.

A Proteomics Approach to Membrane Trafficking

NARCIS (Netherlands)

Groen, A.J.; Vries, de S.C.; Lilley, K.S.

2008-01-01

Membrane trafficking, including that of integral membrane proteins as well as peripherally associated proteins, appears to be a vital process common to all eukaryotes. An important element of membrane trafficking is to determine the protein composition of the various endomembrane compartments. A

Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.

Science.gov (United States)

Sánchez-Gómez, F J; González-Morena, J M; Vida, Y; Pérez-Inestrosa, E; Blanca, M; Torres, M J; Pérez-Sala, D

2017-03-01

Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate. We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques. We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins. These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Human Trafficking of Minors and Childhood Adversity in Florida.

Science.gov (United States)

Reid, Joan A; Baglivio, Michael T; Piquero, Alex R; Greenwald, Mark A; Epps, Nathan

2017-02-01

To examine the link between human trafficking of minors and childhood adversity. We compared the prevalence of adverse childhood experiences (ACEs) and cumulative childhood adversity (ACE score) among a sample of 913 juvenile justice-involved boys and girls in Florida for whom the Florida child abuse hotline accepted human trafficking abuse reports between 2009 and 2015 with those of a matched sample. ACE composite scores were higher and 6 ACEs indicative of child maltreatment were more prevalent among youths who had human trafficking abuse reports. Sexual abuse was the strongest predictor of human trafficking: the odds of human trafficking was 2.52 times greater for girls who experienced sexual abuse, and there was a 8.21 times greater risk for boys who had histories of sexual abuse. Maltreated youths are more susceptible to exploitation in human trafficking. Sexual abuse in connection with high ACE scores may serve as a key predictor of exploitation in human trafficking for both boys and girls.

Accountability and the Use of Raids to Fight Trafficking

Directory of Open Access Journals (Sweden)

Melissa Ditmore

2012-06-01

Full Text Available Accountability in anti-trafficking efforts is a crucial but often overlooked aspect of deciding whether such efforts are truly rooted in a human rights framework. In a rush to help, and inspired by sensationalised views of what human trafficking is, many campaigns actually harm the very people they are supposed to assist. Law enforcement raids are one such effort, as they do not take into account the very different power dynamics between the actor engaging in the raid, and the person who is subject to the raid. Data from the United States suggests that raids conducted by local law enforcement agencies are an ineffective means of locating and identifying trafficked persons. Research also reveals that raids are all too frequently accompanied by violations of the human rights of trafficked persons and sex workers alike, and can therefore be counterproductive to the underlying goals of anti-trafficking initiatives. Findings suggest that a rights-based and “survivor-centred” approach to trafficking in persons requires the development and promotion of alternative methods of identifying and protecting the rights of trafficked persons which prioritise the needs, agency, and self-determination of trafficking survivors. They also indicate that preventative approaches, which address the circumstances that facilitate trafficking in persons, should be pursued over law enforcement based responses.

Child Labor Trafficking in the United States: A Hidden Crime

Directory of Open Access Journals (Sweden)

Katherine Kaufka Walts

2017-06-01

Full Text Available Emerging research brings more attention to labor trafficking in the United States. However, very few efforts have been made to better understand or respond to labor trafficking of minors. Cases of children forced to work as domestic servants, in factories, restaurants, peddling candy or other goods, or on farms may not automatically elicit suspicion from an outside observer as compared to a child providing sexual services for money. In contrast to sex trafficking, labor trafficking is often tied to formal economies and industries, which often makes it more difficult to distinguish from "legitimate" work, including among adolescents. This article seeks to provide examples of documented cases of child labor trafficking in the United States, and to provide an overview of systemic gaps in law, policy, data collection, research, and practice. These areas are currently overwhelmingly focused on sex trafficking, which undermines the policy intentions of the Trafficking Victims Protection Act (2000, the seminal statute criminalizing sex and labor trafficking in the United States, its subsequent reauthorizations, and international laws and protocols addressing human trafficking.

Are calcifying matrix vesicles in atherosclerotic lesions of cellular origin?

Science.gov (United States)

Bobryshev, Yuri V; Killingsworth, Murray C; Huynh, Thuan G; Lord, Reginald S A; Grabs, Anthony J; Valenzuela, Stella M

2007-03-01

Over recent years, the role of matrix vesicles in the initial stages of arterial calcification has been recognized. Matrix calcifying vesicles have been isolated from atherosclerotic arteries and the biochemical composition of calcified vesicles has been studied. No studies have yet been carried out to examine the fine structure of matrix vesicles in order to visualize the features of the consequent stages of their calcification in arteries. In the present work, a high resolution ultrastructural analysis has been employed and the study revealed that matrix vesicles in human atherosclerotic lesions are heterogeneous with two main types which we classified. Type I calcified vesicles were presented by vesicles surrounded by two electron-dense layers and these vesicles were found to be resistant to the calcification process in atherosclerotic lesions in situ. Type II matrix vesicles were presented by vesicles surrounded by several electron-dense layers and these vesicles were found to represent calcifying vesicles in atherosclerotic lesions. To test the hypothesis that calcification of matrix vesicles surrounded by multilayer sheets may occur simply as a physicochemical process, independently from the cell regulation, we produced multilamellar liposomes and induced their calcification in vitro in a manner similar to that occurring in matrix vesicles in atherosclerotic lesions in situ.

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

International Nuclear Information System (INIS)

Kita, Ayako; Higa, Mari; Doi, Akira; Satoh, Ryosuke; Sugiura, Reiko

2015-01-01

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2 + gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2 + gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking

Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

Energy Technology Data Exchange (ETDEWEB)

Kita, Ayako; Higa, Mari [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Doi, Akira [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Satoh, Ryosuke [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan); Sugiura, Reiko, E-mail: sugiurar@phar.kindai.ac.jp [Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502 (Japan)

2015-02-13

Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.

