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Sample records for functional intraflagellar transport

  1. Imaging intraflagellar transport in trypanosomes.

    PubMed

    Santi-Rocca, Julien; Chenouard, Nicolas; Fort, Cécile; Lagache, Thibault; Olivo-Marin, Jean-Christophe; Bastin, Philippe

    2015-01-01

    Trypanosoma brucei is a flagellated eukaryotic pathogen responsible for sleeping sickness in central Africa. Because of the presence of a long motile flagellum (>20 μm) and its amenity to genetic manipulation, it is becoming an attractive model to study the assembly and the functions of cilia and flagella. In recent years, several aspects have been investigated, especially intraflagellar transport (IFT) that has been exhaustively characterized at the light microscopy level. In this manuscript, we review various methods to express fluorescent fusion proteins and to record IFT in living trypanosomes in normal or mutant contexts. We present an approach for separating anterograde and retrograde IFT, hence facilitating quantification of train speed, frequency, and size. A statistical analysis to discriminate different subpopulations of IFT trains is also summarized. These methods have proven their efficiency for the study of IFT in trypanosomes and could be applied to any other organism.

  2. Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton

    PubMed Central

    Yuan, Xue; Serra, Rosa A.; Yang, Shuying

    2014-01-01

    Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes. PMID:24961486

  3. Intraflagellar Transport and Functional Analysis of Genes Required for Flagellum Formation in Trypanosomes

    PubMed Central

    Absalon, Sabrina; Blisnick, Thierry; Kohl, Linda; Toutirais, Géraldine; Doré, Gwénola; Julkowska, Daria; Tavenet, Arounie

    2008-01-01

    Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 μm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant. PMID:18094047

  4. Functional genomics of intraflagellar transport-associated proteins in C. elegans.

    PubMed

    Inglis, Peter N; Blacque, Oliver E; Leroux, Michel R

    2009-01-01

    The nematode Caenorhabditis elegans presents numerous advantages for the identification and molecular analysis of intraflagellar transport (IFT)-associated proteins, which play a critical role in the formation of cilia. Many proteins were first described as participating in IFT in this organism, including IFTA-1 (IFT121), DYF-1 (fleer/IFT70), DYF-2 (IFT144), DYF-3 (Qilin), DYF-11 (MIP-T3/IFT54), DYF-13, XBX-1 (dynein light intermediate chain), XBX-2 (dynein light chain), CHE-13 (IFT57/HIPPI), orthologs of Bardet-Biedl syndrome proteins, and potential regulatory protein, IFTA-2 (RABL5/IFT22). Transgenic animals bearing green fluorescent protein (GFP)-tagged proteins can be generated with ease, and in vivo imaging of IFT in both wild-type and cilia mutant strains can be performed quickly. The analyses permit detailed information on the localization and dynamic properties (velocities along the ciliary axoneme) of the relevant proteins, providing insights into their potential functions in processes such as anterograde and retrograde transport and cilium formation, as well as association with distinct modules of the IFT machinery (e.g., IFT subcomplexes A or B). Behavioral studies of the corresponding IFT-associated gene mutants further enable an understanding of the ciliary role of the proteins-e.g., in chemosensation, lipid homeostasis, lifespan control, and signaling-in a multicellular animal. In this chapter, we discuss how C. elegans can be used for the identification and characterization of IFT-associated proteins, focusing on methods for the generation of GFP-tagged IFT reporter strains, time-lapse microscopy, and IFT rate measurements. PMID:20409822

  5. Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila.

    PubMed

    Balmer, Sophie; Dussert, Aurore; Collu, Giovanna M; Benitez, Elvira; Iomini, Carlo; Mlodzik, Marek

    2015-09-28

    The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity. PMID:26364750

  6. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.

    PubMed

    Haycraft, Courtney J; Banizs, Boglarka; Aydin-Son, Yesim; Zhang, Qihong; Michaud, Edward J; Yoder, Bradley K

    2005-10-01

    Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh) signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction. PMID:16254602

  7. Gli2 and Gli3 Localize to Cilia and Require the Intraflagellar Transport Protein Polaris for Processing and Function

    SciTech Connect

    Michaud III, Edward J; Haycraft, Courtney J; Aydin Son, Yesim; Zhang, Qihong; Yoder, Bradley

    2005-01-01

    Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh)signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction.

  8. Canonical and noncanonical intraflagellar transport regulates craniofacial skeletal development.

    PubMed

    Noda, Kazuo; Kitami, Megumi; Kitami, Kohei; Kaku, Masaru; Komatsu, Yoshihiro

    2016-05-10

    The primary cilium is a cellular organelle that coordinates signaling pathways critical for cell proliferation, differentiation, survival, and homeostasis. Intraflagellar transport (IFT) plays a pivotal role in assembling primary cilia. Disruption and/or dysfunction of IFT components can cause multiple diseases, including skeletal dysplasia. However, the mechanism by which IFT regulates skeletogenesis remains elusive. Here, we show that a neural crest-specific deletion of intraflagellar transport 20 (Ift20) in mice compromises ciliogenesis and intracellular transport of collagen, which leads to osteopenia in the facial region. Whereas platelet-derived growth factor receptor alpha (PDGFRα) was present on the surface of primary cilia in wild-type osteoblasts, disruption of Ift20 down-regulated PDGFRα production, which caused suppression of PDGF-Akt signaling, resulting in decreased osteogenic proliferation and increased cell death. Although osteogenic differentiation in cranial neural crest (CNC)-derived cells occurred normally in Ift20-mutant cells, the process of mineralization was severely attenuated due to delayed secretion of type I collagen. In control osteoblasts, procollagen was easily transported from the endoplasmic reticulum (ER) to the Golgi apparatus. By contrast, despite having similar levels of collagen type 1 alpha 1 (Col1a1) expression, Ift20 mutants did not secrete procollagen because of dysfunctional ER-to-Golgi trafficking. These data suggest that in the multipotent stem cells of CNCs, IFT20 is indispensable for regulating not only ciliogenesis but also collagen intracellular trafficking. Our study introduces a unique perspective on the canonical and noncanonical functions of IFT20 in craniofacial skeletal development. PMID:27118846

  9. Purification and Localization of Intraflagellar Transport Particles and Polypeptides.

    PubMed

    Sloboda, Roger D

    2016-01-01

    The growth and maintenance of almost all cilia and flagella are dependent on the proper functioning of the process of intraflagellar transport (IFT). This includes the primary cilia of most human cells that are in the Go phase of the cell cycle. The model system for the study of IFT is the flagella of the biflagellate green alga Chlamydomonas. It is in this organism that IFT was first discovered, and genetic data from a Chlamydomonas mutant first linked the process of IFT to polycystic kidney disease in humans. The information provided in this chapter addresses procedures to purify IFT particles from flagella and localize these particles, and their associated motor proteins, in flagella using light and electron microscopic approaches. PMID:26498782

  10. Components of Intraflagellar Transport complex A (IFT-A) function independently of the cilium to regulate canonical Wnt signaling in Drosophila

    PubMed Central

    Balmer, Sophie; Dussert, Aurore; Collu, Giovanna M.; Benitez, Elvira; Iomini, Carlo; Mlodzik, Marek

    2015-01-01

    SUMMARY The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily-conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins and/or segregation of pathway components in specific subcellular compartments. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or whether cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A (IFT-A) uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity. PMID:26364750

  11. [Elongation of the axoneme and dynamics of intraflagellar transport].

    PubMed

    Fort, Cécile; Bastin, Philippe

    2014-11-01

    Cilia and flagella are essential organelles in most eukaryotes including human beings. In this review, we will discuss the mode of assembly of these complex organelles that depends on a dynamic process called intraflagellar transport or IFT. IFT delivers structural elements at the distal end of the cilium where assembly takes place, thereby allowing the growth of the organelle. We next discuss the different models for control of cilium length and their alterations in ciliopathies, genetic diseases associated to ciliary defects.

  12. Flagellar Length Control System: Testing a Simple Model Based on Intraflagellar Transport and Turnover

    PubMed Central

    Marshall, Wallace F.; Qin, Hongmin; Brenni, Mónica Rodrigo; Rosenbaum, Joel L.

    2005-01-01

    Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle. PMID:15496456

  13. The Role of Intraflagellar Transport in the Photoreceptor Sensory Cilium.

    PubMed

    Taub, Daniel G; Liu, Qin

    2016-01-01

    The photoreceptor is a complex specialized cell in which a major component responsible for visual transduction is the photoreceptor sensory cilium (PSC). Building and maintenance of the PSC requires the transport of large proteins along microtubules that extend from the inner segments to the outer segments. A key process, termed intraflagellar transport (IFT), has been recognized as an essential phenomenon for photoreceptor development and maintenance, and exciting new studies have highlighted its importance in retinal and cilia related diseases. This review focuses on the important roles of IFT players, including motor proteins, IFT proteins, and photoreceptor-specific cargos in photoreceptor sensory cilium. In addition, specific IFT components that are involved in inherited human diseases are discussed.

  14. The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions

    PubMed Central

    Blisnick, Thierry; Buisson, Johanna; Absalon, Sabrina; Marie, Alexandra; Cayet, Nadège; Bastin, Philippe

    2014-01-01

    Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140RNAi mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex. PMID:24989795

  15. The intraflagellar transport dynein complex of trypanosomes is made of a heterodimer of dynein heavy chains and of light and intermediate chains of distinct functions.

    PubMed

    Blisnick, Thierry; Buisson, Johanna; Absalon, Sabrina; Marie, Alexandra; Cayet, Nadège; Bastin, Philippe

    2014-09-01

    Cilia and flagella are assembled by intraflagellar transport (IFT) of protein complexes that bring tubulin and other precursors to the incorporation site at their distal tip. Anterograde transport is driven by kinesin, whereas retrograde transport is ensured by a specific dynein. In the protist Trypanosoma brucei, two distinct genes encode fairly different dynein heavy chains (DHCs; ∼40% identity) termed DHC2.1 and DHC2.2, which form a heterodimer and are both essential for retrograde IFT. The stability of each heavy chain relies on the presence of a dynein light intermediate chain (DLI1; also known as XBX-1/D1bLIC). The presence of both heavy chains and of DLI1 at the base of the flagellum depends on the intermediate dynein chain DIC5 (FAP133/WDR34). In the IFT140(RNAi) mutant, an IFT-A protein essential for retrograde transport, the IFT dynein components are found at high concentration at the flagellar base but fail to penetrate the flagellar compartment. We propose a model by which the IFT dynein particle is assembled in the cytoplasm, reaches the base of the flagellum, and associates with the IFT machinery in a manner dependent on the IFT-A complex.

  16. Intraflagellar transport balances continuous turnover of outer doublet microtubules

    PubMed Central

    Marshall, Wallace F.; Rosenbaum, Joel L.

    2001-01-01

    A central question in cell biology is how cells determine the size of their organelles. Flagellar length control is a convenient system for studying organelle size regulation. Mechanistic models proposed for flagellar length regulation have been constrained by the assumption that flagella are static structures once they are assembled. However, recent work has shown that flagella are dynamic and are constantly turning over. We have determined that this turnover occurs at the flagellar tips, and that the assembly portion of the turnover is mediated by intraflagellar transport (IFT). Blocking IFT inhibits the incorporation of tubulin at the flagellar tips and causes the flagella to resorb. These results lead to a simple steady-state model for flagellar length regulation by which a balance of assembly and disassembly can effectively regulate flagellar length. PMID:11684707

  17. Hedgehog signalling in the mouse requires intraflagellar transport proteins.

    PubMed

    Huangfu, Danwei; Liu, Aimin; Rakeman, Andrew S; Murcia, Noel S; Niswander, Lee; Anderson, Kathryn V

    2003-11-01

    Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, here we identify two mouse mutants, wimple (wim) and flexo (fxo), that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homologue of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homologue of IFT88. Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of Hedgehog signalling. Our data show that IFT machinery has an essential and vertebrate-specific role in Hedgehog signal transduction. PMID:14603322

  18. Isolation of intraflagellar transport particle proteins from Chlamydomonas reinhardtii.

    PubMed

    Richey, Elizabeth; Qin, Hongmin

    2013-01-01

    Cilia, the hair-like protrusions found on most eukaryotic cells, were once considered vestigial organelles. The recent renaissance of research in cilia arose from the discoveries of intraflagellar transport (IFT) and the involvement of IFT particle proteins in human diseases. Many IFT particle proteins have since been identified, and research on IFT particle complexes and their protein components continues to provide insight into the mechanism of IFT and the etiology of ciliopathies. In this chapter, we describe the methods of isolating IFT particles from the flagella of Chlamydomonas reinhardtii. Two methods, sucrose density gradient fractionation and immunoprecipitation, are explained in detail. Troubleshooting information is presented to illustrate the critical steps of the procedure to ensure successful implementation of these methods in individual labs.

  19. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport.

    PubMed

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R; Mans, Dorus A; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E C; Yap, Zhi Min; Letteboer, Stef J F; Taylor, S Paige; Herridge, Warren; Johnson, Colin A; Scambler, Peter J; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M; Beales, Philip L; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M; Witman, George B

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  20. TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport

    PubMed Central

    Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R.; Mans, Dorus A.; Huber, Celine; Boldt, Karsten; Patel, Mitali; van Reeuwijk, Jeroen; Plaza, Jean-Marc; van Beersum, Sylvia E. C.; Yap, Zhi Min; Letteboer, Stef J. F.; Taylor, S. Paige; Herridge, Warren; Johnson, Colin A.; Scambler, Peter J.; Ueffing, Marius; Kayserili, Hulya; Krakow, Deborah; King, Stephen M.; Beales, Philip L.; Al-Gazali, Lihadh; Wicking, Carol; Cormier-Daire, Valerie; Roepman, Ronald; Mitchison, Hannah M.; Witman, George B.; Al-Turki, Saeed; Anderson, Carl; Anney, Richard; Antony, Dinu; Asimit, Jennifer; Ayub, Mohammad; Barrett, Jeff; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Blackwood, Douglas; Bobrow, Martin; Bochukova, Elena; Bolton, Patrick; Boustred, Chris; Breen, Gerome; Brion, Marie-Jo; Brown, Andrew; Calissano, Mattia; Carss, Keren; Chatterjee, Krishna; Chen, Lu; Cirak, Sebhattin; Clapham, Peter; Clement, Gail; Coates, Guy; Collier, David; Cosgrove, Catherine; Cox, Tony; Craddock, Nick; Crooks, Lucy; Curran, Sarah; Daly, Allan; Danecek, Petr; Smith, George Davey; Day-Williams, Aaron; Day, Ian; Durbin, Richard; Edkins, Sarah; Ellis, Peter; Evans, David; Farooqi, I. Sadaf; Fatemifar, Ghazaleh; Fitzpatrick, David; Flicek, Paul; Floyd, Jamie; Foley, A. Reghan; Franklin, Chris; Futema, Marta; Gallagher, Louise; Gaunt, Tom; Geschwind, Daniel; Greenwood, Celia; Grozeva, Detelina; Guo, Xiaosen; Gurling, Hugh; Hart, Deborah; Hendricks, Audrey; Holmans, Peter; Huang, Jie; Humphries, Steve E.; Hurles, Matt; Hysi, Pirro; Jackson, David; Jamshidi, Yalda; Jewell, David; Chris, Joyce; Kaye, Jane; Keane, Thomas; Kemp, John; Kennedy, Karen; Kent, Alastair; Kolb-Kokocinski, Anja; Lachance, Genevieve; Langford, Cordelia; Lee, Irene; Li, Rui; Li, Yingrui; Ryan, Liu; Lönnqvist, Jouko; Lopes, Margarida; MacArthur, Daniel G.; Massimo, Mangino; Marchini, Jonathan; Maslen, John; McCarthy, Shane; McGuffin, Peter; McIntosh, Andrew; McKechanie, Andrew; McQuillin, Andrew; Memari, Yasin; Metrustry, Sarah; Min, Josine; Moayyeri, Alireza; Morris, James; Muddyman, Dawn; Muntoni, Francesco; Northstone, Kate; O'Donovan, Michael; O'Rahilly, Stephen; Onoufriadis, Alexandros; Oualkacha, Karim; Owen, Michael; Palotie, Aarno; Panoutsopoulou, Kalliope; Parker, Victoria; Parr, Jeremy; Paternoster, Lavinia; Paunio, Tiina; Payne, Felicity; Perry, John; Pietilainen, Olli; Plagnol, Vincent; Quail, Michael A.; Quaye, Lydia; Raymond, Lucy; Rehnström, Karola; Brent Richards, J.; Ring, Sue; Ritchie, Graham R S; Savage, David B.; Schoenmakers, Nadia; Semple, Robert K.; Serra, Eva; Shihab, Hashem; Shin, So-Youn; Skuse, David; Small, Kerrin; Smee, Carol; Soler, Artigas María; Soranzo, Nicole; Southam, Lorraine; Spector, Tim; St Pourcain, Beate; St. Clair, David; Stalker, Jim; Surdulescu, Gabriela; Suvisaari, Jaana; Tachmazidou, Ioanna; Tian, Jing; Timpson, Nic; Tobin, Martin; Valdes, Ana; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Wain, Louise; Walter, Klaudia; Wang, Jun; Ward, Kirsten; Wheeler, Ellie; Whittall, Ros; Williams, Hywel; Williamson, Kathy; Wilson, Scott G.; Wong, Kim; Whyte, Tamieka; ChangJiang, Xu; Zeggini, Eleftheria; Zhang, Feng; Zheng, Hou-Feng

    2015-01-01

    The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions. PMID:26044572

  1. Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella.

    PubMed

    Dentler, William

    2005-08-15

    Intraflagellar transport (IFT) of particles along flagellar microtubules is required for the assembly and maintenance of eukaryotic flagella and cilia. In Chlamydomonas, anterograde and retrograde particles viewed by light microscopy average 0.12-microm and 0.06-microm diameter, respectively. Examination of IFT particle structure in growing flagella by electron microscopy revealed similar size aggregates composed of small particles linked to each other and to the membrane and microtubules. To determine the relationship between the number of particles and flagellar length, the rate and frequency of IFT particle movement was measured in nongrowing, growing, and shortening flagella. In all flagella, anterograde and retrograde IFT averaged 1.9 microm/s and 2.7 microm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 mum. The number of flagellar IFT particles was not fixed, but depended on flagellar length. Pauses in IFT particle entry into flagella suggest the presence of a periodic "gate" that permits up to 4 particles/s to enter a flagellum. PMID:16103230

  2. Electron microscopy of flagella, primary cilia, and intraflagellar transport in flat-embedded cells.

    PubMed

    Rogowski, Michaela; Scholz, Dirk; Geimer, Stefan

    2013-01-01

    Intraflagellar transport (IFT) is an evolutionarily highly conserved, microtubule-based, bidirectional transport system found in eukaryotic cilia/flagella and is indispensable for their assembly, maintenance, and sensory functions. Powered by two different motor complexes, linear arrays of protein particles, called IFT trains, are transported from the base to the tip of the cilium/flagellum and back, carrying axonemal precursors to the tip for assembly and turnover products back to the cell body for recycling. The dynamics of IFT can be visualized using various types of live-cell microscopy techniques, but for analyzing the ultrastructure of IFT trains, transmission electron microscopy is indispensable. The focus of this chapter is to describe the application of the flat embedding technique to Chlamydomonas reinhardtii and monolayers of mammalian culture cells. Such flat embeddings are well suited for the analysis of the ultrastructure of the IFT system by standard electron microscopy and electron tomography.

