Yuan, Xu; Yao, Jun; Norris, David; Tran, David D.; Bram, Richard J.; Chen, Gong; Luscher, Bernhard
Accumulation of GABAA receptors (GABAARs) at GABAergic synapses requires the cytoplasmic loop region and C-terminal transmembrane domain of the receptor γ2 subunit. We here report a novel interaction of γ2 with Calcium-Modulating cyclophilin Ligand (CAML), an integral membrane protein that regulates this mechanism. Interaction of GABAARs with CAML depends on both the cytoplasmic region and fourth transmembrane domain of the γ2 subunit, CAML immunoprecipitates with GABAARs from transfected cells and brain lysates and colocalizes with γ2 in ER vesicles in soma and dendrites of neurons. CAML shRNA treatment results in reduced expression of postsynaptic GABAARs, along with significant reductions in GABA-evoked whole-cell currents and GABAergic synaptic function, while glutamatergic transmission is unaffected. Reduced surface expression of GABAARs in CAML mutant neurons is associated with selective deficits in recycling of endocytosed GABAARs to the cell surface. Our results indicate a specific role of CAML in functional expression and endocytic recycling of postsynaptic GABAARs. PMID:18424167
Dietmar; Benke; Khaled; Zemoura; Patrick; J; Maier
Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.
Kleizen, B; Braakman, I; de Jonge, H R
The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Whereas a key role of cAMP-dependent phosphorylation in CFTR-channel gating has been firmly established, more recent studies have provided clear evidence for the existence of a second level of cAMP regulation, i.e. the exocytotic recruitment of CFFR to the plasma membrane and its endocytotic retrieval. Regulated trafficking of the CFTR Cl- channel has sofar been demonstrated only in a subset of CFTR-expressing cell types. However, with the introduction of more sensitive methods to measure CFTR cycling and submembrane localization, it might turn out to be a more general phenomenon that could contribute importantly to both the regulation of CFTR-mediated chloride transport itself and to the regulation of other transporters and CFTR-modulated cellular functions. This review aims to summarize the present state of knowledge regarding polarized and regulated CFTR trafficking and endosomal recycling in epithelial cells, to discuss present gaps in our understanding of these processes at the cellular and molecular level, and to consider its possible implications for cystic fibrosis.
Jackson, David G.
Lymphatic vessels play vital roles in immune surveillance and immune regulation by conveying antigen loaded dendritic cells, memory T cells, macrophages and neutrophils from the peripheral tissues to draining lymph nodes where they initiate as well as modify immune responses. Until relatively recently however, there was little understanding of how entry and migration through lymphatic vessels is organized or the specific molecular mechanisms that might be involved. Within the last decade, the situation has been transformed by an explosion of knowledge generated largely through the application of microscopic imaging, transgenic animals, specific markers and function blocking mAbs that is beginning to provide a rational conceptual framework. This article provides a critical review of the recent literature, highlighting seminal discoveries that have revealed the fascinating ultrastructure of leucocyte entry sites in lymphatic vessels, as well as generating controversies over the involvement of integrin adhesion, chemotactic and haptotactic mechanisms in DC entry under normal and inflamed conditions. It also discusses the major changes in lymphatic architecture that occur during inflammation and the different modes of leucocyte entry and trafficking within inflamed lymphatic vessels, as well as presenting a timely update on the likely role of hyaluronan and the major lymphatic endothelial hyaluronan receptor LYVE-1 in leucocyte transit.
Leo, M Dennis; Bannister, John P; Narayanan, Damodaran; Nair, Anitha; Grubbs, Jordan E; Gabrick, Kyle S; Boop, Frederick A; Jaggar, Jonathan H
Ion channels composed of pore-forming and auxiliary subunits control physiological functions in virtually all cell types. A conventional view is that channels assemble with their auxiliary subunits before anterograde plasma membrane trafficking of the protein complex. Whether the multisubunit composition of surface channels is fixed following protein synthesis or flexible and open to acute and, potentially, rapid modulation to control activity and cellular excitability is unclear. Arterial smooth muscle cells (myocytes) express large-conductance Ca(2+)-activated potassium (BK) channel α and auxiliary β1 subunits that are functionally significant modulators of arterial contractility. Here, we show that native BKα subunits are primarily (∼95%) plasma membrane-localized in human and rat arterial myocytes. In contrast, only a small fraction (∼10%) of total β1 subunits are located at the cell surface. Immunofluorescence resonance energy transfer microscopy demonstrated that intracellular β1 subunits are stored within Rab11A-postive recycling endosomes. Nitric oxide (NO), acting via cGMP-dependent protein kinase, and cAMP-dependent pathways stimulated rapid (≤1 min) anterograde trafficking of β1 subunit-containing recycling endosomes, which increased surface β1 almost threefold. These β1 subunits associated with surface-resident BKα proteins, elevating channel Ca(2+) sensitivity and activity. Our data also show that rapid β1 subunit anterograde trafficking is the primary mechanism by which NO activates myocyte BK channels and induces vasodilation. In summary, we show that rapid β1 subunit surface trafficking controls functional BK channel activity in arterial myocytes and vascular contractility. Conceivably, regulated auxiliary subunit trafficking may control ion channel activity in a wide variety of cell types.
Full Text Available The CFTR (cystic fibrosis transmembrane conductance regulator protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.
Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.
Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868
Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Yang, Jun-Mo [Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710 (Korea, Republic of); Kang, Jong-Sun, E-mail: firstname.lastname@example.org [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of); Hahn, Myong-Joon, E-mail: email@example.com [Department of Molecular Cell Biology, Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746 (Korea, Republic of)
Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.
Full Text Available Abstract Background Polycystin-2 (PC2, encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD, functions as a calcium (Ca2+ permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. Methods We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. Results We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R inhibition with 2-aminoethoxydiphenyl borate (2-APB or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. Conclusions These novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.
Prendergast, Jillian; Umanah, George K E; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G; Cole, Robert N; Huganir, Richard L; Dawson, Ted M; Dawson, Valina L; Schnaar, Ronald L
Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.
Braakman, L.J.; Kleizen, B.; Jonge, H.R. de
The cystic fibrosis transmembrane conductance regulator (CFTR), the ABC transporter encoded by the cystic fibrosis gene, is localized in the apical membrane of epithelial cells where it functions as a cyclic AMP-regulated chloride channel and as a regulator of other ion channels and transporters. Wh
Hoggatt, J; Pelus, L M
Hematopoietic stem cell (HSC) transplantation is a potentially curative treatment for numerous hematological malignancies. The transplant procedure as performed today takes advantage of HSC trafficking; either egress of HSC from the bone marrow to the peripheral blood, that is, mobilization, for acquisition of the hematopoietic graft, and/or trafficking of HSC from the peripheral blood to bone marrow niches in the recipient patient, that is HSC homing. Numerous studies, many of which are reviewed herein, have defined hematopoietic regulatory mechanisms mediated by the 20-carbon lipid family of eicosanoids, and recent evidence strongly supports a role for eicosanoids in regulation of hematopoietic trafficking, adding a new role whereby eicosanoids regulate hematopoiesis. Short-term exposure of HSC to the eicosanoid prostaglandin E(2) increases CXCR4 receptor expression, migration and in vivo homing of HSC. In contrast, cannabinoids reduce hematopoietic progenitor cell (HPC) CXCR4 expression and induce HPC mobilization when administered in vivo. Leukotrienes have been shown to alter CD34(+) cell adhesion, migration and regulate HSC proliferation, suggesting that eicosanoids have both opposing and complimentary roles in the regulation of hematopoiesis. As numerous FDA approved compounds regulate eicosanoid signaling or biosynthesis, the utility of eicosanoid-based therapeutic strategies to improve hematopoietic transplantation can be rapidly evaluated.
We have recently shown that an inhibition of sterol synthesis by fenpropimorph leads to an accumulation of sterol precursors, hydroxypalmitic acid-containing glucosylceramides and detergent resistant membranes in the Golgi bodies instead of the plasma membrane, suggesting that the individual molecules or the microdomains were blocked in the Golgi. These results and others from several eukaryotic models link lipid metabolism with membrane morphodynamics that are involved in membrane trafficking. Focus has been expanded to other lipid families, and numerous evidences are given showing lipids and lipid-modifying enzymes as key regulators of membrane homeostasis which can strongly regulate membrane morphodynamics and therefore trafficking. Beside protein-based machineries, lipid-based machineries are also shown as crucial regulatory forces involved in protein transport and sorting.
Trapani, Valentina; Luongo, Francesca; Arduini, Daniela; Wolf, Federica I
Magnesium is directly involved in the control of cell growth and survival, but its role in cancer biology and therapy is multifaceted; in particular, it is highly controversial whether magnesium levels can affect therapy outcomes. Here we investigated whether magnesium availability can modulate cellular responses to the widely used chemotherapeutic doxorubicin. We used an in vitro model consisting of mammary epithelial HC11 cells and found that high magnesium availability was correlated with diminished sensitivity both in cells chronically adapted to high magnesium concentrations and in acutely magnesium-supplemented cells. This decrease in sensitivity resulted from reduced intracellular doxorubicin accumulation in the face of a similar drug uptake rate. We observed that high-magnesium conditions caused a decrease in intracellular drug retention by altering drug lysosomal sequestration and trafficking. In our model, magnesium supplementation correspondingly modulated expression of the TRPM7 channel, which is known to control cytoskeletal organization and dynamics and may be involved in the proposed mechanism. Our findings suggest that magnesium supplementation in hypomagnesemic cancer patients may hinder response to therapy.
Chen-Izu, Ye; Shaw, Robin M; Pitt, Geoffrey S; Yarov-Yarovoy, Vladimir; Sack, Jon T; Abriel, Hugues; Aldrich, Richard W; Belardinelli, Luiz; Cannell, Mark B; Catterall, William A; Chazin, Walter J; Chiamvimonvat, Nipavan; Deschenes, Isabelle; Grandi, Eleonora; Hund, Thomas J; Izu, Leighton T; Maier, Lars S; Maltsev, Victor A; Marionneau, Celine; Mohler, Peter J; Rajamani, Sridharan; Rasmusson, Randall L; Sobie, Eric A; Clancy, Colleen E; Bers, Donald M
This paper is the second of a series of three reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on Na+ channel function and regulation, Na+ channel structure and function, and Na+ channel trafficking, sequestration and complexing. PMID:25772290
Yuan Wang; Wen-Hui Lin; Xu Chen; Hong-Wei Xue
Inositol polyphosphate 5-phosphatases (5PTases) are enzymes of phosphatidylinositoi metabolism that affect various aspects of plant growth and development. Arabidopsis 5PTasel3 regulates auxin homeostasis and hormone-related cotyledon vein development, and here we demonstrate that its knockout mutant 5pt13 has elevated sensitivity to gravistimulation in root gravitropic responses. The altered responses of 5pt13 mutants to 1-N-naphthylphthalamic acid (an auxin transport inhibitor) indicate that 5PTasel3 might be involved in the regulation of auxin transport. Indeed, the auxin efflux carrier PIN2 is expressed more broadly under 5PTasel3 deficiency, and observations of the internalization of the membrane-selective dye FM4-64 reveal altered vesicle trafficking in 5pt13 mutants. Compared with wild-type, 5pt13 mutant seedlings are less sensitive to the inhibition by brefeldin A of vesicle cycling, seedling growth, and the intracellular cycling of the PINI and PIN2 proteins. Further, auxin redistribution upon gravitropic stimulation is stimulated under 5PTasel3 deficiency. These results suggest that 5PTasel3 may modulate auxin trans-port by regulating vesicle trafficking and thereby play a role in root gravitropism.
Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M
HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.
Full Text Available There is an acute shortage of transplantable human organs worldwide. The current systems of organ procurement cannot supply in the demand. A new approach is imperative. While countries struggle to find ways of motivating more people to become organ donors, the international illegal black market is thriving. A possible solution to the problem in South Africa might be to change current legislation by confirming human organ trafficking a specific crime as is the case in the United Kingdom. However, more available organs for transplantation in the current recognised system are also essential. To achieve this, it is proposed that donors should be rewarded more effectively, or a regulated market in human organs should be allowed.
Bit Na Kang
Full Text Available Association of the neurotransmitter serotonin (5-HT with the pathogenesis of allergic asthma is well recognized and its role as a chemoattractant for eosinophils (Eos in vitro and in vivo has been previously demonstrated. Here we have examined the regulation of 5-HT-induced human and murine Eos trafficking and migration at a cellular and molecular level. Eos from allergic donors and bone marrow-derived murine Eos (BM-Eos were found to predominantly express the 5-HT2A receptor. Exposure to 5-HT or 2,5-dimethoxy-4-iodoamphetamine (DOI, a 5-HT2A/C selective agonist, induced rolling of human Eos and AML14.3D10 human Eos-like cells on vascular cell adhesion molecule (VCAM-1 under conditions of flow in vitro coupled with distinct cytoskeletal and cell shape changes as well as phosphorylation of MAPK. Blockade of 5-HT2A or of ROCK MAPK, PI3K, PKC and calmodulin, but not G(αi-proteins, with specific inhibitors inhibited DOI-induced rolling, actin polymerization and changes in morphology of VCAM-1-adherent AML14.3D10 cells. More extensive studies with murine BM-Eos demonstrated the role of 5-HT in promoting rolling in vivo within inflamed post-capillary venules of the mouse cremaster microcirculation and confirmed that down-stream signaling of 5-HT2A activation involves ROCK, MAPK, PI3K, PKC and calmodulin similar to AML14.3D10 cells. DOI-induced migration of BM-Eos is also dependent on these signaling molecules and requires Ca(2+. Further, activation of 5-HT2A with DOI led to an increase in intracellular Ca(2+ levels in murine BM-Eos. Overall, these data demonstrate that 5-HT (or DOI/5-HT2A interaction regulates Eos trafficking and migration by promoting actin polymerization associated with changes in cell shape/morphology that favor cellular trafficking and recruitment via activation of specific intracellular signaling molecules (ROCK, MAPK, PI3K and the PKC-calmodulin pathway.
Gleason, Ryan J; Vora, Mehul; Li, Ying; Kane, Nanci S; Liao, Kelvin; Padgett, Richard W
Signal transduction of the conserved transforming growth factor-β (TGFβ) family signaling pathway functions through two distinct serine/threonine transmembrane receptors, the type I and type II receptors. Endocytosis orchestrates the assembly of signaling complexes by coordinating the entry of receptors with their downstream signaling mediators. Recently, we showed that the C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6, part of the TGFβ family, is recycled through the retromer complex while the type II receptor, DAF-4 is recycled in a retromer-independent, ARF-6 dependent manner. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, we reported on SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. Here we show that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, multivesicular bodies (MVB), and the late endosome with a reduction in signaling strength. Our studies show that trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, we find that the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling.
Das, Manasmita; Singh, Raman Preet; Datir, Satyajit R; Jain, Sanyog
The present study explores the possibility of exploiting surface functionality as one of the key regulators for modulating the intracellular trafficking and therapeutic performance of drug loaded carbon nanotubes (CNTs). In line with that approach, a series of biofunctionalized multiwalled carbon nanotubes (f-CNTs 1-6) decorated with various functional molecules including antifouling polymer (PEG), tumor recognition modules (folic acid/hyaluronic acid/estradiol), and fluorophores (rhodamine B isothiocyanate/Alexa Fluor) were synthesized. By loading different anticancer agents (methotrexate (MTX), doxorubicin (DOX), and paclitaxel (PTX)) onto each functionalized CNT preparation, we tried to elucidate how the surface functional molecules associated with each f-CNT influence their therapeutic potential. We observed that antiproliferative or apoptotic activity of drug-loaded CNTs critically depends on their mechanistic pathway of cellular internalization and intracellular trafficking, which in turn had an intimate rapport with their surface chemistry. To our knowledge, for the first time, we have embarked on the possibility of using a surface chemistry dependent "switch" to remote-control the second and third order targeting of chemotherapeutic agents supramolecularly complexed/adsorbed on CNTs, which in turn is expected to benefit the development of futuristic nanobots for cancer theranostics.
Kanerva, Kristiina; Maekitie, Laura T. [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); Baeck, Nils [Department of Anatomy, Institute of Biomedicine, University of Helsinki, Helsinki (Finland); Andersson, Leif C., E-mail: firstname.lastname@example.org [Department of Pathology, Haartman Institute, University of Helsinki, Helsinki (Finland); HUSLAB, Helsinki (Finland); Department of Oncology and Pathology, Karolinska Institutet, Stockholm (Sweden)
Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.
Jana Krtková; Elizabeth B Thomas; Germain C. M. Alas; Schraner, Elisabeth M.; Behjatnia, Habib R; Hehl, Adrian B.; Paredez, Alexander R.
ABSTRACT Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmi...
Krtková, Jana; Elizabeth B Thomas; Germain C. M. Alas; Schraner, Elisabeth M.; Behjatnia, Habib R; Hehl, Adrian B.; Paredez, Alexander R.
UNLABELLED Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplas...
Cholon, Deborah M.; O'Neal, Wanda K.; Randell, Scott H.; Riordan, John R.
CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, ΔF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in ΔF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of ΔF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of ΔF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117
Huan, J; Hornick, N I; Goloviznina, N A; Kamimae-Lanning, A N; David, L L; Wilmarth, P A; Mori, T; Chevillet, J R; Narla, A; Roberts, C T; Loriaux, M M; Chang, B H; Kurre, P
We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-β and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.
Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang
-induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent...... alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic...... redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A...
Teske, Christian; Schweitzer, Christine; Palamidessi, Andrea; Aust, Daniela E; Scita, Giorgio; Weitz, Jürgen; Welsch, Thilo
KRAS is the key mutated gene in pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that KRas modulates endocytic uptake. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression in PDAC. Surprisingly, PANC-1 cells lacking KRas exhibited significantly enlarged early and late endosomes containing internalized dextran and epidermal growth factor. Endosome enlargement was accompanied by reduced endosomal degradation. Both KRas silencing and lysosomal blockade caused an upregulation of the master regulator of early endosome biogenesis, RAB5A, which is likely responsible for the expansion of the early endosomal compartment, because simultaneous KRAS/RAB5A knockdown abolished endosome enlargement. In contrast, early endosome shrinkage was seen in MIA PaCa-2 cells despite RAB5A upregulation, indicating that distinct KRas-modulated responses operate in different metabolic subtypes of PDAC. In conclusion, mutant KRAS promotes endosomal degradation in PDAC cell lines, which is impaired by KRAS silencing. Moreover, KRAS silencing activates RAB5A upregulation and drives PDAC subtype-dependent modulation of endosome trafficking. Copyright © 2017. Published by Elsevier Inc.
Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal
Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.
Starr, Tregei; Sun, Yi; Wilkins, Ngozi; Storrie, Brian
We used multiple approaches to investigate the coordination of trans and medial Rab proteins in the regulation of intra-Golgi retrograde trafficking. We reasoned that medially located Rab33b might act downstream of the trans Golgi Rab, Rab6, in regulating intra-Golgi retrograde trafficking. We found that knockdown of Rab33b, like Rab6, suppressed conserved oligomeric Golgi (COG) complex- or Zeste White 10 (ZW10)-depletion induced disruption of the Golgi ribbon in HeLa cells. Moreover, efficient GTP-restricted Rab6 induced relocation of Golgi enzymes to the endoplasmic reticulum (ER) was Rab33b-dependent, but not vice versa, suggesting that the two Rabs act sequentially in an intra-Golgi Rab cascade. In support of this hypothesis, we found that overexpression of GTP-Rab33b induced the dissociation of Rab6 from Golgi membranes in vivo. In addition, the transport of Shiga-like toxin B fragment (SLTB) from the trans to cis Golgi and ER required Rab33b. Surprisingly, depletion of Rab33b had little, if any, immediate effect on cell growth and multiplication. Furthermore, anterograde trafficking of tsO45G protein through the Golgi apparatus was normal. We suggest that the Rab33b/Rab6 regulated intra-Golgi retrograde trafficking pathway must coexist with other Golgi trafficking pathways. In conclusion, we provide the first evidence that Rab33b and Rab6 act to coordinate a major intra-Golgi retrograde trafficking pathway. This coordination may have parallels with Rab conversion/cascade events that regulate endosome, phagosome and exocytic processes.
Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G
Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.
Restituito, Sophie; Couve, Andrés; Bawagan, Hinayana; Jourdain, Sabine; Pangalos, Menelas N; Calver, Andrew R; Freeman, Katie B; Moss, Stephen J
gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.
Meraviglia, Veronica; Ulivi, Alessandro Francesco; Boccazzi, Marta; Valenza, Fabiola; Fratangeli, Alessandra; Passafaro, Maria; Lecca, Davide; Stagni, Fiorenza; Giacomini, Andrea; Bartesaghi, Renata; Abbracchio, Maria P; Ceruti, Stefania; Rosa, Patrizia
The G protein-coupled receptor 17 (GPR17) plays crucial roles in myelination. It is highly expressed during transition of oligodendrocyte progenitor cells to immature oligodendrocytes, but, after this stage, it must be down-regulated to allow generation of mature myelinating cells. After endocytosis, GPR17 is sorted into lysosomes for degradation or recycled to the plasma membrane. Balance between degradation and recycling is important for modulation of receptor levels at the cell surface and thus for the silencing/activation of GPR17-signaling pathways that, in turn, affect oligodendrocyte differentiation. The molecular mechanisms at the basis of these processes are still partially unknown and their characterization will allow a better understanding of myelination and provide cues to interpret the consequences of GPR17 dysfunction in diseases. Here, we demonstrate that the endocytic trafficking of GPR17 is mediated by the interaction of a type I PDZ-binding motif located at the C-terminus of the receptor and SNX27, a recently identified protein of the endosome-associated retromer complex and whose functions in oligodendrocytes have never been studied. SNX27 knock-down significantly reduces GPR17 plasma membrane recycling in differentiating oligodendrocytes while accelerating cells' terminal maturation. Interestingly, trisomy-linked down-regulation of SNX27 expression in the brain of Ts65Dn mice, a model of Down syndrome, correlates with a decrease in GPR17(+) cells and an increase in mature oligodendrocytes, which, however, fail in reaching full maturation, eventually leading to hypomyelination. Our data demonstrate that SNX27 modulates GPR17 plasma membrane recycling and stability, and that disruption of the SNX27/GPR17 interaction might contribute to pathological oligodendrocyte differentiation defects. GLIA 2016. GLIA 2016;64:1437-1460.
Estin, Miriam L; Thompson, Scott B; Traxinger, Brianna; Fisher, Marlie H; Friedman, Rachel S; Jacobelli, Jordan
Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.
During cell division, cells undergo membrane remodeling to achieve changes in their size and shape. In addition, cell division entails local delivery and retrieval of membranes and specific proteins as well as remodeling of cytoskeletons, in particular, upon cytokinetic abscission. Accumulating lines of evidence highlight that endocytic membrane removal from and subsequent membrane delivery to the plasma membrane are crucial for the changes in cell size and shape, and that trafficking of vesicles carrying specific proteins to the abscission site participate in local remodeling of membranes and cytoskeletons. Furthermore, the endosomal sorting complex required for transport (ESCRT) machinery has been shown to play crucial roles in cytokinetic abscission. Here, the author briefly overviews membrane-trafficking events early in cell division, and subsequently focus on regulation and functional significance of membrane trafficking involving Rab11 and Arf6 small GTPases in late cytokinesis phases and assembly of the ESCRT machinery in cytokinetic abscission.
Gault, William J; Olguin, Patricio; Weber, Ursula; Mlodzik, Marek
Cellular morphogenesis, including polarized outgrowth, promotes tissue shape and function. Polarized vesicle trafficking has emerged as a fundamental mechanism by which protein and membrane can be targeted to discrete subcellular domains to promote localized protrusions. Frizzled (Fz)/planar cell polarity (PCP) signaling orchestrates cytoskeletal polarization and drives morphogenetic changes in such contexts as the vertebrate body axis and external Drosophila melanogaster tissues. Although regulation of Fz/PCP signaling via vesicle trafficking has been identified, the interplay between the vesicle trafficking machinery and downstream terminal PCP-directed processes is less established. In this paper, we show that Drosophila CK1-γ/gilgamesh (gish) regulates the PCP-associated process of trichome formation through effects on Rab11-mediated vesicle recycling. Although the core Fz/PCP proteins dictate prehair formation broadly, CK1-γ/gish restricts nucleation to a single site. Moreover, CK1-γ/gish works in parallel with the Fz/PCP effector multiple wing hairs, which restricts prehair formation along the perpendicular axis to Gish. Our findings suggest that polarized Rab11-mediated vesicle trafficking regulated by CK1-γ is required for PCP-directed processes.
Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye
The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.
Full Text Available Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM. However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER and the Golgi apparatus-like encystation-specific vesicles (ESVs. Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA-tagged GlRac (HA-RacCA revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis.
Lek, Angela; Evesson, Frances J; Sutton, R Bryan; North, Kathryn N; Cooper, Sandra T
Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer-1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60-70 residue ferlin-specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue-specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle-associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.
Logan BASHLINE; Juan DU; Ying GU
Cellulose biosynthesis is a topic of intensive research not only due to the significance of cellulose in the integrity of plant cell walls,but also due to the potential of using cellulose,a natural carbon source,in the production ot biofuels.Characterization of the composition,regulation,and trafficking of cellulose synthase complexes (CSCs) is critical to an understanding of cellulose biosynthesis as well as the characterization of additional proteins that contribute to the production of cellulose either through direct interactions with CSCs or through indirect mechanisms.In this review,a highlight of a few proteins that appear to affect cellulose biosynthesis,which includes:KORRIGAN (KOR),Cellulose Synthase-Interactive Protein 1 (CSI1),and the poplar microtubule-associated protein,PttMAP20,will accompany a description of cellulose synthase (CESA) behavior and a discussion of CESA trafficking compartments that might act in the regulation of cellulose biosynthesis.
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Full Text Available Protein export from the endoplasmic reticulum (ER is an initial and rate-limiting step of molecular trafficking and secretion. This is mediated by coat protein II (COPII-coated vesicles, whose formation requires small GTPase Sar1 and 6 Sec proteins including Sec23 and Sec31. Sec31 is a component of the outer layer of COPII coat and has been identified as a phosphoprotein. The initiation and promotion of COPII vesicle formation is regulated by Sar1; however, the mechanism regulating the completion of COPII vesicle formation followed by vesicle release is largely unknown. Hypothesizing that the Sec31 phosphorylation may be such a mechanism, we identified phosphorylation sites in the middle linker region of Sec31. Sec31 phosphorylation appeared to decrease its association with ER membranes and Sec23. Non-phosphorylatable mutant of Sec31 stayed longer at ER exit sites and bound more strongly to Sec23. We also found that CK2 is one of the kinases responsible for Sec31 phosphorylation because CK2 knockdown decreased Sec31 phosphorylation, whereas CK2 overexpression increased Sec31 phosphorylation. Furthermore, CK2 knockdown increased affinity of Sec31 for Sec23 and inhibited ER-to-Golgi trafficking. These results suggest that Sec31 phosphorylation by CK2 controls the duration of COPII vesicle formation, which regulates ER-to-Golgi trafficking.
John R. Sinnamon; Kevin Czaplinski
Localized translation and the requisite trafficking of the mRNA template play significant roles in the nervous system including the establishment of dendrites and axons,axon path-finding,and synaptic plasticity.We provide a brief review on the regulation of localizing mRNA in mammalian neurons through critical posttranslational modifications of the factors involved.These examples highlight the relationship between mRNA trafficking and the translational regulation of trafficked mRNAs and provide insight into how extracellular signals target these events during signal transduction.
Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T; Cosgrove, Dominic
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 colocalization 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 subpools. The apically trafficked pool colocalized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associated with membrane microdomains and SNAP25. Moreover, coimmunoprecipitation 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.
Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan
The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.
Krtková, Jana; Thomas, Elizabeth B; Alas, Germain C M; Schraner, Elisabeth M; Behjatnia, Habib R; Hehl, Adrian B; Paredez, Alexander R
Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia's sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-Rac(CA)) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-Rac(CA)-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. The encystation process is crucial for the transmission of giardiasis and the life cycle of many protists. Encystation for Giardia lamblia involves the assembly of a protective cyst wall
Pentecost, Mickey; Vashisht, Ajay A; Lester, Talia; Voros, Tim; Beaty, Shannon M; Park, Arnold; Wang, Yao E; Yun, Tatyana E; Freiberg, Alexander N; Wohlschlegel, James A; Lee, Benhur
The paramyxovirus matrix (M) protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M) is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp), a leucine-rich nuclear export signal (NES), and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine). To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP) bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP) bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated nuclear
pools targeting each of 140 human trafficking genes were 89 TR-16-094 DISTRIBUTION STATEMENT A: Approved for public release; distribution is...remodeling in alphavirus-infected cells might 492 be important for trafficking of E1/E2. To test this hypothesis, primary human astrocytes were 493...An arrayed library targeting 140 trafficking genes (Dharmacon Human ON-TARGETplus 620 siRNA Library - Membrane Trafficking - SMARTpool, G-105500-05
Full Text Available Abstract Smad proteins are the intracellular mediators of transforming growth factor β (TGF-β signaling. Smads function as transcription factors and their activities require carboxyl-terminal phosphorylation by TGF-β receptor kinases which are embedded in the cell membrane. Therefore, the translocation of activated Smads from the cytoplasm into the nucleus is a rate-limiting step in TGF-β signal transduction into the nucleus. On the other hand, the export of Smads out of the nucleus turns off TGF-β effect. Such spatial control of Smad ensures a tight regulation of TGF-β target genes. Several cross-talk pathways have been shown to affect TGF-β signaling by impairing nuclear translocation of Smad, exemplifying the biological importance of the nuclear transport process. Many laboratories have investigated the underlying molecular mechanism of Smad nucleocytoplasmic translocation, combining genetics, biochemistry and sophisticated live cell imaging approaches. The last few years have witnessed the elucidation of several key players in Smad nuclear transport, most importantly the karyopherins that carry Smads across the nuclear envelope and nuclear pore proteins that facilitate the trans-nuclear envelope movement. The foundation is now set to further elucidate how the nuclear transport process is regulated and exploit such knowledge to manipulate TGF-β signaling. In this review we will discuss the current understanding of the molecular machinery responsible for nuclear import and export of Smads.
Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean
The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720
Vikram Anmol Kanda
Full Text Available Voltage-gated potassium (Kv channels shape the action potentials of excitable cells and regulate membrane potential and ion homeostasis in excitable and nonexcitable cells. With forty known members in the human genome and a variety of homomeric and heteromeric pore-forming alpha subunit interactions, post-translational modifications, cellular locations and expression patterns, the functional repertoire of the Kv alpha subunit family is monumental. This versatility is amplified by a host of interacting proteins, including the single membrane-spanning KCNE ancillary subunits. Here, examining both the secretory and the endocytic pathways, we review recent findings illustrating the surprising virtuosity of the KCNE proteins in orchestrating not just the function, but also the composition, diaspora and retrieval of channels formed by their Kv alpha subunit partners.
Dragone, Leonard L; Shaw, Laura A; Myers, Margaret D; Weiss, Arthur
Src-like adapter proteins (SLAP and SLAP-2) constitute a family of proteins that are expressed in a variety of cell types but are studied most extensively in lymphocytes. They have been shown to associate with proximal components of the T-cell receptor (TCR) and B-cell receptor (BCR) signaling complexes. An interaction of SLAP with c-Cbl leads to the ubiquitination and degradation of phosphorylated components of the TCR- and BCR-signaling complexes. The absence of this process in immature SLAP-deficient T and B cells leads to increased immunoreceptor levels due to decreased intracellular retention and degradation. We propose a model in which SLAP-dependent regulation of immunoreceptor levels allows for finer control of immunoreceptor signaling. Thus, SLAP functions to dampen immunoreceptor signaling, thereby influencing lymphocyte development and repertoire selection.
Tamma, Grazia; Lasorsa, Domenica; Ranieri, Marianna; Mastrofrancesco, Lisa; Valenti, Giovanna; Svelto, Maria
Aquaporin-2 (AQP2) increases the water permeability of renal collecting ducts in response to vasopressin. Vasopressin stimulation is accompanied by a profound remodeling of actin cytoskeleton whose dynamics are regulated by crosstalk between intracellular and extracellular signals. Here, we report that AQP2 contains a conserved RGD domain in its external C-loop. Co-immunoprecipitation experiments demonstrated that AQP2 binds integrin β1 in renal tissue and in MCD4 cells. To investigate the role of this interaction on AQP2 trafficking, cells were exposed to synthetic RGD-containing peptides, GRGDNP or GRGDSP, able to bind certain integrins. Incubation with these peptides increased the membrane expression of AQP2 in the absence of hormonal stimulation as assessed by confocal analysis and cell surface biotinylation. To identify the signals underlying the effects of peptides on AQP2 trafficking, some possible intracellular messengers were evaluated. Exposure of MCD4 cells to GRGDNP increased intracellular cAMP as assessed by FRET studies while GRGDSP increased intracellular calcium concentration. Taken together, these data propose integrins as new players controlling the cellular localization of AQP2, via two distinct signal transduction pathways dependent on cAMP and calcium respectively. Copyright © 2011 S. Karger AG, Basel.
Full Text Available Most immune cells, particularly phagocytes, express various receptors for the Fc-portion of the different immunoglobulin isotypes (Fc receptors, FcRs. By binding to the antibody, they provide a link between the adaptive immune system and the powerful effector functions triggered by innate immune cells such as mast cells, neutrophils, macrophages, and NK cells. Upon ligation of the immune complexes, the downstream signalling pathways initiated by the different receptors are quite similar for different FcR classes leading to the secretion of preformed and de novo synthesized pro-inflammatory mediators. FcR engagement also promotes negative signals through the combined action of several molecules that limit the extent and duration of positive signalling. To this regard, ligand-induced ubiquitination of Fc receptors for IgE (FcεR and IgG (FcγR has become recognized as a key modification that generates signals for the internalization and/or delivery of engaged receptor complexes to lysosomes or cytoplasmic proteasomes for degradation, providing negative-feedback regulation of Fc receptor activity.In this review, we discuss recent advances in our understanding of the molecular mechanisms that ensure the clearance of engaged Fcε and Fcγ receptor complexes from the cell surface with an emphasis given to the cooperation between the ubiquitin pathway and endosomal adaptors including the endosomal sorting complex required for transport (ESCRT in controlling receptor internalization and sorting along the endocytic compartments.
Tanokashira, Daisuke; Motoki, Kazumi; Minegishi, Seiji; Hosaka, Ai; Mamada, Naomi; Tamaoka, Akira; Okada, Takashi; Lakshmana, Madepalli K; Araki, Wataru
The β-secretase called BACE1 is a membrane-associated protease that initiates the generation of amyloid β-protein (Aβ), a key event in Alzheimer's disease (AD). However, the mechanism of intraneuronal regulation of BACE1 is poorly understood. Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons. We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aβ production. Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation. These findings establish that LRP1 specifically downregulates BACE1 by modulating its intraneuronal trafficking and stability through protein interaction and highlight LRP1 as a potential therapeutic target in AD.
Lavorgna, Alfonso; Harhaj, Edward William
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis.
Brignone, Maria S; Lanciotti, Angela; Visentin, Sergio; De Nuccio, Chiara; Molinari, Paola; Camerini, Serena; Diociaiuti, Marco; Petrini, Stefania; Minnone, Gaetana; Crescenzi, Marco; Laudiero, Luisa Bracci; Bertini, Enrico; Petrucci, Tamara C; Ambrosini, Elena
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis. Copyright © 2014. Published by Elsevier Inc.
Xu, Ming; Zhang, Qiufang; Li, Pin-Lan; Nguyen, Thaison; Li, Xiang; Zhang, Yang
Acid sphingomyelinase (ASM; gene symbol Smpd1) has been shown to play a crucial role in autophagy maturation by controlling lysosomal fusion with autophagosomes in coronary arterial smooth muscle cells (CASMCs). However, the underlying molecular mechanism by which ASM controls autophagolysosomal fusion remains unknown. In primary cultured CASMCs, lysosomal Ca2+ induced by 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer) was markedly attenuated by ASM deficiency or TRPML1 gene silencing suggesting that ASM signaling is required for TRPML1 channel activity and subsequent lysosomal Ca(2+) release. In these CASMCs, ASM deficiency or TRPML1 gene silencing markedly inhibited 7-Ket-induced dynein activation. In addition, 7-Ket-induced autophagosome trafficking, an event associated with lysosomal Ca(2+) release and dynein activity, was significantly inhibited in ASM-deficient (Smpd1(-/-)) CASMCs compared to that in Smpd1(+/+) CASMCs. Finally, overexpression of TRPML1 proteins restored 7-Ket-induced lysosomal Ca(2+) release and autophagosome trafficking in Smpd1-/- CASMCs. Collectively, these results suggest that ASM plays a critical role in regulating lysosomal TRPML1-Ca(2+) signaling and subsequent dynein-mediated autophagosome trafficking, which leads its role in controlling autophagy maturation in CASMCs under atherogenic stimulation.
Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia
Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release. PMID:24040321
Carson, Brian P; Del Bas, Josep Maria; Moreno-Navarrete, Jose Maria; Fernandez-Real, Jose Manuel; Mora, Silvia
Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs) in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.
Brian P Carson
Full Text Available Adiponectin is an adipokine secreted by white adipocytes involved in regulating insulin sensitivity in peripheral tissues. Secretion of adiponectin in adipocytes relies on the endosomal system, however, the intracellular machinery involved in mediating adiponectin release is unknown. We have previously reported that intracellular adiponectin partially compartmentalizes with rab 5 and rab11, markers for the early/sorting and recycling compartments respectively. Here we have examined the role of several rab11 downstream effector proteins (rab11 FIPs in regulating adiponectin trafficking and secretion. Overexpression of wild type rab11 FIP1, FIP3 and FIP5 decreased the amount of secreted adiponectin expressed in HEK293 cells, whereas overexpression of rab11 FIP2 or FIP4 had no effect. Furthermore shRNA-mediated depletion of FIP1 enhanced adiponectin release whereas knock down of FIP5 decreased adiponectin secretion. Knock down of FIP3 had no effect. In 3T3L1 adipocytes, endogenous FIP1 co-distributed intracellularly with endogenous adiponectin and FIP1 depletion enhanced adiponectin release without altering insulin-mediated trafficking of the glucose transporter Glut4. While adiponectin receptors internalized with transferrin receptors, there were no differences in transferrin receptor recycling between wild type and FIP1 depleted adipocytes. Consistent with its inhibitory role, FIP1 expression was decreased during adipocyte differentiation, by treatment with thiazolidinediones, and with increased BMI in humans. In contrast, FIP1 expression increased upon exposure of adipocytes to TNFα. In all, our findings identify FIP1 as a novel protein involved in the regulation of adiponectin trafficking and release.
Khairallah, Ramzi J; Khairallah, Maya; Gélinas, Roselle; Bouchard, Bertrand; Young, Martin E; Allen, Bruce G; Lopaschuk, Gary D; Deschepper, Christian F; Des Rosiers, Christine
While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the impact of its chronic activation on cardiac energy metabolism using mice overexpressing a constitutively active cytoplasmic guanylate cyclase (GC(+/0)) in cardiomyocytes. Ex vivo working GC(+/0) heart perfusions with (13)C-labeled substrates revealed an altered pattern of exogenous substrate fuel selection compared to controls, namely a 38+/-9% lower contribution of exogenous fatty acids to acetyl-CoA formation, while that of carbohydrates remains unchanged despite a two-fold increase in glycolysis. The lower contribution of exogenous fatty acids to energy production is not associated with changes in energy demand or supply (contractile function, oxygen consumption, tissue acetyl-CoA or CoA levels, citric acid cycle flux rate) or in the regulation of beta-oxidation (acetyl-CoA carboxylase activity, tissue malonyl-CoA levels). However, GC(+/0) hearts show a two-fold increase in the incorporation of exogenous oleate into triglycerides. Furthermore, the following molecular data are consistent with a concomitant increase in triglyceride hydrolysis: (i) increased abundance of hormone sensitive lipase (HSL) protein (24+/-11%) and mRNA (22+/-4%) as well as (ii) several phosphorylation events related to HSL inhibitory (AMPK) and activation (ERK 1/2) sites, which should contribute to enhance its activity. These changes in exogenous fatty acid trafficking in GC(+/0) hearts appear to be functionally relevant, as demonstrated by their resistance to fasting-induced triglyceride accumulation. While the documented metabolic profile of GC(+/0) mouse hearts is partly reminiscent of hypertrophied hearts, the observed changes in lipid trafficking have not been previously documented, and may
Samuel J. Hobbs
Full Text Available Selectins constitute a family of oligosaccharide binding proteins that play critical roles in regulating the trafficking of leukocytes. In T cells, L-selectin (CD62L controls the capacity for naive and memory T cells to actively survey peripheral lymph nodes, whereas P- and E-selectin capture activated T cells on inflamed vascular endothelium to initiate extravasation into non-lymphoid tissues. The capacity for T cells to interact with all of these selectins is dependent on the enzymatic synthesis of complex O-glycans, and thus, this protein modification plays an indispensable role in regulating the distribution and homing of both naive and previously activated T cells in vivo. In contrast to neutrophils, O-glycan synthesis is highly dynamic in T cell populations and is largely controlled by extracellular stimuli such as antigen recognition or signaling though cytokine receptors. Herein, we review the basic principles of enzymatic synthesis of complex O-glycans, discuss tools and reagents for studying this type of protein modification and highlight our current understanding of how O-glycan synthesis is regulated and subsequently impacts the trafficking potential of diverse T cell populations.