78 FR 40619 - Combating Wildlife Trafficking

Science.gov (United States)

2013-07-05

... derivative parts and products (together known as ``wildlife trafficking'') represent an international crisis... trafficking in accordance with the following objectives: (a) in appropriate cases, the United States shall... Quality; (xii) the Office of Science and Technology Policy; (xiii) the Office of Management and Budget...

ANTROPOLOGIS TENTANG TRAFFICKING TKW DI MALAYSIA: ANTARA ADA DAN TIADA

Directory of Open Access Journals (Sweden)

Tri Marhaeni Pudji Astuti

2011-12-01

Full Text Available Trafficking has existed since the period of kingdoms in Java, going on to the colonialism period, andto the present time. Its meaning is broadening beyond human trading into the matters related to violence,blackmailing, and forcing. Trafficking happens not only within one specific area, but has crossed theborder of countries, indicating the existence of an international net. The mushrooming of trafficking isdue to weak law and political commitment of the concerning countries. Moreover, the bilateral talk tobanish trafficking has not been maximally conducted. The actors of trafficking vary from man-powerbrokers, agents, taxi drivers, and even officers (of transmigration and police offices. Trafficking happens invarious places ranging from luxurious spots or starred-hotels to plantations and areas which accommodatea lot of migrants. The victims are usually in so unfavorable bargaining positions that they are muchdependent on those traffickers. This dependency is the impact of imbalanced gender relation. Based onsome existing cases, it is indicated that the women’s lack of power, strength, information, and educationare often misused by the traffickers to take them as their preys. That is why empowering migrant womenis very crucial. One of the ways is empowering them through their realization that this need comes fromtheir own selves, not from any force outside. Besides, there should be strong commitment from the stateto seriously implement the law against any traffickers. Cooperation between the concerning countriesare also needed, for instance by issuing common regulations to banish trafficking.Keywords: Trafficking, migrant women, receiving country, sending country, trafficker

Identification of human trafficking victims in health care settings.

Science.gov (United States)

Baldwin, Susie B; Eisenman, David P; Sayles, Jennifer N; Ryan, Gery; Chuang, Kenneth S

2011-07-14

An estimated 18,000 individuals are trafficked into the United States each year from all over the world, and are forced into hard labor or commercial sex work. Despite their invisibility, some victims are known to have received medical care while under traffickers' control. Our project aimed to characterize trafficking victims' encounters in US health care settings. The study consisted of semi-structured interviews with six Key Informants who work closely with trafficking victims (Phase I) and 12 female trafficking survivors (Phase II). All survivors were recruited through the Coalition to Abolish Slavery and Trafficking, an NGO in Los Angeles, and all were trafficked into Los Angeles. Interviews were conducted in English and six other languages, with the assistance of professional interpreters. Using a framework analysis approach that focused on victims' encounters in health care settings, we assessed interview transcript content and coded for themes. We used an exploratory pile-sorting technique to aggregate similar ideas and identify overarching domains. The survivors came from 10 countries. Eight had experienced domestic servitude, three had survived sex trafficking, and one had experienced both. Half the survivors reported that they had visited a physician while in their traffickers' control, and another worked in a health care facility. All Key Informants described other victims who had received medical care. For domestic servants, medical visits were triggered by injury and respiratory or systemic illness, while sex trafficking victims were seen by health professionals for sexually transmitted infections and abortion. Trafficking victims were prevented from disclosing their status to health care providers by fear, shame, language barriers, and limited interaction with medical personnel, among other obstacles. This exploration of survivors' experiences in health care settings supports anecdotal reports that US health care providers may unwittingly encounter

Calcium reduces the sodium permeability of luminal membrane vesicles from toad bladder. Studies using a fast-reaction apparatus

International Nuclear Information System (INIS)

Chase, H.S. Jr.; Al-Awqati, Q.

1983-01-01

Regulation of the sodium permeability of the luminal membrane is the major mechanism by which the net rate of sodium transport across tight epithelia is varied. Previous evidence has suggested that the permeability of the luminal membrane might be regulated by changes in intracellular sodium or calcium activities. To test this directly, we isolated a fraction of the plasma membrane from the toad urinary bladder, which contains a fast, amiloride-sensitive sodium flux with characteristics similar to those of the native luminal membrane. Using a flow-quench apparatus to measure the initial rate of sodium efflux from these vesicles in the millisecond time range, we have demonstrated that the isotope exchange permeability of these vesicles is very sensitive to calcium. Calcium reduces the sodium permeability, and the half-maximal inhibitory concentration is 0.5 microM, well within the range of calcium activity found in cells. Also, the permeability of the luminal membrane vesicles is little affected by the ambient sodium concentration. These results, when taken together with studies on whole tissue, suggest that cell calcium may be an important regulator of transepithelial sodium transport by its effect on luminal sodium permeability. The effect of cell sodium on permeability may be mediated by calcium rather than by sodium itself

Membrane Trafficking Modulation during Entamoeba Encystation.

Science.gov (United States)

Herman, Emily; Siegesmund, Maria A; Bottery, Michael J; van Aerle, Ronny; Shather, Maulood Mohammed; Caler, Elisabet; Dacks, Joel B; van der Giezen, Mark

2017-10-09

Entamoeba histolytica is an intestinal parasite that infects 50-100 million people and causes up to 55,000 deaths annually. The transmissive form of E. histolytica is the cyst, with a single infected individual passing up to 45 million cysts per day, making cyst production an attractive target for infection control. Lectins and chitin are secreted to form the cyst wall, although little is known about the underlying membrane trafficking processes supporting encystation. As E. histolytica does not readily form cysts in vitro, we assessed membrane trafficking gene expression during encystation in the closely related model Entamoeba invadens. Genes involved in secretion are up-regulated during cyst formation, as are some trans-Golgi network-to-endosome trafficking genes. Furthermore, endocytic and general trafficking genes are up-regulated in the mature cyst, potentially preserved as mRNA in preparation for excystation. Two divergent dynamin-related proteins found in Entamoeba are predominantly expressed during cyst formation. Phylogenetic analyses indicate that they are paralogous to, but quite distinct from, classical dynamins found in human, suggesting that they may be potential drug targets to block encystation. The membrane-trafficking machinery is clearly regulated during encystation, providing an additional facet to understanding this crucial parasitic process.