  3. Microtubule doublets are double-track railways for intraflagellar transport trains.

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-01

    The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components. PMID:27151870

  4. Microtubule doublets are double-track railways for intraflagellar transport trains.

    PubMed

    Stepanek, Ludek; Pigino, Gaia

    2016-05-01

    The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components.

  5. The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery.

    PubMed

    Toriyama, Michinori; Lee, Chanjae; Taylor, S Paige; Duran, Ivan; Cohn, Daniel H; Bruel, Ange-Line; Tabler, Jacqueline M; Drew, Kevin; Kelly, Marcus R; Kim, Sukyoung; Park, Tae Joo; Braun, Daniela A; Pierquin, Ghislaine; Biver, Armand; Wagner, Kerstin; Malfroot, Anne; Panigrahi, Inusha; Franco, Brunella; Al-Lami, Hadeel Adel; Yeung, Yvonne; Choi, Yeon Ja; Duffourd, Yannis; Faivre, Laurence; Rivière, Jean-Baptiste; Chen, Jiang; Liu, Karen J; Marcotte, Edward M; Hildebrandt, Friedhelm; Thauvin-Robinet, Christel; Krakow, Deborah; Jackson, Peter K; Wallingford, John B

    2016-06-01

    Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients. PMID:27158779

  6. Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length.

    PubMed

    Fort, Cécile; Bonnefoy, Serge; Kohl, Linda; Bastin, Philippe

    2016-08-01

    Intraflagellar transport (IFT) is required for construction of most cilia and flagella. Here, we used electron microscopy, immunofluorescence and live video microscopy to show that IFT is absent or arrested in the mature flagellum of Trypanosoma brucei upon RNA interference (RNAi)-mediated knockdown of IFT88 and IFT140, respectively. Flagella assembled prior to RNAi did not shorten, showing that IFT is not essential for the maintenance of flagella length. Although the ultrastructure of the axoneme was not visibly affected, flagellar beating was strongly reduced and the distribution of several flagellar components was drastically modified. The R subunit of the protein kinase A was no longer concentrated in the flagellum but was largely found in the cell body whereas the kinesin 9B motor was accumulating at the distal tip of the flagellum. In contrast, the distal tip protein FLAM8 was dispersed along the flagellum. This reveals that IFT also functions in maintaining the distribution of some flagellar proteins after construction of the organelle is completed. PMID:27343245

  7. WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia.

    PubMed

    Patel-King, Ramila S; Gilberti, Renée M; Hom, Erik F Y; King, Stephen M

    2013-09-01

    Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

  8. WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia

    PubMed Central

    Patel-King, Ramila S.; Gilberti, Renée M.; Hom, Erik F. Y.; King, Stephen M.

    2013-01-01

    Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle–like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly. PMID:23864713

  9. Intraflagellar transport, cilia, and mammalian Hedgehog signaling: analysis in mouse embryonic fibroblasts.

    PubMed

    Ocbina, Polloneal Jymmiel R; Anderson, Kathryn V

    2008-08-01

    Genetic studies in the mouse have shown that Intraflagellar Transport (IFT) is essential for mammalian Hedgehog (Hh) signal transduction. In this study, we take advantage of wild type and IFT mutant mouse embryonic fibroblasts (MEFs) to characterize additional aspects of the relationship between IFT and Hh signaling. Exposure to Sonic hedgehog (Shh) ligand or expression of an activated allele of Smo, SmoA1, activates an Hh reporter in wild-type MEFs, but not in MEFs derived from embryos that lack IFT172 or the Dync2h1 subunit of the retrograde IFT motor. Similarly, decreased activity of either Sufu or PKA, two negative regulators of Hh signal transduction, activates the pathway in wild-type, but not IFT mutant, MEFs. In contrast to wild-type MEFs, Smo is constitutively present in the cilia of Dync2h1 mutant MEFs. This finding suggests that IFT-dependent trafficking of Hh pathway components through the cilium is essential for their function.

  10. Total internal reflection fluorescence microscopy of intraflagellar transport in Tetrahymena thermophila.

    PubMed

    Jiang, Yu-Yang; Lechtreck, Karl; Gaertig, Jacek

    2015-01-01

    Live imaging has become a powerful tool in studies of ciliary proteins. Tetrahymena thermophila is an established ciliated model with well-developed genetic and biochemical approaches, but its large size, complex shape, and the large number of short and overlapping cilia, have made live imaging of ciliary proteins challenging. Here we describe a method that combines paralysis of cilia by nickel ions and total internal reflection microscopy for live imaging of fluorescent proteins inside cilia of Tetrahymena. Using this method, we quantitatively documented the intraflagellar transport in Tetrahymena.

  11. Why motor proteins team up - Intraflagellar transport in C. elegans cilia

    PubMed Central

    Mijalkovic, Jona; Prevo, Bram; Peterman, Erwin J. G.

    2016-01-01

    ABSTRACT Inside the cell, vital processes such as cell division and intracellular transport are driven by the concerted action of different molecular motor proteins. In C. elegans chemosensory cilia, 2 kinesin-2 family motor proteins, kinesin-II and OSM-3, team up to drive intraflagellar transport (IFT) in the anterograde direction, from base to tip, whereas IFT dynein hitchhikes toward the tip and subsequently drives IFT in the opposite, retrograde direction, thereby recycling both kinesins. While it is evident that at least a retrograde and an anterograde motor are necessary to drive IFT, it has remained puzzling why 2 same-polarity kinesins are employed. Recently, we addressed this question by combining advanced genome-engineering tools with ultrasensitive, quantitative fluorescence microscopy to study IFT with single-molecule sensitivity.1,2 Using this combination of approaches, we uncovered a differentiation in kinesin-2 function, in which the slower kinesin-II operates as an ‘importer’, loading IFT trains into the cilium before gradually handing them over to the faster OSM-3. OSM-3 subsequently acts as a long-range ‘transporter’, driving the IFT trains toward the tip. The two kinesin-2 motors combine their unique motility properties to achieve something neither motor can achieve on its own; that is to optimize the amount of cargo inside the cilium. In this commentary, we provide detailed insight into the rationale behind our research approach and comment on our recent findings. Moreover, we discuss the role of IFT dynein and provide an outlook on future studies. PMID:27384150

  12. SQL-1, homologue of the Golgi protein GMAP210, modulates intraflagellar transport in C. elegans.

    PubMed

    Broekhuis, Joost R; Rademakers, Suzanne; Burghoorn, Jan; Jansen, Gert

    2013-04-15

    Primary cilia are microtubule-based organelles that have important sensory functions. For their function, cilia rely on the delivery of specific proteins, both by intracellular trafficking and intraflagellar transport (IFT). In the cilia of Caenorhabditis elegans, anterograde IFT is mediated by kinesin-II and OSM-3. Previously, we have shown that expression of a dominant active G protein α subunit (GPA-3QL) in amphid channel neurons affects the coordination of kinesin-II and OSM-3 and also affects cilia length, suggesting that environmental signals can modulate these processes. Here, we show that loss-of-function of sql-1 (suppressor of gpa-3QL 1), which encodes the homologue of the mammalian Golgi protein GMAP210, suppresses the gpa-3QL cilia length phenotype. SQL-1 localizes to the Golgi apparatus, where it contributes to maintaining Golgi organization. Loss of sql-1 by itself does not affect cilia length, whereas overexpression of sql-1 results in longer cilia. Using live imaging of fluorescently tagged IFT proteins, we show that in sql-1 mutants OSM-3 moves faster, kinesin-II moves slower and that some complex A and B proteins move at an intermediate velocity, while others move at the same velocity as OSM-3. This indicates that mutation of sql-1 destabilizes the IFT complex. Finally, we show that simultaneous inactivation of sql-1 and activation of gpa-3QL affects the velocity of OSM-3. In summary, we show that in C. elegans the Golgin protein SQL-1 plays an important role in maintaining the stability of the IFT complex.

  13. Dissecting the Sequential Assembly and Localization of Intraflagellar Transport Particle Complex B in Chlamydomonas

    PubMed Central

    Richey, Elizabeth A.; Qin, Hongmin

    2012-01-01

    Intraflagellar transport (IFT), the key mechanism for ciliogenesis, involves large protein particles moving bi-directionally along the entire ciliary length. IFT particles contain two large protein complexes, A and B, which are constructed with proteins in a core and several peripheral proteins. Prior studies have shown that in Chlamydomonas reinhardtii, IFT46, IFT52, and IFT88 directly interact with each other and are in a subcomplex of the IFT B core. However, ift46, bld1, and ift88 mutants differ in phenotype as ift46 mutants are able to form short flagella, while the other two lack flagella completely. In this study, we investigated the functional differences of these individual IFT proteins contributing to complex B assembly, stability, and basal body localization. We found that complex B is completely disrupted in bld1 mutant, indicating an essential role of IFT52 for complex B core assembly. Ift46 mutant cells are capable of assembling a relatively intact complex B, but such complex is highly unstable and prone to degradation. In contrast, in ift88 mutant cells the complex B core still assembles and remains stable, but the peripheral proteins no longer attach to the B core. Moreover, in ift88 mutant cells, while complex A and the anterograde IFT motor FLA10 are localized normally to the transition fibers, complex B proteins instead are accumulated at the proximal ends of the basal bodies. In addition, in bld2 mutant, the IFT complex B proteins still localize to the proximal ends of defective centrioles which completely lack transition fibers. Taken together, these results revealed a step-wise assembly process for complex B, and showed that the complex first localizes to the proximal end of the centrioles and then translocates onto the transition fibers via an IFT88-dependent mechanism. PMID:22900094

  14. Intraflagellar Transport/Hedgehog-Related Signaling Components Couple Sensory Cilium Morphology and Serotonin Biosynthesis in Caenorhabditis elegans

    PubMed Central

    Moussaif, Mustapha; Sze, Ji Ying

    2009-01-01

    Intraflagellar transport in cilia has been proposed as a crucial mediator of Hedgehog signal transduction during embryonic pattern formation in both vertebrates and invertebrates. Here, we show that the Hh receptor Patched-related factor DAF-6 and intraflagellar transport modulate serotonin production in Caenorhabditis elegans animals, by remodeling the architecture of dendritic cilia of a pair of ADF serotonergic chemosensory neurons. Wild-type animals under aversive environment drastically reduce DAF-6 expression in glia-like cells surrounding the cilia of chemosensory neurons, resulting in cilium structural remodeling and upregulation of the serotonin-biosynthesis enzyme tryptophan hydroxylase tph-1 in the ADF neurons. These cellular and molecular modifications are reversed when the environment improves. Mutants of daf-6 or intraflagellar transport constitutively upregulate tph-1 expression. Epistasis analyses indicate that DAF-6/intraflagellar transport and the OCR-2/OSM-9 TRPV channel act in concert, regulating two layers of activation of tph-1 in the ADF neurons. The TRPV signaling turns on tph-1 expression under favorable and aversive conditions, whereas inactivation of DAF-6 by stress results in further upregulation of tph-1 independently of OCR-2/OSM-9 activity. Behavioral analyses suggest that serotonin facilitates larval animals resuming development when the environment improves. Our study revealed the cilium structure of serotonergic neurons as a trigger of regulated serotonin production, and demonstrated that a Hedgehog-related signaling component is dynamically regulated by environment and underscores neuroplasticity of serotonergic neurons in C. elegans under stress and stress recovery. PMID:19339602

  15. TTC26/DYF13 is an intraflagellar transport protein required for transport of motility-related proteins into flagella

    PubMed Central

    Ishikawa, Hiroaki; Ide, Takahiro; Yagi, Toshiki; Jiang, Xue; Hirono, Masafumi; Sasaki, Hiroyuki; Yanagisawa, Haruaki; Wemmer, Kimberly A; Stainier, Didier YR; Qin, Hongmin; Kamiya, Ritsu; Marshall, Wallace F

    2014-01-01

    Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001 PMID:24596149

  16. Different Effects of Tetrahymena IFT172 Domains on Anterograde and Retrograde Intraflagellar Transport

    PubMed Central

    Tsao, Che-Chia

    2008-01-01

    Intraflagellar transport (IFT) particles are multiprotein complexes that move bidirectionally along the cilium/flagellum. The Tetrahymena IFT172 gene encodes a protein with an N-terminal WD domain (WDD) and a C-terminal repeat domain (RPD). Epitope-tagged Ift172p localized to the basal body and in cilia along the axoneme, and IFT172 knockout cells lost cilia and motility. Using serial deletion constructs to rescue the knockout cells, we found that neither the WDD nor the RPD alone is sufficient to assemble cilia. Ift172p containing only the WDD or the RPD failed to enter cilia. Constructs with a partial truncation of the RPD still rescued although cilia were assembled less efficiently, indicating that the WDD and a part of the RPD are sufficient for anterograde transport. Partial truncation of the RPD caused the accumulation of truncated Ift172p itself and of Ift88p at ciliary tips, suggesting that IFT turnaround or retrograde transport was affected. These results implicate different regions of Ift172p in different steps of the IFT process. PMID:18199688

  17. Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

    PubMed Central

    Engel, Benjamin D.; Ludington, William B.

    2009-01-01

    The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly. PMID:19805630

  18. Two classes of short intraflagellar transport train with different 3D structures are present in Chlamydomonas flagella.

    PubMed

    Vannuccini, Elisa; Paccagnini, Eugenio; Cantele, Francesca; Gentile, Mariangela; Dini, Daniele; Fino, Federica; Diener, Dennis; Mencarelli, Caterina; Lupetti, Pietro

    2016-05-15

    Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these 'narrow' and 'wide', and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16 nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.

  19. The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis

    PubMed Central

    Yang, Shuying; Wang, Changdong

    2012-01-01

    Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia, which play important roles in development and homeostasis. IFT80 is a newly defined IFT protein. Partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III with abnormal skeletal development. However, the role and mechanism of IFT80 in osteogenesis is unknown. Here, we first detected IFT80 expression pattern and found that IFT80 was highly expressed in mouse long bone, skull, and during osteoblast differentiation. By using lentivirus-mediated RNA interference (RNAi) technology to silence IFT80 in murine mesenchymal progenitor cell line-C3H10T1/2 and bone marrow derived stromal cells, we found that silencing IFT80 led to either shortening or loss of cilia and the decrease of Arl13b expression - a small GTPase that is localized in cilia. Additionally, silencing IFT80 blocked the expression of osteoblast markers and significantly inhibited ALP activity and cell mineralization. We further found that IFT80 silencing inhibited the expression of Gli2, a critical transcriptional factor in the hedgehog signaling pathway. Overexpression of Gli2 rescued the deficiency of osteoblast differentiation from IFT80-silenced cells, and dramatically promoted osteoblast differentiation. Moreover, introduction of Smo agonist (SAG) promotes osteoblast differentiation, which was partially inhibited by IFT80 silencing. Thus, these results suggested that IFT80 plays an important role in osteogenesis through regulating Hedgehog/Gli signal pathways. PMID:22771375

  20. Subunit Interactions and Organization of the Chlamydomonas reinhardtii Intraflagellar Transport Complex A Proteins*

    PubMed Central

    Behal, Robert H.; Miller, Mark S.; Qin, Hongmin; Lucker, Ben F.; Jones, Alexis; Cole, Douglas G.

    2012-01-01

    Chlamydomonas reinhardtii intraflagellar transport (IFT) particles can be biochemically resolved into two smaller assemblies, complexes A and B, that contain up to six and 15 protein subunits, respectively. We provide here the proteomic and immunological analyses that verify the identity of all six Chlamydomonas A proteins. Using sucrose density gradient centrifugation and antibody pulldowns, we show that all six A subunits are associated in a 16 S complex in both the cell bodies and flagella. A significant fraction of the cell body IFT43, however, exhibits a much slower sedimentation of ∼2 S and is not associated with the IFT A complex. To identify interactions between the six A proteins, we combined exhaustive yeast-based two-hybrid analysis, heterologous recombinant protein expression in Escherichia coli, and analysis of the newly identified complex A mutants, ift121 and ift122. We show that IFT121 and IFT43 interact directly and provide evidence for additional interactions between IFT121 and IFT139, IFT121 and IFT122, IFT140 and IFT122, and IFT140 and IFT144. The mutant analysis further allows us to propose that a subset of complex A proteins, IFT144/140/122, can form a stable 12 S subcomplex that we refer to as the IFT A core. Based on these results, we propose a model for the spatial arrangement of the six IFT A components. PMID:22170070

  1. Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm

    PubMed Central

    San Agustin, Jovenal T.; Pazour, Gregory J.; Witman, George B.

    2015-01-01

    Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88Tg737Rpw mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88−/− mice are completely sterile. They produce ∼350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella. PMID:26424803

  2. Efficient live fluorescence imaging of intraflagellar transport in mammalian primary cilia

    PubMed Central

    Ishikawa, Hiroaki; Marshall, Wallace F.

    2016-01-01

    Intraflagellar transport (IFT) is a motile process critical for building most cilia, including those of mammalian cells. Defects in IFT lead to short or missing cilia, and in animals can cause defects in development, for example in hedgehog mediated signaling, as well as disease symptoms such as polycystic kidney disease or retinal degeneration. Understanding how IFT works is thus a high priority in ciliary biology. Imaging of living cells has played a key role in understanding the mechanism of IFT, and this is particularly the case in mammalian cells where biochemical analysis of IFT is extremely difficult, due to the difficulty of isolating cilia away from the rest of the cell. Imaging IFT in living mammalian cells requires solution to several problems: constructing cell lines that express fluorescent protein tagged IFT proteins, obtaining cell populations with a high degree of ciliation, confocal or TIRF imaging with sufficient time resolution and signal to noise ratio to observe the majority of IFT particles as they travel back and forth inside the cilium, and analyzing the image data to extract quantitative measurements of IFT. We describe optimized solutions to each of these technical challenges. Using the approaches described here, mammalian cultured cells become powerful platforms for quantitative analysis of IFT dynamics. PMID:25837392

  3. Getting to the heart of intraflagellar transport using Trypanosoma and Chlamydomonas models: the strength is in their differences

    PubMed Central

    2013-01-01

    Cilia and flagella perform diverse roles in motility and sensory perception, and defects in their construction or their function are responsible for human genetic diseases termed ciliopathies. Cilia and flagella construction relies on intraflagellar transport (IFT), the bi-directional movement of ‘trains’ composed of protein complexes found between axoneme microtubules and the flagellum membrane. Although extensive information about IFT components and their mode of action were discovered in the green algae Chlamydomonas reinhardtii, other model organisms have revealed further insights about IFT. This is the case of Trypanosoma brucei, a flagellated protist responsible for sleeping sickness that is turning out to be an emerging model for studying IFT. In this article, we review different aspects of IFT, based on studies of Chlamydomonas and Trypanosoma. Data available from both models are examined to ask challenging questions about IFT such as the initiation of flagellum construction, the setting-up of IFT and the mode of formation of IFT trains, and their remodeling at the tip as well as their recycling at the base. Another outstanding question is the individual role played by the multiple IFT proteins. The use of different models, bringing their specific biological and experimental advantages, will be invaluable in order to obtain a global understanding of IFT. PMID:24289478

  4. Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome

    PubMed Central

    Jacobs, Damon T.; Silva, Luciane M.; Allard, Bailey A.; Schonfeld, Michael P.; Chatterjee, Anindita; Talbott, George C.