Full Text Available The paramyxovirus matrix (M protein is a molecular scaffold required for viral morphogenesis and budding at the plasma membrane. Transient nuclear residence of some M proteins hints at non-structural roles. However, little is known regarding the mechanisms that regulate the nuclear sojourn. Previously, we found that the nuclear-cytoplasmic trafficking of Nipah virus M (NiV-M is a prerequisite for budding, and is regulated by a bipartite nuclear localization signal (NLSbp, a leucine-rich nuclear export signal (NES, and monoubiquitination of the K258 residue within the NLSbp itself (NLSbp-lysine. To define whether the sequence determinants of nuclear trafficking identified in NiV-M are common among other Paramyxovirinae M proteins, we generated the homologous NES and NLSbp-lysine mutations in M proteins from the five major Paramyxovirinae genera. Using quantitative 3D confocal microscopy, we determined that the NES and NLSbp-lysine are required for the efficient nuclear export of the M proteins of Nipah virus, Hendra virus, Sendai virus, and Mumps virus. Pharmacological depletion of free ubiquitin or mutation of the conserved NLSbp-lysine to an arginine, which inhibits M ubiquitination, also results in nuclear and nucleolar retention of these M proteins. Recombinant Sendai virus (rSeV-eGFP bearing the NES or NLSbp-lysine M mutants rescued at similar efficiencies to wild type. However, foci of cells expressing the M mutants displayed marked fusogenicity in contrast to wild type, and infection did not spread. Recombinant Mumps virus (rMuV-eGFP bearing the homologous mutations showed similar defects in viral morphogenesis. Finally, shotgun proteomics experiments indicated that the interactomes of Paramyxovirinae M proteins are significantly enriched for components of the nuclear pore complex, nuclear transport receptors, and nucleolar proteins. We then synthesize our functional and proteomics data to propose a working model for the ubiquitin-regulated
Bae, Seong Han; Kim, Dong Hyun; Shin, Seok Kyo; Choi, Jin Sung; Park, Kang-Sik
The Kv3.1 channel plays a crucial role in regulating the high-frequency firing properties of neurons. Here, we determined whether Src regulates the subcellular distributions of the Kv3.1b channel. Co-expression of active Src induced a dramatic redistribution of Kv3.1b to the endoplasmic reticulum. Furthermore, co-expression of the Kv3.1b channel with active Src induced a remarkable decrease in the pool of Kv3.1b at the cell surface. Moreover, the co-expression of active Src results in a significant decrease in the peak current densities of the Kv3.1b channel, and a substantial alteration in the voltage dependence of its steady-state inactivation. Taken together, these results indicate that Src kinase may play an important role in regulating membrane trafficking of Kv3.1b channels.
Valenti, Giovanna; Procino, Giuseppe; Tamma, Grazia; Carmosino, Monica; Svelto, Maria
In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.
Bilan, Frédéric; Nacfer, Magali; Fresquet, Fleur; Norez, Caroline; Melin, Patricia; Martin-Berge, Alice; Costa de Beauregard, Marie-Alyette; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent
The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.
Feng, Tuancheng; Tammineni, Prasad; Agrawal, Chanchal; Jeong, Yu Young; Cai, Qian
β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the major neuronal β-secretase for amyloid-β generation and is degraded in lysosomes. The autophagy-lysosomal system plays a key role in the maintenance of cellular homeostasis in neurons. Recent studies established that nascent autophagosomes in distal axons move predominantly in the retrograde direction toward the soma, where mature lysosomes are mainly located. However, it remains unknown whether autophagy plays a critical role in regulation of BACE1 trafficking and degradation. Here, we report that induction of neuronal autophagy enhances BACE1 turnover, which is suppressed by lysosomal inhibition. A significant portion of BACE1 is recruited to the autophagy pathway and co-migrates robustly with autophagic vacuoles along axons. Moreover, we reveal that autophagic vacuole-associated BACE1 is accumulated in the distal axon of Alzheimer's disease-related mutant human APP transgenic neurons and mouse brains. Inducing autophagy in mutant human APP neurons augments autophagic retention of BACE1 in distal axons, leading to enhanced β-cleavage of APP. This phenotype can be reversed by Snapin-enhanced retrograde transport, which facilitates BACE1 trafficking to lysosomes for degradation. Therefore, our study provides new insights into autophagy-mediated regulation of BACE1 turnover and APP processing, thus building a foundation for future development of potential Alzheimer's disease therapeutic strategies.
Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P
Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane. © FASEB.
Banushi, Blerida; Forneris, Federico; Straatman-Iwanowska, Anna; Strange, Adam; Lyne, Anne-Marie; Rogerson, Clare; Burden, Jemima J.; Heywood, Wendy E.; Hanley, Joanna; Doykov, Ivan; Straatman, Kornelis R.; Smith, Holly; Bem, Danai; Kriston-Vizi, Janos; Ariceta, Gema; Risteli, Maija; Wang, Chunguang; Ardill, Rosalyn E.; Zaniew, Marcin; Latka-Grot, Julita; Waddington, Simon N.; Howe, S. J.; Ferraro, Francesco; Gjinovci, Asllan; Lawrence, Scott; Marsh, Mark; Girolami, Mark; Bozec, Laurent; Mills, Kevin; Gissen, Paul
Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues. PMID:27435297
Brewer, Paul Duffield; Habtemichael, Estifanos N; Romenskaia, Irina; Mastick, Cynthia Corley; Coster, Adelle C F
The trafficking kinetics of Glut4, the transferrin (Tf) receptor, and LRP1 were quantified in adipocytes and undifferentiated fibroblasts. Six steps were identified that determine steady state cell surface Glut4: (i) endocytosis, (ii) degradation, (iii) sorting, (iv) sequestration, (v) release, and (vi) tethering/docking/fusion. Endocytosis of Glut4 is 3 times slower than the Tf receptor in fibroblasts (ken = 0.2 min(-1) versus 0.6 min(-1)). Differentiation decreases Glut4 ken 40% (ken = 0.12 min(-1)). Differentiation also decreases Glut4 degradation, increasing total and cell surface Glut4 3-fold. In fibroblasts, Glut4 is recycled from endosomes through a slow constitutive pathway (kex = 0.025-0.038 min(-1)), not through the fast Tf receptor pathway (kex = 0.2 min(-1)). The kex measured in adipocytes after insulin stimulation is similar (kex = 0.027 min(-1)). Differentiation decreases the rate constant for sorting into the Glut4 recycling pathway (ksort) 3-fold. In adipocytes, Glut4 is also sorted from endosomes into a second exocytic pathway through Glut4 storage vesicles (GSVs). Surprisingly, transfer from endosomes into GSVs is highly regulated; insulin increases the rate constant for sequestration (kseq) 8-fold. Release from sequestration in GSVs is rate-limiting for Glut4 exocytosis in basal adipocytes. AS160 regulates this step. Tethering/docking/fusion of GSVs to the plasma membrane is regulated through an AS160-independent process. Insulin increases the rate of release and fusion of GSVs (kfuseG) 40-fold. LRP1 cycles with the Tf receptor and Glut4 in fibroblasts but predominantly with Glut4 after differentiation. Surprisingly, AS160 knockdown accelerated LRP1 exocytosis in basal and insulin-stimulated adipocytes. These data indicate that AS160 may regulate trafficking into as well as release from GSVs.
Radoshitzky, Sheli R; Pegoraro, Gianluca; Chī, Xi Olì; D Ng, Lián; Chiang, Chih-Yuan; Jozwick, Lucas; Clester, Jeremiah C; Cooper, Christopher L; Courier, Duane; Langan, David P; Underwood, Knashka; Kuehl, Kathleen A; Sun, Mei G; Caì, Yíngyún; Yú, Shu Qìng; Burk, Robin; Zamani, Rouzbeh; Kota, Krishna; Kuhn, Jens H; Bavari, Sina
Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.
Sheli R Radoshitzky
Full Text Available Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2 expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.
Lu, Wei; Ziff, Edward B
PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.
VPS35, a major component of the retromer, plays an important role in the selective endosome-to-Golgi retrieval of membrane proteins. Dysfunction of retromer is a risk factor for neurodegenerative disorders, but its function in developing mouse brain remains poorly understood. Here we provide evidence for VPS35 promoting dendritic growth and maturation, and axonal protein transport in developing mouse hippocampal neurons. Embryonic hippocampal CA1 neurons suppressing Vps35 expression by in utero electroporation of its micro RNAs displayed shortened apical dendrites, reduced dendritic spines, and swollen commissural axons in the neonatal stage, those deficits reflecting a defective protein transport/trafficking in developing mouse neurons. Further mechanistic studies showed that Vps35 depletion in neurons resulted in an impaired retrograde trafficking of BACE1 (β1-secretase and altered BACE1 distribution. Suppression of BACE1 expression in CA1 neurons partially rescued both dendritic and axonal deficits induced by Vps35-deficiency. These results thus demonstrate that BACE1 acts as a critical cargo of retromer in vitro and in vivo, and suggest that VPS35 plays an essential role in regulating apical dendritic maturation and in preventing axonal spheroid formation in developing hippocampal neurons.
Alves, Daiane S; Thulin, Gunilla; Loffing, Johannes; Kashgarian, Michael; Caplan, Michael J
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.
Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François
Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.
Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François
Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383
Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R
Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.
Brant M Wagener
Full Text Available Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2 mutant deficient in Src kinase binding (arr2-P91G/P121E. Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.
Ardura, Juan A.; Wang, Bin; Watkins, Simon C.; Vilardaga, Jean-Pierre; Friedman, Peter A.
Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner. PMID:21832055
Ardura, Juan A; Wang, Bin; Watkins, Simon C; Vilardaga, Jean-Pierre; Friedman, Peter A
Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.
Polarized and directional growth of pollen tubes is the only means by which immotile sperm of flowering plants reach the deeply embedded female gametes for fertilization. Vesicle trafficking is among the most critical cellular activities for pollen tube growth. Vesicle trafficking maintains membrane...
Cam Judy A
Full Text Available Abstract Amyloid-β peptide (Aβ accumulation in the brain is an early, toxic event in the pathogenesis of Alzheimer's disease (AD. Aβ is produced by proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP, by β- and γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Recent studies have shown that members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apolipoprotein E (apoE receptor 2, interact with APP and regulate its endocytic trafficking. Another common feature of these receptors is their ability to bind apoE, which exists in three isoforms in humans and the presence of the ε4 allele represents a genetic risk factor for AD. In this review, we summarize the current understanding of the function of these apoE receptors with a focus on their role in APP trafficking and processing. Knowledge of the interactions between these distinct low-density lipoprotein receptor family members and APP may ultimately influence future therapies for AD.
Solis, Gonzalo P; Schrock, Yvonne; Hülsbusch, Nikola; Wiechers, Marianne; Plattner, Helmut; Stuermer, Claudia A O
The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.
Anna E Daniel
Full Text Available Plasminogen activator inhibitor-1 (PAI-1, a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.
Rameau, Gerald A; Tukey, David S; Garcin-Hosfield, Elsa D; Titcombe, Roseann F; Misra, Charu; Khatri, Latika; Getzoff, Elizabeth D; Ziff, Edward B
Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS.
Full Text Available Abstract Background Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process. Results We show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking. Conclusions Our results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for
Gardner, Thomas J; Hernandez, Rosmel E; Noriega, Vanessa M; Tortorella, Domenico
The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.
Rogan, Mark P
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?
Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.
Watson, Leah J; Rossi, Guendalina; Brennwald, Patrick
Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post-Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP-tag placed on the N-terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post-Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.
Wasik, Anita A; Polianskyte-Prause, Zydrune; Dong, Meng-Qiu; Shaw, Andrey S; Yates, John R; Farquhar, Marilyn G; Lehtonen, Sanna
Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.
Kioumourtzoglou, Dimitrios; Pryor, Paul R; Gould, Gwyn W; Bryant, Nia J
Insulin-stimulated delivery of glucose transporters (GLUT4, also known as SLC2A4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose regulation. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits through numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin (also known as synaptogyrin-2) and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here, we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin-containing to sortilin-containing membranes, and that cellugyrin-containing IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-containing membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs.
Wakeel, Abdul; Kuriakose, Jeeba A; McBride, Jere W
Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.
Hori, Sharon S; Kurland, Irwin J; DiStefano, Joseph J
Evidence indicates that endosomal insulin receptor (IR) trafficking plays a role in regulating insulin signal transduction. To evaluate its importance, we developed a series of biokinetic models for quantifying activated surface and endosomal IR dynamics from published experimental data. Starting with a published two-compartment Fao hepatoma model, a four-pool model was formulated that depicts IR autophosphorylation after receptor binding, IR endosomal internalization/trafficking, insulin dissociation from and dephosphorylation of internalized IR, and recycling of unliganded, dephosphorylated IR to the plasma membrane. Quantification required three additional data sets, two measured, but unmodeled by the same group. A five-pool model created to include endosomal trafficking of the nonphosphorylated insulin-IR complex was fitted using the same data sets, augmented with another published data set. Creation of a six-pool model added the physiologically relevant dissociation of insulin ligand from the activated endosomal IR. More importantly, all three models, validated against additional data not used in model fitting, predict that, mechanistically, internalization of activated IR is a rate-limiting step, at least under the receptor saturating conditions of the fitting data. This rate includes the transit time to a site where insulin dissociation from and/or dephosphorylation of the IR occurs by docking with protein-tyrosine phosphatases (PTPases), or where a sufficient conformational change occurs in the IR, perhaps due to insulin-IR dissociation, where associated PTPases may complete IR dephosphorylation. Our new models indicate that key events in endosomal IR trafficking have significance in mediating IR activity, possibly serving to regulate insulin signal transduction.
Ulrich, Florian; Heisenberg, Carl-Philipp
The migration of single cells and epithelial sheets is of great importance for gastrulation and organ formation in developing embryos and, if misregulated, can have dire consequences e.g. during cancer metastasis. A keystone of cell migration is the regulation of adhesive contacts, which are dynamically assembled and disassembled via endocytosis. Here, we discuss some of the basic concepts about the function of endocytic trafficking during cell migration: transport of integrins from the cell rear to the leading edge in fibroblasts; confinement of signalling to the front of single cells by endocytic transport of growth factors; regulation of movement coherence in multicellular sheets by cadherin turnover; and shaping of extracellular chemokine gradients. Taken together, endocytosis enables migrating cells and tissues to dynamically modulate their adhesion and signalling, allowing them to efficiently migrate through their extracellular environment.
Conte, Ianina L; Hellen, Nicola; Bierings, Ruben; Mashanov, Gregory I; Manneville, Jean-Baptiste; Kiskin, Nikolai I; Hannah, Matthew J; Molloy, Justin E; Carter, Tom
Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.
Wieman, Heather L.; Wofford, Jessica A.
Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization. PMID:17301289
Ayanna J Flegler
Full Text Available CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.
Flegler, Ayanna J; Cianci, Gianguido C; Hope, Thomas J
CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.
Marubashi, Soujiro; Shimada, Hikaru; Fukuda, Mitsunori; Ohbayashi, Norihiko
Two cell type-specific Rab proteins, Rab32 and Rab38 (Rab32/38), have been proposed as regulating the trafficking of melanogenic enzymes, including tyrosinase and tyrosinase-related protein 1 (Tyrp1), to melanosomes in melanocytes. Like other GTPases, Rab32/38 function as switch molecules that cycle between a GDP-bound inactive form and a GTP-bound active form; the cycle is thought to be regulated by an activating enzyme, guanine nucleotide exchange factor (GEF), and an inactivating enzyme, GTPase-activating protein (GAP), which stimulates the GTPase activity of Rab32/38. Although BLOC-3 has already been identified as a Rab32/38-specific GEF that regulates the trafficking of tyrosinase and Tyrp1, no physiological GAP for Rab32/38 in melanocytes has ever been identified, and it has remained unclear whether Rab32/38 is involved in the trafficking of dopachrome tautomerase, another melanogenic enzyme, in mouse melanocytes. In this study we investigated RUTBC1, which was originally characterized as a Rab9-binding protein and GAP for Rab32 and Rab33B in vitro, and the results demonstrated that RUTBC1 functions as a physiological GAP for Rab32/38 in the trafficking of all three melanogenic enzymes in mouse melanocytes. The results of this study also demonstrated the involvement of Rab9A in the regulation of the RUTBC1 localization and in the trafficking of all three melanogenic enzymes. We discovered that either excess activation or inactivation of Rab32/38 achieved by manipulating RUTBC1 inhibits the trafficking of all three melanogenic enzymes. These results collectively indicate that proper spatiotemporal regulation of Rab32/38 is essential for the trafficking of all three melanogenic enzymes in mouse melanocytes.
Hallstrom, Kelly N; McCormick, Beth A
Salmonella enterica Typhimurium employs type III secreted effectors to induce cellular invasion and pathogenesis. We previously reported the secreted effector SipA is in part responsible for inducing the apical accumulation of the host membrane protein PERP, a host factor we have shown is key to the inflammatory response induced by Salmonella. We now report that the S. Typhimurium type III secreted effector SipC significantly contributes to PERP redistribution to the apical membrane surface. To our knowledge, this is the first report demonstrating a role for SipC in directing the trafficking of a host membrane protein to the cell surface. In sum, facilitation of PERP trafficking appears to be a result of type III secreted effector-mediated recruitment of vesicles to the apical surface. Our study therefore reveals a new role for SipC, and builds upon previous reports suggesting recruitment of vesicles to the cell surface is important for Salmonella invasion.
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.
Inada, Noriko; Betsuyaku, Shigeyuki; Shimada, Takashi L; Ebine, Kazuo; Ito, Emi; Kutsuna, Natsumaro; Hasezawa, Seiichiro; Takano, Yoshitaka; Fukuda, Hiroo; Nakano, Akihiko; Ueda, Takashi
RAB5 is a small GTPase that acts in endosomal trafficking. In addition to canonical RAB5 members that are homologous to animal RAB5, land plants harbor a plant-specific RAB5, the ARA6 group, which regulates trafficking events distinct from canonical RAB5 GTPases. Here, we report that plant RAB5, both canonical and plant-specific members, accumulate at the interface between host plants and biotrophic fungal and oomycete pathogens. Biotrophic fungi and oomycetes colonize living plant tissues by establishing specialized infection hyphae, the haustorium, within host plant cells. We found that Arabidopsis thaliana ARA6/RABF1, a plant-specific RAB5, is localized to the specialized membrane that surrounds the haustorium, the extrahaustorial membrane (EHM), formed by the A. thaliana-adapted powdery mildew fungus Golovinomyces orontii Whereas the conventional RAB5 ARA7/RABF2b was also localized to the EHM, endosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and RAB5-activating proteins were not, which suggests that the EHM has modified endosomal characteristic. The recruitment of host RAB5 to the EHM was a property shared by the barley-adapted powdery mildew fungus Blumeria graminis f.sp. hordei and the oomycete Hyaloperonospora arabidopsidis, but the extrahyphal membrane surrounding the hypha of the hemibiotrophic fungus Colletotrichum higginsianum at the biotrophic stage was devoid of RAB5. The localization of RAB5 to the EHM appears to correlate with the functionality of the haustorium. Our discovery sheds light on a novel relationship between plant RAB5 and obligate biotrophic pathogens.
Diana P Wehrendt
Full Text Available It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4 had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF, an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface.
Leslie K Climer
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.
Cuesta-Marbán, Álvaro; Botet, Javier; Czyz, Ola; Cacharro, Luis M.; Gajate, Consuelo; Hornillos, Valentín; Delgado, Javier; Zhang, Hui; Amat-Guerri, Francisco; Acuña, A. Ulises; McMaster, Christopher R.; Revuelta, José Luis; Zaremberg, Vanina; Mollinedo, Faustino
The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane. PMID:23335509
Cuesta-Marbán, Álvaro; Botet, Javier; Czyz, Ola; Cacharro, Luis M; Gajate, Consuelo; Hornillos, Valentín; Delgado, Javier; Zhang, Hui; Amat-Guerri, Francisco; Acuña, A Ulises; McMaster, Christopher R; Revuelta, José Luis; Zaremberg, Vanina; Mollinedo, Faustino
The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.
Nozaki, Shohei; Katoh, Yohei; Terada, Masaya; Michisaka, Saki; Funabashi, Teruki; Takahashi, Senye; Kontani, Kenji; Nakayama, Kazuhisa
ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46 -: IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E. © 2017. Published by The Company of Biologists Ltd.
Sun, Yi; Shestakova, Anna; Hunt, Lauren; Sehgal, Siddharth; Lupashin, Vladimir; Storrie, Brian
We used multiple approaches to investigate the role of Rab6 relative to Zeste White 10 (ZW10), a mitotic checkpoint protein implicated in Golgi/endoplasmic reticulum (ER) trafficking/transport, and conserved oligomeric Golgi (COG) complex, a putative tether in retrograde, intra-Golgi trafficking. ZW10 depletion resulted in a central, disconnected cluster of Golgi elements and inhibition of ERGIC53 and Golgi enzyme recycling to ER. Small interfering RNA (siRNA) against RINT-1, a protein linker between ZW10 and the ER soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 18, produced similar Golgi disruption. COG3 depletion fragmented the Golgi and produced vesicles; vesicle formation was unaffected by codepletion of ZW10 along with COG, suggesting ZW10 and COG act separately. Rab6 depletion did not significantly affect Golgi ribbon organization. Epistatic depletion of Rab6 inhibited the Golgi-disruptive effects of ZW10/RINT-1 siRNA or COG inactivation by siRNA or antibodies. Dominant-negative expression of guanosine diphosphate-Rab6 suppressed ZW10 knockdown induced-Golgi disruption. No cross-talk was observed between Rab6 and endosomal Rab5, and Rab6 depletion failed to suppress p115 (anterograde tether) knockdown-induced Golgi disruption. Dominant-negative expression of a C-terminal fragment of Bicaudal D, a linker between Rab6 and dynactin/dynein, suppressed ZW10, but not COG, knockdown-induced Golgi disruption. We conclude that Rab6 regulates distinct Golgi trafficking pathways involving two separate protein complexes: ZW10/RINT-1 and COG.
Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F; Bahnson, Brian J
The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response.
Liem, Karel F; Ashe, Alyson; He, Mu; Satir, Peter; Moran, Jennifer; Beier, David; Wicking, Carol; Anderson, Kathryn V
Two intraflagellar transport (IFT) complexes, IFT-A and IFT-B, build and maintain primary cilia and are required for activity of the Sonic hedgehog (Shh) pathway. A weak allele of the IFT-A gene, Ift144, caused subtle defects in cilia structure and ectopic activation of the Shh pathway. In contrast, strong loss of IFT-A, caused by either absence of Ift144 or mutations in two IFT-A genes, blocked normal ciliogenesis and decreased Shh signaling. In strong IFT-A mutants, the Shh pathway proteins Gli2, Sufu, and Kif7 localized correctly to cilia tips, suggesting that these pathway components were trafficked by IFT-B. In contrast, the membrane proteins Arl13b, ACIII, and Smo failed to localize to primary cilia in the absence of IFT-A. We propose that the increased Shh activity seen in partial loss-of-function IFT-A mutants may be a result of decreased ciliary ACIII and that the loss of Shh activity in the absence of IFT-A is a result of severe disruptions of cilia structure and membrane protein trafficking.
Björk, Susann; Hurt, Carl M; Ho, Vincent K; Angelotti, Timothy
Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins
Weiss, Ido D; Shoham, Hadas; Wald, Ori; Wald, Hanna; Beider, Katia; Abraham, Michal; Barashi, Neta; Galun, Eithan; Nagler, Arnon; Peled, Amnon
Chemokines were shown to govern the trafficking of immune cells and may also play important roles in the survival and activation of these cells. We report here that under physiological conditions, the bone marrow (BM), spleen, blood and liver of Ccr5, but not of Ccr1-deficient mice, contain reduced numbers of NK cells. NK cells in the BM of Ccr5-deficient mice proliferate to a lesser extent compared to WT mice. Furthermore, spleen NK cells derived from Ccr5-deficient mice that were transplanted into irradiated recipients failed to proliferate in the host. Ccr5, but not Ccr1-deficient NK cells, failed to migrate in vitro in response to RANTES and MIP-1β but not MIP-1β or SDF-1 and had reduced activation, lower expression levels of NK cell markers and a slightly reduced capacity to adhere to target cells and stimulate their killing. Using the polyI:C mouse model for NK trafficking, we found that in the absence of Ccr5, but not Ccr1, NK cells failed to accumulate in the liver. In contrast, using the influenza viral infection as a model to evaluate NK cell proliferation, we found that Ccr5-deficient NK cells in the BM had a higher proliferation rate than WT NK cells. These results suggest a role for Ccr5 in NK cell proliferation and circulation under physiological conditions and a complex role for Ccr5 in determining the fate of NK cells under pathological conditions. Copyright © 2011. Published by Elsevier Ltd.
Galli, Mary; Liu, Qiujie; Moss, Britney L; Malcomber, Simon; Li, Wei; Gaines, Craig; Federici, Silvia; Roshkovan, Jessica; Meeley, Robert; Nemhauser, Jennifer L; Gallavotti, Andrea
In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.
Shin, Jae-Won; Discher, Dennis E
Clinical success with human hematopoietic stem cell (HSC) transplantation establishes a paradigm for regenerative therapies with other types of stem cells. However, it remains generally challenging to therapeutically treat tissues after engineering of stem cells in vitro. Recent studies suggest that stem and progenitor cells sense physical features of their niches. Here, we review biophysical contributions to lineage decisions, maturation, and trafficking of blood and immune cells. Polarized cellular contractility and nuclear rheology are separately shown to be functional markers of a hematopoietic hierarchy that predict the ability of a lineage to traffic in and out of the bone marrow niche. These biophysical determinants are regulated by a set of structural molecules, including cytoplasmic myosin-II and nuclear lamins, which themselves are modulated by a diverse range of transcriptional and post-translational mechanisms. Small molecules that target these mechanobiological circuits, along with novel bioengineering methods, could prove broadly useful in programming blood and immune cells for therapies ranging from blood transfusions to immune attack of tumors.
Environmental regulations can have a significant impact on product use, disposal, and recycling. This report summarizes the basic aspects of current federal, state and international regulations which apply to end-of-life photovoltaic (PV) modules and PV manufacturing scrap destined for disposal or recycling. It also discusses proposed regulations for electronics that may set the ground of what is to be expected in this area in the near future. In the US, several states have started programs to support the recycling of electronic equipment, and materials destined for recycling often are excepted from solid waste regulations during the collection, transfer, storage and processing stages. California regulations are described separately because they are different from those of most other states. International agreements on the movement of waste between different countries may pose barriers to cross-border shipments. Currently waste moves freely among country members of the Organization of Economic Cooperation and Development (OECD), and between the US and the four countries with which the US has bilateral agreements. However, it is expected, that the US will adopt the rules of the Basel Convention (an agreement which currently applies to 128 countries but not the US) and that the Convection's waste classification system will influence the current OECD waste-handling system. Some countries adopting the Basel Convention consider end-of-life electronics to be hazardous waste, whereas the OECD countries consider them to be non-hazardous. Also, waste management regulations potentially affecting electronics in Germany and Japan are mentioned in this report.
Wilson, David McKay
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…
Takeshita, W.M.; Gushiken, V.O.; Ferreira-Duarte, A.P.; Pinheiro-Torres, A.S.; Roncalho-Buck, I.A. [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil); Squebola-Cola, D.M.; Mello, G.C.; Anhê, G.F.; Antunes, E. [Department of Pharmacology, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, SP (Brazil); DeSouza, I.A., E-mail: email@example.com [Department of Biology and Physiology, Faculty of Medicine of Jundiai (FMJ), Jundiai, SP (Brazil)
Pulmonary neutrophil infiltration produced by Staphylococcal enterotoxin A (SEA) airway exposure is accompanied by marked granulocyte accumulation in bone marrow (BM). Therefore, the aim of this study was to investigate the mechanisms of BM cell accumulation, and trafficking to circulating blood and lung tissue after SEA airway exposure. Male BALB/C mice were intranasally exposed to SEA (1 μg), and at 4, 12 and 24 h thereafter, BM, circulating blood, bronchoalveolar lavage (BAL) fluid and lung tissue were collected. Adhesion of BM granulocytes and flow cytometry for MAC-1, LFA1-α and VLA-4 and cytokine and/or chemokine levels were assayed after SEA-airway exposure. Prior exposure to SEA promoted a marked PMN influx to BAL and lung tissue, which was accompanied by increased counts of immature and/or mature neutrophils and eosinophils in BM, along with blood neutrophilia. Airway exposure to SEA enhanced BM neutrophil MAC-1 expression, and adhesion to VCAM-1 and/or ICAM-1-coated plates. Elevated levels of GM-CSF, G-CSF, INF-γ, TNF-α, KC/CXCL-1 and SDF-1α were detected in BM after SEA exposure. SEA exposure increased production of eosinopoietic cytokines (eotaxin and IL-5) and BM eosinophil VLA-4 expression, but it failed to affect eosinophil adhesion to VCAM-1 and ICAM-1. In conclusion, BM neutrophil accumulation after SEA exposure takes place by integrated action of cytokines and/or chemokines, enhancing the adhesive responses of BM neutrophils and its trafficking to lung tissues, leading to acute lung injury. BM eosinophil accumulation in SEA-induced acute lung injury may occur via increased eosinopoietic cytokines and VLA-4 expression. - Highlights: • Airway exposure to SEA causes acute lung inflammation. • SEA induces accumulation of bone marrow (BM) in immature and mature neutrophils. • SEA increases BM granulocyte or BM PMN adhesion to ICAM-1 and VCAM-1, and MAC-1 expression. • SEA induces BM elevations of CXCL-1, INF-γ, TNF-α, GM-CSF, G-CSF and
Full Text Available RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.
Chan, Audrey S M; Clairfeuille, Thomas; Landao-Bassonga, Euphemie; Kinna, Genevieve; Ng, Pei Ying; Loo, Li Shen; Cheng, Tak Sum; Zheng, Minghao; Hong, Wanjin; Teasdale, Rohan D; Collins, Brett M; Pavlos, Nathan J
The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that sorting nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development. © 2016 Chan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Full Text Available Neurons rely on the long-range transport of several signalling molecules such as neurotrophins and their receptors, which are required for neuronal development, function and survival. However, the nature of the machinery controlling the trafficking of signalling endosomes containing activated neurotrophin receptors has not yet been completely elucidated. We aimed to identify new players involved in the dynamics of neurotrophin signalling endosomes using a high-throughput unbiased siRNA screening approach to quantify the intracellular accumulation of two fluorescently tagged reporters: the binding fragment of tetanus neurotoxin (HCT, and an antibody directed against the neurotrophin receptor p75NTR. This screen performed in motor neurons differentiated from mouse embryonic stem (ES cells identified a number of candidate genes encoding molecular motors and motor adaptor proteins involved in regulating the intracellular trafficking of these probes. Bicaudal D homolog 1 (BICD1, a molecular motor adaptor with pleiotropic roles in intracellular trafficking, was selected for further analyses, which revealed that BICD1 regulates the intracellular trafficking of HCT and neurotrophin receptors and likely plays an important role in nervous system development and function.
Full Text Available Background: We recently reported that aquaporin 5 (AQP5, a water channel never identified in the kidney before, co-localizes with pendrin at the apical membrane of type-B intercalated cells in the kidney cortex. Since co-expression of AQP5 and pendrin in the apical membrane domain is a common feature of several other epithelia such as cochlear and bronchial epithelial cells, we evaluated here whether this strict membrane association may reflect a co-regulation of the two proteins. To investigate this possibility, we analyzed AQP5 and pendrin expression and trafficking in mice under chronic K+ depletion, a condition that results in an increased ability of renal tubule to reabsorb bicarbonate, often leads to metabolic alkalosis and is known to strongly reduce pendrin expression. Methods: Mice were housed in metabolic cages and pair-fed with either a standard laboratory chow or a K+-deficient diet. AQP5 abundance was assessed by western blot in whole kidney homogenates and AQP5 and pendrin were localized by confocal microscopy in kidney sections from those mice. In addition, the short-term effect of changes in external pH on pendrin trafficking was evaluated by fluorescence resonance energy transfer (FRET in MDCK cells, and the functional activity of pendrin was tested in the presence and absence of AQP5 in HEK 293 Phoenix cells. Results: Chronic K+ depletion caused a strong reduction in pendrin and AQP5 expression. Moreover, both proteins shifted from the apical cell membrane to an intracellular compartment. An acute pH shift from 7.4 to 7.0 caused pendrin internalization from the plasma membrane. Conversely, a pH shift from 7.4 to 7.8 caused a significant increase in the cell surface expression of pendrin. Finally, pendrin ion transport activity was not affected by co-expression with AQP5. Conclusions: The co-regulation of pendrin and AQP5 membrane expression under chronic K+-deficiency indicates that these two molecules could cooperate as an
Procino, Giuseppe; Milano, Serena; Tamma, Grazia; Dossena, Silvia; Barbieri, Claudia; Nicoletti, Maria Celeste; Ranieri, Marianna; Di Mise, Annarita; Nofziger, Charity; Svelto, Maria; Paulmichl, Markus; Valenti, Giovanna
We recently reported that aquaporin 5 (AQP5), a water channel never identified in the kidney before, co-localizes with pendrin at the apical membrane of type-B intercalated cells in the kidney cortex. Since co-expression of AQP5 and pendrin in the apical membrane domain is a common feature of several other epithelia such as cochlear and bronchial epithelial cells, we evaluated here whether this strict membrane association may reflect a co-regulation of the two proteins. To investigate this possibility, we analyzed AQP5 and pendrin expression and trafficking in mice under chronic K(+) depletion, a condition that results in an increased ability of renal tubule to reabsorb bicarbonate, often leads to metabolic alkalosis and is known to strongly reduce pendrin expression. Mice were housed in metabolic cages and pair-fed with either a standard laboratory chow or a K(+)-deficient diet. AQP5 abundance was assessed by western blot in whole kidney homogenates and AQP5 and pendrin were localized by confocal microscopy in kidney sections from those mice. In addition, the short-term effect of changes in external pH on pendrin trafficking was evaluated by fluorescence resonance energy transfer (FRET) in MDCK cells, and the functional activity of pendrin was tested in the presence and absence of AQP5 in HEK 293 Phoenix cells. Chronic K(+) depletion caused a strong reduction in pendrin and AQP5 expression. Moreover, both proteins shifted from the apical cell membrane to an intracellular compartment. An acute pH shift from 7.4 to 7.0 caused pendrin internalization from the plasma membrane. Conversely, a pH shift from 7.4 to 7.8 caused a significant increase in the cell surface expression of pendrin. Finally, pendrin ion transport activity was not affected by co-expression with AQP5. The co-regulation of pendrin and AQP5 membrane expression under chronic K(+)-deficiency indicates that these two molecules could cooperate as an osmosensor to rapidly detect and respond to alterations
Chapleau, Christopher A; Larimore, Jennifer L; Theibert, Anne; Pozzo-Miller, Lucas
The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation.
Nicholas de Villiers
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’.
Nicholas de Villiers
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 ...
Yu, Hsiao-Man Ivy; Jin, Ying; Fu, Jiang; Hsu, Wei
Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for proper sorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse orthologue required for axis determination. Gpr177 is a transcriptional target of Wnt which is activated to assist its subcellular distribution in a feedback regulatory loop. We therefore proposed that reciprocal regulation of Wnt and Gpr177 is essential for the Wnt-dependent developmental and patho...
Yu, Hsiao-Man Ivy; Jin, Ying; Fu, Jiang; Hsu, Wei
Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for proper sorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse ortholog required for axis determination. Gpr177 is a transcriptional target of Wnt that is activated to assist its subcellular distribution in a feedback regulatory loop. We, therefore, proposed that reciprocal regulation of Wnt and Gpr177 is essential for the Wnt-dependent developmental and pathogenic processes. Here, we examine the expression pattern of Gpr177 in mouse development. Gpr177 is expressed in a variety of tissues and cell types during organogenesis. Furthermore, Gpr177 is a glycoprotein primarily accumulating in the Golgi apparatus in signal-producing cells. The glycosylation of Gpr177 is necessary for proper transportation in the secretory pathway. Our findings suggest that the Gpr177-mediated regulation of Wnt is crucial for organogenesis in health and disease.
Brandie M Cross
Full Text Available An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca(2+-ATPase, and the Ca(2+ influx channel Orai1, has previously been shown to contribute to elevated Ca(2+ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca(2+ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca(2+ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca(2+ entry (SICE, which mediates the massive basolateral Ca(2+ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.
Phillips-Krawczak, Christine A; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G; Li, Haiying; Dick, Christopher J; Gomez, Timothy S; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F; Geng, Linda N; Kaufmann, Scott H; Hein, Marco Y; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; Sluis, Bart van de; Billadeau, Daniel D; Burstein, Ezra
COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.
Wieman, Heather L.; Horn, Sarah R.; Jacobs, Sarah R.; Altman, Brian J.; Kornbluth, Sally; Rathmell, Jeffrey C.
Cell surface localization of the glucose transporter, Glut1, is a cytokine-controlled process essential to support the metabolism and survival of hematopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. Here we show a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence t...
Olesen, Emma Tina Bisgaard; Poulsen, Søren Brandt; MacAulay, Nanna;
Whole body water homeostasis is regulated by signaling cascades activated following stimulation of a range of 7-transmembrane receptors in the kidney. These receptors signal through heterotrimeric G proteins, and include the type 2 vasopressin receptor (V2R) and prostaglandin receptors EP2 and EP4...
Baster, P.; Robert, S.; Kleine-Vehn, J.; Vanneste, S.; Kania, U.; Grunewald, W.; Rybel, de B.P.M.; Beeckman, T.; Friml, J.
The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels.
Baster, P.; Robert, S.; Kleine-Vehn, J.; Vanneste, S.; Kania, U.; Grunewald, W.; Rybel, de B.P.M.; Beeckman, T.; Friml, J.
The distribution of the phytohormone auxin regulates many aspects of plant development including growth response to gravity. Gravitropic root curvature involves coordinated and asymmetric cell elongation between the lower and upper side of the root, mediated by differential cellular auxin levels. Th
Full Text Available This work deals with the modulator of a matrix converter and its communication with the superset regulator. A switching algorithm is briefly introduced. The input voltage measurement method is presented. In the last part of the paper, the testing of communication between the superset regulator and the modulator in FPGA technology are also presented.
Felix, Ricardo; Calderón-Rivera, Aida; Andrade, Arturo
Voltage-gated Ca(2+) (CaV) channels mediate Ca(2+) ions influx into cells in response to depolarization of the plasma membrane. They are responsible for initiation of excitation-contraction and excitation-secretion coupling, and the Ca(2+) that enters cells through this pathway is also important in the regulation of protein phosphorylation, gene transcription, and many other intracellular events. Initial electrophysiological studies divided CaV channels into low-voltage-activated (LVA) and high-voltage-activated (HVA) channels. The HVA CaV channels were further subdivided into L, N, P/Q, and R-types which are oligomeric protein complexes composed of an ion-conducting CaVα1 subunit and auxiliary CaVα2δ, CaVβ, and CaVγ subunits. The functional consequences of the auxiliary subunits include altered functional and pharmacological properties of the channels as well as increased current densities. The latter observation suggests an important role of the auxiliary subunits in membrane trafficking of the CaVα1 subunit. This includes the mechanisms by which CaV channels are targeted to the plasma membrane and to appropriate regions within a given cell. Likewise, the auxiliary subunits seem to participate in the mechanisms that remove CaV channels from the plasma membrane for recycling and/or degradation. Diverse studies have provided important clues to the molecular mechanisms involved in the regulation of CaV channels by the auxiliary subunits, and the roles that these proteins could possibly play in channel targeting and membrane Stabilization.
Full Text Available The analytical survey on research and trial of cases on human trafficking and those related to trafficking (for the period 2006-2009 has ascertained that in 29% of all the cases on child trafficking (Article 206, Penal Code, children were subjected to labour exploitation or forced labour, and in 32% of all the cases on human trafficking, people were subjected to labour exploitation or forced labour. Notwithstanding this, of all the criminal cases on child trafficking submitted to court, prosecutors demanded that the actions of the perpetrators should be reclassified under Article 168 of the Penal Code in 6.5% of cases, whereas in 13% the court reclassified the actions after it examined the substance of the case. If we refer to the same indexes but in the context of human trafficking, prosecutors reclassified 4% of cases under Article 168 of the Penal Code, whereas courts reclassified the actions of the perpetrators based on other components of crime, including forced labour, in 30% of cases.
Gu, Yuanzheng; Barry, Joshua; Gu, Chen
Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.
Chen, Esther J; Kaiser, Chris A
The delivery to the plasma membrane of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by the quality of the nitrogen source in the growth medium. In an effort to define how different nitrogen sources control Gap1p sorting, we find that mutations in GDH1 and GLN1 that decrease the flux through the glutamate and glutamine synthesis pathways result in increased Gap1p sorting to the plasma membrane. Conversely, deletion of MKS1, which increases glutamate and glutamine synthesis, decreases Gap1p sorting to the plasma membrane. Glutamate and glutamine are not unusual in their ability to regulate Gap1p sorting, because the addition of all natural amino acids and many amino acid analogs to the growth medium results in increased Gap1p sorting to the vacuole. Importantly, amino acids have the capacity to signal Gap1p sorting to the vacuole regardless of whether they can be used as a source of nitrogen. Finally, we show that rapamycin does not affect Gap1p sorting, indicating that Gap1p sorting is not directly influenced by the TOR pathway. Together, these data show that amino acids are a signal for sorting Gap1p to the vacuole and imply that the nitrogen-regulated Gap1p sorting machinery responds to amino acid-like compounds rather than to the overall nutritional status associated with growth on a particular nitrogen source.
Full Text Available Guanylate-binding proteins (GBPs belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5 carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins.