Vesicles Are Persistent Features of Different Plastids.

Science.gov (United States)

Lindquist, Emelie; Solymosi, Katalin; Aronsson, Henrik

2016-10-01

Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

KENYA’S CONSTITUTION AND CHILD TRAFFICKING AS A SECURITY THREAT

Directory of Open Access Journals (Sweden)

E.O.S. ODHIAMBO

2012-01-01

Full Text Available Human trafficking also referred to as modern-day slavery is seen as a security threat. Traditional security approaches to human trafficking call for analysis of trafficking as a threat to the Kenyan state and to Kenya’s control of its borders. Traditional security analyses of trafficking emphasize border security, migration controls, and international law enforcement cooperation. This article discusses three forms of child trafficking: sexual exploitation, forced labor and child soldiers and argues that the newly promulgated Kenyan constitution in chapter three on citizenship has a provision that can be interpreted as encouraging child trafficking.

The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.

Science.gov (United States)

Takahara, Terunao; Inoue, Kuniko; Arai, Yumika; Kuwata, Keiko; Shibata, Hideki; Maki, Masatoshi

2017-10-13

Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Post-transcriptional trafficking and regulation of neuronal gene expression.

Science.gov (United States)

Goldie, Belinda J; Cairns, Murray J

2012-02-01

Intracellular messenger RNA (mRNA) traffic and translation must be highly regulated, both temporally and spatially, within eukaryotic cells to support the complex functional partitioning. This capacity is essential in neurons because it provides a mechanism for rapid input-restricted activity-dependent protein synthesis in individual dendritic spines. While this feature is thought to be important for synaptic plasticity, the structures and mechanisms that support this capability are largely unknown. Certainly specialized RNA binding proteins and binding elements in the 3' untranslated region (UTR) of translationally regulated mRNA are important, but the subtlety and complexity of this system suggests that an intermediate "specificity" component is also involved. Small non-coding microRNA (miRNA) are essential for CNS development and may fulfill this role by acting as the guide strand for mediating complex patterns of post-transcriptional regulation. In this review we examine post-synaptic gene regulation, mRNA trafficking and the emerging role of post-transcriptional gene silencing in synaptic plasticity.

KENYA’S CONSTITUTION AND CHILD TRAFFICKING AS A SECURITY THREAT

OpenAIRE

E.O.S. ODHIAMBO; J. KASSILLY; L.T. MAITO; K. ONKWARE; W. A. OBOKA

2012-01-01

Human trafficking also referred to as modern-day slavery is seen as a security threat. Traditional security approaches to human trafficking call for analysis of trafficking as a threat to the Kenyan state and to Kenya’s control of its borders. Traditional security analyses of trafficking emphasize border security, migration controls, and international law enforcement cooperation. This article discusses three forms of child trafficking: sexual exploitation, forced labor and child soldiers and ...

Extracellular Vesicles: How the External and Internal Environment Can Shape Cell-To-Cell Communication.

Science.gov (United States)

Neven, Kristof Y; Nawrot, Tim S; Bollati, Valentina

2017-03-01

To summarize the scientific evidence regarding the effects of environmental exposures on extracellular vesicle (EV) release and their contents. As environmental exposures might influence the aging phenotype in a very strict way, we will also report the role of EVs in the biological aging process. EV research is a new and quickly developing field. With many investigations conducted so far, only a limited number of studies have explored the potential role EVs play in the response and adaptation to environmental stimuli. The investigations available to date have identified several exposures or lifestyle factors able to modify EV trafficking including air pollutants, cigarette smoke, alcohol, obesity, nutrition, physical exercise, and oxidative stress. EVs are a very promising tool, as biological fluids are easily obtainable biological media that, if successful in identifying early alterations induced by the environment and predictive of disease, would be amenable to use for potential future preventive and diagnostic applications.

Ovarian Cystadenoma in a Trafficked Patient.

Science.gov (United States)

Titchen, Kanani E; Katz, Douglas; Martinez, Kidian; White, Krishna

2016-05-01

The topic of child sex trafficking is receiving increased attention both in the lay press and in research articles. Recently, a number of physician organizations have issued policy statements calling for the education and involvement of physicians in combating this form of "modern-day slavery." Primary care and emergency medicine physicians have led these efforts, but a number of these victims may present to surgeons. Surgeons are in a unique position to identify trafficked patients; during the process of undraping, intubation, and surgical preparation, signs of trafficking such as tattoos, scars, dental injuries, and bruising may be evident. In addition, these patients may have specific needs in terms of anesthesia and postoperative care due to substance abuse. Here, we report the case of an 18-year-old girl with a history of sexual exploitation who presents for cystadenoma excision. To our knowledge, this is the first report of a sex-trafficked pediatric patient presenting for surgery. Copyright © 2016 by the American Academy of Pediatrics.

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

Science.gov (United States)

Zou, Wei; Yadav, Smita; DeVault, Laura; Jan, Yuh Nung; Sherwood, David R.

2015-01-01

Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth. PMID:26394140

Differential trafficking of oxidized LDL and oxidized LDL immune complexes in macrophages: impact on oxidative stress.