    2016-01-01

    ABSTRACT Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease. PMID:27482817

  5. Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD

    PubMed Central

    Hessel, Justina; Heldrich, Jonna; Fuller, Jennifer; Staudt, Michelle R.; Radisch, Sharon; Hollmann, Charleen; Harvey, Ben-Gary; Kaner, Robert J.; Salit, Jacqueline; Yee-Levin, Jenny; Sridhar, Sriram; Pillai, Sreekumar; Hilton, Holly; Wolff, Gerhard; Bitter, Hans; Visvanathan, Sudha; Fine, Jay; Stevenson, Christopher S.; Crystal, Ronald G.; Tilley, Ann E.

    2014-01-01

    Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies. PMID:24465567

  6. Dysfunction of intraflagellar transport-A causes hyperphagia-induced obesity and metabolic syndrome.

    PubMed

    Jacobs, Damon T; Silva, Luciane M; Allard, Bailey A; Schonfeld, Michael P; Chatterjee, Anindita; Talbott, George C; Beier, David R; Tran, Pamela V

    2016-07-01

    Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease. PMID:27482817

  7. Zebrafish ift57, ift88, and ift172 intraflagellar transport mutants disrupt cilia but do not affect hedgehog signaling.

    PubMed

    Lunt, Shannon C; Haynes, Tony; Perkins, Brian D

    2009-07-01

    Cilia formation requires intraflagellar transport (IFT) proteins. Recent studies indicate that mammalian Hedgehog (Hh) signaling requires cilia. It is unclear, however, if the requirement for cilia and IFT proteins in Hh signaling represents a general rule for all vertebrates. Here we examine zebrafish ift57, ift88, and ift172 mutants and morphants for defects in Hh signaling. Although ift57 and ift88 mutants and morphants contained residual maternal protein, the cilia were disrupted. In contrast to previous genetic studies in mouse, mutations in zebrafish IFT genes did not affect the expression of Hh target genes in the neural tube and forebrain and had no quantitative effect on Hh target gene expression. Zebrafish IFT mutants also exhibited no dramatic changes in the craniofacial skeleton, somite formation, or motor neuron patterning. Thus, our data indicate the requirement for cilia in the Hh signal transduction pathway may not represent a universal mechanism in vertebrates.

  8. Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in Chlamydomonas flagella

    PubMed Central

    Harris, J. Aaron; Liu, Yi; Yang, Pinfen; Kner, Peter; Lechtreck, Karl F.

    2016-01-01

    The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia. PMID:26631555

  9. Combined NGS Approaches Identify Mutations in the Intraflagellar Transport Gene IFT140 in Skeletal Ciliopathies with Early Progressive Kidney Disease

    PubMed Central

    Schmidts, Miriam; Frank, Valeska; Eisenberger, Tobias; al Turki, Saeed; Bizet, Albane A.; Antony, Dinu; Rix, Suzanne; Decker, Christian; Bachmann, Nadine; Bald, Martin; Vinke, Tobias; Toenshoff, Burkhard; Donato, Natalia Di; Neuhann, Theresa; Hartley, Jane L.; Maher, Eamonn R.; Bogdanović, Radovan; Peco-Antić, Amira; Mache, Christoph; Hurles, Matthew E.; Joksić, Ivana; Guć-Šćekić, Marija; Dobricic, Jelena; Brankovic-Magic, Mirjana; Bolz, Hanno J.; Pazour, Gregory J.; Beales, Philip L.; Scambler, Peter J.; Saunier, Sophie; Mitchison, Hannah M.; Bergmann, Carsten

    2014-01-01

    Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy. PMID:23418020

  10. Overall Architecture of the Intraflagellar Transport (IFT)-B Complex Containing Cluap1/IFT38 as an Essential Component of the IFT-B Peripheral Subcomplex.

    PubMed

    Katoh, Yohei; Terada, Masaya; Nishijima, Yuya; Takei, Ryota; Nozaki, Shohei; Hamada, Hiroshi; Nakayama, Kazuhisa

    2016-05-20

    Intraflagellar transport (IFT) is essential for assembly and maintenance of cilia and flagella as well as ciliary motility and signaling. IFT is mediated by multisubunit complexes, including IFT-A, IFT-B, and the BBSome, in concert with kinesin and dynein motors. Under high salt conditions, purified IFT-B complex dissociates into a core subcomplex composed of at least nine subunits and at least five peripherally associated proteins. Using the visible immunoprecipitation assay, which we recently developed as a convenient protein-protein interaction assay, we determined the overall architecture of the IFT-B complex, which can be divided into core and peripheral subcomplexes composed of 10 and 6 subunits, respectively. In particular, we identified TTC26/IFT56 and Cluap1/IFT38, neither of which was included with certainty in previous models of the IFT-B complex, as integral components of the core and peripheral subcomplexes, respectively. Consistent with this, a ciliogenesis defect of Cluap1-deficient mouse embryonic fibroblasts was rescued by exogenous expression of wild-type Cluap1 but not by mutant Cluap1 lacking the binding ability to other IFT-B components. The detailed interaction map as well as comparison of subcellular localization of IFT-B components between wild-type and Cluap1-deficient cells provides insights into the functional relevance of the architecture of the IFT-B complex. PMID:26980730

  11. Crystal structures of IFT70/52 and IFT52/46 provide insight into intraflagellar transport B core complex assembly

    PubMed Central

    Taschner, Michael; Kotsis, Fruzsina; Braeuer, Philipp; Kuehn, E. Wolfgang

    2014-01-01

    Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330–370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281–329 in a complex that interacts directly with IFT70/IFT52330–381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains. PMID:25349261

  12. Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits.

    PubMed

    Lucker, Ben F; Behal, Robert H; Qin, Hongmin; Siron, Laura C; Taggart, W David; Rosenbaum, Joel L; Cole, Douglas G

    2005-07-29

    Required for the assembly and maintenance of eukaryotic cilia and flagella, intraflagellar transport (IFT) consists of the bidirectional movement of large protein particles between the base and the distal tip of the organelle. Anterograde movement of particles away from the cell body is mediated by kinesin-2, whereas retrograde movement away from the flagellar tip is powered by cytoplasmic dynein 1b/2. IFT particles contain multiple copies of two distinct protein complexes, A and B, which contain at least 6 and 11 protein subunits, respectively. In this study, we have used increased ionic strength to remove four peripheral subunits from the IFT complex B of Chlamydomonas reinhardtii, revealing a 500-kDa core that contains IFT88, IFT81, IFT74/72, IFT52, IFT46, and IFT27. This result demonstrates that the complex B subunits, IFT172, IFT80, IFT57, and IFT20 are not required for the core subunits to stay associated. Chemical cross-linking of the complex B core resulted in multiple IFT81-74/72 products. Yeast-based two-hybrid and three-hybrid analyses were then used to show that IFT81 and IFT74/72 directly interact to form a higher order oligomer consistent with a tetrameric complex. Similar analysis of the vertebrate IFT81 and IFT74/72 homologues revealed that this interaction has been evolutionarily conserved. We hypothesize that these proteins form a tetrameric complex, (IFT81)2(IFT74/72)2, which serves as a scaffold for the formation of the intact IFT complex B. PMID:15955805

  13. Ciliary intraflagellar transport protein 80 balances canonical versus non-canonical hedgehog signaling for osteoblast differentiation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mutation of different IFT proteins cause numerous different clinical bone disorders accompanied with or without the disruption of cilia formation. Currently, there is no any effective treatment for these disorders due to lack of understanding in the function and mechanism of these proteins. IFT80 is...

  14. Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin-binding IFT-B2 complex.

    PubMed

    Taschner, Michael; Weber, Kristina; Mourão, André; Vetter, Melanie; Awasthi, Mayanka; Stiegler, Marc; Bhogaraju, Sagar; Lorentzen, Esben

    2016-04-01

    Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues.

  15. Intraflagellar transport proteins 172, 80, 57, 54, 38, and 20 form a stable tubulin-binding IFT-B2 complex.

    PubMed

    Taschner, Michael; Weber, Kristina; Mourão, André; Vetter, Melanie; Awasthi, Mayanka; Stiegler, Marc; Bhogaraju, Sagar; Lorentzen, Esben

    2016-04-01

    Intraflagellar transport (IFT) relies on the IFT complex and is required for ciliogenesis. The IFT-B complex consists of 9-10 stably associated core subunits and six "peripheral" subunits that were shown to dissociate from the core structure at moderate salt concentration. We purified the six "peripheral"IFT-B subunits of Chlamydomonas reinhardtiias recombinant proteins and show that they form a stable complex independently of the IFT-B core. We suggest a nomenclature of IFT-B1 (core) and IFT-B2 (peripheral) for the two IFT-B subcomplexes. We demonstrate that IFT88, together with the N-terminal domain of IFT52, is necessary to bridge the interaction between IFT-B1 and B2. The crystal structure of IFT52N reveals highly conserved residues critical for IFT-B1/IFT-B2 complex formation. Furthermore, we show that of the three IFT-B2 subunits containing a calponin homology (CH) domain (IFT38, 54, and 57), only IFT54 binds αβ-tubulin as a potential IFT cargo, whereas the CH domains of IFT38 and IFT57 mediate the interaction with IFT80 and IFT172, respectively. Crystal structures of IFT54 CH domains reveal that tubulin binding is mediated by basic surface-exposed residues. PMID:26912722

  16. Chlamydomonas Kinesin-II–dependent Intraflagellar Transport (IFT): IFT Particles Contain Proteins Required for Ciliary Assembly in Caenorhabditis elegans Sensory Neurons

    PubMed Central

    Cole, Douglas G.; Diener, Dennis R.; Himelblau, Amy L.; Beech, Peter L.; Fuster, Jason C.; Rosenbaum, Joel L.

    1998-01-01

    We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979–992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia. PMID:9585417

  17. A novel dynein light intermediate chain colocalizes with the retrograde motor for intraflagellar transport at sites of axoneme assembly in chlamydomonas and Mammalian cells.

    PubMed

    Perrone, Catherine A; Tritschler, Douglas; Taulman, Patrick; Bower, Raqual; Yoder, Bradley K; Porter, Mary E

    2003-05-01

    The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.

  18. Intraflagellar transport—the “new motility” 20 years later

    PubMed Central

    Kozminski, Keith G.

    2012-01-01

    Intraflagellar transport is the rapid, bidirectional movement of protein complexes along the length of most eukaryotic cilia and flagella. Discovery of this intracellular process in Chlamydomonas reinhardtii 20 years ago led to a rapid discovery of cellular mechanisms that underlie a large number of human ciliopathies. Described herein are the events that led to this discovery. PMID:22379118

  19. Functional insights of nucleocytoplasmic transport in plants

    PubMed Central

    Tamura, Kentaro; Hara-Nishimura, Ikuko

    2014-01-01

    Plant nucleocytoplasmic transport beyond the nuclear envelope is important not only for basic cellular functions but also for growth, development, hormonal signaling, and responses to environmental stimuli. Key components of this transport system include nuclear transport receptors and nucleoporins. The functional and physical interactions between receptors and the nuclear pore in the nuclear membrane are indispensable for nucleocytoplasmic transport. Recently, several groups have reported various plant mutants that are deficient in factors involved in nucleocytoplasmic transport. Here, we summarize the current state of knowledge about nucleocytoplasmic transport in plants, and we review the plant-specific regulation and roles of this process in plants. PMID:24765097

  20. Functional Analysis of Arabidopsis Sucrose Transporters

    SciTech Connect

    John M. Ward

    2009-03-31

    Sucrose is the main photosynthetic product that is transported in the vasculature of plants. The long-distance transport of carbohydrates is required to support the growth and development of net-importing (sink) tissues such as fruit, seeds and roots. This project is focused on understanding the transport mechanism sucrose transporters (SUTs). These are proton-coupled sucrose uptake transporters (membrane proteins) that are required for transport of sucrose in the vasculature and uptake into sink tissues. The accomplishments of this project included: 1) the first analysis of substrate specificity for any SUT. This was accomplished using electrophysiology to analyze AtSUC2, a sucrose transporter from companion cells in Arabidopsis. 2) the first analysis of the transport activity for a monocot SUT. The transport kinetics and substrate specificity of HvSUT1 from barley were studied. 3) the first analysis of a sucrose transporter from sugarcane. and 4) the first analysis of transport activity of a sugar alcohol transporter homolog from plants, AtPLT5. During this period four primary research papers, funded directly by the project, were published in refereed journals. The characterization of several sucrose transporters was essential for the current effort in the analysis of structure/function for this gene family. In particular, the demonstration of strong differences in substrate specificity between type I and II SUTs was important to identify targets for site-directed mutagenesis.

  1. Mapping the functional yeast ABC transporter interactome

    PubMed Central

    Snider, Jamie; Hanif, Asad; Lee, Mid Eum; Jin, Ke; Yu, Analyn R.; Graham, Chris; Chuk, Matthew; Damjanovic, Dunja; Wierzbicka, Marta; Tang, Priscilla; Balderes, Dina; Wong, Victoria; Jessulat, Matthew; Darowski, Katelyn D.; Luis, Bryan-Joseph San; Shevelev, Igor; Sturley, Stephen L; Boone, Charles; Greenblatt, Jack F.; Zhang, Zhaolei; Paumi, Christian M.; Babu, Mohan; Park, Hay-Oak; Michaelis, Susan; Stagljar, Igor

    2013-01-01

    ABC transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used Membrane Yeast Two-Hybrid (MYTH) technology to map the protein interactome of all non-mitochondrial ABC transporters in the model organism Saccharomy cescerevisiae, and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated interactome. We show that ABC transporters physically associate with proteins involved in a surprisingly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters. PMID:23831759

  2. Structure, function, and plasticity of GABA transporters

    PubMed Central

    Scimemi, Annalisa

    2014-01-01

    GABA transporters belong to a large family of neurotransmitter:sodium symporters. They are widely expressed throughout the brain, with different levels of expression in different brain regions. GABA transporters are present in neurons and in astrocytes and their activity is crucial to regulate the extracellular concentration of GABA under basal conditions and during ongoing synaptic events. Numerous efforts have been devoted to determine the structural and functional properties of GABA transporters. There is also evidence that the expression of GABA transporters on the cell membrane and their lateral mobility can be modulated by different intracellular signaling cascades. The strength of individual synaptic contacts and the activity of entire neuronal networks may be finely tuned by altering the density, distribution and diffusion rate of GABA transporters within the cell membrane. These findings are intriguing because they suggest the existence of complex regulatory systems that control the plasticity of GABAergic transmission in the brain. Here we review the current knowledge on the structural and functional properties of GABA transporters and highlight the molecular mechanisms that alter the expression and mobility of GABA transporters at central synapses. PMID:24987330

  3. Thiamin function, metabolism, uptake, and transport.

    PubMed

    Manzetti, Sergio; Zhang, Jin; van der Spoel, David

    2014-02-11

    Vitamins are crucial components in the diet of animals and many other living organisms. One of these essential nutrients, thiamin, is known to be involved in several cell functions, including energy metabolism and the degradation of sugars and carbon skeletons. Other roles that are connected to this vitamin are neuronal communication, immune system activation, signaling and maintenance processes in cells and tissues, and cell-membrane dynamics. Because of the key functions of thiamin, uptake and transport through the body are crucial. Its uptake route is relatively complex, encompassing a variety of protein families, including the solute carrier anion transporters, the alkaline phosphatase transport system, and the human extraneuronal monoamine transporter family, some of which are multispecific proteins. There are two known structures of protein (subunits) involved in thiamin uptake in prokaryotes. Binding of thiamin to these proteins is strongly guided by electrostatic interactions. The lack of structural information about thiamin binding proteins for higher organisms remains a bottleneck for understanding the uptake process of thiamin in atomic detail. This review includes recent data on thiamin metabolism, related deficiencies and pathologies, and the latest findings on thiamin binding transporters.

  4. Particulate distribution function evolution for ejecta transport

    SciTech Connect

    Hammerberg, James Edward; Plohr, Bradley J

    2010-01-01

    The time evolution of the ejecta distribution function in a gas is discussed in the context of the recent experiments of W. Buttler and M. Zellner for well characterized Sn surfaces. Evolution equations are derived for the particulate distribution function when the dominant gas-particle interaction in is particulate drag. In the approximation of separability of the distribution function in velocity and size, the solution for the time dependent distribution function is a Fredholm integral equation of the first kind whose kernel is expressible in terms of the vacuum time dependent velocity distribution function measured with piezo probes or Asay foils. The solution of this equation in principle gives the size distribution function. We discuss the solution of this equation and the results of the Buttler - Zellner experiments. These suggest that correlations in velocity and size are necessary for a complete description of the transport dala. The solutions presented also represent an analytic test problem for the calculated distribution function in ejecta transport implementations.

  5. HDL Function, Dysfunction, and Reverse Cholesterol Transport

    PubMed Central

    Fisher, Edward A.; Feig, Jonathan E.; Hewing, Bernd; Hazen, Stanley L.; Smith, Jonathan D.