... trafficking in persons means— (1) Sex trafficking in which a commercial sex act is induced by force, fraud, or... bondage, or slavery. Sex trafficking means the recruitment, harboring, transportation, provision, or... 48 Federal Acquisition Regulations System 2 2010-10-01 2010-10-01 false Combating Trafficking...
Full Text Available Lactation is characterized by massive transcellular flux of calcium, from the basolateral side of the mammary alveolar epithelium (blood into the ductal lumen (milk. Regulation of calcium transport during lactation is critical for maternal and neonatal health. The monoamine serotonin (5-HT is synthesized by the mammary gland and functions as a homeostatic regulation of lactation. Genetic ablation of tryptophan hydroxylase 1 (Tph1, which encodes the rate-limiting enzyme in non-neuronal serotonin synthesis, causes a deficiency in circulating serotonin. As a consequence maternal calcium concentrations decrease, mammary epithelial cell morphology is altered, and cell proliferation is decreased during lactation. Here we demonstrate that serotonin deficiency decreases the expression and disrupts the normal localization of calcium transporters located in the apical (PMCA2 and basolateral (CaSR, ORAI-1 membranes of the lactating mammary gland. In addition, serotonin deficiency decreases the mRNA expression of calcium transporters located in intracellular compartments (SERCA2, SPCA1 and 2. Mammary expression of serotonin receptor isoform 2b and its downstream pathways (PLCβ3, PKC and MAP-ERK1/2 are also decreased by serotonin deficiency, which might explain the numerous phenotypic alterations described above. In most cases, addition of exogenous 5-hydroxy-L-tryptophan to the Tph1 deficient mice rescued the phenotype. Our data supports the hypothesis that serotonin is necessary for proper mammary gland structure and function, to regulate blood and mammary epithelial cell transport of calcium during lactation. These findings can be applicable to the treatment of lactation-induced hypocalcemia in dairy cows and can have profound implications in humans, given the wide-spread use of selective serotonin reuptake inhibitors as antidepressants during pregnancy and lactation.
Nielsen, Nathalie Hélix
abnormalities induced by “loss of function” Kv7.1 mutations increase the risk of polymorphic ventricular arrhythmias. These cardiac arrhythmias, typically in the form of torsades de pointes, may underlie ventricular fibrillation, recurrent syncope, and sudden death. To date, nearly 300 Kv7.1 mutations have been...... to the regulation of the Kv7.1 channel, which displays a consensus site in the N-terminus for this kinase. Our study, with the support of others, tends to demonstrate that the Kv7.1 channel forms a macromolecular signaling complex with its interactions partners in order to allow a fast response to external signals....
Ratajczak, M Z; Adamiak, M
Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein microdomains, known as lipid rafts, which float freely in the membrane bilayer. These structures have an important role in assembling signaling molecules (e.g., Rac-1, RhoH and Lyn) together with surface receptors, such as the CXCR4 receptor for α-chemokine stromal-derived factor-1, the α4β1-integrin receptor (VLA-4) for vascular cell adhesion molecule-1 and the c-kit receptor for stem cell factor, which together regulate several aspects of hematopoietic stem/progenitor cell (HSPC) biology. Here, we discuss the role of lipid raft integrity in the retention and quiescence of normal HSPCs in bone marrow niches as well as in regulating HSPC mobilization and homing. We will also discuss the pathological consequences of the defect in lipid raft integrity seen in paroxysmal nocturnal hemoglobinuria and the emerging evidence for the involvement of lipid rafts in hematological malignancies.
Di Benedetto, Adriana; Sun, Li; Zambonin, Carlo G.; Tamma, Roberto; Nico, Beatrice; Calvano, Cosima D.; Colaianni, Graziana; Ji, Yaoting; Mori, Giorgio; Grano, Maria; Lu, Ping; Colucci, Silvia; Yuen, Tony; New, Maria I.; Zallone, Alberta; Zaidi, Mone
We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr–EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors. PMID:25378700
Full Text Available Heme oxygenase (HO degrades heme in concert with NADPH cytochrome P450 reductase (CPR which donates electrons to the reaction. Earlier studies reveal the importance of the hydrophobic carboxy-terminus of HO-1 for anchorage to the endoplasmic reticulum (ER which facilitates the interaction with CPR. In addition, HO-1 has been shown to undergo regulated intramembrane proteolysis of the carboxy-terminus during hypoxia and subsequent translocation to the nucleus. Translocated nuclear HO-1 was demonstrated to alter binding of transcription factors and to alter gene expression. Little is known about the homologous membrane anchor of the HO-2 isoform. The current work is the first systematic analysis in a eukaryotic system that demonstrates the crucial role of the membrane anchor of HO-2 for localization at the endoplasmic reticulum, oligomerization and interaction with CPR. We show that although the carboxy-terminal deletion mutant of HO-2 is found in the nucleus, translocation of HO-2 to the nucleus does not occur under conditions of hypoxia. Thus, we demonstrate that proteolytic regulation and nuclear translocation under hypoxic conditions is specific for HO-1. In addition we show for the first time that CPR prevents this translocation and promotes oligomerization of HO-1. Based on these findings, CPR may modulate gene expression via the amount of nuclear HO-1. This is of particular relevance as CPR is a highly polymorphic gene and deficiency syndromes of CPR have been described in humans.
Nowak, Matthias; Machate, Anja; Yu, Shuizi Rachel; Gupta, Mansi; Brand, Michael
Forty years ago, it was proposed that during embryonic development and organogenesis, morphogen gradients provide positional information to the individual cells within a tissue leading to specific fate decisions. Recently, much insight has been gained into how such morphogen gradients are formed and maintained; however, which cellular mechanisms govern their interpretation within target tissues remains debated. Here we used in vivo fluorescence correlation spectroscopy and automated image analysis to assess the role of endocytic sorting dynamics on fibroblast growth factor 8 (Fgf8) morphogen gradient interpretation. By interfering with the function of the ubiquitin ligase Cbl, we found an expanded range of Fgf target gene expression and a delay of Fgf8 lysosomal transport. However, the extracellular Fgf8 morphogen gradient remained unchanged, indicating that the observed signalling changes are due to altered gradient interpretation. We propose that regulation of morphogen signalling activity through endocytic sorting allows fast feedback-induced changes in gradient interpretation during the establishment of complex patterns.
Petrich, Aline; Mann, Anika; Kliewer, Andrea; Nagel, Falko; Strigli, Anne; Märtens, Jan Carlo; Pöll, Florian; Schulz, Stefan
The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.
Wiener, Heidi; Su, Wenjuan; Liot, Caroline; Hancock, John F.
Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)–dependent, fashion. Silencing VPS35 increased N-Ras’s association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras–dependent melanoma cells. PMID:27502489
Full Text Available ThermoTRP channels (thermoTRPs define a subfamily of the transient receptor potential (TRP channels that are activated by changes in the environmental temperature, from noxious cold to injurious heat. Acting as integrators of several stimuli and signalling pathways, dysfunction of these channels contributes to several pathological states. The surface expression of thermoTRPs is controlled by both, the constitutive and regulated vesicular trafficking. Modulation of receptor surface density during pathological processes is nowadays considered as an interesting therapeutic approach for management of diseases, such as chronic pain, in which an increased trafficking is associated with the pathological state. This review will focus on the recent advances trafficking of the thermoTRP channels, TRPV1, TRPV2, TRPV4, TRPM3, TRPM8 and TRPA1, into/from the plasma membrane. Particularly, regulated membrane insertion of thermoTRPs channels contributes to a fine tuning of final channel activity, and indeed, it has resulted in the development of novel therapeutic approaches with successful clinical results such as disruption of SNARE-dependent exocytosis by botulinum toxin or botulinomimetic peptides.
Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno
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. PMID:27092250
Dunn, Henry A; Chahal, Harpreet S; Caetano, Fabiana A; Holmes, Kevin D; Yuan, George Y; Parikh, Ruchi; Heit, Bryan; Ferguson, Stephen S G
Corticotropin-releasing factor (CRF) is a neuropeptide commonly associated with the hypothalamic-pituitary adrenal axis stress response. Upon release, CRF activates two G protein-coupled receptors (GPCRs): CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2). Although both receptors contribute to mood regulation, CRFR1 antagonists have demonstrated anxiolytic and antidepressant-like properties that may be exploited in the generation of new pharmacological interventions for mental illnesses. Previous studies have demonstrated CRFR1 capable of heterologously sensitizing serotonin 2A receptor (5-HT2AR) signaling: another GPCR implicated in psychiatric disease. Interestingly, this phenomenon was dependent on Postsynaptic density 95 (PSD-95)/Disc Large/Zona Occludens (PDZ) interactions on the distal carboxyl termini of both receptors. In the current study, we demonstrate that endogenous PSD-95 can be co-immunoprecipitated with CRFR1 from cortical brain homogenate, and this interaction appears to be primarily via the PDZ-binding motif. Additionally, PSD-95 colocalizes with CRFR1 within the dendritic projections of cultured mouse neurons in a PDZ-binding motif-dependent manner. In HEK 293 cells, PSD-95 overexpression inhibited CRFR1 endocytosis, whereas PSD-95 shRNA knockdown enhanced CRFR1 endocytosis. Although PSD-95 does not appear to play a significant role in CRF-mediated cAMP or ERK1/2 signaling, PSD-95 was demonstrated to suppress β-arrestin2 recruitment: providing a potential mechanism for PSD-95's inhibition of endocytosis. In revisiting previously documented heterologous sensitization, PSD-95 shRNA knockdown did not prevent CRFR1-mediated enhancement of 5-HT2AR signaling. In conclusion, we have identified and characterized a novel functional relationship between CRFR1 and PSD-95 that may have implications in the design of new treatment strategies for mental illness. Copyright © 2016 Elsevier Inc. All rights reserved.
Bichet, Delphine; Blin, Sandy; Feliciangeli, Sylvain; Chatelain, Franck C; Bobak, Nicole; Lesage, Florian
Among K2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined "silent subunits". Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.
Hennigan, R F; Moon, C A; Parysek, L M; Monk, K R; Morfini, G; Berth, S; Brady, S; Ratner, N
Neurofibromatosis type 2 patients develop schwannomas, meningiomas and ependymomas resulting from mutations in the tumor suppressor gene, NF2, encoding a membrane-cytoskeleton adapter protein called merlin. Merlin regulates contact inhibition of growth and controls the availability of growth factor receptors at the cell surface. We tested if microtubule-based vesicular trafficking might be a mechanism by which merlin acts. We found that schwannoma cells, containing merlin mutations and constitutive activation of the Rho/Rac family of GTPases, had decreased intracellular vesicular trafficking relative to normal human Schwann cells. In Nf2-/- mouse Schwann (SC4) cells, re-expression of merlin as well as inhibition of Rac or its effector kinases, MLK and p38(SAPK), each increased the velocity of Rab6 positive exocytic vesicles. Conversely, an activated Rac mutant decreased Rab6 vesicle velocity. Vesicle motility assays in isolated squid axoplasm further demonstrated that both mutant merlin and active Rac specifically reduce anterograde microtubule-based transport of vesicles dependent upon the activity of p38(SAPK) kinase. Taken together, our data suggest loss of merlin results in the Rac-dependent decrease of anterograde trafficking of exocytic vesicles, representing a possible mechanism controlling the concentration of growth factor receptors at the cell surface.
Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki
Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1.
Jonathan G Hanley
Full Text Available The precise regulation of AMPA receptor (AMPAR number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits during learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signalling pathways that modulate actin polymerization and depolymerisation. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.
Meghaan P Walsh
Full Text Available Although tumors naturally prime adaptive immune responses, tolerance may limit the capacity to control progression and can compromise effectiveness of immune-based therapies for cancer. Post-proline cleaving enzymes (PPCE modulate protein function through N-terminal dipeptide cleavage and inhibition of these enzymes has been shown to have anti-tumor activity. We investigated the mechanism by which Val-boroPro, a boronic dipeptide that inhibits post-proline cleaving enzymes, mediates tumor regression and tested whether this agent could serve as a novel immune adjuvant to dendritic cell vaccines in two different murine syngeneic murine tumors. In mice challenged with MB49, which expresses the HY antigen complex, T cell responses primed by the tumor with and without Val-boroPro were measured using interferon gamma ELISPOT. Antibody depletion and gene-deficient mice were used to establish the immune cell subsets required for tumor regression. We demonstrate that Val-boroPro mediates tumor eradication by accelerating the expansion of tumor-specific T cells. Interestingly, T cells primed by tumor during Val-boroPro treatment demonstrate increased capacity to reject tumors following adoptive transfer without further treatment of the recipient. Val-boroPro -mediated tumor regression requires dendritic cells and is associated with enhanced trafficking of dendritic cells to tumor draining lymph nodes. Finally, dendritic cell vaccination combined with Val-boroPro treatment results in complete regression of established tumors. Our findings demonstrate that Val-boroPro has antitumor activity and a novel mechanism of action that involves more robust DC trafficking with earlier priming of T cells. Finally, we show that Val-boroPro has potent adjuvant properties resulting in an effective therapeutic vaccine.
Safaei, Roohangiz; Adams, Preston L; Mathews, Ryan A; Manorek, Gerald; Howell, Stephen B
The copper (Cu) exporter ATP7B mediates cellular resistance to cisplatin (cDDP) by increasing drug efflux. ATP7B binds and sequesters cDDP in into secretory vesicles. Upon cDDP exposure ATP7B traffics from the trans-Golgi network (TGN) to the periphery of the cell in a manner that requires the cysteine residues in its metal binding domains (MBD). To elucidate the role of the various domains of ATP7B in its cDDP-induced trafficking we expressed a series of mCherry-tagged variants of ATP7B in HEK293T cells and analyzed their subcellular localization in basal media and after a 1 h exposure to 30 μM cDDP. The wild type ATP7B and a variant in which the cysteines in the CXXC motifs of MBD 1-5 were converted to serines trafficked out of the trans-Golgi (TGN) when exposed to cDDP. Conversion of the cysteines in all 6 of the CXXC motifs to serines, or in only the sixth MBD, rendered ATP7B incapable of trafficking on exposure to cDDP. Truncation of MBD1-5 or MBD1-6 resulted in the loss of TGN localization. Addition of the first 63 amino acids of ATP7B to these variants restored TGN localization to a great extent and enabled the MBD1-5 variant to undergo cDDP-induced trafficking. A variant of ATP7B in which the aspartate 1027 residue in the phosphorylation domain was converted to glutamine localized to the TGN but was incapable of cDDP-induced trafficking. These results demonstrate that the CXXC motif in the sixth MBD and the catalytic activity of ATP7B are required for cDDP-induced trafficking as they are for Cu-induced redistribution of ATP7B; this provides further evidence that cDDP mimics Cu with respect to the molecular mechanisms by they control the subcellular distribution of ATP7B.
Heo, Jae Bok; Rho, Hee Sun; Kim, Se Won; Hwang, Sung Min; Kwon, Hyun Jin; Nahm, Min Yeop; Bang, Woo Young; Bahk, Jeong Dong
The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.
Funakoshi, Yuji; Chou, Margaret M.; Kanaho, Yasunori; Julie G Donaldson
Plasma membrane proteins that enter cells by clathrin-independent endocytosis (CIE) are sorted either to lysosomes for degradation or recycled back to the plasma membrane. Expression of some MARCH E3 ubiquitin ligases promotes trafficking of CIE cargo proteins to lysosomes by ubiquitylating the proteins. Here, we show that co-expression of the ubiquitin-specific protease TRE17/USP6 counteracts the MARCH-dependent targeting of CIE cargo proteins, but not that of transferrin receptor, to lysoso...
The phosphoinositide lipid PI(4,5)P(2) is now established as a key cofactor in signaling to the actin cytoskeleton and in vesicle trafficking. PI(4,5)P(2) accumulates at membrane rafts and promotes local co-recruitment and activation of specific signaling components at the cell membrane. PI(4,5)P(2) rafts may thus be platforms for local regulation of morphogenetic activity at the cell membrane. Raft PI(4,5)P(2) is regulated by lipid kinases (PI5-kinases) and lipid phosphatases (e.g. synaptojanin). In addition, GAP43-like proteins have recently emerged as a group of PI(4,5)P(2) raft-modulating proteins. These locally abundant proteins accumulate at inner leaflet plasmalemmal rafts where they bind to and co-distribute with PI(4,5)P(2), and promote actin cytoskeleton accumulation and dynamics. In keeping with their proposed role as positive modulators of PI(4,5)P(2) raft function, GAP43-like proteins confer competence for regulated morphogenetic activity on cells that express them. Their function has been investigated extensively in the nervous system, where their expression promotes neurite outgrowth, anatomical plasticity and nerve regeneration. Extrinsic signals and intrinsic factors may thus converge to modulate PI(4,5)P(2) rafts, upstream of regulated activity at the cell surface.
Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V
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.
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.
Chun, Se-Eun; Thakkar, Nilay; Oh, Yunseok; Park, Ji Eun; Han, Songhee; Ryoo, Gongmi; Hahn, Hyunggu; Maeng, Sang Hyun; Lim, Young-Ran; Han, Byung Woo; Lee, Wooin
Organic anion transporting polypeptide 1B3 (OATP1B3) is a major influx transporter mediating the hepatic uptake of various endogenous substrates as well as clinically important drugs such as statins and anticancer drugs. However, molecular mechanisms controlling the membrane trafficking of OATP1B3 have been largely unknown. Several reports recently indicated the presence of a distinct, cancer-type OATP1B3 variant lacking the N-terminal 28 amino acids compared to OATP1B3 expressed in non-malignant hepatocytes. Interestingly, the cancer-type OATP1B3 variant is located predominantly in the cytoplasm, implicating the involvement of the N-terminal region of OATP1B3 in its membrane trafficking. In the current study, we set out to experimentally validate the importance of the N-terminal region of OATP1B3 and to identify responsible sequence motif(s) in that region. A number of truncation or point mutants of OATP1B3 were transiently expressed in HEK293T, HCT-8 or MDCK II cells and their expression in cytoplasmic and surface membrane fractions were analyzed by immunoblotting. Our results indicated that the N-terminal sequence of OATP1B3, in particular, at the amino acid positions between 12 and 28, may be indispensable in its membrane trafficking. Moreover, our results using a fusion construct indicated that the first 50 amino acids of OATP1B3 are sufficient for its membrane localization. The importance of the N-terminal region in membranous localization was shared among the other OATP1B subfamily members, OATP1B1 and rat Oatp1b2. Our efforts to identify the responsible amino acid(s) or structure motif(s) in the N-terminal region did not pinpoint individual amino acids or motifs with putative secondary structures. Our current findings however demonstrate that the N-terminal region is important for the membrane localization of the OATP1B subfamily members and should facilitate future investigations of the mechanisms involved in the regulation and membrane trafficking of
Leung, Yuk M; Kang, Youhou; Gao, Xiaodong; Xia, Fuzhen; Xie, Huanli; Sheu, Laura; Tsuk, Sharon; Lotan, Ilana; Tsushima, Robert G; Gaisano, Herbert Y
Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.
Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo
Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Full Text Available BACKGROUND: MicroRNAs (miRNAs are small RNAs that recognize and regulate mRNA target genes. Multiple lines of evidence indicate that they are key regulators of numerous critical functions in development and disease, including cancer. However, defining the place and function of miRNAs in complex regulatory networks is not straightforward. Systems approaches, like the inference of a module network from expression data, can help to achieve this goal. METHODOLOGY/PRINCIPAL FINDINGS: During the last decade, much progress has been made in the development of robust and powerful module network inference algorithms. In this study, we analyze and assess experimentally a module network inferred from both miRNA and mRNA expression data, using our recently developed module network inference algorithm based on probabilistic optimization techniques. We show that several miRNAs are predicted as statistically significant regulators for various modules of tightly co-expressed genes. A detailed analysis of three of those modules demonstrates that the specific assignment of miRNAs is functionally coherent and supported by literature. We further designed a set of experiments to test the assignment of miR-200a as the top regulator of a small module of nine genes. The results strongly suggest that miR-200a is regulating the module genes via the transcription factor ZEB1. Interestingly, this module is most likely involved in epithelial homeostasis and its dysregulation might contribute to the malignant process in cancer cells. CONCLUSIONS/SIGNIFICANCE: Our results show that a robust module network analysis of expression data can provide novel insights of miRNA function in important cellular processes. Such a computational approach, starting from expression data alone, can be helpful in the process of identifying the function of miRNAs by suggesting modules of co-expressed genes in which they play a regulatory role. As shown in this study, those modules can then be
Full Text Available Abstract Background A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV, resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro. Presentation of the hypothesis It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression. Testing the hypothesis The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology. Implication of the hypothesis If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe
Mor, Amir; White, Michael A; Fontoura, Beatriz M A
In eukaryotic cells, the cytoplasm and the nucleus are separated by a double-membraned nuclear envelope (NE). Thus, transport of molecules between the nucleus and the cytoplasm occurs via gateways termed the nuclear pore complexes (NPCs), which are the largest intracellular channels in nature. While small molecules can passively translocate through the NPC, large molecules are actively imported into the nucleus by interacting with receptors that bind nuclear pore complex proteins (Nups). Regulatory factors then function in assembly and disassembly of transport complexes. Signaling pathways, cell cycle, pathogens, and other physiopathological conditions regulate various constituents of the nuclear transport machinery. Here, we will discuss several findings related to modulation of nuclear transport during physiological and pathological conditions, including tumorigenesis, viral infection, and congenital syndrome. We will also explore chemical biological approaches that are being used as probes to reveal new mechanisms that regulate nucleocytoplasmic trafficking and that are serving as starting points for drug development.
Remko Offringa; and Fang Huang
In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.
Offringa, Remko; Huang, Fang
In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.
Full Text Available Patients with Down syndrome (DS invariably develop Alzheimer’s disease (AD pathology in their 40s. We have recently found that overexpression of a chromosome 21-encoded microRNA-155 results in decreased levels of the membrane trafficking component, SNX27, diminishing glutamate receptor recycling and thereby impairing synaptic functions in DS. Here, we report a function of SNX27 in regulating β-amyloid (Aβ generation by modulating γ-secretase activity. Downregulation of SNX27 using RNAi increased Aβ production, whereas overexpression of full-length SNX27, but not SNX27ΔPDZ, reversed the RNAi-mediated Aβ elevation. Moreover, genetic deletion of Snx27 promoted Aβ production and neuronal loss, whereas overexpression of SNX27 using an adeno-associated viral (AAV vector reduced hippocampal Aβ levels in a transgenic AD mouse model. SNX27 associates with the γ-secretase complex subunit presenilin 1; this interaction dissociates the γ-secretase complex, thus decreasing its proteolytic activity. Our study establishes a molecular mechanism for Aβ-dependent pathogenesis in both DS and AD.
Full Text Available Mutations in the ABCC6 ABC-transporter are causative of pseudoxanthoma elasticum (PXE. The loss of functional ABCC6 protein in the basolateral membrane of the kidney and liver is putatively associated with altered secretion of a circulatory factor. As a result, systemic changes in elastic tissues are caused by progressive mineralization and degradation of elastic fibers. Premature arteriosclerosis, loss of skin and vascular tone, and a progressive loss of vision result from this ectopic mineralization. However, the identity of the circulatory factor and the specific role of ABCC6 in disease pathophysiology are not known. Though recessive loss-of-function alleles are associated with alterations in ABCC6 expression and function, the molecular pathologies associated with the majority of PXE-causing mutations are also not known. Sequence analysis of orthologous ABCC6 proteins indicates the C-terminal sequences are highly conserved and share high similarity to the PDZ sequences found in other ABCC subfamily members. Genetic testing of PXE patients suggests that at least one disease-causing mutation is located in a PDZ-like sequence at the extreme C-terminus of the ABCC6 protein. To evaluate the role of this C-terminal sequence in the biosynthesis and trafficking of ABCC6, a series of mutations were utilized to probe changes in ABCC6 biosynthesis, membrane stability and turnover. Removal of this PDZ-like sequence resulted in decreased steady-state ABCC6 levels, decreased cell surface expression and stability, and mislocalization of the ABCC6 protein in polarized cells. These data suggest that the conserved, PDZ-like sequence promotes the proper biosynthesis and trafficking of the ABCC6 protein.
Smith, Jennifer L; McBride, Christie M; Nataraj, Parvathi S; Bartos, Daniel C; January, Craig T; Delisle, Brian P
The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.
Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan
Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.
Full Text Available The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1. Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1+ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1+ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.
Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B;
. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser...... heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4...
Vaz, Sandra H; Jørgensen, Trine N; Cristóvão-Ferreira, Sofia; Duflot, Sylvie; Ribeiro, Joaquim A; Gether, Ulrik; Sebastião, Ana M
The γ-aminobutyric acid (GABA) transporters (GATs) are located in the plasma membrane of neurons and astrocytes and are responsible for termination of GABAergic transmission. It has previously been shown that brain derived neurotrophic factor (BDNF) modulates GAT-1-mediated GABA transport in nerve terminals and neuronal cultures. We now report that BDNF enhances GAT-1-mediated GABA transport in cultured astrocytes, an effect mostly due to an increase in the V(max) kinetic constant. This action involves the truncated form of the TrkB receptor (TrkB-t) coupled to a non-classic PLC-γ/PKC-δ and ERK/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope was incorporated into the second extracellular loop. An increase in plasma membrane of HA-rGAT-1 as well as of rGAT-1 was observed when both HA-GAT-1-transduced astrocytes and rGAT-1-overexpressing astrocytes were treated with BDNF. The effect of BDNF results from inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1 rather than from facilitation of the monensin-sensitive recycling of GAT-1 molecules back to the plasma membrane. We therefore conclude that BDNF enhances the time span of GAT-1 molecules at the plasma membrane of astrocytes. BDNF may thus play an active role in the clearance of GABA from synaptic and extrasynaptic sites and in this way influence neuronal excitability.
Kimmelmann, Stefan; Knorr, Wolfgang
The high efficient universal Buck Boost Solar Array Regulator (SAR) module concept is applicable for a wide range of input and output voltages. The single point failure tolerant SAR module contains 3 power converters for the transfer of the SAR power to the battery dominated power bus. The converters are operating parallel in a 2 out of 3 redundancy and are driven by two different controllers. The output power of one module can be adjusted up to 1KW depending on the requirements. The maximum power point tracker (MPPT) is placed on a separate small printed circuit board and can be used if no external tracker signal is delivered. Depending on the mode and load conditions an efficiency of more than 97% is achievable. The stable control performance is achieved by implementing the magnetic current sense detection. The sensed power coil current is used in Buck and Boost control mode.
Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B; Fenton, Robert A; MacAulay, Nanna
Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser(321), and Ser(322). To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel.
Segarra, Marta; Ohnuki, Hidetaka; Maric, Dragan; Salvucci, Ombretta; Hou, Xu; Kumar, Anil; Li, Xuri; Tosato, Giovanna
Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of ...
Norez, C; Vandebrouck, C; Bertrand, J; Noel, S; Durieu, E; Oumata, N; Galons, H; Antigny, F; Chatelier, A; Bois, P; Meijer, L; Becq, F
The most common mutation in cystic fibrosis (CF), F508del, causes defects in trafficking, channel gating and endocytosis of the CF transmembrane conductance regulator (CFTR) protein. Because CF is an orphan disease, therapeutic strategies aimed at improving mutant CFTR functions are needed to target the root cause of CF. Human CF airway epithelial cells were treated with roscovitine 100 μM for 2 h before CFTR maturation, expression and activity were examined. The mechanism of action of roscovitine was explored by recording the effect of depleting endoplasmic reticulum (ER) Ca(2+) on the F508del-CFTR/calnexin interaction and by measuring proteasome activity. Of the cyclin-dependent kinase (CDK) inhibitors investigated, roscovitine was found to restore the cell surface expression and defective channel function of F508del-CFTR in human CF airway epithelial cells. Neither olomoucine nor (S)-CR8, two very efficient CDK inhibitors, corrected F508del-CFTR trafficking demonstrating that the correcting effect of roscovitine was independent of CDK inhibition. Competition studies with inhibitors of the ER quality control (ERQC) indicated that roscovitine acts on the calnexin pathway and on the degradation machinery. Roscovitine was shown (i) to partially inhibit the interaction between F508del-CFTR and calnexin by depleting ER Ca(2+) and (ii) to directly inhibit the proteasome activity in a Ca(2+) -independent manner. Roscovitine is able to correct the defective function of F508del-CFTR by preventing the ability of the ERQC to interact with and degrade F508del-CFTR via two synergistic but CDK-independent mechanisms. Roscovitine has potential as a pharmacological therapy for CF. © 2014 The British Pharmacological Society.
Full Text Available UNC50 has long been recognized as a Golgi apparatus protein in yeast, and is involved in nicotinic receptor trafficking in Caenorhabditis elegans, but little is known about UNC50 gene function in human biology despite it being conserved from yeast to high eukaryotes.We investigated the relation between UNC50 and human hepatocellular carcinoma (HCC and the potential mechanisms underlying HCC development.UNC50 mRNA expression patterns in 12 HCC and adjacent non-cancerous tissues determined using northern blotting were confirmed by real-time PCR in another 44 paired tissues. Microarray experiments were used to screen for global effects of UNC50 knockdown in the Hep3B cell line, and were confirmed by real-time PCR, western blotting, flow cytometry, and tetrazolium assay in both UNC50 overexpression and knockdown Hep3B cells.UNC50 expression levels were upregulated in HCC tissues in comparison with the adjacent non-cancerous tissues. UNC50 knockdown reduced mRNA levels of the downstream targets of the epidermal growth factor receptor (EGFR pathway: cyclin D1 (CCND1, EGF, matrix metalloproteinase-7 (MMP7, aldose reductase-like 1 (AKR1B10, cell surface-associated mucin 1 (MUC1, and gastrin (GAST. Moreover, UNC50 influenced EGF, inducing cell cycle entry by affecting cell surface EGFR amounts.UNC50 may plays some roles in HCC progression by affecting the EGFR pathway.
Full Text Available Abstract Background Epigenetic regulators (histone acetyltransferases, methyltransferases, chromatin-remodelling enzymes, etc play a fundamental role in the control of gene expression by modifying the local state of chromatin. However, due to their recent discovery, little is yet known about their own regulation. This paper addresses this point, focusing on alternative splicing regulation, a mechanism already known to play an important role in other protein families, e.g. transcription factors, membrane receptors, etc. Results To this end, we compiled the data available on the presence/absence of alternative splicing for a set of 160 different epigenetic regulators, taking advantage of the relatively large amount of unexplored data on alternative splicing available in public databases. We found that 49 % (70 % in human of these genes express more than one transcript. We then studied their alternative splicing patterns, focusing on those changes affecting the enzyme's domain composition. In general, we found that these sequence changes correspond to different mechanisms, either repressing the enzyme's function (e.g. by creating dominant-negative inhibitors of the functional isoform or creating isoforms with new functions. Conclusion We conclude that alternative splicing of epigenetic regulators can be an important tool for the function modulation of these enzymes. Considering that the latter control the transcriptional state of large sets of genes, we propose that epigenetic regulation of gene expression is itself strongly regulated by alternative splicing.
Bloch, Daria; Monshausen, Gabriele; Gilroy, Simon; Yalovsky, Shaul
Growth of plant cells involves tight regulation of the cytoskeleton and vesicle trafficking by processes including the action of the ROP small G proteins together with pH-modulated cell wall modifications. Yet, little is known on how these systems are coordinated. In a paper recently published in Plant Cell and Environment we show that ROPs/RACs function synergistically with NH4NO3-modulated pH fluctuations to regulate root hair growth. Root hairs expand exclusively at their apical end in a strictly polarized manner by a process known as tip growth. The highly polarized secretion at the apex is maintained by a complex network of factors including the spatial organization of the actin cytoskeleton, tip-focused ion gradients and by small G proteins. Expression of constitutively active ROP mutants disrupts polar growth, inducing the formation of swollen root hairs. Root hairs are also known to elongate in an oscillating manner, which is correlated with oscillatory H(+) fluxes at the tip. Our analysis shows that root hair elongation in wild type plants and swelling in transgenic plants expressing a constitutively active ROP11 (rop11(CA)) is sensitive to the presence of NH4(+) at concentrations higher than 1 mM and on NO3(-). The NH4(+) and NO3(-) ions did not affect the localization of ROP in the membrane but modulated pH fluctuations at the root hair tip. Actin organization and reactive oxygen species distribution were abnormal in rop11CA root hairs but were similar to wild type root hairs when seedlings were grown on medium lacking NH4(+) and / or NO3(-). These observations suggest that the nitrogen source-modulated pH fluctuations may function synergistically with ROP regulated signaling during root hair tip growth. Interestingly, under certain growth conditions, expression of rop11 (CA) suppressed ammonium toxicity, similar to auxin resistant mutants. In this Addendum article we discuss these findings and their implications.
Heisler, Lora K; Jobst, Erin E; Sutton, Gregory M; Zhou, Ligang; Borok, Erzsebet; Thornton-Jones, Zoe; Liu, Hong Yan; Zigman, Jeffrey M; Balthasar, Nina; Kishi, Toshiro; Lee, Charlotte E; Aschkenasi, Carl J; Zhang, Chen-Yu; Yu, Jia; Boss, Olivier; Mountjoy, Kathleen G; Clifton, Peter G; Lowell, Bradford B; Friedman, Jeffrey M; Horvath, Tamas; Butler, Andrew A; Elmquist, Joel K; Cowley, Michael A
The neural pathways through which central serotonergic systems regulate food intake and body weight remain to be fully elucidated. We report that serotonin, via action at serotonin1B receptors (5-HT1BRs), modulates the endogenous release of both agonists and antagonists of the melanocortin receptors, which are a core component of the central circuitry controlling body weight homeostasis. We also show that serotonin-induced hypophagia requires downstream activation of melanocortin 4, but not melanocortin 3, receptors. These results identify a primary mechanism underlying the serotonergic regulation of energy balance and provide an example of a centrally derived signal that reciprocally regulates melanocortin receptor agonists and antagonists in a similar manner to peripheral adiposity signals.
Tschische, Pia; Moser, Elisabeth; Thompson, Dawn; Vischer, Henry F; Parzmair, Gerald P; Pommer, Veronika; Platzer, Wolfgang; Schwarzbraun, Thomas; Schaider, Helmut; Smit, Martine J; Martini, Lene; Whistler, Jennifer L; Waldhoer, Maria
Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.
Naramoto, Satoshi; Otegui, Marisa S; Kutsuna, Natsumaro; de Rycke, Riet; Dainobu, Tomoko; Karampelias, Michael; Fujimoto, Masaru; Feraru, Elena; Miki, Daisuke; Fukuda, Hiroo; Nakano, Akihiko; Friml, Jiří
GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants.
Vaz, Sandra H; Jørgensen, Trine Nygaard; Cristóvão-Ferreira, Sofia
/MAPK pathway and requires active adenosine A(2A) receptors. Transport through GAT-3 is not affected by BDNF. To elucidate if BDNF affects trafficking of GAT-1 in astrocytes, we generated and infected astrocytes with a functional mutant of the rat GAT-1 (rGAT-1) in which the hemagglutinin (HA) epitope...
Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R
The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways.
Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko
The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.
Li, Yanyan; Tang, Moxun; Yu, Jianshe
Gene transcription is a stochastic process in single cells, in which genes transit randomly between active and inactive states. Transcription of many inducible genes is also tightly regulated: It is often stimulated by extracellular signals, activated through signal transduction pathways and later repressed by negative regulations. In this work, we study the nonlinear dynamics of the mean transcription level of inducible genes modulated by the interplay of the intrinsic transcriptional randomness and the repression by negative regulations. In our model, we integrate negative regulations into gene activation process, and make the conventional assumption on the production and degradation of transcripts. We show that, whether or not the basal transcription is temporarily terminated when cells are stimulated, the mean transcription level grows in the typical up and down pattern commonly observed in immune response genes. With the help of numerical simulations, we clarify the delicate impact of the system parameters on the transcription dynamics, and demonstrate how our model generates the distinct temporal gene-induction patterns in mouse fibroblasts discerned in recent experiments.
Mondal, Payel; Khamo, John S; Krishnamurthy, Vishnu V; Cai, Qi; Zhang, Kai
Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.
Larsen, DeLaine D; Choy, Regina Wai-Yan; Park, Minjong
Synaptic activity is modulated by the activation of neuromodulator receptors present in dendrites of neurons. The majority of neuromodulator receptors are G protein coupled receptors (GPCRs), in which membrane trafficking regulates their activities. Membrane trafficking of neuromodulator receptors and their signaling occurs on a rapid time scale and emerging studies indicate that neuromodulator receptors function not just from the plasma membrane but also from the endocytic compartments. Here, we describe a live cell imaging approach using spinning disk confocal microscopy to investigate the effect of neuromodulator receptor activation on synaptic activity by measuring calcium dynamics in primary rat striatal neurons. The advantages of spinning disk confocal microscopy and recent improvements in the genetically encoded calcium sensor, GCaMP6, provide an imaging approach to image both the receptor membrane trafficking to endocytic compartments, and calcium dynamics at a high spatial and temporal resolution. We believe this approach of imaging both the neuromodulator receptor membrane trafficking and synaptic activity using GCaMP6 is a powerful tool to address many questions regarding possible roles of membrane trafficking of neuromodulator receptors in synaptic activity.
Mondal, Payel; Khamo, John S.; Krishnamurthy, Vishnu V.; Cai, Qi; Zhang, Kai
Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer’s disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases. PMID:28163671
Full Text Available Human trafficking is an occurrence that, even in our time, is present in alarming proportions, in its actuality and consequences. It is a phenomenon with a long history and has been qualified as a serious international problem and is the object of interest for a large number of international subjects. However, the key international document that defines this phenomenon is the Convention against Transnational Organized Crime from Palermo 2000; specifically its Protocol to Prevent, Suppress and Punish Trafficking in Persons, especially Women and Children. After its adoption, intensive actions were undertaken to regulate the phenomenon on the level of national legislature. It's done so in the local legislature too. According to the criminal law of the republic of Serbia, besides the concrete law against human trafficking, a number of other crimes are connected to human trafficking. This paper deals with the most important ones. The purpose of this paper is to review the legislature on the phenomenon in the domestic law, then the accordance of incrimination with international standards, as well as to indicate the need for further changes in domestic legislature.
Donowitz, M; Janecki, A; Akhter, S; Cavet, M E; Sanchez, F; Lamprecht, G; Zizak, M; Kwon, W L; Khurana, S; Yun, C H; Tse, C M
NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.
Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas
Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.
Carroll, Reed C; Zukin, R Suzanne
Dynamic regulation of synaptic efficacy is thought to play a crucial role in formation of neuronal connections and in experience-dependent modification of neural circuitry. The molecular and cellular mechanisms by which synaptic changes are triggered and expressed are the focus of intense interest. This articles reviews recent evidence that NMDA receptors undergo dynamically regulated targeting and trafficking, and that the physical transport of NMDA receptors in and out of the synaptic membrane contributes to several forms of long-lasting synaptic plasticity. The identification of targeting and internalization sequences in NMDA-receptor subunits has begun the unraveling of some mechanisms that underlie activity-dependent redistribution of NMDA receptors. Given that NMDA receptors are widely expressed throughout the CNS, regulation of NMDA-receptor trafficking provides a potentially important way to modulate efficacy of synaptic transmission.
Full Text Available Synaptic plasticity is widely regarded as the cellular basis of learning and memory. Understanding the molecular mechanism of synaptic plasticity has been one of center pieces of neuroscience research for more than three decades. It has been well known that the trafficking of α-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid- (AMPA- type, N-methyl-D-aspartate- (NMDA- type glutamate receptors to and from synapses is a key molecular event underlying many forms of synaptic plasticity. Kainate receptors are another type of glutamate receptors playing important roles in synaptic transmission. In addition, GABA receptors also play important roles in modulating the synaptic plasticity. Kinesin superfamily proteins (also known as KIFs transport various cargos in both anterograde and retrograde directions through the interaction with different adaptor proteins. Recent studies indicate that KIFs regulate the trafficking of NMDA receptors, AMPA receptors, kainate receptors, and GABA receptors and thus play important roles in neuronal activity. Here we review the essential functions of KIFs in the trafficking of neuroreceptor and synaptic plasticity.
Hruda N Mallick
Full Text Available Regulation of body temperature and sleep are two physiological mechanisms that are vital for our survival. Interestingly neural structures implicated in both these functions are common. These areas include the medial preoptic area, the lateral preoptic area, the ventrolateral preoptic area, the median preoptic nucleus and the medial septum, which form part of the basal forebrain.When given a choice, rats prefer to stay at an ambient temperature of 270C, though the maximum sleep was observed when they were placed at 300C. Ambient temperature around 270C should be considered as the thermoneutral temperature for rats in all sleep studies. At this temperature the diurnal oscillations of sleep and body temperature are properly expressed. The warm sensitive neurons of the preoptic area mediate the increase in sleep at 300C. Promotion of sleep during the rise in ambient temperature from 270C to 300C, serve a thermoregulatory function. Autonomous thermoregulatory changes in core body temperature and skin temperature could act as an input signal to modulate neuronal activity in sleep-promoting brain areas. The studies presented here show that the neurons of the basal forebrain play a key role in regulating sleep. Basal forebrain thermoregulatory system is a part of the global homeostatic sleep regulatory mechanism, which is auto-regulated.