Directory of Open Access Journals (Sweden)

Mohammed M Al Gadban

2010-09-01

Full Text Available Oxidized low-density lipoproteins (oxLDL and oxLDL-containing immune complexes (oxLDL-IC contribute to formation of lipid-laden macrophages (foam cells. It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI. This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.Fluorescently labeled lipid and protein moieties of oxLDL co-localized within endosomal and lysosomal compartments in U937 human monocytic cells. In contrast, the lipid moiety of oxLDL-IC was detected in the endosomal compartment, whereas its apolipoprotein moiety advanced to the lysosomal compartment. Cells treated with oxLDL-IC prior to oxLDL demonstrated co-localization of internalized lipid moieties from both oxLDL and oxLDL-IC in the endosomal compartment. This sequential treatment likely inhibited oxLDL lipid moieties from trafficking to the lysosomal compartment. In RAW 264.7 macrophages, oxLDL-IC but not oxLDL induced GFP-tagged heat shock protein 70 (HSP70 and HSP70B', which co-localized with the lipid moiety of oxLDL-IC in the endosomal compartment. This suggests that HSP70 family members might prevent the degradation of the internalized lipid moiety of oxLDL-IC by delaying its advancement to the lysosome. The data also showed that mitochondrial membrane potential was decreased and generation of reactive oxygen and nitrogen species was increased in U937 cell treated with oxLDL compared to oxLDL-IC.Findings suggest that lipid and apolipoprotein moieties of oxLDL-IC traffic to separate cellular compartments, and that HSP70/70B' might sequester the lipid moiety of oxLDL-IC in the endosomal compartment. This mechanism could

Human organ trafficking in the cyber space

Directory of Open Access Journals (Sweden)

Vuletić Dejan

2009-01-01

Full Text Available The accelerated growth of the information-communication technology use brought about cyber crime as a new form of crime connected with the misuse of computer network. Human trafficking and human organ trafficking are changing in line with the state-of-art technological achievements i.e. becoming more and more characteristic of cyber space. Passing appropriate regulations at both national and international levels presents an important step in solving the problem of human organ trafficking through Internet.

Devastating consequences of sex trafficking on women's health.

Science.gov (United States)

McTavish, Fr James

2017-11-01

Sex trafficking has devastating consequences on the physical and mental well-being of millions of women around the world. These trafficking victims often come in contact with medical personnel, and these encounters with suitably prepared staff can be a step toward healing of the victims. The Catholic Church, especially through Pope Francis, is making strenuous efforts to curb the spread of sex trafficking. Same-sex feelings and behavior may arise post-trafficking in individuals, although this does not appear to be mentioned thus far in the literature. Here, we are most likely dealing with a type of "pseudo-lesbianism" post-trauma. The trafficking survivor can be helped to understand some of the likely roots of her feelings such as anti-male sentiments following abuse. She needs to be patiently, and expertly, accompanied to process the trauma she has experienced, and learn how to meet her genuine needs for female affection and affirmation in healthy, chaste, and non-erotic ways. Around the world, millions of female victims of human trafficking are forced into sex "work," often resulting in serious physical and mental-health problems. Healthcare staff should be alert to spot victims of sex trafficking and be ready to assist them. The Catholic Church, especially through Pope Francis, has been vocal in denouncing this form of modern slavery. Some female victims of sex trafficking may experience same-sex feelings afterward. Healing for such young women involves helping them to process their traumatic experiences, as well as patiently accompanying them as they seek to develop healthy, chaste friendships with other females and males.

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

Science.gov (United States)

Tombes, R. M.; Simerly, C.; Borisy, G. G.; Schatten, G.

1992-01-01

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.

Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins

DEFF Research Database (Denmark)

Varkey, Jobin; Isas, Jose Mario; Mizuno, Naoko

2010-01-01

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have...... of amphipathic helices alone. Moreover, we frequently observed that a-synuclein caused membrane structures that had the appearance of nascent budding vesicles. The ability to function as a minimal machinery for vesicle budding agrees well with recent findings that a-synuclein plays a role in vesicle trafficking...

Debate: Strategically Working in Parallel to Traffickers

Directory of Open Access Journals (Sweden)

Vincent Tournecuillert

2014-09-01

Full Text Available Let’s be realistic, counter-trafficking teams will never be as effective as the proactive and flexible networks of outlaws that violate the rights of millions of people each year. The ‘bad guys’ operate without the same financial limitations such as bureaucratic red tape and donor criteria, and take advantage of patchy and often uncoordinated border surveillance that is chronically untrained in detecting trafficking in persons.  Non-governmental organisations (NGOs involved in the fight against human trafficking—and in direct contact with presumed victims (their status is not assessed until at a stage later than this initial contact—are in a diametrically opposite situation. They must carefully abide by the national and international legal frameworks that their criminal antagonists ignore. Donors and national authorities operate within the constraints of geographic target areas and funding cycles. Since counter-trafficking actors neither create the markets nor devise the routes for trafficking, their strategic cross-border (or long distance partnerships are always a few steps behind the traffickers, if not many steps behind, and rarely efficient.

Synaptic Vesicle Endocytosis in Different Model Systems

Directory of Open Access Journals (Sweden)

Quan Gan

2018-06-01

Full Text Available Neurotransmission in complex animals depends on a choir of functionally distinct synapses releasing neurotransmitters in a highly coordinated manner. During synaptic signaling, vesicles fuse with the plasma membrane to release their contents. The rate of vesicle fusion is high and can exceed the rate at which synaptic vesicles can be re-supplied by distant sources. Thus, local compensatory endocytosis is needed to replenish the synaptic vesicle pools. Over the last four decades, various experimental methods and model systems have been used to study the cellular and molecular mechanisms underlying synaptic vesicle cycle. Clathrin-mediated endocytosis is thought to be the predominant mechanism for synaptic vesicle recycling. However, recent studies suggest significant contribution from other modes of endocytosis, including fast compensatory endocytosis, activity-dependent bulk endocytosis, ultrafast endocytosis, as well as kiss-and-run. Currently, it is not clear whether a universal model of vesicle recycling exist for all types of synapses. It is possible that each synapse type employs a particular mode of endocytosis. Alternatively, multiple modes of endocytosis operate at the same synapse, and the synapse toggles between different modes depending on its activity level. Here we compile review and research articles based on well-characterized model systems: frog neuromuscular junctions, C. elegans neuromuscular junctions, Drosophila neuromuscular junctions, lamprey reticulospinal giant axons, goldfish retinal ribbon synapses, the calyx of Held, and rodent hippocampal synapses. We will compare these systems in terms of their known modes and kinetics of synaptic vesicle endocytosis, as well as the underlying molecular machineries. We will also provide the future development of this field.