    2012-01-01

    Although high HDL-cholesterol levels are associated with decreased cardiovascular risk in epidemiological studies, recent genetic and pharmacological findings have raised doubts about the beneficial effects of HDL. Raising HDL levels in animal models by infusion or over expression of apolipoprotein A-I has shown clear vascular improvements, such as delayed atherosclerotic lesion progression and accelerated lesion regression, along with increased reverse cholesterol transport. Inflammation and other factors, such as myeloperoxidase mediated oxidation, can impair HDL production and HDL function, in regard to its reverse cholesterol transport, antioxidant, and anti-inflammatory activities. Thus, tests of HDL function, which have not yet been developed as routine diagnostic assays, may prove useful and be a better predictor of cardiovascular risk than HDL-cholesterol levels. PMID:23152494

  6. Functionalization mediates heat transport in graphene nanoflakes

    PubMed Central

    Han, Haoxue; Zhang, Yong; Wang, Nan; Samani, Majid Kabiri; Ni, Yuxiang; Mijbil, Zainelabideen Y.; Edwards, Michael; Xiong, Shiyun; Sääskilahti, Kimmo; Murugesan, Murali; Fu, Yifeng; Ye, Lilei; Sadeghi, Hatef; Bailey, Steven; Kosevich, Yuriy A.; Lambert, Colin J.; Liu, Johan; Volz, Sebastian

    2016-01-01

    The high thermal conductivity of graphene and few-layer graphene undergoes severe degradations through contact with the substrate. Here we show experimentally that the thermal management of a micro heater is substantially improved by introducing alternative heat-escaping channels into a graphene-based film bonded to functionalized graphene oxide through amino-silane molecules. Using a resistance temperature probe for in situ monitoring we demonstrate that the hotspot temperature was lowered by ∼28 °C for a chip operating at 1,300 W cm−2. Thermal resistance probed by pulsed photothermal reflectance measurements demonstrated an improved thermal coupling due to functionalization on the graphene–graphene oxide interface. Three functionalization molecules manifest distinct interfacial thermal transport behaviour, corroborating our atomistic calculations in unveiling the role of molecular chain length and functional groups. Molecular dynamics simulations reveal that the functionalization constrains the cross-plane phonon scattering, which in turn enhances in-plane heat conduction of the bonded graphene film by recovering the long flexural phonon lifetime. PMID:27125636

  7. Functionalization mediates heat transport in graphene nanoflakes.

    PubMed

    Han, Haoxue; Zhang, Yong; Wang, Nan; Samani, Majid Kabiri; Ni, Yuxiang; Mijbil, Zainelabideen Y; Edwards, Michael; Xiong, Shiyun; Sääskilahti, Kimmo; Murugesan, Murali; Fu, Yifeng; Ye, Lilei; Sadeghi, Hatef; Bailey, Steven; Kosevich, Yuriy A; Lambert, Colin J; Liu, Johan; Volz, Sebastian

    2016-01-01

    The high thermal conductivity of graphene and few-layer graphene undergoes severe degradations through contact with the substrate. Here we show experimentally that the thermal management of a micro heater is substantially improved by introducing alternative heat-escaping channels into a graphene-based film bonded to functionalized graphene oxide through amino-silane molecules. Using a resistance temperature probe for in situ monitoring we demonstrate that the hotspot temperature was lowered by ∼28 °C for a chip operating at 1,300 W cm(-2). Thermal resistance probed by pulsed photothermal reflectance measurements demonstrated an improved thermal coupling due to functionalization on the graphene-graphene oxide interface. Three functionalization molecules manifest distinct interfacial thermal transport behaviour, corroborating our atomistic calculations in unveiling the role of molecular chain length and functional groups. Molecular dynamics simulations reveal that the functionalization constrains the cross-plane phonon scattering, which in turn enhances in-plane heat conduction of the bonded graphene film by recovering the long flexural phonon lifetime. PMID:27125636

  8. Functionalization mediates heat transport in graphene nanoflakes

    NASA Astrophysics Data System (ADS)

    Han, Haoxue; Zhang, Yong; Wang, Nan; Samani, Majid Kabiri; Ni, Yuxiang; Mijbil, Zainelabideen Y.; Edwards, Michael; Xiong, Shiyun; Sääskilahti, Kimmo; Murugesan, Murali; Fu, Yifeng; Ye, Lilei; Sadeghi, Hatef; Bailey, Steven; Kosevich, Yuriy A.; Lambert, Colin J.; Liu, Johan; Volz, Sebastian

    2016-04-01

    The high thermal conductivity of graphene and few-layer graphene undergoes severe degradations through contact with the substrate. Here we show experimentally that the thermal management of a micro heater is substantially improved by introducing alternative heat-escaping channels into a graphene-based film bonded to functionalized graphene oxide through amino-silane molecules. Using a resistance temperature probe for in situ monitoring we demonstrate that the hotspot temperature was lowered by ~28 °C for a chip operating at 1,300 W cm-2. Thermal resistance probed by pulsed photothermal reflectance measurements demonstrated an improved thermal coupling due to functionalization on the graphene-graphene oxide interface. Three functionalization molecules manifest distinct interfacial thermal transport behaviour, corroborating our atomistic calculations in unveiling the role of molecular chain length and functional groups. Molecular dynamics simulations reveal that the functionalization constrains the cross-plane phonon scattering, which in turn enhances in-plane heat conduction of the bonded graphene film by recovering the long flexural phonon lifetime.

  9. IFT-Cargo Interactions and Protein Transport in Cilia.

    PubMed

    Lechtreck, Karl F

    2015-12-01

    The motile and sensory functions of cilia and flagella are indispensable for human health. Cilia assembly requires a dedicated protein shuttle, intraflagellar transport (IFT), a bidirectional motility of multi-megadalton protein arrays along ciliary microtubules. IFT functions as a protein carrier delivering hundreds of distinct proteins into growing cilia. IFT-based protein import and export continue in fully grown cilia and are required for ciliary maintenance and sensing. Large ciliary building blocks might depend on IFT to move through the transition zone, which functions as a ciliary gate. Smaller, freely diffusing proteins, such as tubulin, depend on IFT to be concentrated or removed from cilia. As I discuss here, recent work provides insights into how IFT interacts with its cargoes and how the transport is regulated. PMID:26498262

  10. Effect of Disease Pathologies on Transporter Expression and Function.

    PubMed

    Atilano-Roque, Amandla; Roda, Gavriel; Fogueri, Uma; Kiser, Jennifer J; Joy, Melanie S

    2016-07-01

    Transporters are important determinants of drug absorption, distribution, and excretion. The clinical relevance of drug transporters in drug disposition and toxicology depends on their localization in liver, kidney, and brain. There has been growing evidence regarding the importance of disease status on alterations in metabolizing enzymes and transporter proteins. This review focuses on uptake and efflux transporter proteins in liver, kidney, and brain and discusses mechanisms of altered transporter expression and function secondary to disease. PMID:27385176

  11. A Functional-Phylogenetic Classification System for Transmembrane Solute Transporters

    PubMed Central

    Saier, Milton H.

    2000-01-01

    A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional

  12. A functional-phylogenetic classification system for transmembrane solute transporters.

    PubMed

    Saier, M H

    2000-06-01

    A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional

  13. Facilitative plasma membrane transporters function during ER transit

    PubMed Central

    Takanaga, Hitomi; Frommer, Wolf B.

    2010-01-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. PMID:20354141

  14. Control of machine functions or transport systems

    SciTech Connect

    Woodley, M.D.; Lee, M.J.; Jaeger, J.; King, A.S.

    1983-01-01

    A computer code, COMFORT, has been developed at SLAC for on-line calculation of the strengths of magnetic elements in an electron storage ring or transport beam line, subject to first order fitting constraints on the ring or beam line parameters. This code can also be used off-line as an interactive lattice or beam line design tool.

  15. Scallop DMT functions as a Ca2+ transporter.

    PubMed

    Toyohara, Haruhiko; Yamamoto, Sayuri; Hosoi, Masatomi; Takagi, Masaya; Hayashi, Isao; Nakao, Kenji; Kaneko, Shuji

    2005-05-01

    We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca(2+) transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca(2+) from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe(2+) concentration. Scallop DMT distinctly functions as a Ca(2+) transporter unlike other DMTs, however, it also transports Fe(2+) and Cd(2+) similar to them.

  16. Xenobiotic, bile acid, and cholesterol transporters: function and regulation.

    PubMed

    Klaassen, Curtis D; Aleksunes, Lauren M

    2010-03-01

    regulatory factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology. PMID:20103563

  17. Xenobiotic, Bile Acid, and Cholesterol Transporters: Function and Regulation

    PubMed Central

    Aleksunes, Lauren M.

    2010-01-01

    factors that influence transporter expression and function, including transcriptional activation and post-translational modifications as well as subcellular trafficking. Sex differences, ontogeny, and pharmacological and toxicological regulation of transporters are also addressed. Transporters are important transmembrane proteins that mediate the cellular entry and exit of a wide range of substrates throughout the body and thereby play important roles in human physiology, pharmacology, pathology, and toxicology. PMID:20103563

  18. Dissection of Transporter Function: From Genetics to Structure.

    PubMed

    Diallinas, G

    2016-09-01

    Transporters are transmembrane proteins mediating the selective uptake or efflux of solutes, metabolites, drugs, or ions across cellular membranes. Despite their immense biological importance in cell nutrition, communication, signaling, and homeostasis, their study remains technically difficult mostly due to their lipid-embedded nature. The study of eukaryotic transporters presents additional complexity due to multiple subcellular control mechanisms that operate to ensure proper membrane traffic, membrane localization, and turnover. Model fungi present unique genetic tools to study eukaryotic transporter function. This review highlights how fungal transporter genetics combined with new methodologies for assaying their cellular expression and function as well as recent structural approaches have led to the functional dissection of selected transporter paradigms in Aspergillus nidulans. PMID:27430403

  19. Rab6 functions in polarized transport in Drosophila photoreceptors

    PubMed Central

    Satoh, Takunori; Nakamura, Yuri; Satoh, Akiko K.

    2016-01-01

    ABSTRACT Selective membrane transport pathways are essential for cells in situ to construct and maintain a polarized structure comprising multiple plasma membrane domains, which is essential for their specific cellular functions. Genetic screening in Drosophila photoreceptors harboring multiple plasma membrane domains enables the identification of genes involved in polarized transport pathways. Our genome-wide high-throughput screening identified a Rab6-null mutant with a rare phenotype characterized by a loss of 2 apical transport pathways with an intact basolateral transport. Although the functions of Rab6 in the Golgi apparatus are well known, its function in polarized transport is unexpected. The mutant phenotype and localization of Rab6 strongly indicate that Rab6 regulates transport between the trans-Golgi network (TGN) and recycling endosomes (REs): basolateral cargos are segregated at the TGN before Rab6 functions, but cargos going to multiple apical domains are sorted at REs. Both the medial-Golgi resident protein Metallophosphoesterase (MPPE) and the TGN marker GalT::CFP exhibit diffused co-localized distributions in Rab6-deficient cells, suggesting they are trapped in the retrograde transport vesicles returning to trans-Golgi cisternae. Hence, we propose that Rab6 regulates the fusion of retrograde transport vesicles containing medial, trans-Golgi resident proteins to the Golgi cisternae, which causes Golgi maturation to REs. PMID:27116570

  20. Thermodynamics of Ionic Transport through Functionalized Membranes

    NASA Astrophysics Data System (ADS)

    Rathee, Vikramjit; Qu, Siyi; Dilenschneider, Theodore; Phillip, William A.; Whitmer, Jonathan K.

    Through microphase separation of block copolymers, highly porous solid membranes may be assembled. Further functionalization with amine and sulfonic acid groups has demonstrated promise in exquisitely controlling the flux of charged species, and in particular multivalent ions. Using coarse-grained molecular simulations, we explore the essential thermodynamics underlying salt rejection in charge-functionalized membranes, and develop a model capable of linking the performance of these membranes to their molecular character through free energy calculations.

  1. Lysosome Transport as a Function of Lysosome Diameter

    PubMed Central

    Bandyopadhyay, Debjyoti; Cyphersmith, Austin; Zapata, Jairo A.; Kim, Y. Joseph; Payne, Christine K.

    2014-01-01

    Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter. PMID:24497985

  2. Rare Mutations in Renal Sodium and Potassium Transporter Genes Exhibit Impaired Transport Function

    PubMed Central

    Welling, Paul A.

    2014-01-01

    Purpose of review Recent efforts to explore the genetic underpinnings of hypertension revealed rare mutations in kidney salt transport genes contribute to blood pressure variation and hypertension susceptibility in the general population. The current review focuses on these latest findings, highlighting a discussion about the rare mutations and how they affect transport function. Recent findings Rare mutations that confer a low blood pressure trait and resistance to hypertension have recently been extensively studied. Physiological and biochemical analyses of the effected renal salt transport molecules (NKCC2 (SLC12A1), ROMK (KCNJ1), and NCC (SLC12A3)) revealed that most of the mutations do, in fact, cause a loss of transport function. The mutations disrupt transport by many different mechanisms, including altering biosynthetic processing, trafficking, ion transport, and regulation. Summary New insights into the genetic basis of hypertension have recently emerged, supporting a major role of rare, rather than common, gene variants. Many different rare mutations have been found to affect the functions of different salt transporter genes by different mechanisms, yet all confer the same blood pressure phenotype. These studies reinforce the critical roles of the kidney, and renal salt transport in blood pressure regulation and hypertension. PMID:24253496

  3. WDR19: An ancient, retrograde, intraflagellar ciliary protein is mutated in autosomal recessive retinitis pigmentosa and in Senior-Loken syndrome

    PubMed Central

    Coussa, RG; Otto, EA; Gee, H-Y; Arthurs, P; Ren, H; Lopez, I; Keser, V; Fu, Q; Faingold, R; Khan, A; Schwartzentruber, J; Majewski, J; Hildebrandtand, F; Koenekoop, RK

    2014-01-01

    Autosomal recessive retinitis pigmentosa (arRP) is a clinically and genetically heterogeneous retinal disease that causes blindness. Our purpose was to identify the causal gene, describe the phenotype and delineate the mutation spectrum in a consanguineous Quebec arRP family. We performed Arrayed Primer Extension (APEX) technology to exclude ~500 arRP mutations in ~20 genes. Homozygosity mapping [single nucleotide polymorphism (SNP) genotyping] identified 10 novel significant homozygous regions. We performed next generation sequencing and whole exome capture. Sanger sequencing provided cosegregation. We screened another 150 retinitis pigmentosa (RP) and 200 patients with Senior-Løken Syndrome (SLS). We identified a novel missense mutation in WDR19, c.2129T>C which lead to a p.Leu710Ser. We found the same mutation in a second Quebec arRP family. Interestingly, two of seven affected members of the original family developed ‘sub-clinical’ renal cysts. We hypothesized that more severe WDR19 mutations may lead to severe ciliopathies and found seven WDR19 mutations in five SLS families. We identified a new gene for both arRP and SLS. WDR19 is a ciliary protein associated with the intraflagellar transport machinery. We are currently investigating the full extent of the mutation spectrum. Our findings are crucial in expanding the understanding of childhood blindness and identifying new genes. PMID:23683095

  4. The SLC6 transporters: perspectives on structure, functions, regulation, and models for transporter dysfunction

    PubMed Central

    Rudnick, Gary; Krämer, Reinhard; Blakely, Randy D.; Murphy, Dennis L.

    2014-01-01

    The human SLC6 family is composed of approximately 20 structurally related symporters (co-transporters) that use the transmembrane electrochemical gradient to actively import their substrates into cells. Approximately half of the substrates of these transporters are amino acids, with others transporting biogenic amines and/or closely related compounds, such as nutrients and compatible osmolytes. In this short review, five leaders in the field discuss a number of currently important research themes that involve SLC6 transporters, highlighting the integrative role they play across a wide spectrum of different functions. The first essay, by Gary Rudnick, describes the molecular mechanism of their coupled transport which is being progressively better understood based on new crystal structures, functional studies, and modeling. Next, the question of multiple levels of transporter regulation is discussed by Reinhard Krämer, in the context of osmoregulation and stress response by the related bacterial betaine transporter BetP. The role of selected members of the human SLC6 family that function as nutrient amino acid transporters is then reviewed by François Verrey. He discusses how some of these transporters mediate the active uptake of (essential) amino acids into epithelial cells of the gut and the kidney tubule to support systemic amino acid requirements, whereas others are expressed in specific cells to support their specialized metabolism and/or growth. The most extensively studied members of the human SLC6 family are neurotransmitter reuptake transporters, many of which are important drug targets for the treatment of neuropsychiatric disorders. Randy Blakely discusses the role of posttranscriptional modifications of these proteins in regulating transporter subcellular localization and activity state. Finally, Dennis Murphy reviews how natural gene variants and mouse genetic models display consistent behavioral alterations that relate to altered extracellular

  5. Functional droplets that recognize, collect, and transport debris on surfaces

    PubMed Central

    Bai, Ying; Chang, Chia-Chih; Choudhary, Umesh; Bolukbasi, Irem; Crosby, Alfred J.; Emrick, Todd

    2016-01-01

    We describe polymer-stabilized droplets capable of recognizing and picking up nanoparticles from substrates in experiments designed for transporting hydroxyapatite nanoparticles that represent the principal elemental composition of bone. Our experiments, which are inspired by cells that carry out materials transport in vivo, used oil-in-water droplets that traverse a nanoparticle-coated substrate driven by an imposed fluid flow. Nanoparticle capture is realized by interaction of the particles with chemical functionality embedded within the polymeric stabilizing layer on the droplets. Nanoparticle uptake efficiency is controlled by solution conditions and the extent of functionality available for contact with the nanoparticles. Moreover, in an elementary demonstration of nanoparticle transportation, particles retrieved initially from the substrate were later deposited “downstream,” illustrating a pickup and drop-off technique that represents a first step toward mimicking point-to-point transportation events conducted in living systems.

  6. Transportation functions of the Civilian Radioactive Waste Management System

    SciTech Connect

    Shappert, L.B.; Attaway, C.R.; Pope, R.B.; Best, R.E.; Danese, F.L.; Dixon, L.D.; Jones, R.H.; Klimas, M.J.; Peterson, R.W.

    1992-03-01

    Within the framework of Public Law 97.425 and provisions specified in the Code of Federal Regulations, Title 10 Part 961, the US Department of Energy has the responsibility to accept and transport spent fuel and high-level waste from various organizations which have entered into a contract with the federal government in a manner that protects the health and safety of the public and workers. In implementing these requirements, the Office of Civilian Radioactive Waste Management (OCRWM) has, among other things, supported the identification of functions that must be performed by a transportation system (TS) that will accept the waste for transport to a federal facility for storage and/or disposal. This document, through the application of system engineering principles, identifies the functions that must be performed to transport waste under this law.

  7. Adenine nucleotide transporters in organelles: novel genes and functions.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2011-04-01

    In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.

  8. Work functions and transport properties of finite metallic hexaboride nanorods

    NASA Astrophysics Data System (ADS)

    Wang, Lu; Luo, Guangfu; Sabirianov, Renat F.; Mei, Wai-Ning; Valencia, Daniel; Sierra Llavina, Carlos H.; Lu, Jun-Qiang; Cheung, Chin Li

    2014-03-01

    We performed density functional theory calculations of finite metallic hexaboride LaB6 nanorods, which are regarded as good thermoelectric materials for their low work functions. Our purpose is to facilitate the research and manufacture of metal hexaboride probes, thus we study extensively the work functions and electron transport properties of these finite nanorods. The work functions were deducted from the calculated electrostatic potential and the Fermi energy. We found that these finite LaB6 nanorods have low work functions similar to their infinite counterpart. To further investigate the electron transport properties, we adopted the combined Landauer-Buttiker formalism and non-equilibrium Green's function technique to compute the transmission coefficients near the Fermi level and found that the finite LaB6 nanorods can be converted from metallic to semiconducting by applying a gate voltage larger than 10 V.

  9. Structure and Function of SLC4 Family HCO3- Transporters

    PubMed Central

    Liu, Ying; Yang, Jichun; Chen, Li-Ming

    2015-01-01

    The solute carrier SLC4 family consists of 10 members, nine of which are HCO3- transporters, including three Na+-independent Cl−/HCO3- exchangers AE1, AE2, and AE3, five Na+-coupled HCO3- transporters NBCe1, NBCe2, NBCn1, NBCn2, and NDCBE, as well as “AE4” whose Na+-dependence remains controversial. The SLC4 HCO3- transporters play critical roles in pH regulation and transepithelial movement of electrolytes with a broad range of demonstrated physiological relevances. Dysfunctions of these transporters are associated with a series of human diseases. During the past decades, tremendous amount of effort has been undertaken to investigate the topological organization of the SLC4 transporters in the plasma membrane. Based upon the proposed topology models, mutational and functional studies have identified important structural elements likely involved in the ion translocation by the SLC4 transporters. In the present article, we review the advances during the past decades in understanding the structure and function of the SLC4 transporters. PMID:26648873

  10. Hedgehog Signaling Regulates the Ciliary Transport of Odorant Receptors in Drosophila.

    PubMed

    Sanchez, Gonzalo M; Alkhori, Liza; Hatano, Eduardo; Schultz, Sebastian W; Kuzhandaivel, Anujaianthi; Jafari, Shadi; Granseth, Björn; Alenius, Mattias

    2016-01-26

    Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. Here, we show that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). We demonstrate that this is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). We further demonstrate that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand our knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking.