Segarra, Marta; Maric, Dragan; Salvucci, Ombretta; Hou, Xu; Kumar, Anil; Li, Xuri; Tosato, Giovanna
Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis. PMID:23007403
Chimen, Myriam; McGettrick, Helen M; Apta, Bonita; Kuravi, Sahithi J; Yates, Clara M; Kennedy, Amy; Odedra, Arjun; Alassiri, Mohammed; Harrison, Matthew; Martin, Ashley; Barone, Francesca; Nayar, Saba; Hitchcock, Jessica R; Cunningham, Adam F; Raza, Karim; Filer, Andrew; Copland, David A; Dick, Andrew D; Robinson, Joseph; Kalia, Neena; Walker, Lucy S K; Buckley, Christopher D; Nash, Gerard B; Narendran, Parth; Rainger, G Ed
During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3 zeta delta (14.3.3.ζδ) protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin-induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type 1 diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to that of healthy age-matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Control of patient T cell trafficking is re-established by treatment with exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic ischemia-reperfusion injury, Salmonella infection, uveitis and Sjögren's syndrome, PEPITEM reduced T cell recruitment into inflamed tissues.
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.
Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane
The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic
Peterson, Dennis C. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
This document will detail the training curriculum for the Counter-Trafficking System Development (CTSD) Analysis Modules and Lesson Plans are derived from the United States Military, Department of Energy doctrine and Lawrence Livermore National Laboratory (LLNL), Global Security (GS) S Program.
Navarro, Rocio; Compte, Marta; Álvarez-Vallina, Luis;
respond to a series of proinflammatory stimuli and are able to sense different types of danger through expression of functional pattern recognition receptors, contributing to the onset of innate immune responses. In this context, PC not only secrete a variety of chemokines, but they also overexpress...... adhesion molecules such as ICAM-1 and VCAM-1 involved in the control of immune cell trafficking across vessel walls. In addition to their role in innate immunity, pericytes are involved in adaptive immunity. It has been reported that interaction with PC anergizes T cells, attributed, at least in part...
Small GTPases that belong to the ras sub-families of Rab, Arf, and Rho, and the large GTPase dynamin, regulate intracellular trafficking. This issue of Seminars of Cell and Developmental Biology highlights topics regarding mechanisms by which these GTPases regulate the different steps of vesicular transport: vesicle formation, scission, targeting and fusion. In addition, the emerging roles of GTPases in coordination of individual transport steps as well as coordination of intracellular trafficking with other cellular processes are reviewed. Finally, common structures and mechanisms underlying the function of the ras-like GTPases and the importance of their function to human health and disease are discussed.
Zeegers, Nicolle; Althoff, Martina
Is the Nordic model of combating the trafficking of women for sexual purposes to be followed by all member states of the eu? At the moment, the member states still differ considerably in their legislative approaches towards prostitution and the extent to which this is linked to the combat against se
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.
Zeegers, Nicolle; Althoff, Martina
Is the Nordic model of combating the trafficking of women for sexual purposes to be followed by all member states of the eu? At the moment, the member states still differ considerably in their legislative approaches towards prostitution and the extent to which this is linked to the combat against se
Zeegers, Nicolle; Althoff, Martina
Is the Nordic model of combating the trafficking of women for sexual purposes to be followed by all member states of the eu? At the moment, the member states still differ considerably in their legislative approaches towards prostitution and the extent to which this is linked to the combat against
Full Text Available Abstract Background Myf5 is one member of the basic helix-loop-helix family of transcription factors, and it functions as a myogenic factor that is important for the specification and differentiation of muscle cells. The expression of myf5 is somite- and stage-dependent during embryogenesis through a delicate regulation. However, this complex regulatory mechanism of myf5 is not clearly understood. Results We isolated a 156-kb bacterial artificial chromosome clone that includes an upstream 80-kb region and a downstream 70-kb region of zebrafish myf5 and generated a transgenic line carrying this 156-kb segment fused to a green fluorescent protein (GFP reporter gene. We find strong GFP expression in the most rostral somite and in the presomitic mesoderm during segmentation stages, similar to endogenous myf5 expression. Later, the GFP signals persist in caudal somites near the tail bud but are down-regulated in the older, rostral somites. During the pharyngula period, we detect GFP signals in pectoral fin buds, dorsal rostral myotomes, hypaxial myotomes, and inferior oblique and superior oblique muscles, a pattern that also corresponds well with endogenous myf5 transcripts. To characterize the specific upstream cis-elements that regulate this complex and dynamic expression pattern, we also generated several transgenic lines that harbor various lengths within the upstream 80-kb segment. We find that (1 the -80 kb/-9977 segment contains a fin and cranial muscle element and a notochord repressor; (2 the -9977/-6213 segment contains a strong repressive element that does not include the notochord-specific repressor; (3 the -6212/-2938 segment contains tissue-specific elements for bone and spinal cord; (4 the -2937/-291 segment contains an eye enhancer, and the -2937/-2457 segment is required for notochord and myocyte expression; and (5 the -290/-1 segment is responsible for basal transcription in somites and the presomitic mesoderm. Conclusion We suggest
Ghosh, Soumitra; Padmanabhan, Bhavna; Godbole, Adwait Anand; Tare, Priyanka; Ahmed, Wareed; Vasu, Kommireddy; China, Arnab; Kumar, Rupesh; Mitra, Anirban; Nagaraja, Valakunja
Mycobacterium tuberculosis (Mtb) is a formidable pathogen which has the ability to survive the hostile environment of the host by evading the host defense system. The re-configuration of its transcriptional and metabolic process allows the pathogen to confront the adverse environment within the host macrophages. The factors that assist the transcription and modulate the DNA topology would have to play a key role in the regulation of global gene expression of the organism. How transcription of these essential housekeeping genes alters in response to growth conditions and environmental stress has not been addressed together in a set of experimental conditions in Mtb. Now, we have mapped the transcription start sites (TSS) and promoters of several genes that play a central role in the regulation of DNA topology and transcription in Mtb. Using in vivo reporter assays, we validated the activity of the identified promoter elements in different growth conditions. The variation in transcript abundance of these essential genes was also analyzed in growth phase-dependent manner. These data provide the first glimpse into the specific adaptive changes in the expression of genes involved in transcription and DNA topology modulation in Mtb.
Full Text Available BACKGROUND: The 60(+ members of the mammalian Rab protein family group into subfamilies postulated to share common functionality. The Rab VI subfamily contains 5 Rab proteins, Rab6a/a', Rab6b, Rab6c and Rab41. High-level knockdown of Rab6a/a' has little effect on the tightly organized Golgi ribbon in HeLa cells as seen by fluorescence microscopy. In striking contrast, we found Rab41 was strongly required for normal Golgi ribbon organization. METHODS/RESULTS: Treatment of HeLa cells with Rab41 siRNAs scattered the Golgi ribbon into clustered, punctate Golgi elements. Overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a similar Golgi phenotype. By electron microscopy, Rab41 depletion produced short, isolated Golgi stacks. Golgi-associated vesicles accumulated. At low expression levels, wild type and GTP-locked Rab41 showed little concentration in the Golgi region, but puncta were observed and most were in ruffled regions at the cell periphery. There was 25% co-localization of GTP-locked Rab41 with the ER marker, Sec61p. GDP-locked Rab41, as expected, displayed an entirely diffuse cytoplasmic distribution. Depletion of Rab41 or overexpression of GDP-locked Rab41 partially inhibited ER-to-Golgi transport of VSV-G protein. However, Rab41 knockdown had little, if any, effect on endosome-to-Golgi transport of SLTB. Additionally, after a 2-day delay, treatment with Rab41 siRNA inhibited cell growth, while overexpression of GDP-locked Rab41, but not wild type or GTP-locked Rab41, produced a rapid, progressive cell loss. In double knockdown experiments with Rab6, the Golgi ribbon was fragmented, a result consistent with Rab41 and Rab6 acting in parallel. CONCLUSION: We provide the first evidence for distinctive Rab41 effects on Golgi organization, ER-to-Golgi trafficking and cell growth. When combined with the evidence that Rab6a/a' and Rab6b have diverse roles in Golgi function, while Rab6c regulates mitotic function, our
Navarro, Rocio; Compte, Marta; Álvarez-Vallina, Luis
adhesion molecules such as ICAM-1 and VCAM-1 involved in the control of immune cell trafficking across vessel walls. In addition to their role in innate immunity, pericytes are involved in adaptive immunity. It has been reported that interaction with PC anergizes T cells, attributed, at least in part...... respond to a series of proinflammatory stimuli and are able to sense different types of danger through expression of functional pattern recognition receptors, contributing to the onset of innate immune responses. In this context, PC not only secrete a variety of chemokines, but they also overexpress......Pericytes (PC) are mural cells that surround endothelial cells (EC) in small blood vessels. PC have traditionally been endowed with structural functions, being essential for vessel maturation and stabilization. However, accumulating evidence suggest that PC also display immune properties. They can...
Full Text Available Analysis of court cases shows how hard it is forvictims of trafficking to win the right to remain in the UK. Case law is inconsistent and more research and data collection are urgently needed.
Full Text Available Human trafficking is considered as a crime against humanity. To conduct the due process of law towards cases related with human trafficking, the law enforcement officers cannot work by themselves. They really need assistance from many parties – such as active report from the society – as a valuable information to disclose such cases. Law enforcement conducted towards woman and child trafficking is still ineffective. It is proven by many existing cases, that low number of processed cases before the court and minimum sanction convicted to the perpetrators is clearly evident. Factors which are deemed to have correlation with low attempt of law enforcement towards legal case on this case, among others are: Lack of the Government’s commitment to fight against the crime of human trafficking, in the event that the ineffectiveness in utilization of prevailing laws and regulation; Lack of capacity of professionalism of law enforcement agency (and relevant parties in handling women and child trafficking at the field. This may be caused by lack of knowledge on infringed regulation. For that matter, those law enforcement agency shall be given socialization and an SOP (standardized operational procedure, so that there will be no inconsistency in handling the existing cases.
Poulsen, Ebbe T.; Iannuzzi, Filomena; Rasmussen, Helle F.; Maier, Thorsten J.; Enghild, Jan J.; Jørgensen, Arne L.; Matrone, Carmela
Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD.
S.J.E. Langeslag (Sandra); J.W. van Strien (Jan)
textabstractIt has been suggested that emotion regulation improves with aging. Here, we investigated age differences in emotion regulation by studying modulation of the late positive potential (LPP) by emotion regulation instructions. The electroencephalogram of younger (18-26 years) and older (60-7
Vanessa M. D’Costa
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.
Full Text Available Trafficking in children is particularly severe form of exploitation and breach of the children rights, while security and welfare of children that are exposed to trafficking are obligations of state authorities, services and organizations of civil society. System of protection and support to children victims of trafficking should contain following: criteria for proper identification of child-victim of trafficking, mechanisms for immediate referring of a child to specialized services, procedures for appointing a guardian who will secure that procedures and decisions are in accordance with the best interest of child, measures for regulating of residential status, assistance with reparation and reintegration as well as measures for protection of children witnesses and victims of trafficking. Finally, it should include a proper access to justice. In the article, recommendations are proposed for improvement of identification system, proper evaluation of needs and planning services and protection measures as well as measures and activities which should secure long term solutions in accordance with rights of the child and her/his best interests.
Full Text Available Trafficking in human beings is an illegal act which interferes with the order foreseen by international regulations and national laws and endangers the values vital to society. However, all forms of human trafficking are not of the same danger to society and therefore do not represent the same risk, threat or danger to the safety of the state, society or individuals. In this paper, the author tends to classify the existing forms and aspects of trafficking in human beings as a manifestation in security practice in accordance with the level of social endangerment, geographical level of implementation, bio-physical characteristics of the victims, relation of the victim toward the position in which he/she finds himself/herself and the form of exploitation of the victim, including a short description of their most significant characteristics.
Tukey, David S; Ferreira, Jainne M; Antoine, Shannon O; D'amour, James A; Ninan, Ipe; Cabeza de Vaca, Soledad; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K; Hartner, Diana T; Guarini, Carlo B; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F; Khatri, Megna; Marzan, Dave S; Mahajan, Shahana S; Wang, Jing; Froemke, Robert C; Carr, Kenneth D; Aoki, Chiye; Ziff, Edward B
The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.
Mulholland, Patrick J.; Carpenter-Hyland, Ezekiel P.; Hearing, Matthew C.; Becker, Howard C.; Woodward, John J.; Chandler, L. Judson
Delayed-rectifier Kv2.1 potassium channels regulate somatodendritic excitability during periods of repetitive, high-frequency activity. Recent evidence suggests Kv2.1 channel modulation is linked to glutamatergic neurotransmission. Since NMDA-type glutamate receptors are critical regulators of synaptic plasticity, we investigated NMDA receptor modulation of Kv2.1 channels in rodent hippocampus and cortex. Bath application of NMDA potently unclustered and dephosphorylated Kv2.1 and produced a ...
Espinosa, Allan V.; Shinohara, Motoo; Porchia,Leonardo M; Chung, Yun Jae; McCarty, Samantha; Saji, Motoyasu; Ringel, Matthew D.
Metastasis suppressors and other regulators of cell motility play an important role in tumor invasion and metastases. We previously identified that activation of the G protein coupled receptor 54 (GPR54) by the metastasis suppressor metastin inhibits cell migration in association with overexpression of Regulator of calcineurin 1 (RCAN1), an endogenous regulator of calcineurin. Calcineurin inhibitors also blocked cell migration in vitro and RCAN1 protein levels were reduced in nodal metastases...
Moore, Jessica L; Kaplan, Dana M; Barron, Christine E
Sex trafficking is an increasingly recognized global health crisis affecting every country and region in the world. Domestic minor sex trafficking is a subset of commercial sexual exploitation of children, defined as engagement of minors (<18 years of age) in sexual acts for items of value (eg, food, shelter, drugs, money) involving children victimized within US borders. These involved youth are at risk for serious immediate and long-term physical and mental health consequences. Continued efforts are needed to improve preventive efforts, identification, screening, appropriate interventions, and subsequent resource provision for victimized and high-risk youth.
constants, the molecular mechanisms, and the proposed physiological roles of TCR trafficking in resting and stimulated T cells. In resting T cells, the TCR slowly and constitutively cycles between the plasma membrane and the intracellular compartment. Constitutive TCR cycling is dependent on the di......Dynamic regulation of TCR expression levels plays important roles in modulating T-cell responses during T-cell development and in mature T cells. TCR expression levels are determined by the rate constants for synthesis, endocytosis, recycling, and degradation. This review examines the rate....../or might ensure an internal store of TCR that can be rerouted to the immunological synapse during the encounter with an antigen-presenting cell....
Olesen, Christina Wilkens
of cell types. It contributes importantly to the normal physiological function of many human organs, and its dysfunction has been linked to several pathologies including cardiovascular diseases and breast cancer. Expression of a truncated constitutive active HER2 receptor, p95HER2, is associated with poor...... and mechanisms of NBCn1 trafficking, membrane retention and degradation in MCF-7 cells and other epithelial cell types. In Paper-I we review existing evidence regarding the possible roles of the SLC4 and SLC26 families of HCO3 - transporters in breast, colon and lung cancer. Bioinformatic analysis of publicly...
Jiang, Quanbao; Sánchez-Barricarte, Jesús
Based on existing data and prior research, this paper reviews studies of and investigations into the trafficking of women in China. First, the “industrial chain” of human trafficking is analyzed. Second, the analysis indicates that the “buyers market” exhibits a strong demand for trafficking in women. The scale of trafficking is escalating: originally the crime was mainly committed in a few provinces, but has now spread to nearly every province in China. Furthermore, human traf...
Resat, Haluk; Ewald, Jonathan A.; Dixon, David A.; Wiley, H. S.
Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and about 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multi-compartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physio-chemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor- alpha (TGF-a). Our simulations predict that when EGFR is activated with TGF-a, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias towards the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor down-regulation induced by either EGF or TGF-a. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.
Sawaki, Risa; Katayama, Jun'ichi
Attentional capture for distractors is enhanced by increasing the difficulty of discrimination between the standard and the target in the three-stimulus oddball paradigm. In this study, we investigated the cognitive mechanism of this modulation of attentional capture. Event-related brain potentials were recorded from participants while they…
Kwon, Sang-Ho; Oh, Sekyung; Nacke, Marisa; Mostov, Keith E; Lipschutz, Joshua H
Exosomes, 40-150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Tong, Huawei; Li, Qian; Zhang, Zi Chao; Li, Yi; Han, Junhai
Neurexins are cell adhesion molecules involved in synaptic formation and synaptic transmission. Mutations in neurexin genes are linked to autism spectrum disorders (ASDs), which are frequently associated with sleep problems. However, the role of neurexin-mediated synaptic transmission in sleep regulation is unclear. Here, we show that lack of the Drosophila α-neurexin homolog significantly reduces the quantity and quality of nighttime sleep and impairs sleep homeostasis. We report that neurexin expression in Drosophila mushroom body (MB) αβ neurons is essential for nighttime sleep. We demonstrate that reduced nighttime sleep in neurexin mutants is due to impaired αβ neuronal output, and show that neurexin functionally couples calcium channels (Cac) to regulate synaptic transmission. Finally, we determine that αβ surface (αβs) neurons release both acetylcholine and short neuropeptide F (sNPF), whereas αβ core (αβc) neurons release sNPF to promote nighttime sleep. Our findings reveal that neurexin regulates nighttime sleep by mediating the synaptic transmission of αβ neurons. This study elucidates the role of synaptic transmission in sleep regulation, and might offer insights into the mechanism of sleep disturbances in patients with autism disorders. PMID:27905548
Sui, Jinliang; Cotard, Shakira; Andersen, Jennifer; Zhu, Ping; Staunton, Jane; Lee, Margaret; Lin, Stephen
Cystic fibrosis is an inherited, life-threatening disease associated with mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, F508del CFTR, is found in 90% of CF patients. The loss of a single amino acid (phenylalanine at position 508) results in malformed CFTR with defective trafficking to the plasma membrane and impaired channel function. A functional assay with cells expressing F508del CFTR has been previously described by others using genetically engineered halide-sensitive yellow fluorescent protein to screen for CFTR modulators. We adapted this yellow fluorescent protein assay to 384-well plate format with a high-throughput screening plate reader, and optimized the assay in terms of data quality, resolution, and throughput, with target-specific protocols. The optimized assay was validated with reference compounds from cystic fibrosis foundation therapeutics. On the basis of the Z-factor range (≥0.5) and the potential productivity, this assay is well suited for high-throughput screening. It was successfully used to screen for active single agent and synergistic combinations of single agent modulators of F508del CFTR from a library collection of current active pharmaceutical ingredients (supported by Cystic Fibrosis Foundation Therapeutics).
Full Text Available Common microarray and next-generation sequencing data analysis concentrate on tumor subtype classification, marker detection, and transcriptional regulation discovery during biological processes by exploring the correlated gene expression patterns and their shared functions. Genetic regulatory network (GRN based approaches have been employed in many large studies in order to scrutinize for dysregulation and potential treatment controls. In addition to gene regulation and network construction, the concept of the network modulator that has significant systemic impact has been proposed, and detection algorithms have been developed in past years. Here we provide a unified mathematic description of these methods, followed with a brief survey of these modulator identification algorithms. As an early attempt to extend the concept to new RNA regulation mechanism, competitive endogenous RNA (ceRNA, into a modulator framework, we provide two applications to illustrate the network construction, modulation effect, and the preliminary finding from these networks. Those methods we surveyed and developed are used to dissect the regulated network under different modulators. Not limit to these, the concept of “modulation” can adapt to various biological mechanisms to discover the novel gene regulation mechanisms.
Full Text Available Rho family guanosine triphosphatases (GTPases, such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs, which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD, a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG. Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.
Yang, Yunfan; Sun, Lei; Tala; Gao, Jinmin; Li, Dengwen; Zhou, Jun; Liu, Min
Rho family guanosine triphosphatases (GTPases), such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs), which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD), a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG). Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.
Casado, Fanny L.; Singh, Kameshwar P.; Gasiewicz, Thomas A.
The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the Per-ARNT-Sim family of proteins. These proteins sense molecules and stimuli from the cellular/tissue environment and initiate signaling cascades to elicit appropriate cellular responses. Recent literature reports suggest an important function of AhR in hematopoietic stem cell (HSC) biology. However, the molecular mechanisms by which AhR signaling regulates HSC functions are unknown. In previous studies, we and othe...
Lee, Ting-Wei; Kao, Yu-Hsun; Lee, Ting-I; Chen, Yi-Jen
Receptor for advanced glycation end products (RAGE) signalling plays a critical role in the pathogenesis of cardiovascular disease. Calcitriol modulates cardiac RAGE expression. This study explored the mechanisms underlying the effect of calcitriol on RAGE and soluble RAGE (sRAGE) expression in cardiomyocytes. Western blot, ELISA, fluorometric assay and PCR analyses were used to evaluate the RAGE, sRAGE, endogenous secretory RAGE (esRAGE), Jun N-terminal kinase (JNK), and a disintegrin and metalloprotease 10 (ADAM10) expression and enzyme activity in HL-1 atrial myocytes without and with calcitriol (10 and 100 nM), nuclear factor-κB (NF-κB) inhibitor (50 μg/mL), or ADAM10 inhibitor (5 μM) incubation for 48 h. Calcitriol (10 nM) significantly reduced RAGE protein expression and increased sRAGE concentrations in HL-1 cardiomyocytes compared with control cells. These changes were associated with increased protein expression and enzyme activity of ADAM10 and higher mRNA expression of esRAGE. In the presence of ADAM10 inhibitor, however, the suppressive effect of calcitriol on RAGE was diminished. Methylglyoxal (500 μM for 10 min)-mediated JNK phosphorylation was attenuated in the presence of calcitriol (10 nM). Moreover, control and NF-κB inhibitor-treated HL-1 cells had similar RAGE and sRAGE expression, suggesting that calcitriol-mediated RAGE modulation was independent of NF-κB signalling. We showed that RAGE downregulation and increased sRAGE production by calcitriol were mediated through ADAM10 activation in cardiomyocytes. The results suggest that calcitriol has therapeutic potential in treating RAGE-mediated cardiovascular complications. © 2017 Stichting European Society for Clinical Investigation Journal Foundation.
Paul A ST JOHN
Nicotinic acetylcholine receptors (nAChRs) play critical roles throughout the body. Precise regulation of the cellular location and availability of nAChRs on neurons and target cells is critical to their proper function. Dynamic, post-translational regulation of nAChRs, particularly control of their movements among the different compartments of cells, is an important aspect of that regulation. A combination of new information and new techniques has the study of nAChR trafficking poised for new breakthroughs.
Full Text Available BACKGROUND: Several lines of investigation support the notion that endocytosis is crucial for Alzheimer's disease (AD pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1 distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1 in Aβ biogenesis. PRINCIPAL FINDINGS: We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aβ levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPβ and βCTF. Moreover, dyn1 knockdown (KD leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α-secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT or PS1 N-terminal fragment (NTF at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in βCTF and Aβ levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aβ, suggesting that the Aβ-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aβ levels in dyn1(-/- primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts. CONCLUSIONS: In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its
Chandrasekar, M.; Senthilkumar, T.
A passive thermal regulation technique with fins in conjunction with cotton wicks is developed in the present work for controlling the temperature of PV module during its operation. Experiments were conducted with the developed technique in the location of Tiruchirappalli (78.6°E and 10.8°N), Tamil Nadu, India with flat 25 Wp PV module and its viability was confirmed. The PV module temperature got reduced by 12 % while the electrical yield is increased by 14 % with the help of the developed cooling system. Basic energy balance equation applicable for PV module was used to evaluate the module temperatures and a fair agreement was obtained between the theoretical and experimental values for the cases of with cooling and without cooling.
Full Text Available The trafficking protein particle complex 4 (TRAPPC4 is implicated in vesicle-mediated transport, but its association with disease has rarely been reported. We explored its potential interaction with ERK2, part of the ERK1/2 complex in the Extracellular Signal-regulated Kinase/ Mitogen-activated Protein Kinase (ERK-MAPK pathway, by a yeast two-hybrid screen and confirmed by co-immunoprecipitation (Co-IP and glutathione S-transferase (GST pull-down. Further investigation found that when TRAPPC4 was depleted, activated ERK1/2 specifically decreased in the nucleus, which was accompanied with cell growth suppression and apoptosis in colorectal cancer (CRC cells. Overexpression of TRAPPC4 promoted cell viability and caused activated ERK1/2 to increase overall, but especially in the nucleus. TRAPPC4 was expressed more highly in the nucleus of CRC cells than in normal colonic epithelium or adenoma which corresponded with nuclear staining of pERK1/2. We demonstrate here that TRAPPC4 may regulate cell proliferation and apoptosis in CRC by interaction with ERK2 and subsequently phosphorylating ERK1/2 as well as modulating the subcellular location of pERK1/2 to activate the relevant signaling pathway.
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
Matys, V; Kel-Margoulis, O V; Fricke, E; Liebich, I; Land, S; Barre-Dirrie, A; Reuter, I; Chekmenev, D; Krull, M; Hornischer, K; Voss, N; Stegmaier, P; Lewicki-Potapov, B; Saxel, H; Kel, A E; Wingender, E
The TRANSFAC database on transcription factors, their binding sites, nucleotide distribution matrices and regulated genes as well as the complementing database TRANSCompel on composite elements have been further enhanced on various levels. A new web interface with different search options and integrated versions of Match and Patch provides increased functionality for TRANSFAC. The list of databases which are linked to the common GENE table of TRANSFAC and TRANSCompel has been extended by: Ensembl, UniGene, EntrezGene, HumanPSD and TRANSPRO. Standard gene names from HGNC, MGI and RGD, are included for human, mouse and rat genes, respectively. With the help of InterProScan, Pfam, SMART and PROSITE domains are assigned automatically to the protein sequences of the transcription factors. TRANSCompel contains now, in addition to the COMPEL table, a separate table for detailed information on the experimental EVIDENCE on which the composite elements are based. Finally, for TRANSFAC, in respect of data growth, in particular the gain of Drosophila transcription factor binding sites (by courtesy of the Drosophila DNase I footprint database) and of Arabidopsis factors (by courtesy of DATF, Database of Arabidopsis Transcription Factors) has to be stressed. The here described public releases, TRANSFAC 7.0 and TRANSCompel 7.0, are accessible under http://www.gene-regulation.com/pub/databases.html.
Richards, Tiffany A
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.
Khowaja, Shaneela Sadaruddin; Tharani, Ambreen Jawed; Agha, Ajmal; Karamaliani, Rozina Sherali
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.
Loss, Omar; Wu, Chun Ting; Riccio, Antonella; Saiardi, Adolfo
The binding of neurotrophins to tropomyosin receptor kinase receptors initiates several signaling pathways, including the activation of phospholipase C-γ, which promotes the release of diacylglycerol and inositol 1,4,5-trisphosphate (IP3). In addition to recycling back to inositol, IP3 serves as a precursor for the synthesis of higher phosphorylated inositols, such as inositol 1,3,4,5,6-pentakisphosphate (IP5) and inositol hexakisphosphate (IP6). Previous studies on the effect of neurotrophins on inositol signaling were limited to the analysis of IP3 and its dephosphorylation products. Here we demonstrate that nerve growth factor (NGF) regulates the levels of IP5 and IP6 during PC12 differentiation. Furthermore, both NGF and brain-derived neurotrophic factor alter IP5 and IP6 intracellular ratio in differentiated PC12 cells and primary neurons. Neurotrophins specifically regulate the expression of IP5-2 kinase (IP5-2K), which phosphorylates IP5 into IP6. IP5-2K is rapidly induced after NGF treatment, but its transcriptional levels sharply decrease in fully differentiated PC12 cells. Reduction of IP5-2K protein levels by small interfering RNA has an effect on the early stages of PC12 cell differentiation, whereas fully differentiated cells are not affected. Conversely, perturbation of IP5-2K levels by overexpression suggests that both differentiated PC12 cells and sympathetic neurons require low levels of the enzyme for survival. Therefore maintaining appropriate intracellular levels of inositol polyphosphates is necessary for neuronal survival and differentiation. PMID:23864704
Mahajan, Divyanshu; Boh, Boon Kim; Zhou, Yan; Chen, Li; Cornvik, Tobias Carl; Hong, Wanjin; Lu, Lei
Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.
William R. DI PIETRO
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.
Garrofé-Ochoa, Xènia; Cosialls, Ana M; Ribas, Judit; Gil, Joan; Boix, Jacint
Chemical inhibitors of cyclin-dependent kinase (CDK), like roscovitine, are promising drugs in the context of new cancer therapies. Roscovitine and related compounds, like seliciclib and olomoucine, are effective inducers of apoptosis in many proliferating cells in culture. These compounds are known to activate the intrinsic or mitochondrial pathway of apoptosis. In order to better characterize this intrinsic pathway, a transcriptional analysis was performed using the reverse transcriptase-multiplex ligation-dependent probe amplification procedure (RT-MLPA). In five cell lines, we detected an early and marked reduction of most transcripts, which is consistent with the disruption of transcription that results from the inhibition of CDK7 and CDK9. However, the mRNA of p53-upregulated modulator of apoptosis (PUMA) gene escaped from this transcription inhibition in neuroblastoma cells with a functional p53 protein. The increase of PUMA mRNA was not found in roscovitine-treated cell lines defective in p53, which underwent apoptosis like their p53 proficient counterparts. In addition, in SH-SY5Y cells, sublethal and lethal concentrations of roscovitine produced equivalent increases of PUMA mRNA and protein. In conclusion, the increased expression of PUMA was not associated with apoptosis induction. On the contrary, mRNA and protein depletion of MCL-1 gene correlated the best with cell demise. Moreover, NOXA protein suffered a far minor decrease than MCL-1. Because of the selective neutralization of NOXA by MCL-1, we hypothesize that the disruption of this balance is a critical event in apoptosis induction by roscovitine and related compounds.
Fiume, Elisa; de Klein, Niek; Rhee, Seung Yon; Magnani, Enrico
Transcription factors often form protein complexes and give rise to intricate transcriptional networks. The regulation of transcription factor multimerization plays a key role in the fine-tuning of the underlying transcriptional pathways and can be exploited to modulate synthetic transcriptional mod
ZHANG Min; ZHU Jing; GUO Zheng; LI Xia; YANG Da; WANG Lei; RAO Shaoqi
Identifying disease-relevant genes and functional modules, based on gene expression profiles and gene functional knowledge, is of high importance for studying disease mechanisms and subtyping disease phenotypes. Using gene categories of biological process and cellular component in Gene Ontology, we propose an approach to selecting functional modules enriched with differentially expressed genes, and identifying the feature functional modules of high disease discriminating abilities. Using the differentially expressed genes in each feature module as the feature genes, we reveal the relevance of the modules to the studied diseases. Using three datasets for prostate cancer, gastric cancer, and leukemia, we have demonstrated that the proposed modular approach is of high power in identifying functionally integrated feature gene subsets that are highly relevant to the disease mechanisms. Our analysis has also shown that the critical disease-relevant genes might be better recognized from the gene regulation network, which is constructed using the characterized functional modules, giving important clues to the concerted mechanisms of the modules responding to complex disease states. In addition, the proposed approach to selecting the disease-relevant genes by jointly considering the gene functional knowledge suggests a new way for precisely classifying disease samples with clear biological interpretations, which is critical for the clinical diagnosis and the elucidation of the pathogenic basis of complex diseases.
Lise Sofie H. Nissen-Meyer
Full Text Available The system N transporter SN1 (also known as SNAT3 is enriched on perisynaptic astroglial cell membranes. SN1 mediates electroneutral and bidirectional glutamine transport, and regulates the intracellular as well as the extracellular concentrations of glutamine. We hypothesize that SN1 participates in the glutamate/GABA-glutamine cycle and regulates the amount of glutamine supplied to the nerve terminals for replenishment of the neurotransmitter pools of glutamate and GABA. We also hypothesize that its activity on the plasma membrane is regulated by PKC-mediated phosphorylation and that SN1 activity has an impact on synaptic plasticity. This review discusses inconcistencies reported in the regulation of SN1 by PKC and presents a consolidated model for regulation and degradation of SN1 and the subsequent functional implications. As SN1 function is likely also regulated by PKC-mediated phosphorylation in peripheral organs, the same mechanisms may, thus, have impact on e.g. pH regulation in the kidney, urea formation in the liver and insulin secretion in the pancreas.
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.
Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA
The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.
Cai, Zhao; Liu, Yang; Chen, Yicai; Yam, Joey Kuok Hoong; Chew, Su Chuen; Chua, Song Lin; Wang, Ke; Givskov, Michael; Yang, Liang
The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs) quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator.
Full Text Available The alternative sigma factor RpoN regulates many cell functions, such as motility, quorum sensing, and virulence in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa. P. aeruginosa often evolves rpoN-negative variants during the chronic infection in cystic fibrosis patients. It is unclear how RpoN interacts with other regulatory mechanisms to control virulence of P. aeruginosa. In this study, we show that RpoN modulates the function of PqsR, a quorum sensing receptor regulating production of virulence factors including the phenazine pyocyanin. The ∆rpoN mutant is able to synthesize 4-quinolone signal molecule HHQ but unable to activate PqsR and Pseudomonas quinolone signal (pqs quorum sensing. The ∆rpoN mutant produces minimal level of pyocyanin and is unable to produce the anti-staphylococcal agents. Providing pqsR in trans in the ∆rpoN mutant restores its pqs quorum sensing and virulence factor production to the wild-type level. Our study provides evidence that RpoN has a regulatory effect on P. aeruginosa virulence through modulating the function of the PqsR quorum sensing regulator.
Xie, Zhengzhi; Ma, Xiaoqiang; Gang, David R
Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules-groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants.
Full Text Available BACKGROUND: N-MYC down-regulated-like (NDL proteins interact with the Gβ subunit (AGB1 of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. METHODOLOGY/PRINCIPAL FINDINGS: Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. CONCLUSION/SIGNIFICANCE: NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.
Full Text Available Abstract Secreted factors and cell surface receptors can be internalized by endocytosis and translocated to the cytoplasm. Instead of being recycled or proteolysed, they sometimes translocate to the nucleus. Nuclear import generally involves a nuclear localization signal contained either in the secreted factor or its transmembrane receptor, that is recognized by the importins machinery. In the nucleus, these molecules regulate transcription of specific target genes by direct binding to transcription factors or general coregulators. In addition to the transcription regulation, nuclear secreted proteins and receptors seem to be involved in other important processes for cell life and cellular integrity such as DNA replication, DNA repair and RNA metabolism. Nuclear secreted proteins and transmembrane receptors now appear to induce new signaling pathways to regulate cell proliferation and differentiation. Their nuclear localization is often transient, appearing only during certain phases of the cell cycle. Nuclear secreted and transmembrane molecules regulate the proliferation and differentiation of a large panel of cell types during embryogenesis and adulthood and are also potentially involved in wound healing. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Nuclear localization of these molecules has been correlated with tumor progression and poor prognosis for patient survival. Nuclear growth factors and receptors may be responsible for resistance to radiotherapy.
Deng, Pan-Yue; Rotman, Ziv; Blundon, Jay A.; Cho, Yongcheol; Cui, Jianmin; Cavalli, Valeria; Zakharenko, Stanislav S; Klyachko, Vitaly A.
Loss of FMRP causes Fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely r...
Chien, Ming-Wei; Lin, Ming-Hong; Huang, Shing-Hwa; Fu, Shin-Huei; Hsu, Chao-Yuan; Yen, B Lin-Ju; Chen, Jiann-Torng; Chang, Deh-Ming; Sytwu, Huey-Kang
Glucosamine has immunomodulatory effects on autoimmune diseases. However, the mechanism(s) through which glucosamine modulates different T cell subsets and diseases remain unclear. We demonstrate that glucosamine impedes Th1, Th2, and iTreg but promotes Th17 differentiation through down-regulating N-linked glycosylation of CD25 and subsequently inhibiting its downstream Stat5 signaling in a dose-dependent manner. The effect of glucosamine on T helper cell differentiation was similar to that induced by anti-IL-2 treatment, further supporting an IL-2 signaling-dependent modulation. Interestingly, excess glucose rescued this glucosamine-mediated regulation, suggesting a functional competition between glucose and glucosamine. High-dose glucosamine significantly decreased Glut1 N-glycosylation in Th1-polarized cells. This finding suggests that both down-regulated IL-2 signaling and Glut1-dependent glycolytic metabolism contribute to the inhibition of Th1 differentiation by glucosamine. Finally, glucosamine treatment inhibited Th1 cells in vivo, prolonged the survival of islet grafts in diabetic recipients, and exacerbated the severity of EAE. Taken together, our results indicate that glucosamine interferes with N-glycosylation of CD25, and thereby attenuates IL-2 downstream signaling. These effects suggest that glucosamine may be an important modulator of T cell differentiation and immune homeostasis.
Olesen, Christina Wilkens
of the sorting and regulation of NBCn1 in breast and kidney epithelial cells. We found that NBCn1 protein turnover was very slow and that the transporter did not undergo constitutive endocytosis over a 24 h period, suggesting that the protein is unusually stable in the epithelial cell plasma membrane under......Numerous cellular processes are highly pH-dependent in the physiological range, and regulation of intracellular pH (pHi) is therefore fundamental for maintaining normal cell function. The electroneutral Na+;HCO3- co-transporter NBCn1 (SLC4A7) plays essential roles in pHi homeostasis in a wide range...... of cell types. It contributes importantly to the normal physiological function of many human organs, and its dysfunction has been linked to several pathologies including cardiovascular diseases and breast cancer. Expression of a truncated constitutive active HER2 receptor, p95HER2, is associated with poor...
Tukey, David S; Ziff, Edward B
Regulation of striatal medium spiny neuron synapses underlies forms of motivated behavior and pathological drug seeking. A primary mechanism for increasing synaptic strength is the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynapse, a process mediated by GluA1 AMPAR subunit phosphorylation. We have examined the role of converging glutamate and dopamine inputs in regulating biochemical cascades upstream of GluA1 phosphorylation. We focused on the role of Ca(2+)-permeable AMPARs (CPARs), which lack the GluA2 AMPAR subunit. Under conditions that prevented depolarization, stimulation of CPARs activated neuronal nitric oxide synthase and production of cGMP. CPAR-dependent cGMP production was sufficient to induce synaptic insertion of GluA1, detected by confocal microscopy, through a mechanism dependent on GluA1 Ser-845 phosphorylation. Dopamine D1 receptors, in contrast, stimulate GluA1 extra synaptic insertion. Simultaneous activation of dopamine D1 receptors and CPARs induced additive increases in GluA1 membrane insertion, but only CPAR stimulation augmented CPAR-dependent GluA1 synaptic insertion. This incorporation into the synapse proceeded through a sequential two-step mechanism; that is, cGMP-dependent protein kinase II facilitated membrane insertion and/or retention, and protein kinase C activity was necessary for synaptic insertion. These data suggest a feed-forward mechanism for synaptic priming whereby an initial stimulus acting independently of voltage-gated conductance increases striatal neuron excitability, facilitating greater neuronal excitation by a subsequent stimulus.
The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular
Full Text Available In a previous study, we showed that the silencing of the heavy subunit (FHC offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC comparing it with K562 transduced with scrambled RNA (K562shRNA. Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, "Cell Death and Survival, Hematological System Development and Function, Hematopoiesis", is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.
Biamonte, Flavia; Zolea, Fabiana; Bisognin, Andrea; Di Sanzo, Maddalena; Saccoman, Claudia; Scumaci, Domenica; Aversa, Ilenia; Panebianco, Mariafranca; Faniello, Maria Concetta; Bortoluzzi, Stefania; Cuda, Giovanni; Costanzo, Francesco
In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562shFHC) comparing it with K562 transduced with scrambled RNA (K562shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs. PMID:25815883
Friedrich, Steinhäusler; Lyudmila, Zaitseva
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.
Liu, Tong; Wu, Changgong; Jain, Mohit Raja; Nagarajan, Narayani; Yan, Lin; Dai, Huacheng; Cui, Chuanlong; Baykal, Ahmet; Pan, Stacey; Ago, Tetsuro; Sadoshima, Junichi; Li, Hong
Thioredoxin 1 (Trx1) is а antioxidant protein that regulates protein disulfide bond reduction, transnitrosylation, denitrosylation and other redox post-translational modifications. In order to better understand how Trx1 modulates downstream protective cellular signaling events following cardiac ischemia, we conducted an expression proteomics study of left ventricles (LVs) after thoracic aortic constriction stress treatment of transgenic mice with cardiac-specific over-expression of Trx1, an animal model that has been proven to withstand more stress than its non-transgenic littermates. Although previous redox post-translational modifications proteomics studies found that several cellular protein networks are regulated by Trx1-mediated disulfide reduction and transnitrosylation, we found that Trx1 regulates the expression of a limited number of proteins. Among the proteins found to be upregulated in this study was SET and MYND domain-containing protein 1 (SMYD1), a lysine methyltransferase highly expressed in cardiac and other muscle tissues and an important regulator of cardiac development. The observation of SMYD1 induction by Trx1 following thoracic aortic constriction stress is consistent with the retrograde fetal gene cardiac protection hypothesis. The results presented here suggest for the first time that, in addition to being a master redox regulator of protein disulfide bonds and nitrosation, Trx1 may also modulate lysine methylation, a non-redox post-translational modification, via the regulation of SMYD1 expression. Such crosstalk between redox signaling and a non-redox PTM regulation may provide novel insights into the functions of Trx1 that are independent from its immediate function as a protein reductase.
Economic and social inequalities and political conflicts have led to the movement of persons within each country and across the borders in South Asia. Globalization has encouraged free mobility of capital, technology, experts and sex tourism. Illiteracy, dependency, violence, social stigma, cultural stereotypes, gender disparity and endemic poverty, among other factors, place women and children in powerless, non-negotiable situations that have contributed to the emergence and breeding of the cavernous problem of sex trafficking in the entire region. This alarming spread of sex trafficking has fuelled the spread of HIV infection in South Asia, posing a unique and serious threat to community health, poverty alleviation and other crucial aspects of human development. Although the SAARC (South Asian Association for Regional Cooperation) Convention on Trafficking in Women and Children has been an important breakthrough, most of the countries in the region do not have anti-trafficking legislation or means to protect the victims. Countries of the region should make a concerted effort to treat trafficking victims as "victims" of human rights violations in all anti-trafficking strategies and actions.