Prevention of Human Trafficking in Ethiopia: Assessing The Legal Framework

Directory of Open Access Journals (Sweden)

Zelalem Shiferaw Woldemichael

2017-12-01

Full Text Available Recent findings have indicated that both in-country trafficking (trafficking of individuals from rural areas to relatively affluent towns and cities and external trafficking (trafficking of individuals from a given country to foreign countries are prevalent in Ethiopia. In 2012, the government acceded to the Protocol to Suppress and Punish Trafficking in Persons Especially Women and Children supplementing the United Nations Convention against Transnational Organized Crime (The UN Trafficking Protocol, here after. With a view to giving effect to the requirements of this instrument, the government passed in to law Proclamation No. 909/2015 (The Prevention and Suppression of Trafficking in Persons and Smuggling of Migrants Proclamation, which is the most comprehensive of all laws adopted in Ethiopia to deal with human trafficking. Taking in to account the fact that human trafficking is exacerbated by the absence of regulatory framework on the employment of Ethiopian nationals in foreign countries, the govern-ment has also brought in to practice Proclamation No. 923/2016 (Ethiopia’s Overseas Employment Proclamation. This article has examined whether the above-mentioned laws of Ethiopia comply with international standards in dealing with prevention strategies.

TRACE-ing human trafficking : Project Findings

NARCIS (Netherlands)

Rijken, Conny; Pijnenburg, Annick

2016-01-01

Human trafficking is one of the largest criminal enterprises in the world. It is a multi-billion-dollar crime of global scale. This is because human trafficking as a criminal enterprise continues to evolve as a high profit-low risk business for perpetrators and challenges policy makers, law

Sexual slavery without borders: trafficking for commercial sexual exploitation in India

Science.gov (United States)

Joffres, Christine; Mills, Edward; Joffres, Michel; Khanna, Tinku; Walia, Harleen; Grund, Darrin

2008-01-01

Trafficking in women and children is a gross violation of human rights. However, this does not prevent an estimated 800 000 women and children to be trafficked each year across international borders. Eighty per cent of trafficked persons end in forced sex work. India has been identified as one of the Asian countries where trafficking for commercial sexual exploitation has reached alarming levels. While there is a considerable amount of internal trafficking from one state to another or within states, India has also emerged as a international supplier of trafficked women and children to the Gulf States and South East Asia, as well as a destination country for women and girls trafficked for commercial sexual exploitation from Nepal and Bangladesh. Trafficking for commercial sexual exploitation is a highly profitable and low risk business that preys on particularly vulnerable populations. This paper presents an overview of the trafficking of women and girls for sexual exploitation (CSE) in India; identifies the health impacts of CSE; and suggest strategies to respond to trafficking and related issues. PMID:18817576

Sexual slavery without borders: trafficking for commercial sexual exploitation in India.

Science.gov (United States)

Joffres, Christine; Mills, Edward; Joffres, Michel; Khanna, Tinku; Walia, Harleen; Grund, Darrin

2008-09-25

Trafficking in women and children is a gross violation of human rights. However, this does not prevent an estimated 800 000 women and children to be trafficked each year across international borders. Eighty per cent of trafficked persons end in forced sex work. India has been identified as one of the Asian countries where trafficking for commercial sexual exploitation has reached alarming levels. While there is a considerable amount of internal trafficking from one state to another or within states, India has also emerged as a international supplier of trafficked women and children to the Gulf States and South East Asia, as well as a destination country for women and girls trafficked for commercial sexual exploitation from Nepal and Bangladesh. Trafficking for commercial sexual exploitation is a highly profitable and low risk business that preys on particularly vulnerable populations. This paper presents an overview of the trafficking of women and girls for sexual exploitation (CSE) in India; identifies the health impacts of CSE; and suggest strategies to respond to trafficking and related issues.

Additive effects on the energy barrier for synaptic vesicle fusion cause supralinear effects on the vesicle fusion rate

DEFF Research Database (Denmark)

Schotten, Sebastiaan; Meijer, Marieke; Walter, Alexander Matthias

2015-01-01

supralinear effects on the fusion rate. To test this prediction experimentally, we developed a method to assess the number of releasable vesicles, rate constants for vesicle priming, unpriming, and fusion, and the activation energy for fusion by fitting a vesicle state model to synaptic responses induced......-linear effects of genetic/pharmacological perturbations on synaptic transmission and a novel interpretation of the cooperative nature of Ca2+-dependent release....

Immunomicroscopic localization of aminopeptidase N in the pig enterocyte. Implications for the route of intracellular transport

DEFF Research Database (Denmark)

Hansen, Gert Helge; Sjöström, H; Norén, Ove

1987-01-01

The subcellular localization of aminopeptidase N (EC 3.4.11.2) in the pig enterocyte was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). By indirect immunofluorescence on either frozen or paraffin-embedded sections, a very intense staining in the microvillar....... Labelling was demonstrated in the Golgi apparatus and in a minor fraction of the intracellular smooth vesicles positioned between the Golgi apparatus and the microvillar membrane. These observations are compatible with the view that newly synthesized aminopeptidase N is delivered directly to the microvillar...