  11. Co-existence of Functionally Different Vesicular Neurotransmitter Transporters

    PubMed Central

    Münster-Wandowski, Agnieszka; Zander, Johannes-Friedrich; Richter, Karin; Ahnert-Hilger, Gudrun

    2016-01-01

    The vesicular transmitter transporters VGLUT, VGAT, VMAT2 and VAChT, define phenotype and physiological properties of neuronal subtypes. VGLUTs concentrate the excitatory amino acid glutamate, VGAT the inhibitory amino acid GABA, VMAT2 monoamines, and VAChT acetylcholine (ACh) into synaptic vesicle (SV). Following membrane depolarization SV release their content into the synaptic cleft. A strict segregation of vesicular transporters is mandatory for the precise functioning of synaptic communication and of neuronal circuits. In the last years, evidence accumulates that subsets of neurons express more than one of these transporters leading to synaptic co-release of different and functionally opposing transmitters and modulation of synaptic plasticity. Synaptic co-existence of transporters may change during pathological scenarios in order to ameliorate misbalances in neuronal activity. In addition, evidence increases that transporters also co-exist on the same vesicle providing another layer of regulation. Generally, vesicular transmitter loading relies on an electrochemical gradient ΔμH+ driven by the proton ATPase rendering the lumen of the vesicle with respect to the cytosol positive (Δψ) and acidic (ΔpH). While the activity of VGLUT mainly depends on the Δψ component, VMAT, VGAT and VAChT work best at a high ΔpH. Thus, a vesicular synergy of transporters depending on the combination may increase or decrease the filling of SV with the principal transmitter. We provide an overview on synaptic co-existence of vesicular transmitter transporters including changes in the excitatory/inhibitory balance under pathological conditions. Additionally, we discuss functional aspects of vesicular synergy of transmitter transporters. PMID:26909036

  12. Transport properties of water at functionalized molecular interfaces

    NASA Astrophysics Data System (ADS)

    Feng, Jun; Wong, Ka-Yiu; Dyer, Kippi; Pettitt, B. Montgomery

    2009-09-01

    Understanding transport properties of solvent such as diffusion and viscosity at interfaces with biomacromolecules and hard materials is of fundamental importance to both biology and biotechnology. Our study utilizes equilibrium molecular dynamics simulations to calculate solvent transport properties at a model peptide and microarray surface. Both diffusion and selected components of viscosity are considered. Solvent diffusion is found to be affected near the peptide and surface. The stress-stress correlation function of solvent near the hard surface exhibits long time memory. Both diffusion and viscosity are shown to be closely correlated with the density distribution function of water along the microarray surface.

  13. Sodium-Dependent Phosphate Transporters in Osteoclast Differentiation and Function

    PubMed Central

    Dolder, Silvia; Siegrist, Mark; Wagner, Carsten A.; Biber, Jürg; Hernando, Nati; Hofstetter, Willy

    2015-01-01

    Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies. PMID:25910236

  14. Sodium-dependent phosphate transporters in osteoclast differentiation and function.

    PubMed

    Albano, Giuseppe; Moor, Matthias; Dolder, Silvia; Siegrist, Mark; Wagner, Carsten A; Biber, Jürg; Hernando, Nati; Hofstetter, Willy; Bonny, Olivier; Fuster, Daniel G

    2015-01-01

    Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

  15. Adaptively Learning an Importance Function Using Transport Constrained Monte Carlo

    SciTech Connect

    Booth, T.E.

    1998-06-22

    It is well known that a Monte Carlo estimate can be obtained with zero-variance if an exact importance function for the estimate is known. There are many ways that one might iteratively seek to obtain an ever more exact importance function. This paper describes a method that has obtained ever more exact importance functions that empirically produce an error that is dropping exponentially with computer time. The method described herein constrains the importance function to satisfy the (adjoint) Boltzmann transport equation. This constraint is provided by using the known form of the solution, usually referred to as the Case eigenfunction solution.

  16. Functional interaction between autophagy and ciliogenesis

    PubMed Central

    Pampliega, Olatz; Orhon, Idil; Patel, Bindi; Sridhar, Sunandini; Díaz-Carretero, Antonio; Beau, Isabelle; Codogno, Patrice; Satir, Birgit; Satir, Peter; Cuervo, Ana Maria

    2014-01-01

    Summary Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signaling motivated us to explore the possible functional interactions between this signaling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signaling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. While abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signaling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie the basis of some common ciliopathies. PMID:24089209

  17. The straggling Green's function method for ion transport

    NASA Astrophysics Data System (ADS)

    Walker, Steven Andrew

    For many years work has been conducted on developing a concise theory and method for HZE ion transport capable of being validated in the laboratory. Previous attempts have ignored dispersion and energy downshift associated with nuclear fragmentation and energy and range straggling. Here we present a Green's function approach to ion transport that incorporates these missing elements. This work forms the basis for a new version of GRNTRN, a Green's function transport code. Comparisons of GRNTRN predictions and laboratory results for an 56Fe ion beam with average energy at the target of one GeV/amu or more are presented for various targets. Quantities compared are the energy deposited spectra for an Aluminum target and Graphite-Epoxy mix target, the fraction of primary beam surviving and track average LET for these and various other targets.

  18. Discovery of CLC transport proteins: cloning, structure, function and pathophysiology

    PubMed Central

    Jentsch, Thomas J

    2015-01-01

    Abstract After providing a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl− channel ClC-0 and the discovery of the CLC gene family, I succinctly describe the general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl− channels and vesicular Cl−/H+-exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel. PMID:25590607

  19. Discovery of CLC transport proteins: cloning, structure, function and pathophysiology.

    PubMed

    Jentsch, Thomas J

    2015-09-15

    After providing a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl(-) channel ClC-0 and the discovery of the CLC gene family, I succinctly describe the general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl(-) channels and vesicular Cl(-) /H(+) -exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel. PMID:25590607

  20. Ion transport controlled by nanoparticle-functionalized membranes.

    PubMed

    Barry, Edward; McBride, Sean P; Jaeger, Heinrich M; Lin, Xiao-Min

    2014-12-17

    From proton exchange membranes in fuel cells to ion channels in biological membranes, the well-specified control of ionic interactions in confined geometries profoundly influences the transport and selectivity of porous materials. Here we outline a versatile new approach to control a membrane's electrostatic interactions with ions by depositing ligand-coated nanoparticles around the pore entrances. Leveraging the flexibility and control by which ligated nanoparticles can be synthesized, we demonstrate how ligand terminal groups such as methyl, carboxyl and amine can be used to tune the membrane charge density and control ion transport. Further functionality, exploiting the ligands as binding sites, is demonstrated for sulfonate groups resulting in an enhancement of the membrane charge density. We then extend these results to smaller dimensions by systematically varying the underlying pore diameter. As a whole, these results outline a previously unexplored method for the nanoparticle functionalization of membranes using ligated nanoparticles to control ion transport.

  1. Density Functional Theory with Dissipation: Transport through Single Molecules

    SciTech Connect

    Kieron Burke

    2012-04-30

    A huge amount of fundamental research was performed on this grant. Most of it focussed on fundamental issues of electronic structure calculations of transport through single molecules, using density functional theory. Achievements were: (1) First density functional theory with dissipation; (2) Pseudopotential plane wave calculations with master equation; (3) Weak bias limit; (4) Long-chain conductance; and (5) Self-interaction effects in tunneling.

  2. Adjoint Function: Physical Basis of Variational & Perturbation Theory in Transport

    2009-07-27

    Version 00 Dr. J.D. Lewins has now released the following legacy book for free distribution: Importance: The Adjoint Function: The Physical Basis of Variational and Perturbation Theory in Transport and Diffusion Problems, North-Holland Publishing Company - Amsterdam, 582 pages, 1966 Introduction: Continuous Systems and the Variational Principle 1. The Fundamental Variational Principle 2. The Importance Function 3. Adjoint Equations 4. Variational Methods 5. Perturbation and Iterative Methods 6. Non-Linear Theory

  3. Design Function and Structure of a Monomeric CLC Transporter

    SciTech Connect

    L Robertson; L Kolmakova-Partensky; C Miller

    2011-12-31

    Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual - the generation of the kilowatt pulses with which electric fish stun their prey - to the quotidian - the acidification of endosomes, vacuoles and lysosomes. The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch Cl{sup -} channel and later confirmed by crystal structures of bacterial Cl{sup -}/H{sup +} antiporters, is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl{sup -} and H{sup +} antiport in the transporters - are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent crosslinks across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity, we cannot declare as definitive a 'parallel-pathways picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC Cl{sup -}/H{sup +} exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not

  4. Intracellular delivery of functional proteins via decoration with transporter peptides.

    PubMed

    Siprashvili, Zurab; Reuter, Jason A; Khavari, Paul A

    2004-05-01

    Despite numerous attractive intracellular targets, protein therapeutics have been principally confined to the extracellular space due to the lack of a straightforward way to deliver functional polypeptides to the cell interior. Peptide sequences facilitating intracellular protein delivery have been identified; however, current strategies to apply them require problematic steps, such as generation of new in-frame fusion proteins, covalent chemical conjugation, and denaturation. We have developed a new approach to protein transfer into cells and tissues that relies on single-step decoration by cysteine-flanked, arginine-rich transporter peptides. This approach facilitated cell and tissue delivery of a variety of functional proteins, including antibodies and enzymes. Decoration with transporter peptides thus provides an attractive general means of intracellular delivery of functional proteins in vitro and in tissue.

  5. Stepwise Functional Evolution in a Fungal Sugar Transporter Family.

    PubMed

    Gonçalves, Carla; Coelho, Marco A; Salema-Oom, Madalena; Gonçalves, Paula

    2016-02-01

    Sugar transport is of the utmost importance for most cells and is central to a wide range of applied fields. However, despite the straightforward in silico assignment of many novel transporters, including sugar porters, to existing families, their exact biological role and evolutionary trajectory often remain unclear, mainly because biochemical characterization of membrane proteins is inherently challenging, but also owing to their uncommonly turbulent evolutionary histories. In addition, many important shifts in membrane carrier function are apparently ancient, which further limits our ability to reconstruct evolutionary trajectories in a reliable manner. Here, we circumvented some of these obstacles by examining the relatively recent emergence of a unique family of fungal sugar facilitators, related to drug antiporters. The former transporters, named Ffz, were previously shown to be required for fructophilic metabolism in yeasts. We first exploited the wealth of fungal genomic data available to define a comprehensive but well-delimited family of Ffz-like transporters, showing that they are only present in Dikarya. Subsequently, a combination of phylogenetic analyses and in vivo functional characterization was used to retrace important changes in function, while highlighting the evolutionary events that are most likely to have determined extant distribution of the gene, such as horizontal gene transfers (HGTs). One such HGT event is proposed to have set the stage for the onset of fructophilic metabolism in yeasts, a trait that according to our results may be the metabolic hallmark of close to 100 yeast species that thrive in sugar rich environments. PMID:26474848

  6. Stepwise Functional Evolution in a Fungal Sugar Transporter Family.

    PubMed

    Gonçalves, Carla; Coelho, Marco A; Salema-Oom, Madalena; Gonçalves, Paula

    2016-02-01

    Sugar transport is of the utmost importance for most cells and is central to a wide range of applied fields. However, despite the straightforward in silico assignment of many novel transporters, including sugar porters, to existing families, their exact biological role and evolutionary trajectory often remain unclear, mainly because biochemical characterization of membrane proteins is inherently challenging, but also owing to their uncommonly turbulent evolutionary histories. In addition, many important shifts in membrane carrier function are apparently ancient, which further limits our ability to reconstruct evolutionary trajectories in a reliable manner. Here, we circumvented some of these obstacles by examining the relatively recent emergence of a unique family of fungal sugar facilitators, related to drug antiporters. The former transporters, named Ffz, were previously shown to be required for fructophilic metabolism in yeasts. We first exploited the wealth of fungal genomic data available to define a comprehensive but well-delimited family of Ffz-like transporters, showing that they are only present in Dikarya. Subsequently, a combination of phylogenetic analyses and in vivo functional characterization was used to retrace important changes in function, while highlighting the evolutionary events that are most likely to have determined extant distribution of the gene, such as horizontal gene transfers (HGTs). One such HGT event is proposed to have set the stage for the onset of fructophilic metabolism in yeasts, a trait that according to our results may be the metabolic hallmark of close to 100 yeast species that thrive in sugar rich environments.

  7. Structural and functional heterogeneity in phloem loading and transport

    PubMed Central

    Slewinski, Thomas L.; Zhang, Cankui; Turgeon, Robert

    2013-01-01

    The phloem is often regarded as a relatively straightforward transport system composed of loading (collection), long-distance (transport), and unloading (release) zones. While this simple view is necessary and useful in many contexts, it belies the reality, which is that the phloem is inherently complex. At least three types of sieve element–companion cell complexes are found in minor veins of leaves. Individual species may have more than one type, indicating that they employ multiple loading strategies, even in the same vein. Gene expression data in particular point to heterogeneity in sieve element–companion cell complexes of minor veins, perhaps in all flowering plants. Phloem heterogeneity in the transport phloem is also evident in many species based on anatomical, biochemical and gene expression data. In this regard, members of the Cucurbitaceae are especially complex and interesting. We conclude that a hidden world of specialized phloem function awaits discovery. PMID:23847646

  8. Function of prokaryotic and eukaryotic ABC proteins in lipid transport.

    PubMed

    Pohl, Antje; Devaux, Philippe F; Herrmann, Andreas

    2005-03-21

    ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry. PMID:15749056

  9. Functional characterization of a xylose transporter in Aspergillus nidulans

    PubMed Central

    2014-01-01

    Background The production of bioethanol from lignocellulosic feedstocks will only become economically feasible when the majority of cellulosic and hemicellulosic biopolymers can be efficiently converted into bioethanol. The main component of cellulose is glucose, whereas hemicelluloses mainly consist of pentose sugars such as D-xylose and L-arabinose. The genomes of filamentous fungi such as A. nidulans encode a multiplicity of sugar transporters with broad affinities for hexose and pentose sugars. Saccharomyces cerevisiae, which has a long history of use in industrial fermentation processes, is not able to efficiently transport or metabolize pentose sugars (e.g. xylose). Subsequently, the aim of this study was to identify xylose-transporters from A. nidulans, as potential candidates for introduction into S. cerevisiae in order to improve xylose utilization. Results In this study, we identified the A. nidulans xtrD (xylose transporter) gene, which encodes a Major Facilitator Superfamily (MFS) transporter, and which was specifically induced at the transcriptional level by xylose in a XlnR-dependent manner, while being partially repressed by glucose in a CreA-dependent manner. We evaluated the ability of xtrD to functionally complement the S. cerevisiae EBY.VW4000 strain which is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae, XtrD was targeted to the plasma membrane and its expression was able to restore growth on xylose, glucose, galactose, and mannose as single carbon sources, indicating that this transporter accepts multiple sugars as a substrate. XtrD has a high affinity for xylose, and may be a high affinity xylose transporter. We were able to select a S. cerevisiae mutant strain that had increased xylose transport when expressing the xtrD gene. Conclusions This study characterized the regulation and substrate specificity of an A. nidulans transporter that represents a good candidate for further directed

  10. Acrolein impairs the cholesterol transport functions of high density lipoproteins.

    PubMed

    Chadwick, Alexandra C; Holme, Rebecca L; Chen, Yiliang; Thomas, Michael J; Sorci-Thomas, Mary G; Silverstein, Roy L; Pritchard, Kirkwood A; Sahoo, Daisy

    2015-01-01

    High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.

  11. Transport Function of Rice Amino Acid Permeases (AAPs).

    PubMed

    Taylor, Margaret R; Reinders, Anke; Ward, John M

    2015-07-01

    The transport function of four rice (Oryza sativa) amino acid permeases (AAPs), OsAAP1 (Os07g04180), OsAAP3 (Os06g36180), OsAAP7 (Os05g34980) and OsAAP16 (Os12g08090), was analyzed by expression in Xenopus laevis oocytes and electrophysiology. OsAAP1, OsAAP7 and OsAAP16 functioned, similarly to Arabidopsis AAPs, as general amino acid permeases. OsAAP3 had a distinct substrate specificity compared with other rice or Arabidopsis AAPs. OsAAP3 transported the basic amino acids lysine and arginine well but selected against aromatic amino acids. The transport of basic amino acids was further analyzed for OsAAP1 and OsAAP3, and the results support the transport of both neutral and positively charged forms of basic amino acids by the rice AAPs. Cellular localization using the tandem enhanced green fluorescent protein (EGFP)-red fluorescent protein (RFP) reporter pHusion showed that OsAAP1 and OsAAP3 localized to the plasma membrane after transient expression in onion epidermal cells or stable expression in Arabidopsis. PMID:25907566

  12. Functional Monomerization of a ClC-Type Fluoride Transporter

    PubMed Central

    Last, Nicholas B.; Miller, Christopher

    2016-01-01

    Anion channels and antiporters of the ClC superfamily have been found to be exclusively dimeric in nature, even though each individual monomer contains the complete transport pathway. Here, we describe the destabilization through mutagenesis of the dimer interface of a bacterial F−/H+ antiporter, ClCF-eca. Several mutations that produce monomer/dimer equilibrium of the normally dimeric transporter were found, simply by shortening a hydrophobic side chain in some cases. One mutation, L376W, leads to a wholly monomeric variant that shows full activity. Furthermore, we discovered a naturally destabilized homologue, ClCF-rla, which undergoes partial monomerization in detergent without additional mutations. These results, in combination with the previous functional monomerization of the distant relative ClC-ec1, demonstrate that the monomer alone is the functional unit for several clades of the ClC superfamily. PMID:26449639

  13. Approximate Green's function methods for HZE transport in multilayered materials

    NASA Technical Reports Server (NTRS)

    Wilson, John W.; Badavi, Francis F.; Shinn, Judy L.; Costen, Robert C.

    1993-01-01

    A nonperturbative analytic solution of the high charge and energy (HZE) Green's function is used to implement a computer code for laboratory ion beam transport in multilayered materials. The code is established to operate on the Langley nuclear fragmentation model used in engineering applications. Computational procedures are established to generate linear energy transfer (LET) distributions for a specified ion beam and target for comparison with experimental measurements. The code was found to be highly efficient and compared well with the perturbation approximation.

  14. Controlled Transport of Functionalized Nanochannel though Lipid Membrane

    NASA Astrophysics Data System (ADS)

    Dutt, Meenakshi; Kuksenok, Olga; Balazs, Anna C.

    2012-02-01

    Via the Dissipative Particle Dynamics approach, we study the directed transport of a transmembrane nanochannel to a desired location within a lipid bilayer. Each nanochannel encompasses an ABA architecture, with a hydrophobic shaft (B) with two hydrophilic ends (A). One of the ends of the nanochannel is functionalized with hydrophilic functional groups, or hairs. The hydrophilic hairs serve a dual role: (a) control transport across the membrane barrier, and (b) enable the channel relocation to a specific membrane site. Our system comprises a lipid membrane with an embedded transmembrane nanochannel with the hairs extending into solution. First, we hold a suitably functionalized pipette above the membrane while the nanochannel freely diffuses within the membrane. For an optimal range of parameters, we demonstrate that the hairs find the pipette and spontaneously anchor onto it. We then show that by moving the pipette for a range of velocities, we can effectively transport the channel to any location within the membrane. This prototype assembly can provide guidelines for designing a number of systems for biomimetic applications.