Janda, Jaroslav; Nfonsam, Valentine; Calienes, Fernanda; Sligh, James E; Jandova, Jana
Mitochondria are the major source of reactive oxygen species (ROS) in fibroblasts which are thought to be crucial regulators of wound healing with a potential to affect the expression of nuclear genes involved in this process. ROS generated by mitochondria are involved in all stages of tissue repair process but the regulation of ROS-generating system in fibroblasts still remains poorly understood. The purpose of this study was to better understand molecular mechanisms of how the regulation of ROS levels generated by mitochondria may influence the process of wound repair. Cybrid model system of mtDNA variations was used to study the functional consequences of altered ROS levels on wound healing responses in a uniform nuclear background of cultured ρ(0) fibroblasts. Mitochondrial ROS in cybrids were modulated by antioxidants that quench ROS to examine their ability to close the wound. Real-time PCR arrays were used to investigate whether ROS generated by specific mtDNA variants have the ability to alter expression of some key nuclear-encoded genes central to the wound healing response and oxidative stress. Our data suggest levels of mitochondrial ROS affect expression of some nuclear encoded genes central to wound healing response and oxidative stress and modulation of mitochondrial ROS by antioxidants positively affects in vitro process of wound closure. Thus, regulation of mitochondrial ROS-generating system in fibroblasts can be used as effective natural redox-based strategy to help treat non-healing wounds.
Goncharov, Tatiana; Niessen, Kyle; de Almagro, Maria Cristina; Izrael-Tomasevic, Anita; Fedorova, Anna V; Varfolomeev, Eugene; Arnott, David; Deshayes, Kurt; Kirkpatrick, Donald S; Vucic, Domagoj
The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.
Balatsky, Galya I [Los Alamos National Laboratory; Severe, William R [Los Alamos National Laboratory; Wallace, Richard K [Los Alamos National Laboratory
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
Karl Ravet; Brigitte Touraine; Sun A. Kim; Francoise Cellier; Sébastien Thomine; Mary Lou Guerinot; Jean-Francois Briat; Frédéric Gaymard
Ferritins are major players in plant iron homeostasis. Surprisingly, their overexpression in transgenic plants led only to a moderate increase in seed iron content, suggesting the existence of control checkpoints for iron loading and storage in seeds. This work reports the identification of two of these checkpoints. First, measurement of seed metal con-tent during fruit development in Arabidopsis thaliana reveals a similar dynamic of loading for Fe, Mn, Cu, and Zn. The step controlling metal loading into the seed occurs by the regulation of transport from the hull to the seed. Second, metal loading and ferritin abundance were monitored in different genetic backgrounds affected in vacuolar iron transport (AtVIT1, AtNRAMP3, AtNRAMP4) or plastid iron storage (AtFER1 to 4). This approach revealed (1) a post-translational reg-ulation of ferritin accumulation in seeds, and (2) that ferritin stability depends on the balance of iron allocation between vacuoles and plastids. Thus, the success of ferritin overexpression strategies for iron biofortification, a promising approach to reduce iron-deficiency anemia in developing countries, would strongly benefit from the identification and engineering of mechanisms enabling the translocation of high amounts of iron into seed plastids.
Huang, Yanyan; Lin, Youhui; Ran, Xiang; Ren, Jinsong; Qu, Xiaogang
Here we propose a new concept for the fabrication of a semipermeable enzymatic nanoreactor as an efficient modulator to reversibly switch the pH of an aqueous environment. We used amino-functionalized, expanded mesoporous silica nanoparticles (EMSN) as a model nanocarrier to load enzymes. In order to protect enzymes from the interference of a complicated environment, polyelectrolyte multilayers (PEMs) were coated on the surface of the EMSN through layer by layer (LbL) assembly. These PEMs can serve as semipermeable membranes, allowing small molecules to diffuse in and out freely while trapping the enzymes in the nanoreactors. Compared with traditional electrochemical stimulation or optical control methods, our enzymatic regulation platform is easy to operate without complicated instruments. In addition, this system can cover a wide range of pH values and conveniently regulate pH values by simply controlling the concentrations of catalysts or reactants. Meanwhile, this strategy could be generalized to other enzymes or nanocarriers to achieve reversible pH regulation for different purposes. The switched pH values can be implemented for the modulation of the conformational changes of nucleic acids and activation of the charge conversion in drug delivery applications.Here we propose a new concept for the fabrication of a semipermeable enzymatic nanoreactor as an efficient modulator to reversibly switch the pH of an aqueous environment. We used amino-functionalized, expanded mesoporous silica nanoparticles (EMSN) as a model nanocarrier to load enzymes. In order to protect enzymes from the interference of a complicated environment, polyelectrolyte multilayers (PEMs) were coated on the surface of the EMSN through layer by layer (LbL) assembly. These PEMs can serve as semipermeable membranes, allowing small molecules to diffuse in and out freely while trapping the enzymes in the nanoreactors. Compared with traditional electrochemical stimulation or optical control methods
... REGULATIONS General Definitions § 598.314 Specially designated narcotics trafficker. The term specially... Central Intelligence, the Director of the Federal Bureau of Investigation, the Administrator of the Drug... persons determined to fall within this definition who have been designated pursuant to this part....
Racine, Sarah E; Forbush, Kelsie T; Wildes, Jennifer E; Hagan, Kelsey E; Pollack, Lauren O; May, Casey
Emotion regulation difficulties are implicated in the development and maintenance of anorexia nervosa (AN). However, research has been limited by an almost exclusive reliance on self-report. This study is the first to use the emotion-modulated startle paradigm (EMSP) to investigate emotional reactivity and voluntary emotion regulation in individuals with AN. Twenty women with AN viewed negative, positive, neutral, and food images and were asked to enhance, suppress, or maintain their emotional responses mid-way through picture presentation. Startle eyeblink magnitudes in response to startle probes administered prior, and subsequent, to regulation instructions indexed emotional reactivity and regulation, respectively. On emotional reactivity trials, startle magnitudes were greater for negative, positive, and food images, compared to neutral images. Participants had difficulty suppressing startle responses to negative and food images, as indicated by non-significant suppress-maintain comparisons. In contrast, startle responses to enhance and suppress cues during presentation of pleasant images were comparable and significantly lower than maintain cues. Findings converge with self-report data to suggest that patients with AN have difficulties with voluntary emotion regulation. The EMSP may be a promising trans-diagnostic method for examining emotion regulation difficulties that underlie risk for eating disorders and other psychiatric conditions.
Jiménez-García, Lidia; Herranz, Sandra; Higueras, María Angeles; Luque, Alfonso; Hortelano, Sonsoles
Tumor microenvironment has been described to play a key role in tumor growth, progression, and metastasis. Macrophages are a major cellular constituent of the tumor stroma, and particularly tumor associated macrophages (TAMs or M2-like macrophages) exert important immunosuppressive activity and a pro-tumoral role within the tumor microenvironment. Alternative-reading frame (ARF) gene is widely inactivated in human cancer. We have previously demonstrated that ARF deficiency severely impairs inflammatory response establishing a new role for ARF in the regulation of innate immunity. On the basis of these observations, we hypothesized that ARF may also regulates tumor growth through recruitment and modulation of the macrophage phenotype in the tumor microenvironment. Xenograft assays of B16F10 melanoma cells into ARF-deficient mice resulted in increased tumor growth compared to those implanted in WT control mice. Tumors from ARF-deficient mice exhibited significantly increased number of TAMs as well as microvascular density. Transwell assays showed crosstalk between tumor cells and macrophages. On the one hand, ARF-deficient macrophages modulate migratory ability of the tumor cells. And on the other, tumor cells promote the skewing of ARF-/- macrophages toward a M2-type polarization. In conclusion, these results demonstrate that ARF deficiency facilitates the infiltration of macrophages into the tumor mass and favors their polarization towards a M2 phenotype, thus promoting tumor angiogenesis and tumor growth. This work provides novel information about the critical role of ARF in the modulation of tumor microenvironment.
Rosa C Baños
Full Text Available Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.
David, Jens-Peter; Andersen, Martin N; Olesen, Søren-Peter
has not been unequivocally resolved yet. We employed trafficking-deficient K 7.1 and KCNE1 mutants to investigate I trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes......The voltage-gated potassium channel K 7.1 is regulated by non-pore forming regulatory KCNE ß-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current I . However, where the subunits assemble and which of the subunits determines localization of the I -complex...
Crockett Elahé T
-selectin does not appear to be critical for neutrophil infiltration and I/R injury in the liver, they may regulate CXC-chemokine production. Blockage of these adhesion molecules may improve survival and remote organ injury that often accompanies liver I/R injury, through chemokine regulation.
Sze, Heven [Univ. of Maryland, College Park, MD (United States). Dept. of Cell Biology & Molecular Genetics
Multicellular, as well as unicellular, organisms have evolved mechanisms to regulate ion and pH homeostasis in response to developmental cues and to a changing environment. The working hypothesis is that the balance of fluxes mediated by diverse transporters at the plasma membrane and in subcellular organelles determines ionic cellular distribution, which is critical for maintenance of membrane potential, pH control, osmolality, transport of nutrients, and protein activity. An emerging theme in plant cell biology is that cells respond and adapt to diverse cues through changes of the dynamic endomembrane system. Yet we know very little about the transporters that might influence the operation of the secretory system in plants. Here we focus on transporters that influence alkali cation and pH homeostasis, mainly in the endomembrane/ secretory system. The endomembrane system of eukaryote cells serves several major functions: i) sort cargo (e.g. enzymes, transporters or receptors) to specific destinations, ii) modulate the protein and lipid composition of membrane domains through remodeling, and iii) determine and alter the properties of the cell wall through synthesis and remodeling. We had uncovered a novel family of predicted cation/H+ exchangers (CHX) and K+ efflux antiporters (KEA) that are prevalent in higher plants, but rare in metazoans. We combined phylogenetic and transcriptomic analyses with molecular genetic, cell biological and biochemical studies, and have published the first reports on functions of plant CHXs and KEAs. CHX studied to date act at the endomembrane system where their actions are distinct from the better-studied NHX (Na/K-H+ exchangers). Arabidopsis thaliana CHX20 in guard cells modulate stomatal opening, and thus is significant for vegetative survival. Other CHXs ensure reproductive success on dry land, as they participate in organizing pollen walls, targeting of pollen tubes to the ovule or promoting
Kowalczyk, Katarzyna M; Petersen, Janni
Target of Rapamycin (TOR) signalling allows eukaryotic cells to adjust cell growth in response to changes in their nutritional and environmental context. The two distinct TOR complexes (TORC1/2) localise to the cell's internal membrane compartments; the endoplasmic reticulum (ER), Golgi apparatus and lysosomes/vacuoles. Here, we show that Ppk32, a SCYL family pseudo-kinase, is a novel regulator of TOR signalling. The absence of ppk32 expression confers resistance to TOR inhibition. Ppk32 inhibition of TORC1 is critical for cell survival following Brefeldin A (BFA) induced stress. Treatment of wild type cells with either the TORC1 specific inhibitor rapamycin or the general TOR inhibitor Torin1 confirmed that a reduction in TORC1 activity promoted recovery from BFA induced stress. Phosphorylation of Ppk32 on two residues that are conserved within the SCYL pseudo-kinase family are required for this TOR inhibition. Phosphorylation on these sites controls Ppk32 protein levels and sensitivity to BFA. BFA induced ER stress does not account for the response to BFA that we report here, however BFA is also known to induce Golgi stress and impair traffic to lysosomes. In summary, Ppk32 reduce TOR signalling in response to BFA induced stress to support cell survival.
There is growing recognition and evidence that health care professionals regularly encounter-though they may not identify-victims of human trafficking in a variety of health care settings. Identifying and responding appropriately to trafficking victims or survivors requires not only training in trauma-informed care but also consideration of the legal and ethical issues that arise when serving this vulnerable population. This essay examines three areas of law that are relevant to this case scenario: criminal law, with a focus on conspiracy; service provider regulations, with a focus on mandatory reporting laws; and human rights law. In addition to imposing a legal mandate, the law can inform ethical considerations about how health care professionals should respond to human trafficking.
Löfke, Christian; Luschnig, Christian; Kleine-Vehn, Jürgen
Cell-to-cell communication is absolutely essential for multicellular organisms. Both animals and plants use chemicals called hormones for intercellular signaling. However, multicellularity of plants and animals has evolved independently, which led to establishment of distinct strategies in order to cope with variations in an ever-changing environment. The phytohormone auxin is crucial to plant development and patterning. PIN auxin efflux carrier-driven polar auxin transport regulates plant development as it controls asymmetric auxin distribution (auxin gradients), which in turn modulates a wide range of developmental processes. Internal and external cues trigger a number of posttranslational PIN auxin carrier modifications that were demonstrated to decisively influence variations in adaptive growth responses. In this review, we highlight recent advances in the analysis of posttranslational modification of PIN auxin efflux carriers, such as phosphorylation and ubiquitylation, and discuss their eminent role in directional vesicle trafficking, PIN protein de-/stabilization and auxin transport activity. We conclude with updated models, in which we attempt to integrate the mechanistic relevance of posttranslational modifications of PIN auxin carriers for the dynamic nature of plant development.
The main theme of this article is market development and trafficking as a business. It touches upon most of the aspects of the phenomenon, which have been encountered elsewhere, and translates them into the relatively unfamiliar context of many of the Asian and South-East Asian economies. Equally, the literature cited is also probably unfamiliar. Themes touched upon include democratization, inter-state relations, human rights, and scale and perspectives, together with the problems of definitions, theory, and the reliability of data. The directions and characteristics of trafficking flows together with routes and border control are also considered. Coordinated official responses to criminality and criminal organizations, as well as to trafficked individuals, are beginning to emerge. There is a note of caution sounded that contextual and cultural perspectives, particularly on sex workers, must be viewed somewhat differently to those in Western societies. The article concludes that as long as countries in Asia maintain their policies of restrictive immigration, trafficking can be expected to continue and almost certainly increase. This is because accelerating development creates demand for labor at various skill levels and because even in times of recession migrants and brokers will seek to side-step attempts to expel immigrants and restrict access to labor markets. The elimination of trafficking is unlikely to be realistically achieved through legislation and declarations of intent but by improvements in the socioeconomic status of the population.
Baltierra-Uribe, Shantal Lizbeth; García-Vásquez, Manuel de Jesús; Castrejón-Jiménez, Nayeli Shantal; Estrella-Piñón, Mayra Patricia; Luna-Herrera, Julieta; García-Pérez, Blanca Estela
Endothelial cells are susceptible to infection by mycobacteria, but the endocytic mechanisms that mycobacteria exploit to enter host cells and their mechanisms of intracellular transport are completely unknown. Using pharmacological inhibitors, we determined that the internalization of Mycobacterium tuberculosis (MTB), Mycobacterium smegmatis (MSM), and Mycobacterium abscessus (MAB) is dependent on the cytoskeleton and is differentially inhibited by cytochalasin D, nocodazole, cycloheximide, wortmannin, and amiloride. Using confocal microscopy, we investigated their endosomal trafficking by analyzing Rab5, Rab7, LAMP-1, and cathepsin D. Our results suggest that MSM exploits macropinocytosis to enter endothelial cells and that the vacuoles containing these bacteria fuse with lysosomes. Conversely, the entry of MTB seems to depend on more than one endocytic route, and the observation that only a subset of the intracellular bacilli was associated with phagolysosomes suggests that these bacteria are able to inhibit endosomal maturation to persist intracellularly. The route of entry for MAB depends mainly on microtubules, which suggests that MAB uses a different trafficking pathway. However, MAB is also able to inhibit endosomal maturation and can replicate intracellularly. Together, these findings provide the first evidence that mycobacteria modulate proteins of host endothelial cells to enter and persist within these cells.
Full Text Available Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care.
Zimmerman, Cathy; Kiss, Ligia; Houssain, Mazeda; Watts, Charlotte
Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care.
Elbuluk, Malik E.; Gerber, Scott; Hammoud, Ahmad; Patterson, Richard L.
DC/DC converters that are capable of operating at cryogenic temperatures are anticipated to play an important role in the power systems of future NASA deep space missions. Design of these converters to survive cryogenic temperatures will improve the power system performance, and reduce development and launch costs. At the NASA Glenn Research Center Low Temperature Electronics Laboratory, several commercial off-the-shelf dc/dc converter modules were evaluated for their low temperature performance. Various parameters were investigated as a function of temperature, in the range of 20 C to -190 C. Data pertaining to the efficiency and voltage regulation of the tested converters is presented and discussed.
Farinha, Carlos M; Canato, Sara
CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.
Full Text Available The deposition of β-amyloid (Aβ into senile plaques and the impairment of somatostatin-mediated neurotransmission are key pathological events in the onset of Alzheimer's disease (AD. Insulin-degrading-enzyme (IDE is one of the main extracellular protease targeting Aβ, and thus it represents an interesting pharmacological target for AD therapy. We show that the active form of somatostatin-14 regulates IDE activity by affecting its expression and secretion in microglia cells. A similar effect can also be observed when adding octreotide. Following a previous observation where somatostatin directly interacts with IDE, here we demonstrate that somatostatin regulates Aβ catabolism by modulating IDE proteolytic activity in IDE gene-silencing experiments. As a whole, these data indicate the relevant role played by somatostatin and, potentially, by analogue octreotide, in preventing Aβ accumulation by partially restoring IDE activity.
Çamdere, Gamze; Guacci, Vincent; Stricklin, Jeremiah; Koshland, Douglas
Cohesin tethers together regions of DNA, thereby mediating higher order chromatin organization that is critical for sister chromatid cohesion, DNA repair and transcriptional regulation. Cohesin contains a heterodimeric ATP-binding Cassette (ABC) ATPase comprised of Smc1 and Smc3 ATPase active sites. These ATPases are required for cohesin to bind DNA. Cohesin's DNA binding activity is also promoted by the Eco1 acetyltransferase and inhibited by Wpl1. Recently we showed that after cohesin stably binds DNA, a second step is required for DNA tethering. This second step is also controlled by Eco1 acetylation. Here, we use genetic and biochemical analyses to show that this second DNA tethering step is regulated by cohesin ATPase. Furthermore, our results also suggest that Eco1 promotes cohesion by modulating the ATPase cycle of DNA-bound cohesin in a state that is permissive for DNA tethering and refractory to Wpl1 inhibition.
Full Text Available Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway.
Rossi, Giuliana; Antonini, Stefania; Bonfanti, Chiara; Monteverde, Stefania; Vezzali, Chiara; Tajbakhsh, Shahragim; Cossu, Giulio; Messina, Graziella
Summary Nfix belongs to a family of four highly conserved proteins that act as transcriptional activators and/or repressors of cellular and viral genes. We previously showed a pivotal role for Nfix in regulating the transcriptional switch from embryonic to fetal myogenesis. Here, we show that Nfix directly represses the Myostatin promoter, thus controlling the proper timing of satellite cell differentiation and muscle regeneration. Nfix-null mice display delayed regeneration after injury, and this deficit is reversed upon in vivo Myostatin silencing. Conditional deletion of Nfix in satellite cells results in a similar delay in regeneration, confirming the functional requirement for Nfix in satellite cells. Moreover, mice lacking Nfix show reduced myofiber cross sectional area and a predominant slow twitching phenotype. These data define a role for Nfix in postnatal skeletal muscle and unveil a mechanism for Myostatin regulation, thus providing insights into the modulation of its complex signaling pathway. PMID:26923583
Nagendran, Monica; Arora, Prateek; Gori, Payal; Mulay, Aditya; Ray, Shinjini; Jacob, Tressa; Sonawane, Mahendra
The patterning and morphogenesis of body appendages - such as limbs and fins - is orchestrated by the activities of several developmental pathways. Wnt signalling is essential for the induction of limbs. However, it is unclear whether a canonical Wnt signalling gradient exists and regulates the patterning of epithelium in vertebrate appendages. Using an evolutionarily old appendage - the median fin in zebrafish - as a model, we show that the fin epithelium exhibits graded changes in cellular morphology along the proximo-distal axis. This epithelial pattern is strictly correlated with the gradient of canonical Wnt signalling activity. By combining genetic analyses with cellular imaging, we show that canonical Wnt signalling regulates epithelial cell morphology by modulating the levels of laminins, which are extracellular matrix components. We have unravelled a hitherto unknown mechanism involved in epithelial patterning, which is also conserved in the pectoral fins - evolutionarily recent appendages that are homologous to tetrapod limbs.
Guardiola, Ombretta; Lafuste, Peggy; Brunelli, Silvia; Iaconis, Salvatore; Touvier, Thierry; Mourikis, Philippos; De Bock, Katrien; Lonardo, Enza; Andolfi, Gennaro; Bouché, Ann; Liguori, Giovanna L; Shen, Michael M; Tajbakhsh, Shahragim; Cossu, Giulio; Carmeliet, Peter; Minchiotti, Gabriella
Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. However, our understanding of the molecular mechanisms underlying satellite cell activation is still largely undefined. Here, we show that Cripto, a regulator of early embryogenesis, is a novel regulator of muscle regeneration and satellite cell progression toward the myogenic lineage. Conditional inactivation of cripto in adult satellite cells compromises skeletal muscle regeneration, whereas gain of function of Cripto accelerates regeneration, leading to muscle hypertrophy. Moreover, we provide evidence that Cripto modulates myogenic cell determination and promotes proliferation by antagonizing the TGF-β ligand myostatin. Our data provide unique insights into the molecular and cellular basis of Cripto activity in skeletal muscle regeneration and raise previously undescribed implications for stem cell biology and regenerative medicine.
Logan, T K; Walker, Robert; Hunt, Gretchen
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.
Thomas Danielsen, E.; E. Møller, Morten; Yamanaka, Naoki;
and developmental timing. Here, we use our screen as a resource to identify mechanisms regulating intracellular levels of cholesterol, a substrate for steroidogenesis. We identify a conserved fatty acid elongase that underlies a mechanism that adjusts cholesterol trafficking and steroidogenesis with nutrition...... are regulated by TOR and feedback signaling that couples steroidogenesis with growth and ensures proper maturation timing. These results reveal genes regulating steroidogenesis during development that likely modulate disease mechanisms....
Kanaley, Jill A; Shadid, Samyah; Sheehan, Michael T; Guo, ZengKui; Jensen, Michael D
We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-¹³C]palmitate (0400-0900 h) and [U-¹³C]oleate (0800-1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass⁻¹·min⁻¹) clamp (0800-1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-¹³C]palmitate incorporation into imTG-palmitate and palmitoylcarnitine were similar to those we reported in overnight postabsorptive adults (saline control); the intramyocellular palmitoylcarnitine enrichment was not different from and correlated with imTG-palmitate enrichment for both the morning (r = 0.38, P = 0.02) and afternoon (r = 0.44, P = 0.006) biopsy samples. Plasma FFA concentrations, flux, and the incorporation of plasma oleate into imTG-oleate during hyperinsulinemia were ~1/10th of that observed in the previous saline control studies (P women than men, suggesting that sex differences in intramyocellular palmitate trafficking may occur under hyperinsulinemic conditions. We conclude that plasma FFA trafficking into imTG during hyperinsulinemia is markedly suppressed, and these newly incorporated FFA fatty acids do not readily enter the LCAC preoxidative pools. Hyperinsulinemia does not seem to inhibit the entry of fatty acids from imTG pools that were labeled under fasting conditions, possibly reflecting the presence of two distinct imTG pools that are differentially regulated by insulin.
McClain, Natalie M; Garrity, Stacy E
Human trafficking affects a surprisingly large number of adolescents around the globe. Women and girls make up the majority of sex trafficking victims. Nurses must be aware of sex trafficking as a form of sexual violence in the adolescent population. Nurses can play a role in identifying, intervening, and advocating for victims of human trafficking as they currently do for patients that are the victims of other types of violent crimes.
Hennink, Monique; Simkhada, Padam
This study has developed a conceptual framework to provide a clearer understanding of the process and context of sex trafficking from Nepal. Quantitative data were analysed from case records of 202 sex-trafficked women at rehabilitation centres in Nepal. In-depth interviews with 42 sex trafficked women, mostly residing at rehabilitation centres in Kathmandu, provide contextual information on the process and circumstances of sex trafficking. The results of this study provide a clearer understa...
Wilcox, Daniel Joseph
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...
Chen, Jiangtian; Reiher, Wencke; Hermann-Luibl, Christiane; Sellami, Azza; Cognigni, Paola; Kondo, Shu; Helfrich-Förster, Charlotte; Veenstra, Jan A; Wegener, Christian
Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.
Full Text Available Cystic fibrosis (CF is caused by genetic mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR protein. These mutations can impact the synthesis and transfer of the CFTR protein to the apical membrane of epithelial cells, as well as influencing the gating or conductance of chloride and bicarbonate ions through the channel. CFTR dysfunction results in ionic imbalance of epithelial secretions in several organ systems, such as the pancreas, gastrointestinal tract, liver and the respiratory system. Since discovery of the CFTR gene in 1989, research has focussed on targeting the underlying genetic defect to identify a disease-modifying treatment for CF. Investigated management strategies have included gene therapy and the development of small molecules that target CFTR mutations, known as CFTR modulators. CFTR modulators are typically identified by high-throughput screening assays, followed by preclinical validation using cell culture systems. Recently, one such modulator, the CFTR potentiator ivacaftor, was approved as an oral therapy for CF patients with the G551D-CFTR mutation. The clinical development of ivacaftor not only represents a breakthrough in CF care but also serves as a noteworthy example of personalised medicine.
Full Text Available Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF, an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.
Full Text Available Abstract Background The transforming growth factor beta is known to have pleiotropic effects, including differentiation, proliferation and apoptosis. However the underlying mechanisms remain poorly understood. The regulation and effect of TGF-β signaling is complex and highly depends on specific protein context. In liver, we have recently showed that the disintegrin and metalloproteinase ADAM12 interacts with TGF-β receptors and modulates their trafficking among membranes, a crucial point in TGF-β signaling and development of fibrosis. The present study aims to better understand how ADAM12 impacts on TGF-β receptors trafficking and TGF-β signaling. Findings We extracted qualitative biological observations from experimental data and defined a family of models producing a behavior compatible with the presence of ADAM12. We computationally explored the properties of this family of models which allowed us to make novel predictions. We predict that ADAM12 increases TGF-β receptors internalization rate between the cell surface and the endosomal membrane. It also appears that ADAM12 modifies TGF-β signaling shape favoring a permanent response by removing the transient component observed under physiological conditions. Conclusion In this work, confronting differential models with qualitative biological observations, we obtained predictions giving new insights into the role of ADAM12 in TGF-β signaling and hepatic fibrosis process.
Kaunhoven, Rebekah Jane; Dorjee, Dusana
Pre-adolescence is a key developmental period in which complex intrinsic volitional methods of self-regulation are acquired as a result of rapid maturation within the brain networks underlying the self-regulatory processes of attention control and emotion regulation. Fostering adaptive self-regulation skills during this stage of development has strong implications for physical health, emotional and socio-economic outcomes during adulthood. There is a growing interest in mindfulness-based programmes for pre-adolescents with initial findings suggesting self-regulation improvements, however, neurodevelopmental studies on mindfulness with pre-adolescents are scarce. This analytical review outlines an integrative neuro-developmental approach, which combines self-report and behavioural assessments with event related brain potentials (ERPs) to provide a systemic multilevel understanding of the neurocognitive mechanisms of mindfulness in pre-adolescence. We specifically focus on the N2, error related negativity (ERN), error positivity (Pe), P3a, P3b and late positive potential (LPP) ERP components as indexes of mindfulness related modulations in non-volitional bottom-up self-regulatory processes (salience detection, stimulus driven orienting and mind wandering) and volitional top-down self-regulatory processes (endogenous orienting and executive attention).
Baima, Simona; Forte, Valentina; Possenti, Marco; Peñalosa, Andrés; Leoni, Guido; Salvi, Sergio; Felici, Barbara; Ruberti, Ida; Morelli, Giorgio
The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 (ACL5) and bushy and dwarf2 (BUD2) is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper (HD-ZIP) III and key auxin signaling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD-ZIP III protein Arabidopsis thaliana homeobox8 tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signaling genes such as like auxin resistant2 (LAX2) and LAX3. We propose that proper formation and differentiation of xylem depend on a balance between positive and negative feedback loops operating through HD-ZIP III genes.
Zhang, Yu; Yu, Tian; Liu, Yang; Qian, Kun; Yu, Bu-Wei
GABAergic neurons within the ventrolateral preoptic area (VLPO) play an important role in sleep-wakefulness regulation. Propofol, a widely used systemic anesthetic, has lately been reported to excite noradrenaline (NA)-inhibited type of VLPO neurons. Present study tested if acetylcholine system takes part in the propofol modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in mechanically dissociated rat VLPO neurons using a conventional whole-cell patch clamp technique. Propofol reversibly decreased mIPSC frequency without affecting the current amplitude, indicating that propofol acts presynaptically to decrease the probability of spontaneous GABA release. The propofol action on GABAergic mIPSC frequency was completely blocked by atropine, a nonselective muscarinic acetylcholine (mACh) receptor antagonist, and pirenzepine, a selective M1 receptor antagonist. These results suggest that propofol acts on M1 receptors on GABAergic nerve terminals projecting to VLPO neurons to inhibit spontaneous GABA release. The M1 receptor-mediated modulation of GABAergic transmission onto VLPO neurons may contribute to the regulation of loss of consciousness induced by propofol.
Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent
Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.
Fernando, Thilini; Flibotte, Stephane; Xiong, Sheng; Yin, Jianghua; Yzeiraj, Edlira; Moerman, Donald G.; Meléndez, Alicia; Savage-Dunn, Cathy
Organismal growth and body size are influenced by both genetic and environmental factors. We have utilized the strong molecular genetic techniques available in the nematode C. elegans to identify genetic determinants of body size. In C. elegans, DBL-1, a member of the conserved family of secreted growth factors known as the Transforming Growth Factor β superfamily, is known to play a major role in growth control. The mechanisms by which other determinants of body size function, however, is less well understood. To identify additional genes involved in body size regulation, a genetic screen for small mutants was previously performed. One of the genes identified in that screen was sma-21. We now demonstrate that sma-21 encodes ADT-2, a member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family of secreted metalloproteases. ADAMTS proteins are believed to remodel the extracellular matrix and may modulate the activity of extracellular signals. Genetic interactions suggest that ADT-2 acts in parallel with or in multiple size regulatory pathways. We demonstrate that ADT-2 is required for normal levels of expression of a DBL-1-responsive transcriptional reporter. We further demonstrate that adt-2 regulatory sequences drive expression in glial-like and vulval cells, and that ADT-2 activity is required for normal cuticle collagen fibril organization. We therefore propose that ADT-2 regulates body size both by modulating TGFβ signaling activity and by maintaining normal cuticle structure. PMID:21256840
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
W Lee Pang
Full Text Available Copper (Cu is an important enzyme co-factor that is also extremely toxic at high intracellular concentrations, making active efflux mechanisms essential for preventing Cu accumulation. Here, we have investigated the mechanistic role of metallochaperones in regulating Cu efflux. We have constructed a computational model of Cu trafficking and efflux based on systems analysis of the Cu stress response of Halobacterium salinarum. We have validated several model predictions via assays of transcriptional dynamics and intracellular Cu levels, discovering a completely novel function for metallochaperones. We demonstrate that in addition to trafficking Cu ions, metallochaperones also function as buffers to modulate the transcriptional responsiveness and efficacy of Cu efflux. This buffering function of metallochaperones ultimately sets the upper limit for intracellular Cu levels and provides a mechanistic explanation for previously observed Cu metallochaperone mutation phenotypes.
Chaanine, Antoine H; Kohlbrenner, Erik; Gamb, Scott I; Guenzel, Adam J; Klaus, Katherine; Fayyaz, Ahmed U; Nair, K Sreekumaran; Hajjar, Roger J; Redfield, Margaret M
The forkhead box O3a (FOXO3a) transcription factor has been shown to regulate glucose metabolism, muscle atrophy, and cell death in postmitotic cells. Its role in regulation of mitochondrial and myocardial function is not well studied. Based on previous work, we hypothesized that FOXO3a, through BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3), modulates mitochondrial morphology and function in heart failure (HF). We modulated the FOXO3a-BNIP3 pathway in normal and phenylephrine (PE)-stressed adult cardiomyocytes (ACM) in vitro and developed a cardiotropic adeno-associated virus serotype 9 encoding dominant-negative FOXO3a (AAV9.dn-FX3a) for gene delivery in a rat model of HF with preserved ejection fraction (HFpEF). We found that FOXO3a upregulates BNIP3 expression in normal and PE-stressed ACM, with subsequent increases in mitochondrial Ca(2+), leading to decreased mitochondrial membrane potential, mitochondrial fragmentation, and apoptosis. Whereas dn-FX3a attenuated the increase in BNIP3 expression and its consequences in PE-stressed ACM, AAV9.dn-FX3a delivery in an experimental model of HFpEF decreased BNIP3 expression, reversed adverse left ventricular remodeling, and improved left ventricular systolic and, particularly, diastolic function, with improvements in mitochondrial structure and function. Moreover, AAV9.dn-FX3a restored phospholamban phosphorylation at S16 and enhanced dynamin-related protein 1 phosphorylation at S637. Furthermore, FOXO3a upregulates maladaptive genes involved in mitochondrial apoptosis, autophagy, and cardiac atrophy. We conclude that FOXO3a activation in cardiac stress is maladaptive, in that it modulates Ca(2+) cycling, Ca(2+) homeostasis, and mitochondrial dynamics and function. Our results suggest an important role of FOXO3a in HF, making it an attractive potential therapeutic target. Copyright © 2016 the American Physiological Society.
Sweatt, J David
Hebbian plasticity, including long-term potentiation and long-term depression, has long been regarded as important for local circuit refinement in the context of memory formation and stabilization. However, circuit development and stabilization additionally relies on non-Hebbian, homeostatic, forms of plasticity such as synaptic scaling. Synaptic scaling is induced by chronic increases or decreases in neuronal activity. Synaptic scaling is associated with cell-wide adjustments in postsynaptic receptor density, and can occur in a multiplicative manner resulting in preservation of relative synaptic strengths across the entire neuron's population of synapses. Both active DNA methylation and demethylation have been validated as crucial regulators of gene transcription during learning, and synaptic scaling is known to be transcriptionally dependent. However, it has been unclear whether homeostatic forms of plasticity such as synaptic scaling are regulated via epigenetic mechanisms. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously largely considered separately: DNA methylation, homeostatic plasticity, and glutamate receptor trafficking. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously considered separately: glutamate receptor trafficking, DNA methylation, and homeostatic plasticity.
Tanaka, Leonardo Y; Laurindo, Francisco R M
Vascular remodeling, i.e. whole-vessel structural reshaping, determines lumen caliber in (patho)physiology. Here we review mechanisms underlying vessel remodeling, with emphasis in redox regulation. First, we discuss confusing terminology and focus on strictu sensu remodeling. Second, we propose a mechanobiological remodeling paradigm based on the concept of tensional homeostasis as a setpoint regulator. We first focus on shear-mediated models as prototypes of remodeling closely dominated by highly redox-sensitive endothelial function. More detailed discussions focus on mechanosensors, integrins, extracellular matrix, cytoskeleton and inflammatory pathways as potential of mechanisms potentially coupling tensional homeostasis to redox regulation. Further discussion of remodeling associated with atherosclerosis and injury repair highlights important aspects of redox vascular responses. While neointima formation has not shown consistent responsiveness to antioxidants, vessel remodeling has been more clearly responsive, indicating that despite the multilevel redox signaling pathways, there is a coordinated response of the whole vessel. Among mechanisms that may orchestrate redox pathways, we discuss roles of superoxide dismutase activity and extracellular protein disulfide isomerase. We then discuss redox modulation of aneurysms, a special case of expansive remodeling. We propose that the redox modulation of vascular remodeling may reflect (1) remodeling pathophysiology is dominated by a particularly redox-sensitive cell type, e.g., endothelial cells (2) redox pathways are temporospatially coordinated at an organ level across distinct cellular and acellular structures or (3) the tensional homeostasis setpoint is closely connected to redox signaling. The mechanobiological/redox model discussed here can be a basis for improved understanding of remodeling and helps clarifying mechanisms underlying prevalent hard-to-treat diseases.
Full Text Available Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. Here, we monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor KIF17 by Ca2+/calmodulin-dependent protein kinases (CaMK. Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport.
Prendergast, Jillian; Umanah, George K. E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.; Schnaar, Ronald L.
Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside G...
Pripfl, Jürgen; Lamm, Claus
Recent neuroscience theories suggest that different kinds of self-regulation may share a common psychobiological mechanism. However, empirical evidence for a domain general self-regulation mechanism is scarce. The aim of this study was to investigate whether focused anodal transcranial direct current stimulation (tDCS), facilitating the activity of the dorsolateral prefrontal cortex (dlPFC), acts on a domain general self-regulation mechanism and thus modulates both affective and appetitive self-regulation. Twenty smokers participated in this within-subject sham controlled study. Effects of anodal left, anodal right and sham tDCS over the dlPFC on affective picture appraisal and nicotine craving-cue appraisal were assessed. Anodal right tDCS over the dlPFC reduced negative affect in emotion appraisal, but neither modulated regulation of positive emotion appraisal nor of craving appraisal. Anodal left stimulation did not induce any significant effects. The results of our study show that domain specific self-regulation networks are at work in the prefrontal cortex. Focused tDCS modulation of this specific self-regulation network could probably be used during the first phase of nicotine abstinence, during which negative affect might easily result in relapse. These findings have implications for neuroscience models of self-regulation and are of relevance for the development of brain stimulation based treatment methods for neuropsychiatric disorders associated with self-regulation deficits. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.
There is now solid evidence that cell-to-cell trafficking of certain proteins and RNAs plays a critical role in trans-cellular regulation of gene expression to coordinate cellular differentiation and development. Such trafficking also is critical for viral infection and plant defense. The mechanisms of trafficking remain poorly understood. Although some proteins may move between cells by diffusion, many proteins and RNAs move in a highly regulated fashion. Regulation is likely achieved through interactions between distinct protein or RNA motifs and cellular factors. Some motifs and factors have been identified. One of the major focuses for future studies is to identify all motifs and their cognate factors and further elucidate their roles in trafficking between specific cells. With increasing information from such studies, we should be able to develop an understanding of the mechanisms that regulate trafficking of various proteins and RNAs across all and specific cellular boundaries. On the basis of such mechanistic knowledge, we can further investigate how the trafficking machinery has evolved to regulate developmental and physiological processes in a plant, how pathogens have co-evolved to use this machinery for systemic spread in a plant, and how plants use this machinery for counterdefense.
Full Text Available Abstract Background Numerous epidemiological studies have documented that obesity is associated with hepatocellular carcinoma (HCC. The aim of this study was to investigate the biological actions regulated by leptin, the obesity biomarker molecule, and its receptors in HCC and the correlation between leptin and human telomerase reverse transcriptase (hTERT, a known mediator of cellular immortalization. Methods We investigated the relationship between leptin, leptin receptors and hTERT mRNA expression in HCC and healthy liver tissue samples. In HepG2 cells, chromatin immunoprecipitation assay was used to study signal transducer and activator of transcription-3 (STAT3 and myc/mad/max transcription factors downstream of leptin which could be responsible for hTERT regulation. Flow cytometry was used for evaluation of cell cycle modifications and MMP1, 9 and 13 expression after treatment of HepG2 cells with leptin. Blocking of leptin's expression was achieved using siRNA against leptin and transfection with liposomes. Results We showed, for the first time, that leptin's expression is highly correlated with hTERT expression levels in HCC liver tissues. We also demonstrated in HepG2 cells that leptin-induced up-regulation of hTERT and TA was mediated through binding of STAT3 and Myc/Max/Mad network proteins on hTERT promoter. We also found that leptin could affect hepatocellular carcinoma progression and invasion through its interaction with cytokines and matrix mettaloproteinases (MMPs in the tumorigenic microenvironment. Furthermore, we showed that histone modification contributes to leptin's gene regulation in HCC. Conclusions We propose that leptin is a key regulator of the malignant properties of hepatocellular carcinoma cells through modulation of hTERT, a critical player of oncogenesis.
Chakravarthi, B V S K; Goswami, M T; Pathi, S S; Robinson, A D; Cieślik, M; Chandrashekar, D S; Agarwal, S; Siddiqui, J; Daignault, S; Carskadon, S L; Jing, X; Chinnaiyan, A M; Kunju, L P; Palanisamy, N; Varambally, S
MicroRNA-101, a tumor suppressor microRNA (miR), is often downregulated in cancer and is known to target multiple oncogenes. Some of the genes that are negatively regulated by miR-101 expression include histone methyltransferase EZH2 (enhancer of zeste homolog 2), COX2 (cyclooxygenase-2), POMP (proteasome maturation protein), CERS6, STMN1, MCL-1 and ROCK2, among others. In the present study, we show that miR-101 targets transcriptional coactivator SUB1 homolog (Saccharomyces cerevisiae)/PC4 (positive cofactor 4) and regulates its expression. SUB1 is known to have diverse role in vital cell processes such as DNA replication, repair and heterochromatinization. SUB1 is known to modulate transcription and acts as a mediator between the upstream activators and general transcription machinery. Expression profiling in several cancers revealed SUB1 overexpression, suggesting a potential role in tumorigenesis. However, detailed regulation and function of SUB1 has not been elucidated. In this study, we show elevated expression of SUB1 in aggressive prostate cancer. Knockdown of SUB1 in prostate cancer cells resulted in reduced cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Gene expression analyses coupled with chromatin immunoprecipitation revealed that SUB1 binds to the promoter regions of several oncogenes such as PLK1 (Polo-like kinase 1), C-MYC, serine-threonine kinase BUB1B and regulates their expression. Additionally, we observed SUB1 downregulated CDKN1B expression. PLK1 knockdown or use of PLK1 inhibitor can mitigate oncogenic function of SUB1 in benign prostate cancer cells. Thus, our study suggests that miR-101 loss results in increased SUB1 expression and subsequent activation of known oncogenes driving prostate cancer progression and metastasis. This study therefore demonstrates functional role of SUB1 in prostate cancer, and identifies its regulation and potential downstream therapeutic targets of SUB1 in prostate
Lee, Cho-Yin; Lou, Jizhong; Wen, Kuo-Kuang; McKane, Melissa; Eskin, Suzanne G.; Rubenstein, Peter A.; Chien, Shu; Ono, Shoichiro; Zhu, Cheng; McIntire, Larry V.