MILD CHOLESTEROL DEPLETION REDUCES AMYLOID-β PRODUCTION BY IMPAIRING APP TRAFFICKING TO THE CELL SURFACE

Science.gov (United States)

Guardia-Laguarta, Cristina; Coma, Mireia; Pera, Marta; Clarimón, Jordi; Sereno, Lidia; Agulló, José M.; Molina-Porcel, Laura; Gallardo, Eduard; Deng, Amy; Berezovska, Oksana; Hyman, Bradley T.; Blesa, Rafael; Gómez-Isla, Teresa; Lleó, Alberto

2009-01-01

It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and γ-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Aβ40 and Aβ42 in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of γ-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts. PMID:19457132

Human trafficking: an evaluation of Canadian medical students' awareness and attitudes.

Science.gov (United States)

Wong, Janice C; Hong, Jonathan; Leung, Pearl; Yin, Penny; Stewart, Donna E

2011-04-01

Human trafficking is a human rights violation prevalent globally. Current guidelines highlight healthcare professionals' key role in responding to human trafficking, emphasizing the importance of medical education in raising awareness of trafficking. To assess pre-clerkship medical students' awareness of human trafficking and attitudes towards learning about trafficking in the medical curriculum at Canada's largest medical school. An anonymous, classroom-based questionnaire was designed, piloted and administered to first- and second-year medical students at one large Canadian medical school with a diverse student population. The questionnaire sought demographic data and information on students' self-perceived awareness of human trafficking and interest in learning about trafficking and other community health issues. 262 medical students completed the questionnaire (70.0% response). Most participants reported that they were not knowledgeable (48.5%) or only somewhat knowledgeable (45.4%) about human trafficking. 88.9% of participants were not familiar with signs and symptoms of trafficked persons. While students' responses indicated that they prioritized other social issues, a majority of participants (76.0%) thought that trafficking was important to learn about in medical school, especially identifying trafficked persons and their health needs. These medical students of one Canadian medical school demonstrated limited familiarity with the issue of human trafficking but largely felt that they should be taught more about this issue during their medical education. This assessment of early medical students' awareness of human trafficking is relevant to medical educators and the organizations that could develop the required educational curricula and resources.

New mechanisms for non-porative ultrasound stimulation of cargo delivery to cell cytosol with targeted perfluorocarbon nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Soman, N R; Marsh, J N; Lanza, G M; Wickline, S A [Washington University School of Medicine, Consortium for Translational Research in Advanced Imaging and Nanomedicine, CTRAIN, Campus Box 8215, St Louis, MO 63110 (United States)], E-mail: saw@wuphys.wustl.edu

2008-05-07

The cell membrane constitutes a major barrier for non-endocytotic intracellular delivery of therapeutic molecules from drug delivery vehicles. Existing approaches to breaching the cell membrane include cavitational ultrasound (with microbubbles), electroporation and cell-penetrating peptides. We report the use of diagnostic ultrasound for intracellular delivery of therapeutic bulky cargo with the use of molecularly targeted liquid perfluorocarbon (PFC) nanoparticles. To demonstrate the concept, we used a lipid with a surrogate polar head group, nanogold-DPPE, incorporated into the nanoparticle lipid monolayer. Melanoma cells were incubated with nanogold particles and this was followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa). Cells not exposed to ultrasound showed gold particles partitioned only in the outer bilayer of the cell membrane with no evidence of the intracellular transit of nanogold. However, the cells exposed to ultrasound exhibited numerous nanogold-DPPE components inside the cell that appeared polarized inside intracellular vesicles demonstrating cellular uptake and trafficking. Further, ultrasound-exposed cells manifested no incorporation of calcein or the release of lactate dehydrogenase. These observations are consistent with a mechanism that suggests that ultrasound is capable of stimulating the intracellular delivery of therapeutic molecules via non-porative mechanisms. Therefore, non-cavitational adjunctive ultrasound offers a novel paradigm in intracellular cargo delivery from PFC nanoparticles.

New mechanisms for non-porative ultrasound stimulation of cargo delivery to cell cytosol with targeted perfluorocarbon nanoparticles

International Nuclear Information System (INIS)

Soman, N R; Marsh, J N; Lanza, G M; Wickline, S A

2008-01-01

The cell membrane constitutes a major barrier for non-endocytotic intracellular delivery of therapeutic molecules from drug delivery vehicles. Existing approaches to breaching the cell membrane include cavitational ultrasound (with microbubbles), electroporation and cell-penetrating peptides. We report the use of diagnostic ultrasound for intracellular delivery of therapeutic bulky cargo with the use of molecularly targeted liquid perfluorocarbon (PFC) nanoparticles. To demonstrate the concept, we used a lipid with a surrogate polar head group, nanogold-DPPE, incorporated into the nanoparticle lipid monolayer. Melanoma cells were incubated with nanogold particles and this was followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa). Cells not exposed to ultrasound showed gold particles partitioned only in the outer bilayer of the cell membrane with no evidence of the intracellular transit of nanogold. However, the cells exposed to ultrasound exhibited numerous nanogold-DPPE components inside the cell that appeared polarized inside intracellular vesicles demonstrating cellular uptake and trafficking. Further, ultrasound-exposed cells manifested no incorporation of calcein or the release of lactate dehydrogenase. These observations are consistent with a mechanism that suggests that ultrasound is capable of stimulating the intracellular delivery of therapeutic molecules via non-porative mechanisms. Therefore, non-cavitational adjunctive ultrasound offers a novel paradigm in intracellular cargo delivery from PFC nanoparticles

New mechanisms for non-porative ultrasound stimulation of cargo delivery to cell cytosol with targeted perfluorocarbon nanoparticles

Science.gov (United States)

Soman, N. R.; Marsh, J. N.; Lanza, G. M.; Wickline, S. A.