  15. Membrane Receptors and Transporters Involved in the Function and Transport of Vitamin A and Its Derivatives

    PubMed Central

    Sun, Hui

    2011-01-01

    The eye is the human organ most sensitive to vitamin A deficiency because of vision's absolute and heavy dependence on vitamin A for light perception. Studies of the molecular basis of vision have provided important insights into the intricate mechanistic details of the function, transport and recycling of vitamin A and its derivatives (retinoid). This review focuses on retinoid-related membrane receptors and transporters. Three kinds of mammalian membrane receptors and transporters are discussed: opsins, best known as vitamin A-based light sensors in vision; ABCA4, an ATP-dependent transporter specializes in the transport of vitamin A derivative; and STRA6, a recently identified membrane receptor that mediates cellular uptake of vitamin A. The evolutionary driving forces for their existence and the wide spectrum of human diseases associated with these proteins are discussed. Lessons learned from the study of the visual system might be useful for understanding retinoid biology and retinoid-related diseases in other organ systems as well. PMID:21704730

  16. A Green's function method for heavy ion beam transport

    NASA Technical Reports Server (NTRS)

    Shinn, J. L.; Wilson, J. W.; Schimmerling, W.; Shavers, M. R.; Miller, J.; Benton, E. V.; Frank, A. L.; Badavi, F. F.

    1995-01-01

    The use of Green's function has played a fundamental role in transport calculations for high-charge high-energy (HZE) ions. Two recent developments have greatly advanced the practical aspects of implementation of these methods. The first was the formulation of a closed-form solution as a multiple fragmentation perturbation series. The second was the effective summation of the closed-form solution through nonperturbative techniques. The nonperturbative methods have been recently extended to an inhomogeneous, two-layer transport media to simulate the lead scattering foil present in the Lawrence Berkeley Laboratories (LBL) biomedical beam line used for cancer therapy. Such inhomogeneous codes are necessary for astronaut shielding in space. The transport codes utilize the Langley Research Center atomic and nuclear database. Transport code and database evaluation are performed by comparison with experiments performed at the LBL Bevalac facility using 670 A MeV 20Ne and 600 A MeV 56Fe ion beams. The comparison with a time-of-flight and delta E detector measurement for the 20Ne beam and the plastic nuclear track detectors for 56Fe show agreement up to 35%-40% in water and aluminium targets, respectively.

  17. A Green's function method for heavy ion beam transport.

    PubMed

    Shinn, J L; Wilson, J W; Schimmerling, W; Shavers, M R; Miller, J; Benton, E V; Frank, A L; Badavi, F F

    1995-08-01

    The use of Green's function has played a fundamental role in transport calculations for high-charge high-energy (HZE) ions. Two recent developments have greatly advanced the practical aspects of implementation of these methods. The first was the formulation of a closed-form solution as a multiple fragmentation perturbation series. The second was the effective summation of the closed-form solution through nonperturbative techniques. The nonperturbative methods have been recently extended to an inhomogeneous, two-layer transport media to simulate the lead scattering foil present in the Lawrence Berkeley Laboratories (LBL) biomedical beam line used for cancer therapy. Such inhomogeneous codes are necessary for astronaut shielding in space. The transport codes utilize the Langley Research Center atomic and nuclear database. Transport code and database evaluation are performed by comparison with experiments performed at the LBL Bevalac facility using 670 A MeV 20Ne and 600 A MeV 56Fe ion beams. The comparison with a time-of-flight and delta E detector measurement for the 20Ne beam and the plastic nuclear track detectors for 56Fe show agreement up to 35%-40% in water and aluminium targets, respectively. PMID:7480630

  18. Molecular approach to intracellular cargo transport

    NASA Astrophysics Data System (ADS)

    Yildiz, Ahmet

    2010-03-01

    Landmark discoveries in the study of cytoplasmic motors have been made through advances in single molecule biophysics and detailed mechanistic models exist for kinesin and dynein. However, the function of motors in physiological conditions has not been carefully tested. In cells, more than few dyneins can attach to the same cargo and interact with the opposite polarity motors of kinesin. To study the molecular crosstalk between the motors, we have used intraflagellar transport (IFT) in Chlamydomonas reinhardtii as a model system. Ultrahigh spatio-temporal tracking of single cargo movement showed that IFT particles move for long distances unidirectionally with 8 nm increments, agreeing with measured step sizes of kinesin and dynein. To measure how many motors transport each cargo, we have linked large polystyrene beads to internal IFT particles through a transmembrane protein. Force measurements indicated that, on average, 3-4 motors transport cargoes in each direction. The results showed that IFT motors are tightly coordinated and might be involved in recycling each other to the appropriate end of the flagellum.

  19. Chloroplast Iron Transport Proteins - Function and Impact on Plant Physiology.

    PubMed

    López-Millán, Ana F; Duy, Daniela; Philippar, Katrin

    2016-01-01

    Chloroplasts originated about three billion years ago by endosymbiosis of an ancestor of today's cyanobacteria with a mitochondria-containing host cell. During evolution chloroplasts of higher plants established as the site for photosynthesis and thus became the basis for all life dependent on oxygen and carbohydrate supply. To fulfill this task, plastid organelles are loaded with the transition metals iron, copper, and manganese, which due to their redox properties are essential for photosynthetic electron transport. In consequence, chloroplasts for example represent the iron-richest system in plant cells. However, improvement of oxygenic photosynthesis in turn required adaptation of metal transport and homeostasis since metal-catalyzed generation of reactive oxygen species (ROS) causes oxidative damage. This is most acute in chloroplasts, where radicals and transition metals are side by side and ROS-production is a usual feature of photosynthetic electron transport. Thus, on the one hand when bound by proteins, chloroplast-intrinsic metals are a prerequisite for photoautotrophic life, but on the other hand become toxic when present in their highly reactive, radical generating, free ionic forms. In consequence, transport, storage and cofactor-assembly of metal ions in plastids have to be tightly controlled and are crucial throughout plant growth and development. In the recent years, proteins for iron transport have been isolated from chloroplast envelope membranes. Here, we discuss their putative functions and impact on cellular metal homeostasis as well as photosynthetic performance and plant metabolism. We further consider the potential of proteomic analyses to identify new players in the field. PMID:27014281

  20. Analysis of Transport Through a Fault Using Transfer Functions

    NASA Astrophysics Data System (ADS)

    Su, G. W.; Salve, R.

    2003-12-01

    Understanding flow and transport in unsaturated fractured rock (i.e., matrix and fracture flow, and fracture-matrix interactions) is important for performance assessment and the design of the proposed radioactive waste repository at Yucca Mountain, Nevada. A key factor affecting performance of the proposed repository is the transport of radionuclides through unsaturated fractured rock that lies between the repository horizon and water table located ~300 m below. Of particular importance is the need for an understanding of diffusive mass transfer between high-permeability, advection-dominated domains and low-permeability domains. An in situ field experiment was conducted in the Exploratory Studies Facility at Yucca Mountain, Nevada to examine flow and transport in the vicinity of a fault located in unsaturated fractured rock. This experiment involved the release of ~75,000 liters of ponded water over a period of 14 months directly into a near-vertical fault located in the fractured welded tuff of the Topopah Spring Tuff unit. Seepage rates were monitored in a large cavity (niche) excavated 20 m below where the water was released along the fault. Seven months after water was introduced, two conservative tracers (pentafluorobenzoic acid [PFBA] and bromide) were simultaneously released along the fault over a period of nine days. After the release of the tracers, seepage water was continuously collected from three locations in the niche and analyzed for the injected tracers. The results from this field experiment demonstrate that flow and transport near a fault located in unsaturated fractured rock is complex due to mechanisms such as dynamic flow behavior. Continuum-based models may not be applicable when these types of mechanisms affect flow and transport. Measured breakthrough curves from this field experiment are analyzed using transfer functions as an alternative to the continuum-based models, and the applicability of using transfer functions to describe transport

  1. Current approaches to enhance glutamate transporter function and expression.

    PubMed

    Fontana, Andréia C K

    2015-09-01

    L-glutamate is the predominant excitatory neurotransmitter in the CNS and has a central role in a variety of brain functions. The termination of glutamate neurotransmission by excitatory amino acid transporters (EAATs) is essential to maintain glutamate concentration low in extracellular space and avoid excitotoxicity. EAAT2/GLT-1, being the most abundant subtype of glutamate transporter in the CNS, plays a key role in regulation of glutamate transmission. Dysfunction of EAAT2 has been correlated with various pathologies such as traumatic brain injury, stroke, amyotrophic lateral sclerosis, Alzheimer's disease, among others. Therefore, activators of the function or enhancers of the expression of EAAT2/GLT-1 could serve as a potential therapy for these conditions. Translational activators of EAAT2/GLT-1, such as ceftriaxone and LDN/OSU-0212320, have been described to have significant protective effects in animal models of amyotrophic lateral sclerosis and epilepsy. In addition, pharmacological activators of the activity of EAAT2/GLT-1 have been explored for decades and are currently emerging as promising tools for neuroprotection, having potential advantages over expression activators. This review describes the current status of the search for EAAT2/GLT-1 activators and addresses challenges and limitations that this approach might encounter. Termination of glutamate neurotransmission by glutamate transporter EAAT2 is essential to maintain homeostasis in the brain and to avoid excitotoxicity. Dysfunction of EAAT2 has been correlated with various neurological pathologies. Therefore, activators of the function or enhancers of the expression of EAAT2 (green arrows) could serve as a potential therapy for these conditions. This review describes the current status of the search for EAAT2 activators and addresses challenges and limitations of this approach. PMID:26096891

  2. Diverse functional roles of monosaccharide transporters and their homologs in vascular plants: a physiological perspective.

    PubMed

    Slewinski, Thomas L

    2011-07-01

    Vascular plants contain two gene families that encode monosaccharide transporter proteins. The classical monosaccharide transporter(-like) gene superfamily is large and functionally diverse, while the recently identified SWEET transporter family is smaller and, thus far, only found to transport glucose. These transporters play essential roles at many levels, ranging from organelles to the whole plant. Many family members are essential for cellular homeostasis and reproductive success. Although most transporters do not directly participate in long-distance transport, their indirect roles greatly impact carbon allocation and transport flux to the heterotrophic tissues of the plant. Functional characterization of some members from both gene families has revealed their diverse roles in carbohydrate partitioning, phloem function, resource allocation, plant defense, and sugar signaling. This review highlights the broad impacts and implications of monosaccharide transport by describing some of the functional roles of the monosaccharide transporter(-like) superfamily and the SWEET transporter family.

  3. Functional expression of sodium-glucose transporters in cancer

    PubMed Central

    Scafoglio, Claudio; Hirayama, Bruce A.; Kepe, Vladimir; Liu, Jie; Ghezzi, Chiara; Satyamurthy, Nagichettiar; Moatamed, Neda A.; Huang, Jiaoti; Koepsell, Hermann; Barrio, Jorge R.; Wright, Ernest M.

    2015-01-01

    Glucose is a major metabolic substrate required for cancer cell survival and growth. It is mainly imported into cells by facilitated glucose transporters (GLUTs). Here we demonstrate the importance of another glucose import system, the sodium-dependent glucose transporters (SGLTs), in pancreatic and prostate adenocarcinomas, and investigate their role in cancer cell survival. Three experimental approaches were used: (i) immunohistochemical mapping of SGLT1 and SGLT2 distribution in tumors; (ii) measurement of glucose uptake in fresh isolated tumors using an SGLT-specific radioactive glucose analog, α-methyl-4-deoxy-4-[18F]fluoro-d-glucopyranoside (Me4FDG), which is not transported by GLUTs; and (iii) measurement of in vivo SGLT activity in mouse models of pancreatic and prostate cancer using Me4FDG-PET imaging. We found that SGLT2 is functionally expressed in pancreatic and prostate adenocarcinomas, and provide evidence that SGLT2 inhibitors block glucose uptake and reduce tumor growth and survival in a xenograft model of pancreatic cancer. We suggest that Me4FDG-PET imaging may be used to diagnose and stage pancreatic and prostate cancers, and that SGLT2 inhibitors, currently in use for treating diabetes, may be useful for cancer therapy. PMID:26170283

  4. Characteristics and Possible Functions of Mitochondrial Ca2+ Transport Mechanisms

    PubMed Central

    Gunter, Thomas E.; Sheu, Shey-Shing

    2009-01-01

    Mitochondria produce around 92% of the ATP used in the typical animal cell by oxidative phosphorylation using energy from their electrochemical proton gradient. Intramitochondrial free Ca2+ concentration ([Ca2+]m) has been found to be an important component of control of the rate of this ATP production. In addition, [Ca2+]m also controls the opening of a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP), which plays a role in mitochondrial control of programmed cell death or apoptosis. Therefore, [Ca2+]m can control whether the cell has sufficient ATP to fulfill its functions and survive or is condemned to death. Ca2+ is also one of the most important second messengers within the cytosol, signaling changes in cellular response through Ca2+ pulses or transients. Mitochondria can also sequester Ca2+ from these transients so as to modify the shape of Ca2+ signaling transients or control their location within the cell. All of this is controlled by the action of four or five mitochondrial Ca2+ transport mechanisms and the PTP. The characteristics of these mechanisms of Ca2+ transport and a discussion of how they might function are described in this paper. PMID:19161975

  5. Platelet Serotonin Transporter Function Predicts Default-Mode Network Activity

    PubMed Central

    Kasess, Christian H.; Meyer, Bernhard M.; Hofmaier, Tina; Diers, Kersten; Bartova, Lucie; Pail, Gerald; Huf, Wolfgang; Uzelac, Zeljko; Hartinger, Beate; Kalcher, Klaudius; Perkmann, Thomas; Haslacher, Helmuth; Meyer-Lindenberg, Andreas; Kasper, Siegfried; Freissmuth, Michael; Windischberger, Christian; Willeit, Matthäus; Lanzenberger, Rupert; Esterbauer, Harald; Brocke, Burkhard; Moser, Ewald; Sitte, Harald H.; Pezawas, Lukas

    2014-01-01

    Background The serotonin transporter (5-HTT) is abundantly expressed in humans by the serotonin transporter gene SLC6A4 and removes serotonin (5-HT) from extracellular space. A blood-brain relationship between platelet and synaptosomal 5-HT reuptake has been suggested, but it is unknown today, if platelet 5-HT uptake can predict neural activation of human brain networks that are known to be under serotonergic influence. Methods A functional magnetic resonance study was performed in 48 healthy subjects and maximal 5-HT uptake velocity (Vmax) was assessed in blood platelets. We used a mixed-effects multilevel analysis technique (MEMA) to test for linear relationships between whole-brain, blood-oxygen-level dependent (BOLD) activity and platelet Vmax. Results The present study demonstrates that increases in platelet Vmax significantly predict default-mode network (DMN) suppression in healthy subjects independent of genetic variation within SLC6A4. Furthermore, functional connectivity analyses indicate that platelet Vmax is related to global DMN activation and not intrinsic DMN connectivity. Conclusion This study provides evidence that platelet Vmax predicts global DMN activation changes in healthy subjects. Given previous reports on platelet-synaptosomal Vmax coupling, results further suggest an important role of neuronal 5-HT reuptake in DMN regulation. PMID:24667541

  6. Filtered density function approach for reactive transport in groundwater

    NASA Astrophysics Data System (ADS)

    Suciu, Nicolae; Schüler, Lennart; Attinger, Sabine; Knabner, Peter

    2016-04-01

    Spatial filtering may be used in coarse-grained simulations (CGS) of reactive transport in groundwater, similar to the large eddy simulations (LES) in turbulence. The filtered density function (FDF), stochastically equivalent to a probability density function (PDF), provides a statistical description of the sub-grid, unresolved, variability of the concentration field. Besides closing the chemical source terms in the transport equation for the mean concentration, like in LES-FDF methods, the CGS-FDF approach aims at quantifying the uncertainty over the whole hierarchy of heterogeneity scales exhibited by natural porous media. Practically, that means estimating concentration PDFs on coarse grids, at affordable computational costs. To cope with the high dimensionality of the problem in case of multi-component reactive transport and to reduce the numerical diffusion, FDF equations are solved by particle methods. But, while trajectories of computational particles are modeled as stochastic processes indexed by time, the concentration's heterogeneity is modeled as a random field, with multi-dimensional, spatio-temporal sets of indices. To overcome this conceptual inconsistency, we consider FDFs/PDFs of random species concentrations weighted by conserved scalars and we show that their evolution equations can be formulated as Fokker-Planck equations describing stochastically equivalent processes in concentration-position spaces. Numerical solutions can then be approximated by the density in the concentration-position space of an ensemble of computational particles governed by the associated Itô equations. Instead of sequential particle methods we use a global random walk (GRW) algorithm, which is stable, free of numerical diffusion, and practically insensitive to the increase of the number of particles. We illustrate the general FDF approach and the GRW numerical solution for a reduced complexity problem consisting of the transport of a single scalar in groundwater

  7. Density Functional Theory Calculations of Mass Transport in UO2

    SciTech Connect

    Andersson, Anders D.; Dorado, Boris; Uberuaga, Blas P.; Stanek, Christopher R.

    2012-06-26

    In this talk we present results of density functional theory (DFT) calculations of U, O and fission gas diffusion in UO{sub 2}. These processes all impact nuclear fuel performance. For example, the formation and retention of fission gas bubbles induce fuel swelling, which leads to mechanical interaction with the clad thereby increasing the probability for clad breach. Alternatively, fission gas can be released from the fuel to the plenum, which increases the pressure on the clad walls and decreases the gap thermal conductivity. The evolution of fuel microstructure features is strongly coupled to diffusion of U vacancies. Since both U and fission gas transport rates vary strongly with the O stoichiometry, it is also important to understand O diffusion. In order to better understand bulk Xe behavior in UO{sub 2{+-}x} we first calculate the relevant activation energies using DFT techniques. By analyzing a combination of Xe solution thermodynamics, migration barriers and the interaction of dissolved Xe atoms with U, we demonstrate that Xe diffusion predominantly occurs via a vacancy-mediated mechanism. Since Xe transport is closely related to diffusion of U vacancies, we have also studied the activation energy for this process. In order to explain the low value of 2.4 eV found for U migration from independent damage experiments (not thermal equilibrium) the presence of vacancy clusters must be included in the analysis. Next we investigate species transport on the (111) UO{sub 2} surface, which is motivated by the formation of small voids partially filled with fission gas atoms (bubbles) in UO{sub 2} under irradiation. Surface diffusion could be the rate-limiting step for diffusion of such bubbles, which is an alternative mechanism for mass transport in these materials. As expected, the activation energy for surface diffusion is significantly lower than for bulk transport. These results are further discussed in terms of engineering-scale fission gas release models

  8. An Improved Green's Function for Ion Beam Transport

    NASA Technical Reports Server (NTRS)

    Tweed, J.; Wilson, J. W.; Tripathi, R. K.

    2003-01-01

    Ion beam transport theory allows testing of material transmission properties in the laboratory environment generated by particle accelerators. This is a necessary step in materials development and evaluation for space use. The approximations used in solving the Boltzmann transport equation for the space setting are often not sufficient for laboratory work and those issues are the main emphasis of the present work. In consequence, an analytic solution of the linear Boltzmann equation is pursued in the form of a Green's function allowing flexibility in application to a broad range of boundary value problems. It has been established that simple solutions can be found for the high charge and energy (HZE) by ignoring nuclear energy downshifts and dispersion. Such solutions were found to be supported by experimental evidence with HZE ion beams when multiple scattering was added. Lacking from the prior solutions were range and energy straggling and energy downshift with dispersion associated with nuclear events. Recently, we have found global solutions including these effects providing a broader class of HZE ion solutions.