The dynamic turnover of the actin cytoskeleton is regulated cooperatively by force and biochemical signaling. We previously demonstrated that actin depolymerization under force is governed by catch-slip bonds mediated by force-induced K113:E195 salt-bridges. Yet, the biochemical regulation as well as the functional significance of actin catch bonds has not been elucidated. Using AFM force-clamp experiments, we show that formin controlled by RhoA switches the actin catch-slip bonds to slip-only bonds. SMD simulations reveal that the force does not induce the K113:E195 interaction when formin binds to actin K118 and E117 residues located at the helical segment extending to K113. Actin catch-slip bonds are suppressed by single residue replacements K113E and E195K that interrupt the force-induced K113:E195 interaction; and this suppression is rescued by a K113E/E195K double mutant (E/K) restoring the interaction in the opposite orientation. These results support the biological significance of actin catch bonds, as they corroborate reported observations that RhoA and formin switch force-induced actin cytoskeleton alignment and that either K113E or E195K induces yeast cell growth defects rescued by E/K. Our study demonstrates how the mechano-regulation of actin dynamics is modulated by biochemical signaling molecules, and suggests that actin catch bonds may be important in cell functions.
Subauste, M. Cecilia; Pertz, Olivier; Adamson, Eileen D.; Turner, Christopher E.; Junger, Sachiko; Hahn, Klaus M.
Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction. PMID:15138291
Robert W Moon
Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.
Bosveld, Floris; Guirao, Boris; Wang, Zhimin; Rivière, Mathieu; Bonnet, Isabelle; Graner, François; Bellaïche, Yohanns
Tumor suppressors and proto-oncogenes play crucial roles in tissue proliferation. Furthermore, de-regulation of their functions is deleterious to tissue architecture and can result in the sorting of somatic rounded clones minimizing their contact with surrounding wild-type (wt) cells. Defects in the shape of somatic clones correlate with defects in proliferation, cell affinity, cell-cell adhesion, oriented cell division and cortical contractility. Combining genetics, live-imaging, laser ablation and computer simulations, we aim to analyze whether distinct or similar mechanisms can account for the common role of tumor suppressors and proto-oncogenes in cell-cell contact regulation. In Drosophila epithelia, the tumor suppressors Fat (Ft) and Dachsous (Ds) regulate cell proliferation, tissue morphogenesis, planar cell polarity and junction tension. By analyzing the evolution over time of ft mutant cells and clones, we show that ft clones reduce their cell-cell contacts with the surrounding wt tissue in the absence of concomitant cell divisions and over-proliferation. This contact reduction depends on opposed changes of junction tensions in the clone bulk and its boundary with neighboring wt tissue. More generally, either clone bulk or boundary junction tension is modulated by the activation of Yorkie, Myc and Ras, yielding similar contact reductions with wt cells. Together, our data highlight mechanical roles for proto-oncogene and tumor suppressor pathways in cell-cell interactions.
Full Text Available P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane.All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e. homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C-linked metabotropic receptors and P2X receptor channels in DRG sensory neurons and microglia.
This article is based on empirical research with West African migrant women working in prostitution in Paris. Given current migration regulations in Western Europe, as well as state policies on prostitution, the traffickers and people considered to be trafficking victims de facto form part of the sa
This article is based on empirical research with West African migrant women working in prostitution in Paris. Given current migration regulations in Western Europe, as well as state policies on prostitution, the traffickers and people considered to be trafficking victims de facto form part of the
Barrows, Jeffrey; Finger, Reginald
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.
Jenna M. Ramaker
APPL-dependent responses within the nervous system. Lastly, targeted expression of our double-tagged constructs (combined with time-lapse imaging revealed that APP family proteins are subject to complex patterns of trafficking and processing that vary dramatically between different neuronal subtypes. In combination, our results provide a new perspective on how the regulation of APP family proteins can be modulated to accommodate a variety of cell type-specific responses within the embryonic and adult nervous system.
Full Text Available The author is analyzing trafficking in human beings as a specific form of women's (illegal migration. The author is presenting detailed analysis of the international standards and recent activities of different international organizations (UN, Council of Europe, European Community, OSCE, concerning prevention of trafficking in human beings, regulation of foreign migrants' status and protection of victims of trafficking. Starting from the analysis of international documents and national legislations dealing with migration and prostitution, the author is proposing changes of existing domestic laws concerning movement and residence of foreigners. The aim of such changes is to harmonize our legislation with international standards and obligations accepted by signing the Palermo Convention.
Deng, Pan-Yue; Rotman, Ziv; Blundon, Jay A; Cho, Yongcheol; Cui, Jianmin; Cavalli, Valeria; Zakharenko, Stanislav S; Klyachko, Vitaly A
Loss of FMRP causes fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx, and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely rescued in postnatal neurons. These presynaptic actions of FMRP are translation independent and are mediated selectively by BK channels via interaction of FMRP with BK channel's regulatory β4 subunits. Information-theoretical analysis demonstrates that loss of these FMRP functions causes marked dysregulation of synaptic information transmission. FMRP-dependent AP broadening is not limited to the hippocampus, but also occurs in cortical pyramidal neurons. Our results thus suggest major translation-independent presynaptic functions of FMRP that may have important implications for understanding FXS neuropathology.
Fair, K; Anderson, M; Bulanova, E; Mi, H; Tropschug, M; Diaz, M O
The PHD fingers of the human MLL and Drosophila trx proteins have strong amino acid sequence conservation but their function is unknown. We have determined that these fingers mediate homodimerization and binding of MLL to Cyp33, a nuclear cyclophilin. These two proteins interact in vitro and in vivo in mammalian cells and colocalize at specific nuclear subdomains. Overexpression of the Cyp33 protein in leukemia cells results in altered expression of HOX genes that are targets for regulation by MLL. These alterations are suppressed by cyclosporine and are not observed in cell lines that express a mutant MLL protein without PHD fingers. These results suggest that binding of Cyp33 to MLL modulates its effects on the expression of target genes.
Chaves, Inês; van der Horst, Gijsbertus T J; Schellevis, Raymond; Nijman, Romana M; Koerkamp, Marian Groot; Holstege, Frank C P; Smidt, Marten P; Hoekman, Marco F M
Circadian rhythms are responsive to external and internal cues, light and metabolism being among the most important. In mammals, the light signal is sensed by the retina and transmitted to the suprachiasmatic nucleus (SCN) master clock , where it is integrated into the molecular oscillator via regulation of clock gene transcription. The SCN synchronizes peripheral oscillators, an effect that can be overruled by incoming metabolic signals . As a consequence, peripheral oscillators can be uncoupled from the master clock when light and metabolic signals are not in phase. The signaling pathways responsible for coupling metabolic cues to the molecular clock are being rapidly uncovered [3-5]. Here we show that insulin-phosphatidylinositol 3-kinase (PI3K)-Forkhead box class O3 (FOXO3) signaling is required for circadian rhythmicity in the liver via regulation of Clock. Knockdown of FoxO3 dampens circadian amplitude, an effect that is rescued by overexpression of Clock. Subsequently, we show binding of FOXO3 to two Daf-binding elements (DBEs) located in the Clock promoter area, implicating Clock as a transcriptional target of FOXO3. Transcriptional oscillation of both core clock and output genes in the liver of FOXO3-deficient mice is affected, indicating a disrupted hepatic circadian rhythmicity. Finally, we show that insulin, a major regulator of FOXO activity [6-9], regulates Clock levels in a PI3K- and FOXO3-dependent manner. Our data point to a key role of the insulin-FOXO3-Clock signaling pathway in the modulation of circadian rhythms.
Mahan, Amy L; Mou, Liping; Shah, Nirali; Hu, Jia-Hua; Worley, Paul F; Ressler, Kerry J
The consolidation of conditioned fear involves upregulation of genes necessary for long-term memory formation. An important question remains as to whether this results in part from epigenetic regulation and chromatin modulation. We examined whether Homer1a, which is required for memory formation, is necessary for Pavlovian cued fear conditioning, whether it is downstream of BDNF-TrkB activation, and whether this pathway utilizes histone modifications for activity-dependent transcriptional regulation. We initially found that Homer1a knock-out mice exhibited deficits in cued fear conditioning (5 tone-shock presentations with 70 dB, 6 kHz tones and 0.5 s, 0.6 mA footshocks). We then demonstrated that: (1) Homer1a mRNA increases after fear conditioning in vivo within both amygdala and hippocampus of wild-type mice; (2) it increases after BDNF application to primary hippocampal and amygdala cultures in vitro; and (3) these increases are dependent on transcription and MAPK signaling. Furthermore, using chromatin immunoprecipitation we found that both in vitro and in vivo manipulations result in decreases in Homer1 promoter H3K9 methylation in amygdala cells but increases in Homer1 promoter H3 acetylation in hippocampal cells. However, no changes were observed in H4 acetylation or H3K27 dimethylation. Inhibition of histone deacetylation by sodium butyrate enhanced contextual but not cued fear conditioning and enhanced Homer1 H3 acetylation in the hippocampus. These data provide evidence for dynamic epigenetic regulation of Homer1a following BDNF-induced plasticity and during a BDNF-dependent learning process. Furthermore, upregulation of this gene may be regulated through distinct epigenetic modifications in the hippocampus and amygdala.
Full Text Available Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43 in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel, we stably transfected connexin43-containing normal rat kidney (NRK cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.
Full Text Available Abstract Background Retinal degenerations, such as age-related macular degeneration (AMD and retinitis pigmentosa (RP, are characterized by photoreceptor loss and anomalous remodeling of the surviving retina that corrupts visual processing and poses a barrier to late-stage therapeutic interventions in particular. However, the molecular events associated with retinal remodeling remain largely unknown. Given our prior evidence of ionotropic glutamate receptor (iGluR reprogramming in retinal degenerations, we hypothesized that the edited glutamate receptor 2 (GluR2 subunit and its trafficking may be modulated in retinal degenerations. Results Adult albino Balb/C mice were exposed to intense light for 24 h to induce light-induced retinal degeneration (LIRD. We found that prior to the onset of photoreceptor loss, protein levels of GluR2 and related trafficking proteins, including glutamate receptor-interacting protein 1 (GRIP1 and postsynaptic density protein 95 (PSD-95, were rapidly increased. LIRD triggered neuritogenesis in photoreceptor survival regions, where GluR2 and its trafficking proteins were expressed in the anomalous dendrites. Immunoprecipitation analysis showed interaction between KIF3A and GRIP1 as well as PSD-95, suggesting that KIF3A may mediate transport of GluR2 and its trafficking proteins to the novel dendrites. However, in areas of photoreceptor loss, GluR2 along with its trafficking proteins nearly vanished in retracted retinal neurites. Conclusions All together, LIRD rapidly triggers GluR2 plasticity, which is a potential mechanism behind functionally phenotypic revisions of retinal neurons and neuritogenesis during retinal degenerations.
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.
Niclas, J; Allan, V J; Vale, R D
Cytoplasmic dynein is a minus end-directed microtubule motor that performs distinct functions in interphase and mitosis. In interphase, dynein transports organelles along microtubules, whereas in metaphase this motor has been implicated in mitotic spindle formation and orientation as well as chromosome segregation. The manner in which dynein activity is regulated during the cell cycle, however, has not been resolved. In this study, we have examined the mechanism by which organelle transport is controlled by the cell cycle in extracts of Xenopus laevis eggs. Here, we show that photocleavage of the dynein heavy chain dramatically inhibits minus end-directed organelle transport and that purified dynein restores this motility, indicating that dynein is the predominant minus end-directed membrane motor in Xenopus egg extracts. By measuring the amount of dynein associated with isolated membranes, we find that cytoplasmic dynein and its activator dynactin detach from the membrane surface in metaphase extracts. The sevenfold decrease in membrane-associated dynein correlated well with the eightfold reduction in minus end-directed membrane transport observed in metaphase versus interphase extracts. Although dynein heavy or intermediate chain phosphorylation did not change in a cell cycle-dependent manner, the dynein light intermediate chain incorporated approximately 12-fold more radiolabeled phosphate in metaphase than in interphase extracts. These studies suggest that cell cycle-dependent phosphorylation of cytoplasmic dynein may regulate organelle transport by modulating the association of this motor with membranes.
Zhu, Boqian; Gong, Yaoyao; Yan, Gaoliang; Wang, Dong; Wang, Qingjie; Qiao, Yong; Hou, Jiantong; Liu, Bo; Tang, Chengchun
Intimal hyperplasia, the key event of arterial restenosis, is a result of cell proliferation and cell migration. Atorvastatin exerts an inhibitory effect on cell proliferation and migration, but the mechanism remains largely unknown. p16, as a well-known tumor suppressor, was also reported to suppress cell growth and migration, but with an unclear mechanism. In this study, we demonstrated that atorvastatin represses cell proliferation and migration in vascular smooth muscle cells (VSMCs) and that this process is mediated by p16. Furthermore, we found that DNA methylation in the p16 promoter was reduced and p16 expression was restored in VSMCs treated with 5-aza-2'-deoxycytidine or atorvastatin. However, the effect was absent when DNA methyltransferase 1 (DNMT1) was knocked down with RNA interference. These observations demonstrated that atorvastatin regulates p16 expression via DNMT1-induced DNA methylation in the p16 promoter. In addition, we found that the mitogen-activated protein kinase (MAPK) pathway was involved in the regulation of p16 by DNMT1, and MAPK inhibitors partially released the effects of atorvastatin on p16 and DNMT1. Finally, we illustrated that atorvastatin inhibits neointima formation and modulates p16 expression in balloon catheter-injured rat carotid artery. Taken together, we demonstrated that atorvastatin inhibits neointima formation through inducing p16 expression by affecting DNA methylation in the p16 promoter region. © 2017 Federation of European Biochemical Societies.
Kitange, Gaspar J.; Mladek, Ann C.; Schroeder, Mark A.; Pokorny, Jenny C.; Carlson, Brett L.; Zhang, Yuji; Nair, Asha A.; Lee, Jeong-Heon; Yan, Huihuang; Decker, Paul A.; Zhang, Zhiguo; Sarkaria, Jann N.
Summary Here we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of 2 key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51 and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin modifying complex that binds within the promoter of MGMT, RAD51 and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. RBBP4/CBP/p300 complex may provide an interesting target for developing therapy sensitizing strategies for GBM and other tumors. PMID:26972001
Gaspar J. Kitange
Full Text Available Here we provide evidence that RBBP4 modulates temozolomide (TMZ sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac and increased H3K9 tri-methylation (H3K9me3. Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.
Ren, An-Jing; Wang, Kai; Zhang, Huan; Liu, Anjun; Ma, Xianhua; Liang, Qing; Cao, Dongmei; Wood, John N; He, David Z; Ding, Yu-Qiang; Yuan, Wen-Jun; Xie, Zhifang; Zhang, Weiping J
In mammals, pain sensation is initiated by the detection of noxious stimuli through specialized transduction ion channels and receptors in nociceptive sensory neurons. Transient receptor potential (TRP) channels are the key sensory transducers that confer nociceptors distinct sensory modalities. However, the regulatory mechanisms about their expression are poorly defined. Here we show that the zinc-finger protein ZBTB20 regulates TRP channels expression in nociceptors. ZBTB20 is highly expressed in nociceptive sensory neurons of dorsal root ganglia. Disruption of ZBTB20 in nociceptors led to a marked decrease in the expression levels of TRPV1, TRPA1 and TRPM8 and the response of calcium flux and whole-cell currents evoked by their respective specific agonists. Phenotypically, the mice lacking ZBTB20 specifically in nociceptors showed a defect in nociception and pain sensation in response to thermal, mechanical and inflammatory stimulation. Our findings point to ZBTB20 as a critical regulator of nociception and pain sensation by modulating TRP channels expression in nociceptors.
Full Text Available Psychosocial stress affects resources for adequate coping with environmental demands. A crucial question in this context is the extent to which acute psychosocial stressors impact empathy and emotion regulation. In the present study, 120 participants were randomly assigned to a control group vs. a group confronted with the Trier Social Stress Test, an established paradigm for the induction of acute psychosocial stress. Empathy for pain as a specific subgroup of empathy was assessed via pain intensity ratings during a pain-picture task. Self-reported emotion regulation skills were measured as predictors using an established questionnaire. Stressed individuals scored significantly lower on the appraisal of pain pictures. A regression model was chosen to find variables that further predict the pain ratings. These findings implicate that acute psychosocial stress might impair empathic processes to observed pain in another person and the ability to accept one’s emotion additionally predicts the empathic reaction. Furthermore, the ability to tolerate negative emotions modulated the relation between stress and pain judgments, and thus influenced core cognitive-affective functions relevant for coping with environmental challenges. In conclusion, our study emphasizes the necessity of reducing negative emotions in terms of empathic distress when confronted with pain of another person under psychosocial stress, in order to be able to retain pro-social behavior.
Buruck, Gabriele; Wendsche, Johannes; Melzer, Marlen; Strobel, Alexander; Dörfel, Denise
Psychosocial stress affects resources for adequate coping with environmental demands. A crucial question in this context is the extent to which acute psychosocial stressors impact empathy and emotion regulation. In the present study, 120 participants were randomly assigned to a control group vs. a group confronted with the Trier Social Stress Test (TSST), an established paradigm for the induction of acute psychosocial stress. Empathy for pain as a specific subgroup of empathy was assessed via pain intensity ratings during a pain-picture task. Self-reported emotion regulation skills were measured as predictors using an established questionnaire. Stressed individuals scored significantly lower on the appraisal of pain pictures. A regression model was chosen to find variables that further predict the pain ratings. These findings implicate that acute psychosocial stress might impair empathic processes to observed pain in another person and the ability to accept one's emotion additionally predicts the empathic reaction. Furthermore, the ability to tolerate negative emotions modulated the relation between stress and pain judgments, and thus influenced core cognitive-affective functions relevant for coping with environmental challenges. In conclusion, our study emphasizes the necessity of reducing negative emotions in terms of empathic distress when confronted with pain of another person under psychosocial stress, in order to be able to retain pro-social behavior.
Williams, Jeffrey M.; Tsai, Billy
Bacterial toxins often translocate across a cellular membrane to gain access into the host cytosol, modifying cellular components in order to exert their toxic effects. To accomplish this feat, these toxins traffic to a membrane penetration site where they undergo conformational changes essential to eject the toxin’s catalytic subunit into the cytosol. In this brief review, we highlight recent findings that elucidate both the trafficking pathways and membrane translocation mechanisms of toxin...
Snelling, Paul C; Lipscomb, Martin; Lockyer, Lesley; Yates, Sue; Young, Pat
European Union (EU) regulations require that university programmes are of specified duration. Additional EU regulations apply specifically to university based nurse education, enacted in the UK by the Nursing and Midwifery Council (NMC). However, little is known about how much time student nurses spend on their studies. In this exploratory study, students undertaking a single module in the pre-registration diploma programme at an English university were asked to keep a log of learning activity for the duration of the module. Twenty-six students completed the log. These students achieved higher grades and attended more lectures than the average for the module. The mean study time was 128.4 h against a regulatory assumption that the module should take 200 h. More than half of the 26 students undertook paid work during the module run, though this work was not associated with poorer performance. Problems in regulation for course duration are discussed and it is suggested that undertaking a 4600 h course in 3 years is problematic. More research is required so that patterns of study can be better understood and student centred programmes meeting regulatory requirements developed.
Full Text Available The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes is expressed to activate inorganic phosphate (Pi metabolism for adaptation to Pi starvation. Pi starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus and enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4, thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system in that Pho85 monitors whether environmental conditions are adequate for cell growth and represses inadequate (untimely responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4 and Rpo21 binding (Rpo21 being the largest subunit of RNA polymerase II using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP species (IP7 that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates the transcription of antisense and intragenic RNAs in the KCS1 locus to down-regulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 m
Barreto, George E; Santos-Galindo, Maria; Garcia-Segura, Luis Miguel
Following brain injury, microglia assume a reactive-like state and secrete pro-inflammatory molecules that can potentiate damage. A therapeutic strategy that may limit microgliosis is of potential interest. In this context, selective estrogen receptor modulators, such as raloxifene and tamoxifen, are known to reduce microglia activation induced by neuroinflammatory stimuli in young animals. In the present study, we have assessed whether raloxifene and tamoxifen are able to affect microglia activation after brain injury in young and aged animals in time points relevant to clinics, which is hours after brain trauma. Volume fraction of MHC-II(+) microglia was estimated according to the point-counting method of Weibel within a distance of 350 μm from the lateral border of the wound, and cellular morphology was measured by fractal analysis. Two groups of animals were studied: (1) young rats, ovariectomized at 2 months of age; and (2) aged rats, ovariectomized at 18 months of age. Fifteen days after ovariectomy animals received a stab wound brain injury and the treatment with estrogenic compounds. Our findings indicate that raloxifene and tamoxifen reduced microglia activation in both young and aged animals. Although the volume fraction of reactive microglia was found lower in aged animals, this was accompanied by important changes in cell morphology, where aged microglia assume a bushier and hyperplasic aspect when compared to young microglia. These data suggest that early regulation of microglia activation provides a mechanism by which selective estrogen receptors modulators (SERMs) may exert a neuroprotective effect in the setting of a brain trauma.
Sparks, Avis E.; Chen, Chiachen; Breslin, Mary B.; Lan, Michael S.
INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity. PMID:27048654
Sparks, Avis E; Chen, Chiachen; Breslin, Mary B; Lan, Michael S
INS-VNTR (insulin-variable number of tandem repeats) and AIRE (autoimmune regulator) have been associated with the modulation of insulin gene expression in thymus, which is essential to induce either insulin tolerance or the development of insulin autoimmunity and type 1 diabetes. We sought to analyze whether each functional domain of AIRE is critical for the activation of INS-VNTR in human thymic epithelial cells. Twelve missense or nonsense mutations in AIRE and two chimeric AIRE constructs were generated. A luciferase reporter assay and a pulldown assay using biotinylated INS-class I VNTR probe were performed to examine the transactivation and binding activities of WT, mutant, and chimeric AIREs on the INS-VNTR promoter. Confocal microscopy analysis was performed for WT or mutant AIRE cellular localization. We found that all of the AIRE mutations resulted in loss of transcriptional activation of INS-VNTR except mutant P252L. Using WT/mutant AIRE heterozygous forms to modulate the INS-VNTR target revealed five mutations (R257X, G228W, C311fsX376, L397fsX478, and R433fsX502) that functioned in a dominant negative fashion. The LXXLL-3 motif is identified for the first time to be essential for DNA binding to INS-VNTR, whereas the intact PHD1, PHD2, LXXLL-3, and LXXLL-4 motifs were important for successful transcriptional activation. AIRE nuclear localization in the human thymic epithelial cell line was disrupted by mutations in the homogenously staining region domain and the R257X mutation in the PHD1 domain. This study supports the notion that AIRE mutation could specifically affect human insulin gene expression in thymic epithelial cells through INS-VNTR and subsequently induce either insulin tolerance or autoimmunity.
Chen, Yong; Boettger, Michael K; Reif, Andreas; Schmitt, Angelika; Uçeyler, Nurcan; Sommer, Claudia
Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1beta), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1beta. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1beta, and IL-10 following CFA, overall corroborating the inhibitor data. These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.
COMBATING HUMAN TRAFFICKING IN COLOMBIA by Daniel Joseph Wilcox March 2015 Thesis Advisor: Rodrigo Nieto-Gómez Second Reader: Maiah Jaskoski...Master ’s Thesis 4. TITLE AND SUBTITLE BARRIERS TO COMBATING HUMAN TRAFFICKING IN COLOMBIA 6. AUTHOR(S) Daniel Joseph Wilcox 7. PERFORMING...maximum 200 words) 12b. DISTRIBUTION CODE Despite intema.tional and domestic policies and programs intended to combat human trafficking , Colombia
Albright, Erin; D'Adamo, Kate
In order to decrease human trafficking, health care workers should support the full decriminalization of prostitution. Similar to trafficking in other forms of labor, preventing trafficking in the sex trade requires addressing the different forms of marginalization that create vulnerable communities. By removing punitive laws that prevent reporting of exploitation and abuse, decriminalization allows sex workers to work more safely, thereby reducing marginalization and vulnerability. Decriminalization can also help destigmatize sex work and help resist political, social, and cultural marginalization of sex workers.
N. M. Lukach
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.
Kanaley, Jill A.; Shadid, Samyah; Sheehan, Michael T.; Guo, ZengKui
We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-13C]palmitate (0400–0900 h) and [U-13C]oleate (0800–1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass−1·min−1) clamp (0800–1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-13C]palmitate incorporation into imTG-palmitate and palmitoylcarnitine were similar to those we reported in overnight postabsorptive adults (saline control); the intramyocellular palmitoylcarnitine enrichment was not different from and correlated with imTG-palmitate enrichment for both the morning (r = 0.38, P = 0.02) and afternoon (r = 0.44, P = 0.006) biopsy samples. Plasma FFA concentrations, flux, and the incorporation of plasma oleate into imTG-oleate during hyperinsulinemia were ∼1/10th of that observed in the previous saline control studies (P < 0.001). At the time of the second biopsy, the enrichment in oleoylcarnitine was <25% of that in imTG-oleate and was not correlated with imTG-oleate enrichment. The intramyocellular nonesterified fatty acid-palmitate-to-imTG-palmitate enrichment ratio was greater (P < 0.05) in women than men, suggesting that sex differences in intramyocellular palmitate trafficking may occur under hyperinsulinemic conditions. We conclude that plasma FFA trafficking into imTG during hyperinsulinemia is markedly suppressed, and these newly incorporated FFA fatty acids do not readily enter the LCAC preoxidative pools. Hyperinsulinemia does not seem to inhibit the entry of fatty acids from imTG pools that were labeled under fasting conditions, possibly reflecting the presence of two distinct imTG pools that are differentially regulated by insulin. PMID:23820622
Liu, Xu; Hu, Pengwei; Huang, Mingkun; Tang, Yang; Li, Yuge; Li, Ling; Hou, Xingliang
The antagonistic crosstalk between gibberellic acid (GA) and abscisic acid (ABA) plays a pivotal role in the modulation of seed germination. However, the molecular mechanism of such phytohormone interaction remains largely elusive. Here we show that three Arabidopsis NUCLEAR FACTOR-Y C (NF-YC) homologues NF-YC3, NF-YC4 and NF-YC9 redundantly modulate GA- and ABA-mediated seed germination. These NF-YCs interact with the DELLA protein RGL2, a key repressor of GA signalling. The NF-YC–RGL2 module targets ABI5, a gene encoding a core component of ABA signalling, via specific CCAAT elements and collectively regulates a set of GA- and ABA-responsive genes, thus controlling germination. These results suggest that the NF-YC–RGL2–ABI5 module integrates GA and ABA signalling pathways during seed germination. PMID:27624486
Lee Hyun Jung
Full Text Available Abstract Background Parkinson disease (PD is characterized by a slow, progressive degeneration of dopaminergic neurons in the substantianigra. The cause of neuronal loss in PD is not well understood, but several genetic loci, including PTEN-induced putative kinase 1 (PINK1, have been linked to early-onset autosomal recessive forms of familial PD. Neuroinflammation greatly contributes to PD neuronal degeneration and pathogenesis. IL-1 is one of the principal cytokines that regulates various immune and inflammatory responses via the activation of the transcription factors NF-κB and activating protein-1. Despite the close relationship between PD and neuroinflammation, the functional roles of PD-linked genes during inflammatory processes remain poorly understood. Methods To explore the functional roles of PINK1 in response to IL-1β stimulation, HEK293 cells, mouse embryonic fibroblasts derived from PINK1-null (PINK1−/− and control (PINK1+/+ mice, and 293 IL-1RI cells stably expressing type 1 IL-1 receptor were used. Immunoprecipitation and western blot analysis were performed to detect protein–protein interaction and protein ubiquitination. To confirm the effect of PINK1 on NF-κB activation, NF-κB-dependent firefly luciferase reporter assay was conducted. Results PINK1 specifically binds two components of the IL-1-mediated signaling cascade, Toll-interacting protein (Tollip and IL-1 receptor-associated kinase 1 (IRAK1. The association of PINK1 with Tollip, a negative regulator of IL-1β signaling, increases upon IL-1β stimulation, which then facilitates the dissociation of Tollip from IRAK1 as well as the assembly of the IRAK1–TNF receptor-associated factor 6 (TRAF6 complex. PINK1 also enhances Lys63-linked polyubiquitination of IRAK1, an essential modification of recruitment of NF-κB essential modulator and subsequent IκB kinase activation, and increases formation of the intermediate signalosome including IRAK1, TRAF6, and
Patel, Ronak B.; Ahn, Roy; Burke, Thomas F.
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 potential trafficking cases by increasing their awareness of this phenomenon, using qualified interpreters, isolating potential victims by providing privacy and using simple clear reassuring statements ensuring security. A multidisciplinary approach can then be mobilized to help these patients. PMID:21293753
Thomsen, Morten Skøtt; Mikkelsen, Jens D
and II α7 nAChR positive allosteric modulators (PAMs) on agonist-induced α7 nAChR up-regulation. We show that the type II PAMs, PNU-120596 (10 μM) or TQS (1 and 10 μM), inhibit up-regulation, as measured by protein levels, induced by the α7 nAChR agonist A-582941 (10 nM or 10 μM), in SH-EP1 cells stably...... and A-582941 induce up-regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs....
Lu, Yu-Ju; Liu, Ya-Chuan; Lin, Meng-Chieh; Chen, Yi-Ting; Lin, Lih-Yuan
Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5' to 3') shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.
Garbe, Annette I; Roscher, Anne; Schüler, Christiane; Lutter, Anne-Helen; Glösmann, Martin; Bernhardt, Ricardo; Chopin, Michael; Hempel, Ute; Hofbauer, Lorenz C; Rammelt, Stefan; Egerbacher, Monika; Erben, Reinhold G; Jessberger, Rolf
Bone remodeling involves tightly regulated bone-resorbing osteoclasts and bone-forming osteoblasts. Determining osteoclast function is central to understanding bone diseases such as osteoporosis and osteopetrosis. Here, we report a novel function of the F-actin binding and regulatory protein SWAP-70 in osteoclast biology. F-actin ring formation, cell morphology, and bone resorption are impaired in Swap-70(-/-) osteoclasts, whereas the expression of osteoclast differentiation markers induced in vitro by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) remains unaffected. Swap-70(-/-) mice develop osteopetrosis with increased bone mass, abnormally dense bone, and impaired osteoclast function. Ectopic expression of SWAP-70 in Swap-70(-/-) osteoclasts in vitro rescues their deficiencies in bone resorption and F-actin ring formation. Rescue requires a functional pleckstrin homology (PH) domain, known to support membrane localization of SWAP-70, and the F-actin binding domain. Transplantation of SWAP-70-proficient bone marrow into Swap-70(-/-) mice restores osteoclast resorption capacity in vivo. The identification of the role of SWAP-70 in promoting osteoclast function through modulating membrane-proximal F-actin rearrangements reveals a new pathway to control osteoclasts and bone homeostasis.
Sripada, Rebecca K; Marx, Christine E; King, Anthony P; Rajaram, Nirmala; Garfinkel, Sarah N; Abelson, James L; Liberzon, Israel
Dehydroepiandrosterone (DHEA) is a neurosteroid with anxiolytic, antidepressant, and antiglucocorticoid properties. It is endogenously released in response to stress, and may reduce negative affect when administered exogenously. Although there have been multiple reports of DHEA's antidepressant and anxiolytic effects, no research to date has examined the neural pathways involved. In particular, brain imaging has not been used to link neurosteroid effects to emotion neurocircuitry. To investigate the brain basis of DHEA's impact on emotion modulation, patients were administered 400 mg of DHEA (N=14) or placebo (N=15) and underwent 3T fMRI while performing the shifted-attention emotion appraisal task (SEAT), a test of emotional processing and regulation. Compared with placebo, DHEA reduced activity in the amygdala and hippocampus, enhanced connectivity between the amygdala and hippocampus, and enhanced activity in the rACC. These activation changes were associated with reduced negative affect. DHEA reduced memory accuracy for emotional stimuli, and also reduced activity in regions associated with conjunctive memory encoding. These results demonstrate that DHEA reduces activity in regions associated with generation of negative emotion and enhances activity in regions linked to regulatory processes. Considering that activity in these regions is altered in mood and anxiety disorders, our results provide initial neuroimaging evidence that DHEA may be useful as a pharmacological intervention for these conditions and invite further investigation into the brain basis of neurosteroid emotion regulatory effects. PMID:23552182
Mizoguchi, Toshihide; Nagasawa, Sakae; Takahashi, Naoyuki; Yagasaki, Hiroshi; Ito, Michio
Dolomite, a mineral composed of calcium magnesium carbonate (CaMg (CO3)2), is used as a food supplement that supplies calcium and magnesium. However, the effect of magnesium supplementation on bone metabolism in patients with osteoporosis is a matter of controversy. We examined the effects of daily supplementation with dolomite on calcium metabolism in ovariectomized (OVX) rats. Dolomite was administered daily to OVX rats for 9 weeks. The same amount of magnesium chloride as that supplied by the dolomite was given to OVX rats as a positive control. Histological examination revealed that ovariectomy decreased trabecular bone and increased adipose tissues in the femoral metaphysis. Dolomite or magnesium supplementation failed to improve these bone histological features. Calcium content in the femora was decreased in OVX rats. Neither calcium nor magnesium content in the femora in OVX rats was significantly increased by dolomite or magnesium administration. Urinary deoxypyridinoline excretion was significantly increased in OVX rats, and was not affected by the magnesium supplementation. Serum concentrations of magnesium were increased, and those of calcium were decreased, in OVX rats supplemented with dolomite or magnesium. However, there was a tendency toward decreased parathyroid hormone secretion and increased calcitonin secretion in OVX rats supplemented with dolomite or magnesium. Serum 1,25-dihydroxyvitamin D(3) and osteocalcin levels were significantly increased in the supplemented OVX rats. These results suggest that increased magnesium intake improves calcium metabolism in favor of increasing bone formation, through the modulation of calcium-regulating hormone secretion.
Deng, Pan-Yue; Rotman, Ziv; Blundon, Jay A.; Cho, Yongcheol; Cui, Jianmin; Cavalli, Valeria; Zakharenko, Stanislav S.; Klyachko, Vitaly A.
SUMMARY Loss of FMRP causes Fragile X syndrome (FXS), but the physiological functions of FMRP remain highly debatable. Here we show that FMRP regulates neurotransmitter release in CA3 pyramidal neurons by modulating action potential (AP) duration. Loss of FMRP leads to excessive AP broadening during repetitive activity, enhanced presynaptic calcium influx and elevated neurotransmitter release. The AP broadening defects caused by FMRP loss have a cell-autonomous presynaptic origin and can be acutely rescued in postnatal neurons. These presynaptic actions of FMRP are translation-independent and are mediated selectively by BK channels via interaction of FMRP with BK channel’s regulatory β4 subunits. Information-theoretical analysis demonstrates that loss of these FMRP functions causes marked dysregulation of synaptic information transmission. FMRP-dependent AP broadening is not limited to the hippocampus, but also occurs in cortical pyramidal neurons. Our results thus suggest major translation-independent presynaptic functions of FMRP that may have important implications for understanding FXS neuropathology. PMID:23439122
Sripada, Rebecca K; Marx, Christine E; King, Anthony P; Rajaram, Nirmala; Garfinkel, Sarah N; Abelson, James L; Liberzon, Israel
Dehydroepiandrosterone (DHEA) is a neurosteroid with anxiolytic, antidepressant, and antiglucocorticoid properties. It is endogenously released in response to stress, and may reduce negative affect when administered exogenously. Although there have been multiple reports of DHEA's antidepressant and anxiolytic effects, no research to date has examined the neural pathways involved. In particular, brain imaging has not been used to link neurosteroid effects to emotion neurocircuitry. To investigate the brain basis of DHEA's impact on emotion modulation, patients were administered 400 mg of DHEA (N=14) or placebo (N=15) and underwent 3T fMRI while performing the shifted-attention emotion appraisal task (SEAT), a test of emotional processing and regulation. Compared with placebo, DHEA reduced activity in the amygdala and hippocampus, enhanced connectivity between the amygdala and hippocampus, and enhanced activity in the rACC. These activation changes were associated with reduced negative affect. DHEA reduced memory accuracy for emotional stimuli, and also reduced activity in regions associated with conjunctive memory encoding. These results demonstrate that DHEA reduces activity in regions associated with generation of negative emotion and enhances activity in regions linked to regulatory processes. Considering that activity in these regions is altered in mood and anxiety disorders, our results provide initial neuroimaging evidence that DHEA may be useful as a pharmacological intervention for these conditions and invite further investigation into the brain basis of neurosteroid emotion regulatory effects.
Full Text Available Understanding the fiber-type specification and metabolic switch in skeletal muscle provides insights into energy metabolism in physiology and diseases. Here, we show that miR-182 is highly expressed in fast-twitch muscle and negatively correlates with blood glucose level. miR-182 knockout mice display muscle loss, fast-to-slow fiber-type switching, and impaired glucose metabolism. Mechanistic studies reveal that miR-182 modulates glucose utilization in muscle by targeting FoxO1 and PDK4, which control fuel selection via the pyruvate dehydrogenase complex (PDHC. Short-term high-fat diet (HFD feeding reduces muscle miR-182 levels by tumor necrosis factor α (TNFα, which contributes to the upregulation of FoxO1/PDK4. Restoration of miR-182 expression in HFD-fed mice induces a faster muscle phenotype, decreases muscle FoxO1/PDK4 levels, and improves glucose metabolism. Together, our work establishes miR-182 as a critical regulator that confers robust and precise controls on fuel usage and glucose homeostasis. Our study suggests that a metabolic shift toward a faster and more glycolytic phenotype is beneficial for glucose control.
Ho, Chin Yee; Jaalouk, Diana E.; Vartiainen, Maria K.; Lammerding, Jan
Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss Muscular Dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome (HGPS).1 The majority of LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and disturbed interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes.1 We report here that lamin A/C-deficient (Lmna−/−) and Lmna N195K mutant cells have impaired nuclear translocation and downstream signaling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function.2 Disturbed nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease etiology for the cardiac phenotype in many laminopathies, whereby lamins A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization. PMID:23644458
Ho, Chin Yee; Jaalouk, Diana E; Vartiainen, Maria K; Lammerding, Jan
Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison-Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna(-/-)) and Lmna(N195K/N195K) mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna(-/-) and Lmna(N195K/N195K) mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.
Jensen, Camilla S; Rasmussen, Hanne Borger; Misonou, Hiroaki
The computational ability of CNS neurons depends critically on the specific localization of ion channels in the somatodendritic and axonal membranes. Neuronal dendrites receive synaptic inputs at numerous spines and integrate them in time and space. The integration of synaptic potentials....... The physiological significance of proper Kv channel localization is emphasized by the fact that defects in the trafficking of Kv channels are observed in several neurological disorders including epilepsy. In this review, we will summarize the current understanding of the mechanisms of Kv channel trafficking...... is regulated by voltage-gated potassium (Kv) channels, such as Kv4.2, which are specifically localized in the dendritic membrane. The synaptic potentials eventually depolarize the membrane of the axon initial segment, thereby activating voltage-gated sodium channels to generate action potentials. Specific Kv...
Marisa S Goo
Full Text Available Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses.
Houston-Kolnik, Jaclyn D; Todd, Nathan R; Wilson, Midge
This study presents the Sex Trafficking Attitudes Scale (STAS), assessing cognitive, behavioral, and affective attitudes toward the sex trafficking of women and girls. Across two studies, exploratory and confirmatory factor analyses revealed and confirmed six subscales: (a) Knowledge About Sex Trafficking, (b) Awareness of Sex Trafficking, (c) Attitudes Toward Ability to Leave Sex Trafficking, (d) Attitudes Toward Helping Survivors, (e) Empathic Reactions Toward Sex Trafficking, and (f) Efficacy to Reduce Sex Trafficking. Results showed support for convergent validity as the subscales were associated with related measures. The STAS holds promise to expand research and inform efforts to support trafficking survivors.
Fulcher, Nanette B; Holliday, Phillip M; Klem, Erich; Cann, Martin J; Wolfgang, Matthew C
Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3', 5'-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.