2008-05-01

The cell membrane constitutes a major barrier for non-endocytotic intracellular delivery of therapeutic molecules from drug delivery vehicles. Existing approaches to breaching the cell membrane include cavitational ultrasound (with microbubbles), electroporation and cell-penetrating peptides. We report the use of diagnostic ultrasound for intracellular delivery of therapeutic bulky cargo with the use of molecularly targeted liquid perfluorocarbon (PFC) nanoparticles. To demonstrate the concept, we used a lipid with a surrogate polar head group, nanogold-DPPE, incorporated into the nanoparticle lipid monolayer. Melanoma cells were incubated with nanogold particles and this was followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa). Cells not exposed to ultrasound showed gold particles partitioned only in the outer bilayer of the cell membrane with no evidence of the intracellular transit of nanogold. However, the cells exposed to ultrasound exhibited numerous nanogold-DPPE components inside the cell that appeared polarized inside intracellular vesicles demonstrating cellular uptake and trafficking. Further, ultrasound-exposed cells manifested no incorporation of calcein or the release of lactate dehydrogenase. These observations are consistent with a mechanism that suggests that ultrasound is capable of stimulating the intracellular delivery of therapeutic molecules via non-porative mechanisms. Therefore, non-cavitational adjunctive ultrasound offers a novel paradigm in intracellular cargo delivery from PFC nanoparticles.

A Comparison of Psychological Symptoms in Survivors of Sex and Labor Trafficking.

Science.gov (United States)

Hopper, E K; Gonzalez, L D

2018-03-20

Human trafficking is a form of interpersonal trauma that has significant mental health impacts on survivors. This study examined psychological symptoms in 131 survivors of sex and labor trafficking, including people trafficked into or within the U.S. High rates of depression (71%) and PTSD (61%) were identified. Two thirds of survivors also met criteria for multiple categories of Complex PTSD (C-PTSD), including affect dysregulation and impulsivity; alterations in attention and consciousness; changes in interpersonal relationships; revictimization; somatic dysregulation; and alterations in self-perception. Although there were not significant differences in the prevalence rates of diagnoses of PTSD or depression between survivors of sex and labor trafficking, important group differences were identified. Compared to survivors of labor trafficking, sex trafficking survivors had higher prevalence rates of pre-trafficking childhood abuse and a higher incidence of physical and sexual violence during trafficking. They reported more severe post-trauma reactions than labor trafficking survivors, including more PTSD and C-PTSD symptoms. They were also more likely to meet criteria for comorbid PTSD and depression, while labor trafficking survivors were more likely than sex trafficking survivors to meet criteria for depression alone. An analysis of gender differences found that trafficking survivors who identified as transgender endorsed more PTSD and C-PTSD symptoms, than male or female survivors. Childhood abuse exposure was linked to PTSD and C-PTSD in trafficking survivors, and trafficking type was predictive of the number of trauma-related symptoms beyond the role of pre-trafficking child abuse. Implications for assessment and intervention with trafficking survivors are discussed.

The role of the nurse in combating human trafficking.

Science.gov (United States)

Sabella, Donna

2011-02-01

Human trafficking, also called modern slavery, happens worldwide--and the United States is no exception. Within our borders, thousands of foreign nationals and U.S. citizens, many of them children, are forced or coerced into sex work or various forms of labor every year. Nurses and other health care providers who encounter victims of trafficking often don't realize it, and opportunities to intervene are lost. Although no one sign can demonstrate with certainty when someone is being trafficked, there are several indicators that clinicians should know. This article provides an overview of human trafficking, describes how to recognize signs that a person is being trafficked and how to safely intervene, and offers an extensive resource list.

Extracellular vesicles: Exosomes, microvesicles, and friends

NARCIS (Netherlands)

Raposo, G.; Stoorvogel, W.|info:eu-repo/dai/nl/074352385

2013-01-01

Cells release into the extracellular environment diverse types of membrane vesicles of endosomal and plasma membrane origin called exosomes and microvesicles, respectively. These extracellular vesicles (EVs) represent an important mode of intercellular communication by serving as vehicles for

Health impacts and research ethics in female trafficking.

Science.gov (United States)

Dhital, S R; Aro, R A; Sapkota, K

2011-04-01

Female trafficking is a social and public health problem, associated with physical and sexual abuse, psychological trauma, injuries from violence, sexually transmitted infections, adverse reproductive outcomes and substance misuse. It faces several challenges ranging from the hidden nature of the problem to ethical and human rights issues. The objectives of this paper are to analyze health impact of trafficking; ethical and research issues and anti-trafficking strategies in the Nepalese context. We collected published and unpublished data assessing the public health, ethical burden and research needs from different sources. Trafficked female involved in sex-industry that face grave situation as depicted and it might a reservoir of sexually transmitted diseases. Ethical issues related to survey of assessing the burden are difficult to carry out. The best ways to prevent and control these problems are to enhance anti- trafficking laws and raise awareness, empower and mobilize females and establish organizational capacity.

Captured ‘Realities’ of Human Trafficking: Analysis of photographs illustrating stories on trafficking into the sex industry in Serbian media

Directory of Open Access Journals (Sweden)

Elena Krsmanovic

2016-09-01

Full Text Available Past research has looked at how the media frames human trafficking, but has seldom included analysis of visual representations. To bridge this gap, this paper scrutinises stereotypical representations of persons trafficked into the sex industry in photographs published in Serbian online media from 2011 to 2014. To uncover characteristics of dominant tropes in this sample, a method of semiotic analysis is applied. The analysis argues that images are dominated by portrayals of trafficked persons that fit into one of two frames: powerless victim or unworthy prostitute. Male figures are rarely presented in these photographs, but when present, they are shown to hurt or control the women depicted alongside them. Chains, padlocks, barcodes, whip marks, and other symbols associated with slavery are present to a lesser extent. However, they testify to the tendency to link human trafficking to slavery and to use the moral potential of the anti-slavery rhetoric. Photographs are too easily seen as authentic, factual transcripts of reality. This paper suggests that these images tell us more about societal fear of insecurity, ideas about gender, erotic obsessions and morality than about human trafficking itself. It also argues that the meaning of trafficking is shaped by the deeply embedded codes of patriarchy and hidden misogyny present in Serbian society.