  9. Magnetic Alignment and Charge Transport Improvement in Functional Soft Materials

    NASA Astrophysics Data System (ADS)

    Majewski, Pawel W.

    The realization of nanostructured functional materials by self-assembly in polymers and polymer nanocomposites is adversely affected by persisting structural defects which greatly diminish the performance of the material. The use of magnetic fields to impose long-range order is investigated in three distinct systems - ion-conducting block copolymers, semiconducting nanowire-polymer composites and lyotropic surfactant mesophases. The alignment process is quantitatively studied with X-ray scattering and microscopic methods. Time and temperature resolved data collected in situ during the magnetic experiments provide an insight into the thermodynamic and kinetic aspects of the process. These data together with simultaneous electrical conductivity measurements allow relating fundamental structural properties (e.g., morphology and long-range order) to transport properties (i.e., conductivity). In particular, it is demonstrated that magnetic fields offer a viable route for improvement of electric conductivity in these systems. More than an order of magnitude increase in conductivity is recorded in magnetically-annealed materials. The resulting aligned nanostructured systems are attractive for ordered solid polymer electrolyte membranes, heterojunction photovoltaic devices and generally help to understand charge transport mechanisms in anisotropic heterogeneous systems.

  10. Multicellularity and the Functional Interdependence of Motility and Molecular Transport

    NASA Astrophysics Data System (ADS)

    Solari, C.; Ganguly, S.; Kessler, J. O.; Michod, R.; Goldstein, R. E.

    2006-03-01

    Benefits, costs and requirements accompany the transition from motile totipotent unicellular organisms to multicellular organisms having cells specialized into reproductive (germ) and vegetative (sterile soma) functions such as motility. In flagellated colonial organisms such as the volvocalean green algae, organized beating by the somatic cells' flagella yields propulsion important in phototaxis and chemotaxis. It has not been generally appreciated that for the larger colonies, flagellar stirring of boundary layers and remote transport are fundamental for maintaining a sufficient rate of metabolite turnover, one not attainable by diffusive transport alone. We describe experiments that quantify the role of advective dynamics in enhancing productivity in germ-soma differentiated colonies. First, experiments with suspended deflagellated colonies of Volvox carteri show that forced advection improves productivity. Second, Particle Imaging Velocimetry of fluid motion around colonies reveals flow fields with very large characteristic velocities U extending to length scales comparable to the colony radius R. For a typical metabolite diffusion constant D, the Peclet number Pe=2UR/D 1, indicative of the dominance of advection over diffusion, with striking augmentation at the cell division stage.

  11. An improved Green's function for ion beam transport.

    PubMed

    Tweed, J; Wilson, J W; Tripathi, R K

    2004-01-01

    Ion beam transport theory allows testing of material transmission properties in the laboratory environment generated by particle accelerators. This is a necessary step in materials development and evaluation for space use. The approximations used in solving the Boltzmann transport equation for the space setting are often not sufficient for laboratory work and those issues are the main emphasis of the present work. In consequence, an analytic solution of the linear Boltzmann equation is pursued in the form of a Green's function allowing flexibility in application to a broad range of boundary value problems. It has been established that simple solutions can be found for high charge and energy (HZE) ions by ignoring nuclear energy downshifts and dispersion. Such solutions were found to be supported by experimental evidence with HZE ion beams when multiple scattering was added. Lacking from the prior solutions were range and energy straggling and energy downshift with dispersion associated with nuclear events. Recently, we have found global solutions including these effects providing a broader class of HZE ion solutions.

  12. Perinatal reduction of functional serotonin transporters results in developmental delay.

    PubMed

    Kroeze, Yvet; Dirven, Bart; Janssen, Stefan; Kröhnke, Marijke; Barte, Ramona M; Middelman, Anthonieke; van Bokhoven, Hans; Zhou, Huiqing; Homberg, Judith R

    2016-10-01

    While there is strong evidence from rodent and human studies that a reduction in serotonin transporter (5-HTT) function in early-life can increase the risk for several neuropsychiatric disorders in adulthood, the effects of reduced 5-HTT function on behavior across developmental stages are underinvestigated. To elucidate how perinatal pharmacological and lifelong genetic inactivation of the 5-HTT affects behavior across development, we conducted a battery of behavioral tests in rats perinatally exposed to fluoxetine or vehicle and in 5-HTT(-/-) versus 5-HTT(+/+) rats. We measured motor-related behavior, olfactory function, grooming behavior, sensorimotor gating, object directed behavior and novel object recognition in the first three postnatal weeks and if possible the tests were repeated in adolescence and adulthood. We also measured developmental milestones such as eye opening, reflex development and body weight. We observed that both pharmacological and genetic inactivation of 5-HTT resulted in a developmental delay. Except for hypo-locomotion, most of the observed early-life effects were normalized later in life. In adolescence and adulthood we observed object directed behavior and decreased novel object recognition in the 5-HTT(-/-) rats, which might be related to the lifelong inactivation of 5-HTT. Together, these data provide an important contribution to the understanding of the effects of perinatal and lifelong 5-HTT inactivation on behavior across developmental stages. PMID:27208789

  13. Functional Analysis of a Putative Dothistromin Toxin MFS Transporter Gene

    PubMed Central

    Bradshaw, Rosie E.; Feng, Zhilun; Schwelm, Arne; Yang, Yongzhi; Zhang, Shuguang

    2009-01-01

    Dothistromin is a non-host selective toxin produced by the pine needle pathogen Dothistroma septosporum. Dothistromin is not required for pathogenicity, but may have a role in competition and niche protection. To determine how D. septosporum tolerates its own toxin, a putative dothistromin transporter, DotC, was investigated. Studies with mutants lacking a functional dotC gene, overproducing DotC, or with a DotC-GFP fusion gene, did not provide conclusive evidence of a role in dothistromin efflux. The mutants revealed a major effect of DotC on dothistromin biosynthesis but were resistant to exogenous dothistromin. Intracellular localization studies suggest that compartmentalization may be important for dothistromin tolerance. PMID:22069539

  14. Diet-induced obesity: dopamine transporter function, impulsivity and motivation

    PubMed Central

    Narayanaswami, V; Thompson, AC; Cassis, LA; Bardo, MT; Dwoskin, LP

    2013-01-01

    OBJECTIVE A rat model of diet-induced obesity (DIO) was used to determine dopamine transporter (DAT) function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity. DESIGN To evaluate neurobehavioral alterations following the development of DIO induced by an 8-week high-fat diet (HF) exposure, striatal D2-receptor density, DAT function and expression, extracellular dopamine concentrations, impulsivity, and motivation for high- and low-fat reinforcers were determined. To determine predictors of DIO, neurobehavioral antecedents including impulsivity, motivation for high-fat reinforcers, DAT function and extracellular dopamine were evaluated before the 8-week HF exposure. METHODS Striatal D2-receptor density was determined by in vitro kinetic analysis of [3H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [3H]dopamine uptake, methamphetamine-evoked [3H]dopamine overflow and no-net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule, respectively. RESULTS Relative to obesity-resistant (OR) rats, obesity-prone (OP) rats exhibited 18% greater body weight following an 8-week HF-diet exposure, 42% lower striatal D2-receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine and twofold greater methamphetamine-evoked [3H]dopamine overflow. OP rats exhibited higher motivation for food, and surprisingly, were less impulsive relative to OR rats. Impulsivity, in vivo DAT function and extracellular dopamine concentration did not predict DIO. Importantly, motivation for high-fat reinforcers predicted the development of DIO. CONCLUSION Human studies are limited by their ability to determine if impulsivity, motivation and DAT function are causes or consequences of DIO. The

  15. Visualizing Functional Motions of Membrane Transporters with Molecular Dynamics Simulations

    PubMed Central

    2013-01-01

    Computational modeling and molecular simulation techniques have become an integral part of modern molecular research. Various areas of molecular sciences continue to benefit from, indeed rely on, the unparalleled spatial and temporal resolutions offered by these technologies, to provide a more complete picture of the molecular problems at hand. Because of the continuous development of more efficient algorithms harvesting ever-expanding computational resources, and the emergence of more advanced and novel theories and methodologies, the scope of computational studies has expanded significantly over the past decade, now including much larger molecular systems and far more complex molecular phenomena. Among the various computer modeling techniques, the application of molecular dynamics (MD) simulation and related techniques has particularly drawn attention in biomolecular research, because of the ability of the method to describe the dynamical nature of the molecular systems and thereby to provide a more realistic representation, which is often needed for understanding fundamental molecular properties. The method has proven to be remarkably successful in capturing molecular events and structural transitions highly relevant to the function and/or physicochemical properties of biomolecular systems. Herein, after a brief introduction to the method of MD, we use a number of membrane transport proteins studied in our laboratory as examples to showcase the scope and applicability of the method and its power in characterizing molecular motions of various magnitudes and time scales that are involved in the function of this important class of membrane proteins. PMID:23298176

  16. Vesicular Monoamine Transporters: Structure-Function, Pharmacology, and Medicinal Chemistry

    PubMed Central

    Wimalasena, Kandatege

    2010-01-01

    Vesicular monoamine transporters (VMAT) are responsible for the uptake of cytosolic monoamines into synaptic vesicles in monoaminergic neurons. Two closely related VMATs with distinct pharmacological properties and tissue distributions have been characterized. VMAT1 is preferentially expressed in neuroendocrine cells and VMAT2 is primarily expressed in the CNS. The neurotoxicity and addictive properties of various psychostimulants have been attributed, at least partly, to their interference with VMAT2 functions. The quantitative assessment of the VMAT2 density by PET scanning has been clinically useful for early diagnosis and monitoring of the progression of Parkinson’s and Alzheimer’s diseases and drug addiction. The classical VMAT2 inhibitor tetrabenazine has long been used for the treatment of chorea associated with Huntington’s disease in UK, Canada and Australia and recently approved in the US. The VMAT2 imaging may also be useful for exploiting the onset of diabetes mellitus, since VMAT2 is also expressed in the β-cells of the pancreas. VMAT1 gene SLC18A1 is a locus with strong evidence of linkage with schizophrenia and thus, the polymorphic forms of the VMAT1 gene may confer susceptibility to schizophrenia. This review summarizes the current understanding of the structure-function relationships of VMAT2, and the role of VMAT2 on addiction and psychostimulant induced neurotoxicity, and the therapeutic and diagnostic applications of specific VMAT2 ligands. The evidence for the linkage of VMAT1 gene with schizophrenia and bipolar disorder I are also discussed. PMID:20135628

  17. Galectin-4 interacts with the drug transporter human concentrative nucleoside transporter 3 to regulate its function.

    PubMed

    Fernández-Calotti, Paula; Casulleras, Olga; Antolin, María; Guarner, Francisco; Pastor-Anglada, Marçal

    2016-02-01

    The intracellular N-terminal domain of the nucleoside and drug transporter human concentrative nucleoside transporter (hCNT)3 was used as bait in a glutathione S-transferase pull-down approach, to identify hCNT3 protein partners, using human colon homogenates as a prey source. Galectin (Gal)-4 was identified as a potential hCNT3 partner in the colon. The biochemical validation of the Gal-4-hCNT3 interaction was verified by targeted pull-down assays and coimmunoprecipitation experiments in HT-29 cells, which endogenously express hCNT3 and Gal-4. Furthermore, Gal-4 was shown to colocalize with hCNT3 in HT-29 cells. The biologic significance of this interaction was obtained from experiments in which Gal-4 was knocked down, showing that this protein is a regulator of hCNT3 trafficking and retention at the cell membrane, reducing its plasma membrane location by 70%. Conversely, the addition of Gal-4 increased hCNT3 location at the plasma membrane by 77%, thereby demonstrating that this lectin modulates hCNT3 function in colonic cells. The integrity of this partnership may be clinically relevant, because hCNT3 may be responsible for the translocation of thiopurines, such as 6-mercaptopurine, a front-line treatment in inflammatory bowel disease. The expression of Gal-4 and hCNT3 proteins is not impaired in inflamed colon from patients with Crohn's disease, thereby anticipating the integrity of this system for drug targeting. PMID:26481311

  18. Membrane Transporters: Structure, Function and Targets for Drug Design

    NASA Astrophysics Data System (ADS)

    Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

    Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

  19. Evolutionary Relationships and Functional Diversity of Plant Sulfate Transporters

    PubMed Central

    Takahashi, Hideki; Buchner, Peter; Yoshimoto, Naoko; Hawkesford, Malcolm J.; Shiu, Shin-Han

    2011-01-01

    Sulfate is an essential nutrient cycled in nature. Ion transporters that specifically facilitate the transport of sulfate across the membranes are found ubiquitously in living organisms. The phylogenetic analysis of known sulfate transporters and their homologous proteins from eukaryotic organisms indicate two evolutionarily distinct groups of sulfate transport systems. One major group named Tribe 1 represents yeast and fungal SUL, plant SULTR, and animal SLC26 families. The evolutionary origin of SULTR family members in land plants and green algae is suggested to be common with yeast and fungal SUL and animal anion exchangers (SLC26). The lineage of plant SULTR family is expanded into four subfamilies (SULTR1–SULTR4) in land plant species. By contrast, the putative SULTR homologs from Chlorophyte green algae are in two separate lineages; one with the subfamily of plant tonoplast-localized sulfate transporters (SULTR4), and the other diverged before the appearance of lineages for SUL, SULTR, and SLC26. There also was a group of yet undefined members of putative sulfate transporters in yeast and fungi divergent from these major lineages in Tribe 1. The other distinct group is Tribe 2, primarily composed of animal sodium-dependent sulfate/carboxylate transporters (SLC13) and plant tonoplast-localized dicarboxylate transporters (TDT). The putative sulfur-sensing protein (SAC1) and SAC1-like transporters (SLT) of Chlorophyte green algae, bryophyte, and lycophyte show low degrees of sequence similarities with SLC13 and TDT. However, the phylogenetic relationship between SAC1/SLT and the other two families, SLC13 and TDT in Tribe 2, is not clearly supported. In addition, the SAC1/SLT family is absent in the angiosperm species analyzed. The present study suggests distinct evolutionary trajectories of sulfate transport systems for land plants and green algae. PMID:22629272

  20. Mitogen-stimulated and rapamycin-sensitive glucose transporter 12 targeting and functional glucose transport in renal epithelial cells.

    PubMed

    Wilson-O'Brien, Amy L; Dehaan, Carrie L; Rogers, Suzanne

    2008-03-01

    We hypothesized that glucose transporter 12 (GLUT12) is involved in regulation of glucose flux in distal renal tubules in response to elevated glucose. We used the Madin-Darby canine kidney polarized epithelial cell model and neutralizing antibodies to analyze GLUT12 targeting and directional GLUT12-mediated glucose transport. At physiological glucose concentrations, GLUT12 was localized to a perinuclear position. High glucose and serum treatment resulted in GLUT12 localization to the apical membrane. This mitogen-stimulated targeting of GLUT12 was inhibited by rapamycin, the specific inhibitor of mammalian target of rapamycin (mTOR). The functional role of GLUT12 was also examined. We constructed a GLUT12 cDNA containing a c-Myc epitope tag in the fifth exofacial loop. Assays of glucose transport at the apical membrane were performed using Transwell filters. By comparing transport assays in the presence of neutralizing anti-c-Myc monoclonal antibody, we specifically measured GLUT12-mediated glucose transport at the apical surface. GLUT12-mediated glucose transport was mitogen dependent and rapamycin sensitive. Our results implicate mTOR signaling in a novel pathway of glucose transporter protein targeting and glucose transport. Activity of the mTOR pathway has been associated with diabetic kidney disease. Our results provide evidence for a link between GLUT12 protein trafficking, glucose transport and signaling molecules central to the control of metabolic disease processes. PMID:18039784

  1. Amphetamine and methamphetamine reduce striatal dopamine transporter function without concurrent dopamine transporter relocalization.

    PubMed

    German, Christopher L; Hanson, Glen R; Fleckenstein, Annette E

    2012-10-01

    Amphetamine (AMPH) and methamphetamine (METH) alter dopamine transporter (DAT) function. In vitro heterologous cell line and synaptosome studies demonstrate AMPH-induced DAT internalization, implicating relocalization in reduced DAT uptake following drug exposure. However, few studies have evaluated DAT localization following in vivo AMPH/METH administration. To determine DAT subcellular localization following drug administration, a centrifugation technique was developed to isolate striatal synaptosomal membrane and vesicle fractions. DAT was distributed between the synaptosomal membrane (60%) and endosomal vesicles (40%), and in vitro application of the protein kinase C activator phorbol 12-myristate 13-acetate to striatal synaptosomes caused DAT internalization into the vesicle fractions. In contrast, neither single nor repeated in vivo AMPH and/or METH administrations altered DAT localization 5, 15, 30, or 60 min post-treatment, despite reduced DAT uptake. Importantly, repeated METH injections uniformly decreased total DAT immunoreactivity within all fractions 7 days post-treatment. These findings suggest that factors other than internalization can contribute to the observed acute and persistent DAT dysfunction and dopaminergic deficits following in vivo AMPH or METH administration.

  2. SLC6 Transporters: Structure, Function, Regulation, Disease Association and Therapeutics

    PubMed Central

    Bala, Pramod Akula; Foster, James; Carvelli, Lucia; Henry, L. Keith

    2012-01-01

    The SLC6 family of secondary active transporters are integral membrane solute carrier proteins characterized by the Na+-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, which include the serotonin, dopamine, norepinephrine, GABA, taurine, creatine, as well as amino acid transporters, are associated with a number of human diseases and disorders making this family a critical target for therapeutic development. In addition, several members of this family are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy. Recent advances providing structural insight into this family have vastly accelerated our ability to study these proteins and their involvement in complex biological processes. PMID:23506866

  3. Functional properties of ion channels and transporters in tumour vascularization

    PubMed Central

    Fiorio Pla, Alessandra; Munaron, Luca

    2014-01-01

    Vascularization is crucial for solid tumour growth and invasion, providing metabolic support and sustaining metastatic dissemination. It is now accepted that ion channels and transporters play a significant role in driving the cancer growth at all stages. They may represent novel therapeutic, diagnostic and prognostic targets for anti-cancer therapies. On the other hand, although the expression and role of ion channels and transporters in the vascular endothelium is well recognized and subject of recent reviews, only recently has their involvement in tumour vascularization been recognized. Here, we review the current literature on ion channels and transporters directly involved in the angiogenic process. Particular interest will be focused on tumour angiogenesis in vivo as well as in the different steps that drive this process in vitro, such as endothelial cell proliferation, migration, adhesion and tubulogenesis. Moreover, we compare the ‘transportome’ system of tumour vascular network with the physiological one. PMID:24493751

  4. PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

    PubMed Central

    Mairinger, Severin; Erker, Thomas; Müller, Markus; Langer, Oliver

    2013-01-01

    Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications. PMID:21434859

  5. Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters.

    PubMed

    Jaehme, Michael; Slotboom, Dirk Jan

    2015-09-01

    Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications. PMID:26352203

  6. Functions of Ion Transport Peptide and Ion Transport Peptide-Like in the Red Flour Beetle Tribolium castaneum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ion transport peptide (ITP) and ITP-like (ITPL) are highly conserved neuropeptides in insects and crustaceans. We investigated the alternatively spliced variants of ITP/ITPL in Tribolium castaneum to understand their functions. We identified three alternatively spliced transcripts named itp, itpl-...