Full Text Available Emotion regulation has an important role in child development and psychopathology. Reappraisal as cognitive regulation technique can be used effectively by children. Moreover, an ERP component known to reflect emotional processing called late positive potential (LPP can be modulated by children using reappraisal and this modulation is also related to children's emotional adjustment. The present study seeks to elucidate the neural generators of such LPP effects. To this end, children aged 8–14 years reappraised emotional faces, while neural activity in an LPP time window was estimated using magnetoencephalography-based source localization. Additionally, neural activity was correlated with two indexes of emotional adjustment and age. Reappraisal reduced activity in the left dorsolateral prefrontal cortex during down-regulation and enhanced activity in the right parietal cortex during up-regulation. Activity in the visual cortex decreased with increasing age, more adaptive emotion regulation and less anxiety. Results demonstrate that reappraisal changed activity within a frontoparietal network in children. Decreasing activity in the visual cortex with increasing age is suggested to reflect neural maturation. A similar decrease with adaptive emotion regulation and less anxiety implies that better emotional adjustment may be associated with an advance in neural maturation.
society , including NGOs, schools, civic associations, and community leaders, to identify the needs of the community, plan effective interventions...increase identification of forced labor trafficking. the City of vienna’s specialist center for unaccompanied minors accommodated 121 children in 2009...trafficked in Belgium are unaccompanied , vulnerable asylum seekers and refugees . according to the Belgian immigration office, the government identified
espanol /cpo_trata�asp Trafficking Point of Contact Fernando García-Robles OAS 1889 F St� NW Washington, DC 20006 USA Tel: 202 458 3202 Email: Mgarcia...Research Materials http://www�oas�org/dsp/ espanol /cpo_armas�asp Trafficking Points of Contact Alison August Treppel OAS 1889 F St� NW Washington, DC
Scanlon, Colleen; Krausa, Laura
Human trafficking is modern-day slavery - an insidious, criminal industry that gener- ates billions of dollars in labor trafficking alone. It knows no boundary of continent, country, race or class; it is a shattering, impartial predator that robs individuals of their basic human dignity.
Rijken, Conny; Pijnenburg, Annick
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 enforcem
Heinig, Kristina; Sage, Fanny; Robin, Catherine; Sperandio, Markus
Fetal haematopoiesis is a highly regulated process in terms of time and location. It is characterized by the emergence of specific cell populations at different extra-and intraembryonic anatomical sites. Trafficking of haematopoietic stem cells (HSCs) between these supportive niches is regulated by
Heinig, Kristina; Sage, Fanny; Robin, Catherine; Sperandio, Markus
Fetal haematopoiesis is a highly regulated process in terms of time and location. It is characterized by the emergence of specific cell populations at different extra- and intraembryonic anatomical sites. Trafficking of haematopoietic stem cells (HSCs) between these supportive niches is regulated by
Le, PhuongThao D
Survivors of human trafficking who return to their community of origin must cope with the trauma they experienced as victims as well as the conditions that contributed to their trafficking vulnerabilities. In this article, I examine the psychosocial adjustment process among women survivors of trafficking who returned to Vietnam. Supplemented by participation observation, thematic analysis of in-depth interviews with survivors revealed that throughout the trafficking process, the women experienced multiple abuses and changes in relationships and environments. The women coped by navigating a process of "reconstructing a sense of self," seeking congruence between their self-understandings and the changing contextual factors while exhibiting three main coping strategies: regulating emotional expression and thought, creating opportunities within constraints, and relating to cultural schemas. The findings underscore the importance of considering contextual factors such as cultural norms and societal values in efforts to assist trafficked survivors reintegrate into their communities.
Since the year 2000 when the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, human trafficking has been regarded as one of the egregious violations of human rights, and global efforts have been made to eradicate it. The anti-trafficking framework has multiple dimensions, and the way the anti-trafficking framework is constructed influences its impact on the victims and non-trafficked migrants. This paper will analyze the impact of the anti-traffi...
Since the year 2000 when the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, human trafficking has been regarded as one of the egregious violations of human rights, and global efforts have been made to eradicate it. The anti-trafficking framework has multiple dimensions, and the way the anti-trafficking framework is constructed influences its impact on the victims and non-trafficked migrants. This paper will analyze the impact of the anti-traffi...
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.
Balatsky, Galya [Los Alamos National Laboratory; Severe, William R [Los Alamos National Laboratory; Schoeneck, Jeffery [DHS
The need to stop illicit trafficking of nuclear and radioactive materials around the world is undeniable and urgent. This issue is particularly evident due to the highly dangerous consequences of the risks involved, the known interest of terrorist groups in acquiring such materials and the vulnerability of theft and diversion of such materials. Yet the phenomenon of nuclear trafficking remains a subject where the unknown dominates what is known on the subject. The trafficking panel at the Institute for Nuclear Materials Management (INMM) Workshop on Reducing the Risk of Radioactive and Nuclear Materials that took place in Albuquerque, New Mexico, March 10-11, 2009, dealt with some of the issues associated with nuclear trafficking. Different points of view on how to better address trafficking and thwart perpetrator efforts were discussed. This paper presents some of these views and addresses practical measures that should be considered to improve the situation.
Contreras, Jorge R; Palanichamy, Jayanth Kumar; Tran, Tiffany M; Fernando, Thilini R; Rodriguez-Malave, Norma I; Goswami, Neha; Arboleda, Valerie A; Casero, David; Rao, Dinesh S
miR-146a is a NF-κB induced microRNA that serves as a feedback regulator of this critical pathway. In mice, deficiency of miR-146a results in hematolymphoid cancer at advanced ages as a consequence of constitutive NF-κB activity. In this study, we queried whether the deficiency of miR-146a contributes to B-cell oncogenesis. Combining miR-146a deficiency with transgenic expression of c-Myc led to the development of highly aggressive B-cell malignancies. Mice transgenic for c-Myc and deficient for miR-146a were characterized by significantly shortened survival, increased lymph node involvement, differential involvement of the spleen and a mature B-cell phenotype. High-throughput sequencing of the tumors revealed significant dysregulation of approximately 250 genes. Amongst these, the transcription factor Egr1 was consistently upregulated in mice deficient for miR-146a. Interestingly, transcriptional targets of Egr1 were enriched in both the high-throughput dataset and in a larger set of miR-146a-deficient tumors. miR-146a overexpression led to downregulation of Egr1 and downstream targets with concomitant decrease in cell growth. Direct targeting of the human EGR1 by miR-146a was seen by luciferase assay. Together our findings illuminate a bona fide role for miR-146a in the modulation of B-cell oncogenesis and reveal the importance of understanding microRNA function in a cell- and disease-specific context.
Contreras, Jorge R.; Palanichamy, Jayanth Kumar; Tran, Tiffany M.; Fernando, Thilini R.; Rodriguez-Malave, Norma I.; Goswami, Neha; Arboleda, Valerie A.; Casero, David; Rao, Dinesh S.
miR-146a is a NF-κB induced microRNA that serves as a feedback regulator of this critical pathway. In mice, deficiency of miR-146a results in hematolymphoid cancer at advanced ages as a consequence of constitutive NF-κB activity. In this study, we queried whether the deficiency of miR-146a contributes to B-cell oncogenesis. Combining miR-146a deficiency with transgenic expression of c-Myc led to the development of highly aggressive B-cell malignancies. Mice transgenic for c-Myc and deficient for miR-146a were characterized by significantly shortened survival, increased lymph node involvement, differential involvement of the spleen and a mature B-cell phenotype. High-throughput sequencing of the tumors revealed significant dysregulation of approximately 250 genes. Amongst these, the transcription factor Egr1 was consistently upregulated in mice deficient for miR-146a. Interestingly, transcriptional targets of Egr1 were enriched in both the high-throughput dataset and in a larger set of miR-146a-deficient tumors. miR-146a overexpression led to downregulation of Egr1 and downstream targets with concomitant decrease in cell growth. Direct targeting of the human EGR1 by miR-146a was seen by luciferase assay. Together our findings illuminate a bona fide role for miR-146a in the modulation of B-cell oncogenesis and reveal the importance of understanding microRNA function in a cell- and disease-specific context. PMID:25906746
Birshtein, Barbara K
The Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V), diversity (D), and joining (J) segments (VDJ joining). B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM) focused on the variable region, and class switch recombination (CSR), which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR) that is located downstream of Cα, the most 3' of the Igh constant region genes. The ~40 kb 3' RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region comprising four enhancers, and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3' RR enhancer region is essential for SHM and CSR (but not for VDJ joining) and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3' RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications, and DNA methylation, in association with transcription factor binding. I propose that these changes provide a foundation by which regulatory elements in modules of the 3' RR function by interacting with each other and with target sequences of the Igh locus.
George E. Barreto
Full Text Available Following brain injury, microglia assume a reactive-like state and secrete pro-inflammatory molecules that can potentiate damage. A therapeutic strategy that may limit microgliosis is of potential interest. In this context, selective estrogen receptor modulators, such as raloxifene and tamoxifen, are known to reduce microglia activation induced by neuroinflammatory stimuli in young animals. In the present study, we have assessed whether raloxifene and tamoxifen are able to affect microglia activation after brain injury in young and aged animals in time points relevant to clinics, which is hours after brain trauma. Volume fraction of MHC-II+ microglia was estimated according to the point-counting method of Weibel within a distance of 350 μm from the lateral border of the wound, and cellular morphology was measured by fractal analysis. Two groups of animals were studied: 1 young rats, ovariectomized at 2 months of age; and 2 aged rats, ovariectomized at 18 months of age. Fifteen days after ovariectomy animals received a stab wound brain injury and the treatment with estrogenic compounds. Our findings indicate that raloxifene and tamoxifen reduced microglia activation in both young and aged animals. Although the volume fraction of reactive microglia was found lower in aged animals, this was accompanied by important changes in cell morphology, where aged microglia assume a bushier and hyperplasic aspect when compared to young microglia. These data suggest that early regulation of microglia activation provides a mechanism by which SERMs may exert a neuroprotective effect in the setting of a brain trauma.
Yang, Kai-Chien; Rutledge, Cody A; Mao, Mao; Bakhshi, Farnaz R; Xie, An; Liu, Hong; Bonini, Marcelo G; Patel, Hemal H; Minshall, Richard D; Dudley, Samuel C
Genome-wide association studies have revealed significant association of caveolin-1 (Cav1) gene variants with increased risk of cardiac arrhythmias. Nevertheless, the mechanism for this linkage is unclear. Using adult Cav1(-/-) mice, we revealed a marked reduction in the left ventricular conduction velocity in the absence of myocardial Cav1, which is accompanied with increased inducibility of ventricular arrhythmias. Further studies demonstrated that loss of Cav1 leads to the activation of cSrc tyrosine kinase, resulting in the downregulation of connexin 43 and subsequent electric abnormalities. Pharmacological inhibition of cSrc mitigates connexin 43 downregulation, slowed conduction, and arrhythmia inducibility in Cav1(-/-) animals. Using a transgenic mouse model with cardiac-specific overexpression of angiotensin-converting enzyme (ACE8/8), we demonstrated that, on enhanced cardiac renin-angiotensin system activity, Cav1 dissociated from cSrc because of increased Cav1 S-nitrosation at Cys(156), leading to cSrc activation, connexin 43 reduction, impaired gap junction function, and subsequent increase in the propensity for ventricular arrhythmias and sudden cardiac death. Renin-angiotensin system-induced Cav1 S-nitrosation was associated with increased Cav1-endothelial nitric oxide synthase binding in response to increased mitochondrial reactive oxidative species generation. The present studies reveal the critical role of Cav1 in modulating cSrc activation, gap junction remodeling, and ventricular arrhythmias. These data provide a mechanistic explanation for the observed genetic link between Cav1 and cardiac arrhythmias in humans and suggest that targeted regulation of Cav1 may reduce arrhythmic risk in cardiac diseases associated with renin-angiotensin system activation. © 2014 American Heart Association, Inc.
De Craene, Johan-Owen; Ripp, Raymond; Lecompte, Odile; Thompson, Julie D; Poch, Olivier; Friant, Sylvie
Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.
De Craene Johan-Owen
Full Text Available Abstract Background Membrane trafficking involves the complex regulation of proteins and lipids intracellular localization and is required for metabolic uptake, cell growth and development. Different trafficking pathways passing through the endosomes are coordinated by the ENTH/ANTH/VHS adaptor protein superfamily. The endosomes are crucial for eukaryotes since the acquisition of the endomembrane system was a central process in eukaryogenesis. Results Our in silico analysis of this ENTH/ANTH/VHS superfamily, consisting of proteins gathered from 84 complete genomes representative of the different eukaryotic taxa, revealed that genomic distribution of this superfamily allows to discriminate Fungi and Metazoa from Plantae and Protists. Next, in a four way genome wide comparison, we showed that this discriminative feature is observed not only for other membrane trafficking effectors, but also for proteins involved in metabolism and in cytokinesis, suggesting that metabolism, cytokinesis and intracellular trafficking pathways co-evolved. Moreover, some of the proteins identified were implicated in multiple functions, in either trafficking and metabolism or trafficking and cytokinesis, suggesting that membrane trafficking is central to this co-evolution process. Conclusions Our study suggests that membrane trafficking and compartmentalization were not only key features for the emergence of eukaryotic cells but also drove the separation of the eukaryotes in the different taxa.
Lee, F. C.; Yu, Y.; Mahmoud, M. F.
Three basic switching regulators: buck, boost, and buck/boost, employing a multi-loop control module (SCM) were characterized by a common small signal block diagram. Employing the unified model, regulator performances such as stability, audiosusceptibility, output impedance and step load transient are analyzed and key performance indexes are expressed in simple analytical forms. More importantly, the performance characteristics of all three regulators are shown to enjoy common properties due to the unique SCM control scheme which nullifies the positive zero and provides adaptive compensation to the moving poles of the boost and buck/boost converters. This allows a simple unified design procedure to be devised for selecting the key SCM control parameters for an arbitrarily given power stage configuration and parameter values, such that all regulator performance specifications can be met and optimized concurrently in a single design attempt.
Full Text Available Synapse formation requires the precise coordination of axon elongation, cytoskeletal stability, and diverse modes of cell signaling. The underlying mechanisms of this interplay, however, remain unclear. Here, we demonstrate that Strip, a component of the striatin-interacting phosphatase and kinase (STRIPAK complex that regulates these processes, is required to ensure the proper development of synaptic boutons at the Drosophila neuromuscular junction. In doing so, Strip negatively regulates the activity of the Hippo (Hpo pathway, an evolutionarily conserved regulator of organ size whose role in synapse formation is currently unappreciated. Strip functions genetically with Enabled, an actin assembly/elongation factor and the presumptive downstream target of Hpo signaling, to modulate local actin organization at synaptic termini. This regulation occurs independently of the transcriptional co-activator Yorkie, the canonical downstream target of the Hpo pathway. Our study identifies a previously unanticipated role of the Strip-Hippo pathway in synaptic development, linking cell signaling to actin organization.
Mark, M D; Wittemann, S; Herlitze, S
1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.
Kajiho, Hiroaki; Kajiho, Yuko; Scita, Giorgio
How cancer disseminates and metastasizes remains an outstanding open question. Emerging evidence indicates that membrane trafficking is frequently harnessed by tumors of epithelial origin to acquire a mesenchymal program of invasiveness. However, the critical molecular hubs used by cancer cells this context have only began to be elucidated. Here, we discussed the results of a recent phenotypic screening that led to the identification of the small GTPase RAB2A, not previously involved in cancer dissemination, as pivotal for the acquisition of pericellular proteolysis, cell dissemination and distant metastatic spreading of human breast cancer. At the cellular levels, RAB2A controls both canonical polarized Golgi-to-Plasma membrane trafficking of the junctional protein E-cadherin, and post-endocytic trafficking of the membrane-bound metalloprotease, MT1-MMP. This finding reveals an unexpected plasticity in the control of diverse trafficking routes exerted by RAB2A through canonical (Golgi stacking) and non-canonical (late endosome recycling) functional interactions, contributing to break established membrane trafficking dogma on the rigorous molecular distinction between polarized Golgi and post endocytic routes. Finally, they suggest that epithelial cancers may specifically select for those molecules that enable them to control multiple trafficking routes, in turn essential for the regulation of activities necessary for acquisition of mesenchymal traits.
Linder, Tomas; Rasmussen, Nina; Samuelsen, Camilla O;
and Deltamed27 alleles revealed a stepwise head domain molecular architecture. Phenotypical analysis of Cdk8 and head module alleles including expression profiling classified the Mediator mutant alleles into one of two groups. Cdk8 module mutants flocculate due to overexpression of adhesive cell...
He, Chao; Larson-Casey, Jennifer L; Gu, Linlin; Ryan, Alan J; Murthy, Shubha; Carter, A Brent
M2 macrophages are implicated in the development of pulmonary fibrosis as they generate profibrotic signals. The polarization process, at least in part, is regulated by epigenetic modulation. Because Cu,Zn-superoxide dismutase-induced H2O2 can polarize macrophages to a profibrotic M2 phenotype, we hypothesized that modulation of the redox state of the cell is involved in the epigenetic modulation of the macrophage phenotype. In this study, we show that signal transducer and activator of transcription 6 (STAT6) regulates Jumonji domain containing (Jmjd) 3, a histone H3 lysine 27 demethylase, and mutation of a redox-sensitive cysteine in STAT6 attenuates jmjd3 expression. Moreover, Jmjd3 deficiency abrogates profibrotic M2 gene expression. Treatment with leflunomide, which reduces mitochondrial reactive oxygen species production and tyrosine phosphorylation, inhibits jmjd3 expression and M2 polarization, as well as development of a fibrotic phenotype. Taken together, these observations provide evidence that the redox regulation of Jmjd3 is a unique regulatory mechanism for Cu,Zn-superoxide dismutase-mediated profibrotic M2 polarization. Furthermore, leflunomide, which reduces reactive oxygen species production and tyrosine phosphorylation, may prove to be therapeutic in the treatment of asbestos-induced pulmonary fibrosis.
Shivers, Robert P; Kooistra, Tristan; Chu, Stephanie W; Pagano, Daniel J; Kim, Dennis H
Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-interleukin-1 receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens--in the cell-autonomous regulation of innate immunity and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.
Yosef Y Kuttner
Full Text Available Knowledge of the structural basis of protein-protein interactions (PPI is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM, whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+. Calmodulin is a regulatory protein that acts as an intracellular Ca(2+ sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+ and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+ binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.
O'Callaghan, Michael G
"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.
Williams, Jeffrey M; Tsai, Billy
Bacterial toxins often translocate across a cellular membrane to gain access into the host cytosol, modifying cellular components in order to exert their toxic effects. To accomplish this feat, these toxins traffic to a membrane penetration site where they undergo conformational changes essential to eject the toxin's catalytic subunit into the cytosol. In this brief review, we highlight recent findings that elucidate both the trafficking pathways and membrane translocation mechanisms of toxins that cross the plasma, endosomal, or endoplasmic reticulum (ER) membrane. These findings not only illuminate the specific nature of the host-toxin interactions during entry, but should also provide additional therapeutic strategies to prevent or alleviate the bacterial toxin-induced diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.
Wieland Felix T
Full Text Available Abstract Background Alzheimer's disease (AD is characterized by cerebral deposition of β-amyloid (Aβ peptides. Aβ is released from ectodomain cleaved amyloid precursor protein (APP via intramembranous proteolysis by γ-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating γ-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated. Results Here, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Aβ production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature γ-secretase complexes isolated using an active-site γ-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Aβ42 production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in γ-secretase modulation. Conclusion These data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis.
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.
Moxley, Joel F.; Jewett, Michael Christopher; Antoniewicz, Maciek R.
Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein protein interactions and transcription factor binding revealed critical insights into cellular behavior. Howe...... that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing strains for industrial applications.......RNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental C-13-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator...... of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow...
Feng, Qiang-Nan; Zhang, Yan; Li, Sha
Vacuolar trafficking routes and their regulators have recently drawn lots of attention in plant cell biology. A recent study reported the discovery of a plant-specific vacuolar trafficking route, i.e., a direct ER-to-vacuole route, through analysis of VHA-a3 subcellular targeting, a key component for the tonoplast V-ATPases. Our recent findings showed that VHA-a3 targets to the tonoplast through a Rab5-mediated but Rab7-independent pathway, shedding new lights on the unconventional vacuolar trafficking route in plant cells. © 2017 Institute of Botany, Chinese Academy of Sciences.
Zhang, X; Bao, L; Li, S
Opiate analgesics such as morphine are often used for pain therapy. However, antinociceptive tolerance and dependence may develop with long-term use of these drugs. It was found that μ-opioid receptors can interact with δ-opioid receptors, and morphine antinociceptive tolerance can be reduced by blocking δ-opioid receptors. Recent studies have shown that μ- and δ-opioid receptors are co-expressed in a considerable number of small neurons in the dorsal root ganglion. The interaction of μ-opioid receptors with δ-opioid receptors in the nociceptive afferents is facilitated by the stimulus-induced cell-surface expression of δ-opioid receptors, and contributes to morphine tolerance. Further analysis of the molecular, cellular and neural circuit mechanisms that regulate the trafficking and interaction of opioid receptors and related signalling molecules in the pain pathway would help to elucidate the mechanism of opiate analgesia and improve pain therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24611685
Hang, Howard C; Wilson, John P; Charron, Guillaume
Protein lipidation and lipid trafficking control many key biological functions in all kingdoms of life. The discovery of diverse lipid species and their covalent attachment to many proteins has revealed a complex and regulated network of membranes and lipidated proteins that are central to fundamental aspects of physiology and human disease. Given the complexity of lipid trafficking and the protein targeting mechanisms involved with membrane lipids, precise and sensitive methods are needed to monitor and identify these hydrophobic molecules in bacteria, yeast, and higher eukaryotes. Although many analytical methods have been developed for characterizing membrane lipids and covalently modified proteins, traditional reagents and approaches have limited sensitivity, do not faithfully report on the lipids of interest, or are not readily accessible. The invention of bioorthogonal ligation reactions, such as the Staudinger ligation and azide-alkyne cycloadditions, has provided new tools to address these limitations, and their use has begun to yield fresh insight into the biology of protein lipidation and lipid trafficking. In this Account, we discuss how these new bioorthogonal ligation reactions and lipid chemical reporters afford new opportunities for exploring the biology of lipid-modified proteins and lipid trafficking. Lipid chemical reporters from our laboratory and several other research groups have enabled improved detection and large-scale proteomic analysis of fatty-acylated and prenylated proteins. For example, fatty acid and isoprenoid chemical reporters in conjunction with bioorthogonal ligation methods have circumvented the limited sensitivity and hazards of radioactive analogues, allowing rapid and robust fluorescent detection of lipidated proteins in all organisms tested. These chemical tools have revealed alterations in protein lipidation in different cellular states and are beginning to provide unique insights in mechanisms of regulation. Notably, the
Meshkovska, Biljana; Siegel, Melissa; Stutterheim, Sarah E; Bos, Arjan E R
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.
Baghaee Ravari, Soodeh
G-quadruplexes (G-QPX) have been the subject of intense research due to their unique structural configuration and potential applications, particularly their functionality in biological process as a novel type of nano--switch. They have been found in critical regions of the human genome such as telomeres, promoter regions, and untranslated regions of RNA. About 50% of human DNA in promoters has G-rich regions with the potential to form G-QPX structures. A G-QPX might act mechanistically as an ON/OFF switch, regulating gene expression, meaning that the formation of G-QPX in a single strand of DNA disrupts double stranded DNA, prevents the binding of transcription factors (TF) to their recognition sites, resulting in gene down-regulation. Although there are numerous studies on biological roles of G-QPXs in oncogenes, their potential formation in neuronal cells, in particular upstream of transcription start sites, is poorly investigated. The main focus of this research is to identify stable G-QPXs in the 97bp active promoter region of the choline acetyltransferase (ChAT) gene, the terminal enzyme involved in synthesis of the neurotransmitter acetylcholine, and to clarify ionic modulation of G-QPX nanostructures through the mechanism of neural action potentials. Different bioinformatics analyses (in silico), including the QGRS, quadparser and G4-Calculator programs, have been used to predict stable G-QPX in the active promoter region of the human ChAT gene, located 1000bp upstream from the TATA box. The results of computational studies (using those three different algorithms) led to the identification of three consecutive intramolecular G-QPX structures in the negative strand (ChAT G17-2, ChAT G17, and ChAT G29) and one intramolecular G-QPX structure in the positive strand (ChAT G30). Also, the results suggest the possibility that nearby G-runs in opposed DNA strands with a short distance of each other may be able to form a stable intermolecular G-QPX involving two DNA
Thomsen, Morten S; Mikkelsen, Jens D
Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist-induced α7 nAChR up-regulation. We show that the type II PAMs, PNU-120596 (10 μM) or TQS (1 and 10 μM), inhibit up-regulation, as measured by protein levels, induced by the α7 nAChR agonist A-582941 (10 nM or 10 μM), in SH-EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL-3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine-induced receptor up-regulation, suggesting that nicotine and A-582941 induce up-regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU-120596 inhibits up-regulation of the α7 nAChR induced by 10 mg/kg A-582941, as measured by [(125)I]-bungarotoxin autoradiography, whereas 1 mg/kg AVL-3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A-582941-induced up-regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist-dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A-582941 induce up-regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.
The Trafficking Protocol has shaped and advanced a global movement against human trafficking; notably through establishing a global definition and creating criminal justice remedies befitting an international crime. Borne out of and including the Protocol, a global anti-trafficking framework has emerged. This framework reflects these two central tenets at international, regional and national levels and includes initiatives by States not party to the Protocol, such as Singapore. However,...
Doyle, Siamsa M; Vain, Thomas; Robert, Stéphanie
The establishment and maintenance of controlled auxin gradients within plant tissues are essential for a multitude of developmental processes. Auxin gradient formation is co-ordinated via local biosynthesis and transport. Cell to cell auxin transport is facilitated and precisely regulated by complex endomembrane trafficking mechanisms that target auxin carrier proteins to their final destinations. In turn, auxin and cross-talk with other phytohormones regulate the endomembrane trafficking of auxin carriers. Dissecting such rapid and complicated processes is challenging for classical genetic experiments due to trafficking pathway diversity, gene functional redundancy, and lethality in loss-of-function mutants. Many of these difficulties can be bypassed via the use of small molecules to modify or disrupt the function or localization of proteins. Here, we will review examples of the knowledge acquired by the use of such chemical tools in this field, outlining the advantages afforded by chemical biology approaches.
Li, Jianchao; Lu, Qing; Zhang, Mingjie
Unconventional myosins are a superfamily of actin-based molecular motors playing diverse roles including cellular trafficking, mechanical supports, force sensing and transmission, etc. The variable neck and tail domains of unconventional myosins function to bind to specific cargoes including proteins and lipid vesicles and thus are largely responsible for the diverse cellular functions of myosins in vivo. In addition, the tail regions, together with their cognate cargoes, can regulate activities of the motor heads. This review outlines the advances made in recent years on cargo recognition and cargo binding-induced regulation of the activity of several unconventional myosins including myosin-I, V, VI and X in cellular trafficking. We approach this topic by describing a series of high-resolution structures of the neck and tail domains of these unconventional myosins either alone or in complex with their specific cargoes, and by discussing potential implications of these structural studies on cellular trafficking of these myosin motors.
Full Text Available Glioblastoma (GBM is the most aggressive primary brain tumor in adults. The mechanisms that confer GBM cells their invasive behavior are poorly understood. The electroneutral Na+-K+-2Cl− co-transporter 1 (NKCC1 is an important cell volume regulator that participates in cell migration. We have shown that inhibition of NKCC1 in GBM cells leads to decreased cell migration, in vitro and in vivo. We now report on the role of NKCC1 on cytoskeletal dynamics. We show that GBM cells display a significant decrease in F-actin content upon NKCC1 knockdown (NKCC1-KD. To determine the potential actin-regulatory mechanisms affected by NKCC1 inhibition, we studied NKCC1 protein interactions. We found that NKCC1 interacts with the actin-regulating protein Cofilin-1 and can regulate its membrane localization. Finally, we analyzed whether NKCC1 could regulate the activity of the small Rho-GTPases RhoA and Rac1. We observed that the active forms of RhoA and Rac1 were decreased in NKCC1-KD cells. In summary, we report that NKCC1 regulates GBM cell migration by modulating the cytoskeleton through multiple targets including F-actin regulation through Cofilin-1 and RhoGTPase activity. Due to its essential role in cell migration NKCC1 may serve as a specific therapeutic target to decrease cell invasion in patients with primary brain cancer.
Deshpande, Neha A; Nour, Nawal M
Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community. The costs to society include the degradation of human and women's rights, poor public health, disrupted communities, and diminished social development. Victims of sex trafficking acquire adverse physical and psychological health conditions and social disadvantages. Thus, sex trafficking is a critical health issue with broader social implications that requires both medical and legal attention. Healthcare professionals can work to improve the screening, identification, and assistance of victims of sex trafficking in a clinical setting and help these women and girls access legal and social services.
Titchen, Kanani E; Katz, Douglas; Martinez, Kidian; White, Krishna
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.
Full Text Available Are trafficked also travellers? The article analyses the traveller as merchandise: migrants, clandestine and trafficked are some of the roles sometimes unwittingly performed by the social actors. During a trip a migrant can therefore become trafficked, i.e. the social actor goes through a continuous transformation of the self: every single transformation has its name. The activity of giving names is a juridical process which involves many international institutions. The example made in this article concerns the Balkan area and central-oriental Europe. The migratory routes will be examined as well as the paths which constitute them. A labyrinth which changes continuously: the paths are modified; they disappear only to re-appear later. The testimony of the trafficked is here gathered and analyzed on the basis of the documents gathered on the field or produced by the public prosecutor’s office in Trieste.
Deshpande, Neha A; Nour, Nawal M
Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community...
Human trafficking is a widespread problem with estimates ranging from 14,000 to 50,000 individuals trafficked into the United States annually. This hidden population involves the commercial sex industry, agriculture, factories, hotel and restaurant businesses, domestic workers, marriage brokers, and some adoption firms. Because 80% of trafficked individuals are women and girls, women’s health care providers may better serve their diverse patient population by increasing their awareness of this problem. The exploitation of people of any race, gender, sexual orientation, or ethnicity is unacceptable at any time, in any place. The members of the American College of Obstetricians and Gynecologists should be aware of this problem and strive to recognize and assist their patients who are victims or who have been victims of human trafficking.
Full Text Available 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 potential trafficking cases by increasing their awareness of this phenomenon, using qualified interpreters, isolating potential victims by providing privacy and using simple clear reassuring statements ensuring security. A multidisciplinary approach can then be mobilized to help these patients. [West J Emerg Med. 2010;11(5; 402-404.
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.
... billions of dollars in illicit revenues each year, contributing to the illegal economy, fueling instability, and undermining security. Also, the prevention of trafficking of live animals helps us control the...
demand a dowry . He took her to India and forced her into prostitution. When she refused she was beaten. She managed to escape and found her way home...survival and are vulnerable to the false promises of work or marriage in another country are trafficked from Bangladesh to Pakistan, India , and other...Middle East countries. Approximately 2,400 to 4,800 women and children are annually trafficked into India . A considerable number of boys are sent to become
Li, Xiao-Jiang; Orr, Adam L.; Li, Shihua
Abstract Impaired mitochondrial function has been well documented in Huntington?s disease. Mutant huntingtin is found to affect mitochondria via various mechanisms including the dysregulation of gene transcription and impairment of mitochondrial function or trafficking. The lengthy and highly branched neuronal processes constitute complex neural networks in which there is a large demand for mitochondria-generated energy. Thus, the impaired mitochondria trafficking in neuronal cells...
Keilberg, Daniela; Wuichet, Kristin; Drescher, Florian; Søgaard-Andersen, Lotte
How cells establish and dynamically change polarity are general questions in cell biology. Cells of the rod-shaped bacterium Myxococcus xanthus move on surfaces with defined leading and lagging cell poles. Occasionally, cells undergo reversals, which correspond to an inversion of the leading-lagging pole polarity axis. Reversals are induced by the Frz chemosensory system and depend on relocalization of motility proteins between the poles. The Ras-like GTPase MglA localizes to and defines the leading cell pole in the GTP-bound form. MglB, the cognate MglA GTPase activating protein, localizes to and defines the lagging pole. During reversals, MglA-GTP and MglB switch poles and, therefore, dynamically localized motility proteins switch poles. We identified the RomR response regulator, which localizes in a bipolar asymmetric pattern with a large cluster at the lagging pole, as important for motility and reversals. We show that RomR interacts directly with MglA and MglB in vitro. Furthermore, RomR, MglA, and MglB affect the localization of each other in all pair-wise directions, suggesting that RomR stimulates motility by promoting correct localization of MglA and MglB in MglA/RomR and MglB/RomR complexes at opposite poles. Moreover, localization analyses suggest that the two RomR complexes mutually exclude each other from their respective poles. We further show that RomR interfaces with FrzZ, the output response regulator of the Frz chemosensory system, to regulate reversals. Thus, RomR serves at the functional interface to connect a classic bacterial signalling module (Frz) to a classic eukaryotic polarity module (MglA/MglB). This modular design is paralleled by the phylogenetic distribution of the proteins, suggesting an evolutionary scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed by the addition of the Frz system to dynamically regulate cell polarity.
Full Text Available How cells establish and dynamically change polarity are general questions in cell biology. Cells of the rod-shaped bacterium Myxococcus xanthus move on surfaces with defined leading and lagging cell poles. Occasionally, cells undergo reversals, which correspond to an inversion of the leading-lagging pole polarity axis. Reversals are induced by the Frz chemosensory system and depend on relocalization of motility proteins between the poles. The Ras-like GTPase MglA localizes to and defines the leading cell pole in the GTP-bound form. MglB, the cognate MglA GTPase activating protein, localizes to and defines the lagging pole. During reversals, MglA-GTP and MglB switch poles and, therefore, dynamically localized motility proteins switch poles. We identified the RomR response regulator, which localizes in a bipolar asymmetric pattern with a large cluster at the lagging pole, as important for motility and reversals. We show that RomR interacts directly with MglA and MglB in vitro. Furthermore, RomR, MglA, and MglB affect the localization of each other in all pair-wise directions, suggesting that RomR stimulates motility by promoting correct localization of MglA and MglB in MglA/RomR and MglB/RomR complexes at opposite poles. Moreover, localization analyses suggest that the two RomR complexes mutually exclude each other from their respective poles. We further show that RomR interfaces with FrzZ, the output response regulator of the Frz chemosensory system, to regulate reversals. Thus, RomR serves at the functional interface to connect a classic bacterial signalling module (Frz to a classic eukaryotic polarity module (MglA/MglB. This modular design is paralleled by the phylogenetic distribution of the proteins, suggesting an evolutionary scheme in which RomR was incorporated into the MglA/MglB module to regulate cell polarity followed by the addition of the Frz system to dynamically regulate cell polarity.
Activation of 3-phosphoinositide-dependent kinase 1 (PDK1) and serum- and glucocorticoid-induced protein kinase 1 (SGK1) by short-chain sphingolipid C4-ceramide rescues the trafficking defect of ΔF508-cystic fibrosis transmembrane conductance regulator (ΔF508-CFTR).
Caohuy, Hung; Yang, Qingfeng; Eudy, Yvonne; Ha, Thien-An; Xu, Andrew E; Glover, Matthew; Frizzell, Raymond A; Jozwik, Catherine; Pollard, Harvey B
Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1 signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that C4-CER initially induces autophosphorylation of SGK1 at Ser(422). SGK1[Ser(P)(422)] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser(241). Then PDK1[Ser(P)(241)] phosphorylates SGK1[Ser(P)(422)] at Thr(256) to generate fully activated SGK1[Ser(422), Thr(P)(256)]. SGK1[Ser(P)(422),Thr(P)(256)] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane. Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy for a life-limiting disorder that affects one child, on average, each day.
Le, PhuongThao Dinh
This study employs qualitative and quantitative approaches to examine the psychosocial issues among women survivors of trafficking who have returned to Vietnam. The quantitative study examines the relationship between trafficking abuse and psychological symptoms among a pilot sample of trafficked women who accessed a post-trafficking project at the Vietnam-China border. The results are consistent with the existing literature suggesting that greater trafficking-related abuse is associated with...
Megan L LeBlanc
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.
Maruyama, Takamitsu; Jiang, Ming; Hsu, Wei
Human genetic analysis has recently identified Gpr177 as a susceptibility locus for bone-mineral-density and osteoporosis. Determining the unknown function of this gene is therefore extremely important to further our knowledge base of skeletal development and disease. The protein encoded by Gpr177 exhibits an ability to modulate the trafficking of Wnt similar to the Drosophila Wls/Evi/Srt. Because of a critical role in Wnt regulation, Gpr177 might be required for several key steps of skeletog...
Feng, Xiujing; Weng, Dan; Zhou, Feifei; Owen, Young D; Qin, Haohan; Zhao, Jingfa; WenYu; Huang, Yahong; Chen, Jiajia; Fu, Haijian; Yang, Nanfei; Chen, Dianhua; Li, Jianxin; Tan, Renxiang; Shen, Pingping
PPARγ has emerged as a master regulator of macrophage polarization and is the molecular target of the thiazolidinedione drugs. Here we show that apigenin binds and activates PPARγ by acting as a modulator. Activation of PPARγ by apigenin blocks p65 translocation into nuclei through inhibition of p65/PPARγ complex translocation into nuclei, thereby decreasing NF-κB activation and favoringM2 macrophage polarization. In HFD and ob/ob mice, apigenin significantly reverses M1 macrophage into M2 and reduces the infiltration of inflammatory cells in liver and adipose tissues, as well as decreases the levels of pro-inflammatory cytokines, thereby alleviating inflammation. Strikingly, apigenin reduces liver and muscular steatosis, decreases the levels of ALT, AST, TC and TG, improving glucose resistance obviously. Unlike rosiglitazone, apigenin does not cause significant weight gain, osteoporosis et al. Our findings identify apigenin as a modulator of PPARγ and a potential lead compound for treatment of metabolic disorders.
Full Text Available Developmental plasticity enables plants to respond to elevated ambient temperatures by adapting their shoot architecture. On the cellular level, the basic-helix-loop-helix (bHLH transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4 coordinates this response by activating hormonal modules that in turn regulate growth. In addition to an unknown temperature-sensing mechanism, it is currently not understood how temperature regulates PIF4 activity. Using a forward genetic approach in Arabidopsis thaliana, we present extensive genetic evidence demonstrating that the DE-ETIOLATED 1 (DET1-CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1-ELONGATED HYPOCOTYL 5 (HY5-dependent photomorphogenesis pathway transcriptionally regulates PIF4 to coordinate seedling growth in response to elevated temperature. Our findings demonstrate that two of the most prevalent environmental cues, light and temperature, share a much larger set of signaling components than previously assumed. Similar to the toolbox concept in animal embryonic patterning, multipurpose signaling modules might have evolved in plants to translate various environmental stimuli into adaptational growth processes.
Full Text Available Recruitment of leukocytes from the bloodstream to inflamed tissues requires a carefully regulated cascade of binding interactions between adhesion molecules on leukocytes and endothelial cells. Adhesive interactions between CD44 and hyaluronan have been implicated in the regulation of immune cell trafficking within various tissues. In this review, the biology of CD44-hyaluronan interactions in cell trafficking is summarized, with special attention to neutrophil recruitment within the liver microcirculation. We describe the molecular mechanisms that regulate adhesion between neutrophil CD44 and endothelial hyaluronan, including recent evidence implicating serum-derived hyaluronan associated protein (SHAP as an important co-factor in the binding of hyaluronan to CD44 under flow conditions. CD44-hyaluronan mediated neutrophil recruitment has been shown to contribute to innate immune responses to invading microbes, as well as to the pathogenesis of many inflammatory diseases, including various liver pathologies. As a result, blockade of neutrophil recruitment by targeting CD44-HA interactions has proven beneficial as an anti-inflammatory treatment strategy in a number of animal models of inflammatory diseases.
Full Text Available The mechanisms of learning involved in brain self-regulation have still to be unveiled to exploit the full potential of this methodology for therapeutic interventions. This skill of volitionally changing brain activity presumably resembles motor skill learning which in turn is accompanied by plastic changes modulating resting state networks. Along these lines, we hypothesized that brain regulation and neurofeedback would similarly modify intrinsic networks at rest while presenting a distinct spatio-temporal pattern. High-resolution EEG preceded and followed a single neurofeedback training intervention of modulating circumscribed sensorimotor low β -activity by motor imagery in eleven healthy participants. They were kept in the deliberative phase of skill acquisition with high demands for learning self-regulation through stepwise increases of task difficulty. By applying the corrected imaginary part of the coherency function, we observed increased functional connectivity of both the primary motor and the primary somatosensory cortex with their respective contralateral homologous cortices in the low β-frequency band which was self-regulated during feedback. At the same time, the primary motor cortex - but none of the surrounding cortical areas - showed connectivity to contralateral supplementary motor and dorsal premotor areas in the high β-band. Simultaneously, the neurofeedback target displayed a specific increase of functional connectivity with an ipsilateral fronto-parietal network in the α-band while presenting a de-coupling with contralateral primary and secondary sensorimotor areas in the very same frequency band.Brain self-regulating modifies resting state connections spatially selective to the neurofeedback target of the dominant hemisphere. These are anatomically distinct with regard to the cortico-cortical connectivity pattern and are functionally specific with regard to the time domain of coherent activity consistent with a Hebbian
Clause, Kristen J; Lawler, Kate Byrnes
As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices.
Clause, Kristen J; Lawler, Kate Byrnes
As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices. To learn more or to help with this cause, visit the Somaly Mam Foundation at www.somaly.org or the U.S. Department of State at www. state.gov.
Wada, Takeyoshi [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Asahi, Toru [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Sawamura, Naoya, E-mail: firstname.lastname@example.org [Faculty of Science and Engineering, Waseda University, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan); Research Organization for Nano & Life Innovation, Waseda University #03C309, TWIns, 2-2 Wakamatsu, Shinjuku, Tokyo, 162-8480 (Japan)
The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. - Highlights: • We found that CRBN is a nucleocytoplasmic shutting protein and identified the key domain for nucleocytoplasmic shuttling. • CRBN associates with the transcription factor Ikaros via the N-terminal domain. • CRBN modulates Ikaros-mediated transcriptional regulation and its downstream target, enkephalin.