INTERNATIONAL COOPERATION AGAINST HUMAN TRAFFICKING

OpenAIRE

Ionita COCHINTU; Laura TUTUNARU; Narcisa Mihaela STOICU; Daniela Cristina VALEA

2011-01-01

Trafficking in human beings, a phenomenon with global dimensions constitutes a serious violation of human rights, dignity and freedom, a social phenomenon with negative consequences for the entire society. Countries have been concerned over the time to find the most effective policy measures to combat and prevent human trafficking, and in this regard the United Nations, the European Union and the Council of Europe have developed a series of international documents which established an interna...

USSOCOM’s Role in Addressing Human Trafficking

Science.gov (United States)

2010-12-02

world. However, despite those alarming numbers, 62 countries have yet to convict a trafficker under laws in compliance with the Palermo Protocol.7...Against Transnational Organized Crime( Palermo Protocol), Palermo , Italy, 2000. 8 US Department of State, 2010 Trafficking In Persons Report...activities with the proceeds from narcotics trafficking. Terrorist groups such as the FARC in Colombia , Al Qaida in Afghanistan, and groups around

Domestic Sex Trafficking of Minors: Medical Student and Physician Awareness.

Science.gov (United States)

Titchen, Kanani E; Loo, Dyani; Berdan, Elizabeth; Rysavy, Mary Becker; Ng, Jessica J; Sharif, Iman

2017-02-01

Our aim was to assess: (1) medical trainee and practicing physician awareness about domestic sex trafficking of minors; and (2) whether respondents believe that awareness of trafficking is important to their practice. We designed an anonymous electronic survey, and a convenience sample was collected from June through October 2013. Voluntary participants were 1648 medical students, residents, and practicing physicians throughout the United States. Data were analyzed for correlations between study cohort characteristics and: (1) agreement with the statement: "knowing about sex trafficking in my state is important to my profession"; (2) knowledge of national statistics regarding the sex trafficking of minors; and (3) knowledge of appropriate responses to encountering a trafficked victim. More practicing physicians than residents or medical students: (1) agreed or strongly agreed that knowledge about human trafficking was important to their practice (80.6%, 71.1%, and 69.2%, respectively; P = .0008); (2) correctly estimated the number of US trafficked youth according to the US Department of State data (16.1%, 11.7%, and 7.9%, respectively; P = .0011); and (3) were more likely to report an appropriate response to a trafficked victim (40.4%, 20.4%, and 8.9%, respectively; P = .0001). Although most medical trainees and physicians place importance on knowing about human trafficking, they lack knowledge about the scope of the problem, and most would not know where to turn if they encountered a trafficking victim. There exists a need for standardized trafficking education for physicians, residents, and medical students. Copyright © 2015 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.

Visualizing Mutation-Specific Differences in the Trafficking-Deficient Phenotype of Kv11.1 Proteins Linked to Long QT Syndrome Type 2.

Science.gov (United States)

Hall, Allison R; Anderson, Corey L; Smith, Jennifer L; Mirshahi, Tooraj; Elayi, Claude S; January, Craig T; Delisle, Brian P

2018-01-01

KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K + current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular

Slow sedimentation and deformability of charged lipid vesicles.

Directory of Open Access Journals (Sweden)

Iván Rey Suárez

Full Text Available The study of vesicles in suspension is important to understand the complicated dynamics exhibited by cells in in vivo and in vitro. We developed a computer simulation based on the boundary-integral method to model the three dimensional gravity-driven sedimentation of charged vesicles towards a flat surface. The membrane mechanical behavior was modeled using the Helfrich Hamiltonian and near incompressibility of the membrane was enforced via a model which accounts for the thermal fluctuations of the membrane. The simulations were verified and compared to experimental data obtained using suspended vesicles labelled with a fluorescent probe, which allows visualization using fluorescence microscopy and confers the membrane with a negative surface charge. The electrostatic interaction between the vesicle and the surface was modeled using the linear Derjaguin approximation for a low ionic concentration solution. The sedimentation rate as a function of the distance of the vesicle to the surface was determined both experimentally and from the computer simulations. The gap between the vesicle and the surface, as well as the shape of the vesicle at equilibrium were also studied. It was determined that inclusion of the electrostatic interaction is fundamental to accurately predict the sedimentation rate as the vesicle approaches the surface and the size of the gap at equilibrium, we also observed that the presence of charge in the membrane increases its rigidity.

Effectiveness of Counter-Trafficking Response in Albania

Directory of Open Access Journals (Sweden)

Meçe Merita H.

2016-07-01

Full Text Available Human trafficking is a new phenomenon of Albanian post-socialist society which significantly increased during the difficult years of its transformation from centralized state-led economy to market economy. Both economic and political instability contributed to its size, nature and multiple dynamics. Drawing on a rights-based approach to human trafficking, this paper examines the effectiveness of the counter-trafficking response of the Albanian government with a special emphasis on prevention, protection and prosecution. Using secondary data and reviewing various country strategic documents, it highlights a range of weaknesses and challenges which have hindered its effectiveness over years. It concludes that successful and effective counter-trafficking response requires well rounded and coordinated gender sensitive, victim-centred, holistic and human rights-based efforts. Combined with adequate law enforcement, they will sustainably tackle the full spectrum of this problem.