Wang, Zhongyuan; Wu, Yanping; Wang, Haifeng; Zhang, Yangqing; Mei, Lin; Fang, Xuexun; Zhang, Xudong; Zhang, Fang; Chen, Hongbo; Liu, Ying; Jiang, Yuyang; Sun, Shengnan; Zheng, Yi; Li, Na; Huang, Laiqiang
Expression of receptor for hyaluronan-mediated motility (RHAMM), a breast cancer susceptibility gene, is tightly controlled in normal tissues but elevated in many tumors, contributing to tumorigenesis and metastases. However, how the expression of RHAMM is regulated remains elusive. Statins, inhibitors of mevalonate metabolic pathway widely used for hypercholesterolemia, have been found to also have antitumor effects, but little is known of the specific targets and mechanisms. Moreover, Hippo signaling pathway plays crucial roles in organ size control and cancer development, yet its downstream transcriptional targets remain obscure. Here we show that RHAMM expression is regulated by mevalonate and Hippo pathways converging onto Yes-associated protein (YAP)/TEAD, which binds RHAMM promoter at specific sites and controls its transcription and consequently breast cancer cell migration and invasion (BCCMI); and that simvastatin inhibits BCCMI via targeting YAP-mediated RHAMM transcription. Required for ERK phosphorylation and BCCMI, YAP-activated RHAMM transcription is dependent on mevalonate and sensitive to simvastatin, which modulate RHAMM transcription by modulating YAP phosphorylation and nuclear-cytoplasmic localization. Further, modulation by mevalonate/simvastatin of YAP-activated RHAMM transcription requires geranylgeranylation, Rho GTPase activation, and actin cytoskeleton rearrangement, but is largely independent of MST and LATS kinase activity. These findings from in vitro and in vivo investigations link mevalonate and Hippo pathways with RHAMM as a downstream effector, a YAP-transcription and simvastatin-inhibition target, and a cancer metastasis mediator; uncover a mechanism regulating RHAMM expression and cancer metastases; and reveal a mode whereby simvastatin exerts anticancer effects; providing potential targets for cancer therapeutic agents. PMID:24367099
Full Text Available Worldwide human trafficking is the third most often registered international criminal activity, ranked only after drug and weapon trafficking. This article focusses on three questions: 1 How can human trafficking be measured? 2 What are the causes and indicators of this criminal activity which exploits individuals? 3 Which countries observe a high (or low level of human trafficking inflow? We apply the Multiple Indicators Multiple Causes structural equation model to measure human trafficking inflows in a way which includes all potential causes and indicators in one estimation model. The human trafficking measurement focusses on international human trafficking. We use freely available existing data and thus generate an objective measure of the extent of trafficking. Countries are ranked according to their potential to be a destination country based on various characteristics of the trafficking process.
Vanini, Giancarlo; Nemanis, Kriste; Baghdoyan, Helen A.; Lydic, Ralph
The oral part of the pontine reticular formation (PnO) contributes to the regulation of sleep, anesthesia, and pain. The role of PnO GABA in modulating these states remains incompletely understood. The present study used time to Loss and time to Resumption of Righting Response (LoRR and RoRR) as surrogate measures of loss and resumption of consciousness. This study tested three hypotheses: (1) pharmacologically manipulating GABA levels in rat PnO alters LoRR, RoRR, and nociception; (2) propof...
Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik
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.
During myelin formation OLGs may utilize basic mechanisms of epithelial membrane trafficking, as described and summarized in the introductory chapter (Chapter 1). However, whether specific transport pathways, unique to myelin biogenesis are involved and how such pathways might be regulated in biogen
Levy, Maayan; Thaiss, Christoph A; Zeevi, David; Dohnalová, Lenka; Zilberman-Schapira, Gili; Mahdi, Jemal Ali; David, Eyal; Savidor, Alon; Korem, Tal; Herzig, Yonatan; Pevsner-Fischer, Meirav; Shapiro, Hagit; Christ, Anette; Harmelin, Alon; Halpern, Zamir; Latz, Eicke; Flavell, Richard A; Amit, Ido; Segal, Eran; Elinav, Eran
Host-microbiome co-evolution drives homeostasis and disease susceptibility, yet regulatory principles governing the integrated intestinal host-commensal microenvironment remain obscure. While inflammasome signaling participates in these interactions, its activators and microbiome-modulating mechanisms are unknown. Here, we demonstrate that the microbiota-associated metabolites taurine, histamine, and spermine shape the host-microbiome interface by co-modulating NLRP6 inflammasome signaling, epithelial IL-18 secretion, and downstream anti-microbial peptide (AMP) profiles. Distortion of this balanced AMP landscape by inflammasome deficiency drives dysbiosis development. Upon fecal transfer, colitis-inducing microbiota hijacks this microenvironment-orchestrating machinery through metabolite-mediated inflammasome suppression, leading to distorted AMP balance favoring its preferential colonization. Restoration of the metabolite-inflammasome-AMP axis reinstates a normal microbiota and ameliorates colitis. Together, we identify microbial modulators of the NLRP6 inflammasome and highlight mechanisms by which microbiome-host interactions cooperatively drive microbial community stability through metabolite-mediated innate immune modulation. Therefore, targeted "postbiotic" metabolomic intervention may restore a normal microenvironment as treatment or prevention of dysbiosis-driven diseases.
Tenbrock, K; Kyttaris, VC; Ahlmann, M; Ehrchen, JA; Tolnay, M; Melkonyan, H; Mawrin, C; Roth, J; Sorg, C; Juang, YT; Tsokos, GC
Systemic lupus erythematusus T cells display decreased amounts of TCR zeta mRNA that results in part from limited binding of the transcriptional enhancer Elf-1 to the TCR zeta promoter. We have identified a new cis-binding site for the cAMP response element (CRE) modulator (CREM) on the TCR zeta pro
Busch, Bernhard L.; Schmitz, Gregor; Rossmann, Susanne; Piron, Florence; Ding, Jia; Bendahmane, Abdelhafid; Theres, Klaus
Aerial plant architecture is predominantly determined by shoot branching and leaf morphology, which are governed by apparently unrelated developmental processes, axillary meristem formation, and leaf dissection. Here, we show that in tomato (Solanum lycopersicum), these processes share essential functions in boundary establishment. Potato leaf (C), a key regulator of leaf dissection, was identified to be the closest paralog of the shoot branching regulator Blind (Bl). Comparative genomics revealed that these two R2R3 MYB genes are orthologs of the Arabidopsis thaliana branching regulator REGULATOR OF AXILLARY MERISTEMS1 (RAX1). Expression studies and complementation analyses indicate that these genes have undergone sub- or neofunctionalization due to promoter differentiation. C acts in a pathway independent of other identified leaf dissection regulators. Furthermore, the known leaf complexity regulator Goblet (Gob) is crucial for axillary meristem initiation and acts in parallel to C and Bl. Finally, RNA in situ hybridization revealed that the branching regulator Lateral suppressor (Ls) is also expressed in leaves. All four boundary genes, C, Bl, Gob, and Ls, may act by suppressing growth, as indicated by gain-of-function plants. Thus, leaf architecture and shoot architecture rely on a conserved mechanism of boundary formation preceding the initiation of leaflets and axillary meristems. PMID:22039213
Hougaard, S; Loechel, F; Xu, X
We have investigated the trafficking of the membrane-anchored form of human ADAM 12 (ADAM 12-L) fused to a green fluorescence protein tag. Subcellular localization of the protein in transiently transfected cells was determined by fluorescence microscopy and trypsin sensitivity. Full-length ADAM 12...... the cytoplasmic and transmembrane domains, but not the Src homology 3 domain (SH3) binding sites. These results raise the possibility that a trafficking checkpoint in the trans-Golgi network is one of the cellular mechanisms for regulation of ADAM 12-L function, by allowing a rapid release of ADAM 12-L...
Caiado, Francisco; Real, Carla; Carvalho, Tânia; Dias, Sérgio
Bone marrow (BM) derived vascular precursor cells (BM-PC, endothelial progenitors) are involved in normal and malignant angiogenesis, in ischemia and in wound healing. However, the mechanisms by which BM-PC stimulate the pre-existing endothelial cells at sites of vascular remodelling/recovery, and their contribution towards the formation of new blood vessels are still undisclosed. In the present report, we exploited the possibility that members of the Notch signalling pathway, expressed by BM-PC during endothelial differentiation, might regulate their pro-angiogenic or pro-wound healing properties. We demonstrate that Notch pathway modulates the adhesion of BM-PC to extracellular matrix (ECM) in vitro via regulation of integrin alpha3beta1; and that Notch pathway inhibition on BM-PC impairs their capacity to stimulate endothelial cell tube formation on matrigel and to promote endothelial monolayer recovery following wounding in vitro. Moreover, we show that activation of Notch pathway on BM-PC improved wound healing in vivo through angiogenesis induction. Conversely, inoculation of BM-PC pre-treated with a gamma secretase inhibitor (GSI) into wounded mice failed to induce angiogenesis at the wound site and did not promote wound healing, presumably due to a lower frequency of BM-PC at the wound area. Our data suggests that Notch pathway regulates BM-PC adhesion to ECM at sites of vascular repair and that it also regulates the capacity of BM-PC to stimulate angiogenesis and to promote wound healing. Drug targeting of the Notch pathway on BM-PC may thus represent a novel strategy to modulate neo-angiogenesis and vessel repair.
Full Text Available Bone marrow (BM derived vascular precursor cells (BM-PC, endothelial progenitors are involved in normal and malignant angiogenesis, in ischemia and in wound healing. However, the mechanisms by which BM-PC stimulate the pre-existing endothelial cells at sites of vascular remodelling/recovery, and their contribution towards the formation of new blood vessels are still undisclosed. In the present report, we exploited the possibility that members of the Notch signalling pathway, expressed by BM-PC during endothelial differentiation, might regulate their pro-angiogenic or pro-wound healing properties. We demonstrate that Notch pathway modulates the adhesion of BM-PC to extracellular matrix (ECM in vitro via regulation of integrin alpha3beta1; and that Notch pathway inhibition on BM-PC impairs their capacity to stimulate endothelial cell tube formation on matrigel and to promote endothelial monolayer recovery following wounding in vitro. Moreover, we show that activation of Notch pathway on BM-PC improved wound healing in vivo through angiogenesis induction. Conversely, inoculation of BM-PC pre-treated with a gamma secretase inhibitor (GSI into wounded mice failed to induce angiogenesis at the wound site and did not promote wound healing, presumably due to a lower frequency of BM-PC at the wound area. Our data suggests that Notch pathway regulates BM-PC adhesion to ECM at sites of vascular repair and that it also regulates the capacity of BM-PC to stimulate angiogenesis and to promote wound healing. Drug targeting of the Notch pathway on BM-PC may thus represent a novel strategy to modulate neo-angiogenesis and vessel repair.
Lizcano, Fernando; Romero, Carolina; Vargas, Diana
.... The nuclear receptor Peroxisome Proliferator-activated receptor gamma (PPARγ) is the master regulator of adipose cell differentiation and its functional activation is currently used as a therapeutic approach for Diabetes Mellitus type 2...
Fernando, Lizcano; Carolina, Romero; Vargas, Diana
.... The nuclear receptor Peroxisome Proliferator-activated receptor gamma (PPARγ) is the master regulator of adipose cell differentiation and its functional activation is currently used as a therapeutic approach for Diabetes Mellitus type 2...
Gee, Heon Yung; Kim, Joo Young; Lee, Min Goo
The cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic transmembrane protein that functions as a cAMP-activated anion channel at the apical membrane of epithelial cells. Mutations in CFTR cause cystic fibrosis and are also associated with monosymptomatic diseases in the lung, pancreas, intestines, and vas deferens. Many disease-causing CFTR mutations, including the deletion of a single phenylalanine residue at position 508 (ΔF508-CFTR), result in protein misfolding and trafficking defects. Therefore, intracellular trafficking of wild-type and mutant CFTR has been studied extensively, and results from these studies significantly contribute to our general understanding of mechanisms involved in the cell-surface trafficking of membrane proteins. CFTR is a glycoprotein that undergoes complex N-glycosylation as it passes through Golgi-mediated conventional exocytosis. Interestingly, results from recent studies revealed that CFTR and other membrane proteins can reach the plasma membrane via an unconventional alternative route that bypasses Golgi in specific cellular conditions. Here, we describe methods that have been used to investigate the conventional and unconventional surface trafficking of CFTR. With appropriate modifications, the protocols described in this chapter can also be applied to studies investigating the intracellular trafficking of other plasma membrane proteins.
Full Text Available Trafficking or people commerce is a recruitment, transportation, reception centre, sending, moving or reception somaone with threat, harshness, abduction, forgery, deception, abuse of power, trapping of debt or giving payment or profit, so get approval from people holding to conduct of others, both for conducted in inter-states and state for ceploittation or result people exploited. From understanding above, hence form trafficking can in the form of labor migran legal also illegal, worker of hausehold, worker of commercial seks, wedding orger, spurlous child adoption, beggar, pornography industry, circulation of forbidden drug and sale of body organ. Pursuant to research result, trafficking form that happened Banyumas is expressed. Its for ecample that is husemaid labour of migran and worker of commercial seks. Form of him not yet been expressed. Cause factor the happen of trafficking is economic factor or poorness, education which relative lower, patriakhi culture who then push woman motivate to fulfill requirement of economics and fulfill the him of as especial entrpreneur. The trafficking victims less get protection of law, this matter is caused by law and substanstion regulation completely arrangen protection to victim. The adjacent is theoretically conducted in three aspects, its relocation, repatriating, and reintegration, but not all victims get adjacent pattern.
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.
Roed, Sarah Noerklit; Nøhr, Anne Cathrine; Wismann, Pernille
The signaling capacity of seven-transmembrane/G-protein-coupled receptors (7TM/GPCRs) can be regulated through ligand-mediated receptor trafficking. Classically, the recycling of internalized receptors is associated with resensitization, whereas receptor degradation terminates signaling. We have......) and glucagon (GCGR) receptors. The interaction and cross-talk between coexpressed receptors is a wide phenomenon of the 7TM/GPCR superfamily. Numerous reports show functional consequences for signaling and trafficking of the involved receptors. On the basis of the high structural similarity and tissue...... coexpression, we here investigated the potential cross-talk between GLP-1R and GIPR or GCGR in both trafficking and signaling pathways. Using a real-time time-resolved FRET-based internalization assay, we show that GLP-1R, GIPR, and GCGR internalize with differential properties. Remarkably, upon coexpression...
Darrin V. Bann
Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.
Full Text Available Synaptic transmission relies on several processes, such as the location of a released vesicle, the number and type of receptors, trafficking between the postsynaptic density (PSD and extrasynaptic compartment, as well as the synapse organization. To study the impact of these parameters on excitatory synaptic transmission, we present a computational model for the fast AMPA-receptor mediated synaptic current. We show that in addition to the vesicular release probability, due to variations in their release locations and the AMPAR distribution, the postsynaptic current amplitude has a large variance, making a synapse an intrinsic unreliable device. We use our model to examine our experimental data recorded from CA1 mice hippocampal slices to study the differences between mEPSC and evoked EPSC variance. The synaptic current but not the coefficient of variation is maximal when the active zone where vesicles are released is apposed to the PSD. Moreover, we find that for certain type of synapses, receptor trafficking can affect the magnitude of synaptic depression. Finally, we demonstrate that perisynaptic microdomains located outside the PSD impacts synaptic transmission by regulating the number of desensitized receptors and their trafficking to the PSD. We conclude that geometrical modifications, reorganization of the PSD or perisynaptic microdomains modulate synaptic strength, as the mechanisms underlying long-term plasticity.
Chango, Abalo; Pogribny, Igor P
Fetal life is characterized by a tremendous plasticity and ability to respond to various environmental and lifestyle factors, including maternal nutrition. Identification of the role of dietary factors that can modulate and reshape the cellular epigenome during development, including methyl group donors (e.g., folate, choline) and bioactive compounds (e.g., polyphenols) is of great importance; however, there is insufficient knowledge of a particular effect of each type of modulator and/or their combination on fetal life. To enhance the quality and safety of food products for proper fetal health and disease prevention in later life, a better understanding of the underlying mechanisms of dietary epigenetic modulators during the critical prenatal period is necessary. This review focuses on the influence of maternal dietary components on DNA methylation, histone modification, and microRNAs, and summarizes current knowledge of the effect and importance of dietary components on epigenetic mechanisms that control the proper expression of genetic information. Evidence reveals that some components in the maternal diet can directly or indirectly affect epigenetic mechanisms. Understanding the underlying mechanisms of how early-life nutritional environment affects the epigenome during development is of great importance for the successful prevention of adult chronic diseases through optimal maternal nutrition.
The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.
Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya
The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin.
Ai, Junkui; Wang, Yujuan; Dar, Javid A; Liu, June; Liu, Lingqi; Nelson, Joel B; Wang, Zhou
The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.
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: email@example.com [Cell Microscopy Center, Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht, Heidelberglaan 100, 3584CX Utrecht (Netherlands)
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.
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.
Twigg, Naomi M
The purpose of this study was to identify aftercare services for domestic minor of sex trafficking (DMST) survivors provided by U.S. residential treatment centers. A qualitative research study was conducted with aftercare program personnel from five U.S. residential treatment centers for DMST survivors. Interviews were conducted with staff from five different residential treatment centers providing services exclusively to domestic minor sex trafficking survivors. Participants described the range of services offered to address survivors' posttrafficking needs. Participants' responses assisted in expanding an existing care model to include education re-entry, family reunification, family reconciliation, and emergency substance use services. This study led to the refinement of an aftercare service delivery model and laid the foundation to develop best practice guidelines for providing aftercare services to DMST survivors. Sex trafficking is a global health problem affecting our youth today. Nurses have a vital role in combatting sex trafficking by raising awareness about the problem and restoring the lives of sex trafficking victims by implementing innovative care programs. © 2017 Sigma Theta Tau International.
van der Woerd, Wendy L; Wichers, Catharina G K; Vestergaard, Anna L;
in cystic fibrosis transmembrane conductance regulator (CFTR), associated with cystic fibrosis, impair protein folding and trafficking. The aim of this study was to investigate whether compounds that rescue CFTR F508del trafficking are capable of improving p.I661T-ATP8B1 plasma membrane expression. METHODS...... functionality. Combination therapy of SAHA and compound C4 resulted in an additional improvement of ATP8B1 cell surface abundance. CONCLUSIONS: This study shows that several CFTR correctors can improve trafficking of p.I661T-ATP8B1 to the plasma membrane in vitro. Hence, these compounds may be suitable...... in other protein folding diseases. Using these compounds, we could indeed show improved trafficking to the (apical) plasma membrane of a mutated ATP8B1 protein, carrying the p.I661T missense mutation. This is the most frequently identified mutation in this rare cholestatic disorder. Importantly, ATP8B1...
Child Sex Trafficking in America: A Guide for Parents and Guardians What is Child Sex Trafficking ? Child sex trafficking is one of the most common types of commercial sexual exploitation . Child sex trafficking is a high priority at the National ...
Park, Ae Ran; Fu, Minmin; Shin, Ji Young; Son, Hokyoung; Lee, Yin-Won
Zearalenone (ZEA) is an estrogenic mycotoxin that is produced by several Fusarium species, including Fusarium graminearum. One of the ZEA biosynthetic genes, ZEB2, encodes two isoforms of Zeb2 by alternative transcription, forming an activator (Zeb2L-Zeb2L homooligomer) and an inhibitor (Zeb2L-Zeb2S heterodimer) that directly regulate the ZEA biosynthetic genes in F. graminearum. Cyclic AMP-dependent protein kinase A (PKA) signaling regulates secondary metabolic processes in several filamentous fungi. In this study, we investigated the effects of the PKA signaling pathway on ZEA biosynthesis. Through functional analyses of PKA catalytic and regulatory subunits (CPKs and PKR), we found that the PKA pathway negatively regulates ZEA production. Genetic and biochemical evidence further demonstrated that the PKA pathway specifically represses ZEB2L transcription and also takes part in posttranscriptional regulation of ZEB2L during ZEA production. Our findings reveal the intriguing mechanism that the PKA pathway regulates secondary metabolite production by reprograming alternative transcription.
Yan-fei WANG; Hong TIAN; Chao-shu TANG; Hong-fang JIN; Jun-bao DU
Aim: To explore the role of carbon monoxide (CO) in the regulation of hypoxic pulmonary artery smooth muscle cell (PASMC) proliferation and apoptosis by nitric oxide (NO). Methods: PASMC of Wistar rats was cultured in vitro in the presence of a NO donor, sodium nitroprusside, or an inhibitor of heme oxygenase (HO), zinc protoporphyrin-IX, or under both normoxic and hypoxic conditions.Nitrite and carboxyhemoglobin in PASMC medium were detected with spectrophotometry. The proliferating and apoptotic percentage of PASMC was measured by flow cytometry. The expression of HO-1 mRNA in PASMC was analyzed by fluorescent real-time quantitative PCR, and the proliferating cell nuclear antigen and caspase-3 were examined by immunocytochemical analysis. Results: The results showed that hypoxia suppressed NO generation from PASMC, which promoted hypoxic PASMC proliferation and induced apoptosis. Meanwhile, hy-poxia induced HO-1 expression in PASMC and promoted CO production from PASMC, which inhibited PASMC proliferation and regulated PASMC apoptosis. NO upregulated the expression of HO-1 mRNA in hypoxic PASMC; NO also inhib-ited proliferation and promoted apoptosis of hypoxic PASMC, possibly by regu-lating the production of CO. Conclusion: The results indicated that CO could inhibit proliferation and regulate apoptosis of PASMC, and NO inhibited prolifera-tion and promoted apoptosis of hypoxic PASMC, possibly by regulating the pro-duction of CO.
Menon, Govind; Krishnan, J
While signalling and biochemical modules have been the focus of numerous studies, they are typically studied in isolation, with no examination of the effects of the ambient network. In this paper we formulate and develop a systems framework, rooted in dynamical systems, to understand such effects, by studying the interaction of signalling modules. The modules we consider are (i) basic covalent modification, (ii) monostable switches, (iii) bistable switches, (iv) adaptive modules, and (v) oscillatory modules. We systematically examine the interaction of these modules by analyzing (a) sequential interaction without shared components, (b) sequential interaction with shared components, and (c) oblique interactions. Our studies reveal that the behaviour of a module in isolation may be substantially different from that in a network, and explicitly demonstrate how the behaviour of a given module, the characteristics of the ambient network, and the possibility of shared components can result in new effects. Our global approach illuminates different aspects of the structure and functioning of modules, revealing the importance of dynamical characteristics as well as biochemical features; this provides a methodological platform for investigating the complexity of natural modules shaped by evolution, elucidating the effects of ambient networks on a module in multiple cellular contexts, and highlighting the capabilities and constraints for engineering robust synthetic modules. Overall, such a systems framework provides a platform for bridging the gap between non-linear information processing modules, in isolation and as parts of networks, and a basis for understanding new aspects of natural and engineered cellular networks.
Menon, Govind; Krishnan, J.
While signalling and biochemical modules have been the focus of numerous studies, they are typically studied in isolation, with no examination of the effects of the ambient network. In this paper we formulate and develop a systems framework, rooted in dynamical systems, to understand such effects, by studying the interaction of signalling modules. The modules we consider are (i) basic covalent modification, (ii) monostable switches, (iii) bistable switches, (iv) adaptive modules, and (v) oscillatory modules. We systematically examine the interaction of these modules by analyzing (a) sequential interaction without shared components, (b) sequential interaction with shared components, and (c) oblique interactions. Our studies reveal that the behaviour of a module in isolation may be substantially different from that in a network, and explicitly demonstrate how the behaviour of a given module, the characteristics of the ambient network, and the possibility of shared components can result in new effects. Our global approach illuminates different aspects of the structure and functioning of modules, revealing the importance of dynamical characteristics as well as biochemical features; this provides a methodological platform for investigating the complexity of natural modules shaped by evolution, elucidating the effects of ambient networks on a module in multiple cellular contexts, and highlighting the capabilities and constraints for engineering robust synthetic modules. Overall, such a systems framework provides a platform for bridging the gap between non-linear information processing modules, in isolation and as parts of networks, and a basis for understanding new aspects of natural and engineered cellular networks.
Full Text Available The vasopressin-regulated urea transporter UT-A1, expressed in kidney inner medullary collecting duct (IMCD epithelial cells, plays a critical role in the urinary concentrating mechanisms. As a membrane protein, the function of UT-A1 transport activity relies on its presence in the plasma membrane. Therefore, UT-A1 successfully trafficking to the apical membrane of the polarized epithelial cells is crucial for the regulation of urea transport. This review summarizes the research progress of UT-A1 regulation over the past few years, specifically on the regulation of UT-A1 membrane trafficking by lipid rafts, N-linked glycosylation and a group of accessory proteins.
The vasopressin-regulated urea transporter UT-A1, expressed in kidney inner medullary collecting duct (IMCD) epithelial cells, plays a critical role in the urinary concentrating mechanisms. As a membrane protein, the function of UT-A1 transport activity relies on its presence in the plasma membrane. Therefore, UT-A1 successfully trafficking to the apical membrane of the polarized epithelial cells is crucial for the regulation of urea transport. This review summarizes the research progress of UT-A1 regulation over the past few years, specifically on the regulation of UT-A1 membrane trafficking by lipid rafts, N-linked glycosylation and a group of accessory proteins.
Cho, Hae-Mi; Kang, Young-Ho; Yoo, Hanju; Yoon, Seung-Yong; Kang, Sang-Wook; Chang, Eun-Ju; Song, Youngsup
Regulation of balance between lipid accumulation and energy consumption is a critical step for the maintenance of energy homeostasis. Here, we show that Panax red ginseng extract treatments increased energy expenditures and prevented mice from diet induced obesity. Panax red ginseng extracts strongly activated Hormone Specific Lipase (HSL) via Protein Kinase A (PKA). Since activation of HSL induces lipolysis in WAT and fatty acid oxidation in brown adipose tissue (BAT), these results suggest that Panax red ginseng extracts reduce HFD induced obesity by regulating lipid mobilization. Copyright © 2014 Elsevier Inc. All rights reserved.
Full Text Available The Trafficking Protocol has shaped and advanced a global movement against human trafficking; notably through establishing a global definition and creating criminal justice remedies befitting an international crime. Borne out of and including the Protocol, a global anti-trafficking framework has emerged. This framework reflects these two central tenets at international, regional and national levels and includes initiatives by States not party to the Protocol, such as Singapore. However, the emphasis on these tenets, which comprise only part of a robust anti-trafficking strategy, has rendered the existing framework insufficient to address exploitation.  In full: Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children  K H Heinrich, ‘Ten Years After the Palermo Protocol: Where are Protections for Human Trafficking?’, Human Rights Brief 18, no.1, 2010, retrieved 5 January 2015, http://digitalcommons.wcl.american.edu/cgi/viewcontent.cgi?article=1145&context=hrbrief ; K E Hyland, The Impact of the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, Human Rights Brief 8, no. 2, 2001, retrieved 5 January 2015, http://digitalcommons.wcl.american.edu/cgi/viewcontent.cgi?article=1492&context=hrbrief
Full Text Available Human trafficking is a widely studied phenomenon. Comparing public perceptions of trafficking to institutional (i.e. the academy, governmental and non-governmental organizations perceptions gives a richer understanding of the problem. The data for this study were collected in and around Chisinau, Moldova in the summer of 2004. Public discourse provides a more intimate "portraiture" of the issue, but the public also demonstrated a complex level of understanding of this social problem in this study. Its view is juxtaposed against an institutional view of human trafficking as explored through a literature review. Combining institutional and public perceptions and knowledge of a social problem is helpful in not only establishing a more thorough understanding of the social problem and guiding policy decisions, but in exploring the experiences victims may face at the community level.
Full Text Available In spite of relative prevalence of trafficking in human beings issues in the expert and general public discourse in recent years, recognition of victimization by various specialists that may come across with victims still is being estimated as unsatisfactory. Stereotypes about victims of trafficking in human beings are just one factor that imperils correct and prompt recognition of victims, i.e. victims' identification, as principal prerequisite of their protection and support. Today, there are various efforts to overcome that problem - primarily through the training of professionals and creating the identification guidelines, i.e. lists of indicators of trafficking in human beings victimization; however, these resolves only one part of the problem and reveal some new challenges at the same time.
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.
Danailova-Trainor, Gergana; Laczko, Frank
Poverty is often regarded as the "root cause" of trafficking, but the linkages between poverty, a lack of development and trafficking are complex. For example, there is some evidence to suggest that victims of cross-border trafficking are more likely to originate from middle-income rather than lower-income countries. Trafficking and development have tended to be treated as very separate policy areas and the assessment of the development impact of counter-trafficking programmes is still at an early stage. This paper outlines a possible framework for a more evidence-based approach to understanding the linkages between trafficking, trafficking policy and human development. The paper argues that the human development gains from greater mobility could be significantly enhanced if there was greater coherence between policies to combat trafficking and policies to promote development.
Habtezion, Aida; Nguyen, Linh P; Hadeiba, Husein; Butcher, Eugene C
Leukocyte trafficking to the small and large intestines is tightly controlled to maintain intestinal immune homeostasis, mediate immune responses, and regulate inflammation. A wide array of chemoattractants, chemoattractant receptors, and adhesion molecules expressed by leukocytes, mucosal endothelium, epithelium, and stromal cells controls leukocyte recruitment and microenvironmental localization in intestine and in the gut-associated lymphoid tissues (GALTs). Naive lymphocytes traffic to the gut-draining mesenteric lymph nodes where they undergo antigen-induced activation and priming; these processes determine their memory/effector phenotypes and imprint them with the capacity to migrate via the lymph and blood to the intestines. Mechanisms of T-cell recruitment to GALT and of T cells and plasmablasts to the small intestine are well described. Recent advances include the discovery of an unexpected role for lectin CD22 as a B-cell homing receptor GALT, and identification of the orphan G-protein-coupled receptor 15 (GPR15) as a T-cell chemoattractant/trafficking receptor for the colon. GPR15 decorates distinct subsets of T cells in mice and humans, a difference in species that could affect translation of the results of mouse colitis models to humans. Clinical studies with antibodies to integrin α4β7 and its vascular ligand mucosal vascular addressin cell adhesion molecule 1 are proving the value of lymphocyte trafficking mechanisms as therapeutic targets for inflammatory bowel diseases. In contrast to lymphocytes, cells of the innate immune system express adhesion and chemoattractant receptors that allow them to migrate directly to effector tissue sites during inflammation. We review the mechanisms for innate and adaptive leukocyte localization to the intestinal tract and GALT, and discuss their relevance to human intestinal homeostasis and inflammation.
Jasmeet P Hayes
Full Text Available During times of emotional stress, individuals often engage in emotion regulation to reduce the experiential and physiological impact of negative emotions. Interestingly, emotion regulation strategies also influence memory encoding of the event. Cognitive reappraisal is associated with enhanced memory while expressive suppression is associated with impaired explicit memory of the emotional event. However, the mechanism by which these emotion regulation strategies affect memory is unclear. We used event-related fMRI to investigate the neural mechanisms that give rise to memory formation during emotion regulation. Twenty-five participants viewed negative pictures while alternately engaging in cognitive reappraisal, expressive suppression, or passive viewing. As part of the subsequent memory design, participants returned to the laboratory two weeks later for a surprise memory test. Behavioral results showed a reduction in negative affect and a retention advantage for reappraised stimuli relative to the other conditions. Imaging results showed that successful encoding during reappraisal was uniquely associated with greater co-activation of the left inferior frontal gyrus, amygdala and hippocampus, suggesting a possible role for elaborative encoding of negative memories. This study provides neurobehavioral evidence that engaging in cognitive reappraisal is advantageous to both affective and mnemonic processes.
Robert W Thompson
Full Text Available Cationic amino acid transporters (CAT are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/- mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/- mice developed stronger IFN-gamma responses, nitric oxide (NO production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/- mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/- mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.
Curtis-Ducey, Carol Dianne
Interaction of estrogen receptor [alpha] (ER[alpha]) with 17[beta]-estradiol (E[subscript 2]) facilitates binding of the receptor to estrogen response elements (EREs) in target genes, which in turn leads to recruitment of coregulatory proteins. To better understand how estrogen-responsive genes are regulated, our laboratory identified a number of…
Kepka, M.; Verburg-van Kemenade, B.M.L.; Chadzinska, M.K.
Inflammatory responses have to be carefully controlled, as high concentrations and/or prolonged action of inflammation-related molecules (e.g. reactive oxygen species, nitric oxide and pro-inflammatory cytokines) can be detrimental to host tissue and organs. One of the potential regulators of the in
Full Text Available Intracellular chloride channel protein 1 (CLIC1 participates in inflammatory processes by regulating macrophage phagosomal functions such as pH and proteolysis. Here, we sought to determine if CLIC1 can regulate adaptive immunity by actions on dendritic cells (DCs, the key professional antigen presenting cells. To do this, we first generated bone marrow-derived DCs (BMDCs from germline CLIC1 gene-deleted (CLIC1−/− and wild-type (CLIC1+/+ mice, then studied them in vitro and in vivo. We found phagocytosis triggered cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from CLIC1−/− BMDCs displayed impaired acidification and proteolysis, which could be reproduced if CLIC1+/+, but not CLIC1−/− cells, were treated with IAA94, a CLIC family ion channel blocker. CLIC1−/− BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG and reduced MOG-induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates DC phagosomal pH to ensure optimal processing of antigen for presentation to antigen-specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target to help reduce the adaptive immune response in autoimmune diseases.
Goto, Tsuyoshi; Kim, Young-Il; Takahashi, Nobuyuki; Kawada, Teruo
Obesity causes excess fat accumulation in various tissues, most notoriously in the adipose tissue, along with other insulin-responsive organs such as skeletal muscle and the liver, which predisposes an individual to the development of metabolic abnormalities. The molecular mechanisms underlying obesity-induced metabolic abnormalities have not been completely elucidated; however, in recent years, the search for therapies to prevent the development of obesity and obesity-associated metabolic disorders has increased. It is known that several nuclear receptors, when activated by specific ligands, regulate carbohydrate and lipid metabolism at the transcriptional level. The expression of lipid metabolism-related enzymes is directly regulated by the activity of various nuclear receptors via their interaction with specific response elements in promoters of those genes. Many natural compounds act as ligands of nuclear receptors and regulate carbohydrate and lipid metabolism by regulating the activities of these nuclear receptors. In this review, we describe our current knowledge of obesity, the role of lipid-sensing nuclear receptors in energy metabolism, and several examples of food factors that act as agonists or antagonists of nuclear receptors, which may be useful for the management of obesity and the accompanying energy metabolism abnormalities. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Full Text Available Béla Z Schmidt,1 Jérémy B Haaf,2 Teresinha Leal,2 Sabrina Noel,2 1Stem Cell Biology and Embryology, Department of Development and Regeneration, Katholieke Universiteit Leuven, Leuven, 2Louvain Center for Toxicology and Applied Pharmacology, Université Catholique de Louvain, Brussels, Belgium Abstract: Mutations of the CFTR gene cause cystic fibrosis (CF, the most common recessive monogenic disease worldwide. These mutations alter the synthesis, processing, function, or half-life of CFTR, the main chloride channel expressed in the apical membrane of epithelial cells in the airway, intestine, pancreas, and reproductive tract. Lung disease is the most critical manifestation of CF. It is characterized by airway obstruction, infection, and inflammation that lead to fatal tissue destruction. In spite of great advances in early and multidisciplinary medical care, and in our understanding of the pathophysiology, CF is still considerably reducing the life expectancy of patients. This review highlights the current development in pharmacological modulators of CFTR, which aim at rescuing the expression and/or function of mutated CFTR. While only Kalydeco® and Orkambi® are currently available to patients, many other families of CFTR modulators are undergoing preclinical and clinical investigations. Drug repositioning and personalized medicine are particularly detailed in this review as they represent the most promising strategies for restoring CFTR function in CF. Keywords: high-throughput screening, drug repositioning, personalized medicine, precision medicine, potentiators, correctors
Masek, Pavel; Worden, Kurtresha; Aso, Yoshinori; Rubin, Gerald M; Keene, Alex C
Taste memories allow animals to modulate feeding behavior in accordance with past experience and avoid the consumption of potentially harmful food . We have developed a single-fly taste memory assay to functionally interrogate the neural circuitry encoding taste memories . Here, we screen a collection of Split-GAL4 lines that label small populations of neurons associated with the fly memory center-the mushroom bodies (MBs) . Genetic silencing of PPL1 dopamine neurons disrupts conditioned, but not naive, feeding behavior, suggesting these neurons are selectively involved in the conditioned taste response. We identify two PPL1 subpopulations that innervate the MB α lobe and are essential for aversive taste memory. Thermogenetic activation of these dopamine neurons during training induces memory, indicating these neurons are sufficient for the reinforcing properties of bitter tastant to the MBs. Silencing of either the intrinsic MB neurons or the output neurons from the α lobe disrupts taste conditioning. Thermogenetic manipulation of these output neurons alters naive feeding response, suggesting that dopamine neurons modulate the threshold of response to appetitive tastants. Taken together, these findings detail a neural mechanism underlying the formation of taste memory and provide a functional model for dopamine-dependent plasticity in Drosophila.
Talevi, Riccardo; Zagami, Maria; Castaldo, Marianna; Gualtieri, Roberto
Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.
Full Text Available Abstract Background The expression of gene batteries, genomic units of functionally linked genes which are activated by similar sets of cis- and trans-acting regulators, has been proposed as a major determinant of cell specialization in metazoans. We developed a predictive procedure to screen the mouse and human genomes and transcriptomes for cases of gene-battery-like regulation. Results In a screen that covered ~40 per cent of all annotated protein-coding genes, we identified 21 co-expressed gene clusters with statistically supported sharing of cis-regulatory sequence elements. 66 predicted cases of over-represented transcription factor binding motifs were validated against the literature and fell into three categories: (i previously described cases of gene battery-like regulation, (ii previously unreported cases of gene battery-like regulation with some support in a limited number of genes, and (iii predicted cases that currently lack experimental support. The novel predictions include for example Sox 17 and RFX transcription factor binding sites that were detected in ~10% of all testis specific genes, and HNF-1 and 4 binding sites that were detected in ~30% of all kidney specific genes respectively. The results are publicly available at http://www.wlab.gu.se/lindahl/genebatteries. Conclusion 21 co-expressed gene clusters were enriched for a total of 66 shared cis-regulatory sequence elements. A majority of these predictions represent novel cases of potential co-regulation of functionally coupled proteins. Critical technical parameters were evaluated, and the results and the methods provide a valuable resource for future experimental design.
Full Text Available Brassinosteroids (BRs regulate rice plant architecture, including leaf bending, which affects grain yield. Although BR signaling has been investigated in Arabidopsis thaliana, the components negatively regulating this pathway are less well understood. Here, we demonstrate that Oryza sativa LEAF and TILLER ANGLE INCREASED CONTROLLER (LIC acts as an antagonistic transcription factor of BRASSINAZOLE-RESISTANT 1 (BZR1 to attenuate the BR signaling pathway. The gain-of-function mutant lic-1 and LIC-overexpressing lines showed erect leaves, similar to BZR1-depleted lines, which indicates the opposite roles of LIC and BZR1 in regulating leaf bending. Quantitative PCR revealed LIC transcription rapidly induced by BR treatment. Image analysis and immunoblotting showed that upon BR treatment LIC proteins translocate from the cytoplasm to the nucleus in a phosphorylation-dependent fashion. Phosphorylation assay in vitro revealed LIC phosphorylated by GSK3-like kinases. For negative feedback, LIC bound to the core element CTCGC in the BZR1 promoter on gel-shift and chromatin immunoprecipitation assay and repressed its transcription on transient transformation assay. LIC directly regulated target genes such as INCREASED LEAF INCLINATION 1 (ILI1 to oppose the action of BZR1. Repression of LIC in ILI1 transcription in protoplasts was partially rescued by BZR1. Phenotypic analysis of the crossed lines depleted in both LIC and BZR1 suggested that BZR1 functionally depends on LIC. Molecular and physiology assays revealed that LIC plays a dominant role at high BR levels, whereas BZR1 is dominant at low levels. Thus, LIC regulates rice leaf bending as an antagonistic transcription factor of BZR1. The phenotypes of lic-1 and LIC-overexpressing lines in erect leaves contribute to ideal plant architecture. Improving this phenotype may be a potential approach to molecular breeding for high yield in rice.
In Denmark, human trafficking has emerged as a central issue within the policy field of prostitution during the last decade. Taking a Foucauldian approach from a historical perspective, understanding the policy field of prostitution as a discursive terrain, the article analyses the thinking...... that lies behind policies on prostitution by identifying ruptures and discursive struggles which lead to transformations of the policy field. In particular, this article investigates how the problematisation of human trafficking has created space for a feminist discourse breakthrough within the policy field...
Schmidt, Jens C; Cech, Thomas R
Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric DNA in eukaryotes. Recent work has begun to reveal key aspects of the assembly of the human telomerase complex, its intracellular trafficking involving Cajal bodies, and its recruitment to telomeres. Once telomerase has been recruited to the telomere, it appears to undergo a separate activation step, which may include an increase in its repeat addition processivity. This review covers human telomerase biogenesis, trafficking, and activation, comparing key aspects with the analogous events in other species.
Human trafficking is a major public health problem, both domestically and internationally. Health care providers are often the only professionals to interact with trafficking victims who are still in captivity. The expert assessment and interview skills of providers contribute to their readiness to identify victims of trafficking. The purpose of this article is to provide clinicians with knowledge on trafficking and give specific tools that they may use to assist victims in the clinical setti...