  7. Membranes with functionalized carbon nanotube pores for selective transport

    DOEpatents

    Bakajin, Olgica; Noy, Aleksandr; Fornasiero, Francesco; Park, Hyung Gyu; Holt, Jason K; Kim, Sangil

    2015-01-27

    Provided herein composition and methods for nanoporous membranes comprising single walled, double walled, or multi-walled carbon nanotubes embedded in a matrix material. Average pore size of the carbon nanotube can be 6 nm or less. These membranes are a robust platform for the study of confined molecular transport, with applications in liquid and gas separations and chemical sensing including desalination, dialysis, and fabric formation.

  8. Creatine transporter deficiency: Novel mutations and functional studies.

    PubMed

    Ardon, O; Procter, M; Mao, R; Longo, N; Landau, Y E; Shilon-Hadass, A; Gabis, L V; Hoffmann, C; Tzadok, M; Heimer, G; Sada, S; Ben-Zeev, B; Anikster, Y

    2016-09-01

    X-linked cerebral creatine deficiency (MIM 300036) is caused by deficiency of the creatine transporter encoded by the SLC6A8 gene. Here we report three patients with this condition from Israel. These unrelated patients were evaluated for global developmental delays and language apraxia. Borderline microcephaly was noted in one of them. Diagnosis was prompted by brain magnetic resonance imaging and spectroscopy which revealed normal white matter distribution, but absence of the creatine peak in all three patients. Biochemical testing indicated normal plasma levels of creatine and guanidinoacetate, but an increased urine creatine/creatinine ratio. The diagnosis was confirmed by demonstrating absent ([14])C-creatine transport in fibroblasts. Molecular studies indicated that the first patient is hemizygous for a single nucleotide change substituting a single amino acid (c.619 C > T, p.R207W). Expression studies in HeLa cells confirmed the causative role of the R207W substitution. The second patient had a three base pair deletion in the SLC6A8 gene (c.1222_1224delTTC, p.F408del) as well as a single base change (c.1254 + 1G > A) at a splicing site in the intron-exon junction of exon 8, the latter occurring de novo. The third patient, had a three base pair deletion (c.1006_1008delAAC, p.N336del) previously reported in other patients with creatine transporter deficiency. These three patients are the first reported cases of creatine transporter deficiency in Israel. PMID:27408820

  9. Osmoregulation in zebrafish: ion transport mechanisms and functional regulation

    PubMed Central

    Guh, Ying-Jey; Lin, Chia-Hao; Hwang, Pung-Pung

    2015-01-01

    Fish, like mammals, have to maintain their body fluid ionic and osmotic homeostasis through sophisticated iono-/osmoregulation mechanisms, which are conducted mainly by ionocytes of the gill (the skin in embryonic stages), instead of the renal tubular cells in mammals. Given the advantages in terms of genetic database availability and manipulation, zebrafish is an emerging model for research into regulatory and integrative physiology. At least five types of ionocytes, HR, NaR, NCC, SLC26, and KS cells, have been identified to carry out Na+ uptake/H+ secretion/NH4+ excretion, Ca2+ uptake, Na+/Cl- uptake, K+ secretion, and Cl- uptake/HCO3- secretion, respectively, through distinct sets of transporters. Several hormones, namely isotocin, prolactin, cortisol, stanniocalcin-1, calcitonin, endothelin-1, vitamin D, parathyorid hormone 1, catecholamines, and the renin-angiotensin-system, have been demonstrated to positively or negatively regulate ion transport through specific receptors at different ionocytes stages, at either the transcriptional/translational or posttranslational level. The knowledge obtained using zebrafish answered many long-term contentious or unknown issues in the field of fish iono-/osmoregulation. The homology of ion transport pathways and hormone systems also means that the zebrafish model informs studies on mammals or other animal species, thereby providing insights into related fields. PMID:26600749

  10. SLC transporters as a novel class of tumour suppressors: identity, function and molecular mechanisms.

    PubMed

    Bhutia, Yangzom D; Babu, Ellappan; Ramachandran, Sabarish; Yang, Shengping; Thangaraju, Muthusamy; Ganapathy, Vadivel

    2016-05-01

    The role of plasma membrane transporters in cancer is receiving increasing attention in recent years. Several transporters for essential nutrients are up-regulated in cancer and serve as tumour promoters. Transporters could also function as tumour suppressors. To date, four transporters belonging to the SLC gene family have been identified as tumour suppressors. SLC5A8 is a Na(+)-coupled transporter for monocarboxylates. Among its substrates are the bacterial fermentation products butyrate and propionate and the ubiquitous metabolite pyruvate. The tumour-suppressive function of this transporter relates to the ability of butyrate, propionate and pyruvate to inhibit histone deacetylases (HDAC). SLC5A8 functions as a tumour suppressor in most tissues studied thus far, and provides a molecular link to Warburg effect, a characteristic feature in most cancers. It also links colonic bacteria and dietary fibre to the host. SLC26A3 as a tumour suppressor is restricted to colon; it is a Cl(-)/HCO(-) 3 exchanger, facilitating the efflux of HCO(-) 3 The likely mechanism for the tumour-suppressive function of SLC26A3 is related to intracellular pH regulation. SLC39A1 is a Zn(2+) transporter and its role in tumour suppression has been shown in prostate. Zn(2+) is present at high concentrations in normal prostate where it elicits its tumour-suppressive function. SLC22A18 is possibly an organic cation transporter, but the identity of its physiological substrates is unknown. As such, there is no information on molecular pathways responsible for the tumour-suppressive function of this transporter. It is likely that additional SLC transporters will be discovered as tumour suppressors in the future.

  11. SLC transporters as a novel class of tumour suppressors: identity, function and molecular mechanisms

    PubMed Central

    Bhutia, Yangzom D.; Babu, Ellappan; Ramachandran, Sabarish; Yang, Shengping; Thangaraju, Muthusamy; Ganapathy, Vadivel

    2016-01-01

    The role of plasma membrane transporters in cancer is receiving increasing attention in recent years. Several transporters for essential nutrients are up-regulated in cancer and serve as tumour promoters. Transporters could also function as tumour suppressors. To date, four transporters belonging to the SLC gene family have been identified as tumour suppressors. SLC5A8 is a Na+-coupled transporter for monocarboxylates. Among its substrates are the bacterial fermentation products butyrate and propionate and the ubiquitous metabolite pyruvate. The tumour-suppressive function of this transporter relates to the ability of butyrate, propionate and pyruvate to inhibit histone deacetylases (HDAC). SLC5A8 functions as a tumour suppressor in most tissues studied thus far, and provides a molecular link to Warburg effect, a characteristic feature in most cancers. It also links colonic bacteria and dietary fibre to the host. SLC26A3 as a tumour suppressor is restricted to colon; it is a Cl-/HCO3- exchanger, facilitating the efflux of HCO3-. The likely mechanism for the tumour-suppressive function of SLC26A3 is related to intracellular pH regulation. SLC39A1 is a Zn2+ transporter and its role in tumour suppression has been shown in prostate. Zn2+ is present at high concentrations in normal prostate where it elicits its tumour-suppressive function. SLC22A18 is possibly an organic cation transporter, but the identity of its physiological substrates is unknown. As such, there is no information on molecular pathways responsible for the tumour-suppressive function of this transporter. It is likely that additional SLC transporters will be discovered as tumour suppressors in the future. PMID:27118869

  12. Human organic anion transporter 2 is distinct from organic anion transporters 1 and 3 with respect to transport function.

    PubMed

    Henjakovic, Maja; Hagos, Yohannes; Krick, Wolfgang; Burckhardt, Gerhard; Burckhardt, Birgitta C

    2015-11-15

    Phylogentically, organic anion transporter (OAT)1 and OAT3 are closely related, whereas OAT2 is more distant. Experiments with human embryonic kidney-293 cells stably transfected with human OAT1, OAT2, or OAT3 were performed to compare selected transport properties. Common to OAT1, OAT2, and OAT3 is their ability to transport cGMP. OAT2 interacted with prostaglandins, and cGMP uptake was inhibited by PGE2 and PGF2α with IC50 values of 40.8 and 12.7 μM, respectively. OAT1 (IC50: 23.7 μM), OAT2 (IC50: 9.5 μM), and OAT3 (IC50: 1.6 μM) were potently inhibited by MK571, an established multidrug resistance protein inhibitor. OAT2-mediated cGMP uptake was not inhibited by short-chain monocarboxylates and, as opposed to OAT1 and OAT3, not by dicarboxylates. Consequently, OAT2 showed no cGMP/glutarate exchange. OAT1 and OAT3 exhibited a pH and a Cl- dependence with higher substrate uptake at acidic pH and lower substrate uptake in the absence of Cl-, respectively. Such pH and Cl- dependencies were not observed with OAT2. Depolarization of membrane potential by high K+ concentrations in the presence of the K+ ionophore valinomycin left cGMP uptake unaffected. In addition to cGMP, OAT2 transported urate and glutamate, but cGMP/glutamate exchange could not be demonstrated. These experiments suggest that OAT2-mediated cGMP uptake does not occur via exchange with monocarboxylates, dicarboxylates, and hydroxyl ions. The counter anion for electroneutral cGMP uptake remains to be identified.

  13. Functional characterization of a putative disaccharide membrane transporter in crustacean intestine.

    PubMed

    Likely, Rasheda; Johnson, Eric; Ahearn, Gregory A

    2015-02-01

    Transepithelial absorption of dietary sucrose in the American lobster, Homarus americanus, was investigated by mounting an intestine in a perfusion chamber to characterize mucosal to serosal (MS) (14)C-sucrose transport. These fluxes were measured by adding varying concentrations of (14)C-sucrose to the perfusate and monitoring their appearance in the bathing solution. Transepithelial (14)C-sucrose transport was the combination of a hyperbolic function of luminal concentration, following Michaelis-Menten kinetics, and apparent diffusion. The kinetic constants of the putative sucrose transporter were KM = 20.50 ± 6.00 µM and J max = 1.81 ± 0.50 pmol/cm(2) × min. Phloridzin, an inhibitor of Na(+)-dependent mucosal glucose transport, decreased MS (14)C-sucrose transport. Decreased MS (14)C-sucrose transport also occurred in the presence of luminal trehalose, a disaccharide containing D-glucose moieties. Thin-layer chromatography (TLC) identified the chemical nature of radioactively labeled sugars in the bath following transepithelial transport. TLC revealed (14)C-sucrose was transported across the intestine largely intact with no (14)C-glucose or (14)C-fructose appearing in the serosal bath or luminal perfusate. Only 13% of bath radioactivity was volatile metabolites. Results suggest that disaccharide sugars can be transported intact across crustacean intestine and support the occurrence of a functional disaccharide membrane transporter. PMID:25416426

  14. A novel fungal family of oligopeptide transporters identified by functional metatranscriptomics of soil eukaryotes

    PubMed Central

    Damon, Coralie; Vallon, Laurent; Zimmermann, Sabine; Haider, Muhammad Z; Galeote, Virginie; Dequin, Sylvie; Luis, Patricia; Fraissinet-Tachet, Laurence; Marmeisse, Roland

    2011-01-01

    Functional environmental genomics has the potential to identify novel biological functions that the systematic sequencing of microbial genomes or environmental DNA may fail to uncover. We targeted the functions expressed by soil eukaryotes using a metatranscriptomic approach based on the use of soil-extracted polyadenylated messenger RNA to construct environmental complementary DNA expression libraries. Functional complementation of a yeast mutant defective in di/tripeptide uptake identified a novel family of oligopeptide transporters expressed by fungi. This family has a patchy distribution in the Basidiomycota and Ascomycota and is present in the genome of a Saccharomyces cerevisiae wine strain. High throughput phenotyping of yeast mutants expressing two environmental transporters showed that they both displayed broad substrate specificity and could transport more than 60–80 dipeptides. When expressed in Xenopus oocytes one environmental transporter induced currents upon dipeptide addition, suggesting proton-coupled co-transport of dipeptides. This transporter was also able to transport specifically cysteine. Deletion of the two copies of the corresponding gene family members in the genome of the wine yeast strain severely reduced the number of dipeptides that it could assimilate. These results demonstrate that these genes are functional and can be used by fungi to efficiently scavenge the numerous, low concentration, oligopeptides continuously generated in soils by proteolysis. PMID:21654847

  15. Sensory Ciliogenesis in Caenorhabditis elegans: Assignment of IFT Components into Distinct Modules Based on Transport and Phenotypic Profiles

    PubMed Central

    Ou, Guangshuo; Koga, Makato; Blacque, Oliver E.; Murayama, Takashi; Ohshima, Yasumi; Schafer, Jenny C.; Li, Chunmei; Yoder, Bradley K.

    2007-01-01

    Sensory cilium biogenesis within Caenorhabditis elegans neurons depends on the kinesin-2–dependent intraflagellar transport (IFT) of ciliary precursors associated with IFT particles to the axoneme tip. Here we analyzed the molecular organization of the IFT machinery by comparing the in vivo transport and phenotypic profiles of multiple proteins involved in IFT and ciliogenesis. Based on their motility in wild-type and bbs (Bardet-Biedl syndrome) mutants, IFT proteins were classified into groups with similar transport profiles that we refer to as “modules.” We also analyzed the distribution and transport of fluorescent IFT particles in multiple known ciliary mutants and 49 new ciliary mutants. Most of the latter mutants were snip-SNP mapped and one, namely dyf-14(ks69), was cloned and found to encode a conserved protein essential for ciliogenesis. The products of these ciliogenesis genes could also be assigned to the aforementioned set of modules or to specific aspects of ciliogenesis, based on IFT particle dynamics and ciliary mutant phenotypes. Although binding assays would be required to confirm direct physical interactions, the results are consistent with the hypothesis that the C. elegans IFT machinery has a modular design, consisting of modules IFT-subcomplex A, IFT-subcomplex B, and a BBS protein complex, in addition to motor and cargo modules, with each module contributing to distinct functional aspects of IFT or ciliogenesis. PMID:17314406

  16. Glutamine transporters in mammalian cells and their functions in physiology and cancer.

    PubMed

    Bhutia, Yangzom D; Ganapathy, Vadivel

    2016-10-01

    The SLC (solute carrier)-type transporters (~400 in number) in mammalian cells consist of 52 distinct gene families, grouped solely based on the amino acid sequence (primary structure) of the transporter proteins and not on their transport function. Among them are the transporters for amino acids. Fourteen of them, capable of transporting glutamine across the plasma membrane, are found in four families: SLC1, SLC6, SLC7, and SLC38. However, it is generally thought that the members of the SLC38 family are the principal transporters for glutamine. Some of the glutamine transporters are obligatory exchangers whereas some function as active transporters in one direction. While most glutamine transporters mediate the influx of the amino acid into cells, some actually mediate the efflux of the amino acid out of the cells. Glutamine transporters play important roles in a variety of tissues, including the liver, brain, kidney, and placenta, as clearly evident from the biological and biochemical phenotypes resulting from the deletion of specific glutamine transporters in mice. Owing to the obligatory role of glutamine in growth and proliferation of tumor cells, there is increasing attention on glutamine transporters in cancer biology as potential drug targets for cancer treatment. Selective blockers of certain glutamine transporters might be effective in preventing the entry of glutamine and other important amino acids into tumor cells, thus essentially starving these cells to death. This could represent the beginning of a new era in the discovery of novel anticancer drugs with a previously unexplored mode of action. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

  17. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

    PubMed

    Otero, Maria G; Alloatti, Matías; Cromberg, Lucas E; Almenar-Queralt, Angels; Encalada, Sandra E; Pozo Devoto, Victorio M; Bruno, Luciana; Goldstein, Lawrence S B; Falzone, Tomás L

    2014-04-01

    Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

  18. Functional Characterization of a Hexose Transporter from Root Endophyte Piriformospora indica.

    PubMed

    Rani, Mamta; Raj, Sumit; Dayaman, Vikram; Kumar, Manoj; Dua, Meenakshi; Johri, Atul K

    2016-01-01

    Understanding the mechanism of photosynthate transfer at symbiotic interface by fungal monosaccharide transporter is of substantial importance. The carbohydrate uptake at the apoplast by the fungus is facilitated by PiHXT5 hexose transporter in root endophytic fungus Piriformospora indica. The putative PiHXT5 belongs to MFS superfamily with 12 predicted transmembrane helices. It possess sugar transporter PFAM motif (PF0083) and MFS superfamily domain (PS50850). It contains the signature tags related to glucose transporter GLUT1 of human erythrocyte. PiHXT5 is regulated in response to mutualism as well as glucose concentration. We have functionally characterized PiHXT5 by complementation of hxt-null mutant of Saccharomyces cerevisiae EBY.VW4000. It is involved in transport of multiple sugars ranging from D-glucose, D-fructose, D-xylose, D-mannose, D-galactose with decreasing affinity. The uncoupling experiments indicate that it functions as H(+)/glucose co-transporter. Further, pH dependence analysis suggests that it functions maximum between pH 5 and 6. The expression of PiHXT5 is dependent on glucose concentration and was found to be expressed at low glucose levels (1 mM) which indicate its role as a high affinity glucose transporter. Our study on this sugar transporter will help in better understanding of carbon metabolism and flow in this agro-friendly fungus. PMID:27499747

  19. Functional Characterization of a Hexose Transporter from Root Endophyte Piriformospora indica

    PubMed Central

    Rani, Mamta; Raj, Sumit; Dayaman, Vikram; Kumar, Manoj; Dua, Meenakshi; Johri, Atul K.

    2016-01-01

    Understanding the mechanism of photosynthate transfer at symbiotic interface by fungal monosaccharide transporter is of substantial importance. The carbohydrate uptake at the apoplast by the fungus is facilitated by PiHXT5 hexose transporter in root endophytic fungus Piriformospora indica. The putative PiHXT5 belongs to MFS superfamily with 12 predicted transmembrane helices. It possess sugar transporter PFAM motif (PF0083) and MFS superfamily domain (PS50850). It contains the signature tags related to glucose transporter GLUT1 of human erythrocyte. PiHXT5 is regulated in response to mutualism as well as glucose concentration. We have functionally characterized PiHXT5 by complementation of hxt-null mutant of Saccharomyces cerevisiae EBY.VW4000. It is involved in transport of multiple sugars ranging from D-glucose, D-fructose, D-xylose, D-mannose, D-galactose with decreasing affinity. The uncoupling experiments indicate that it functions as H+/glucose co-transporter. Further, pH dependence analysis suggests that it functions maximum between pH 5 and 6. The expression of PiHXT5 is dependent on glucose concentration and was found to be expressed at low glucose levels (1 mM) which indicate its role as a high affinity glucose transporter. Our study on this sugar transporter will help in better understanding of carbon metabolism and flow in this agro-friendly fungus. PMID:27499747

  20. Pathogenic mutations causing glucose transport defects in GLUT1 transporter: The role of intermolecular forces in protein structure-function.

    PubMed

    Raja, Mobeen; Kinne, Rolf K H

    2015-01-01

    Two families of glucose transporter - the Na(+)-dependent glucose cotransporter-1 (SGLT family) and the facilitated diffusion glucose transporter family (GLUT family) - play a crucial role in the translocation of glucose across the epithelial cell membrane. How genetic mutations cause life-threatening diseases like GLUT1-deficiency syndrome (GLUT1-DS) is not well understood. In this review, we have combined previous functional data with our in silico analyses of the bacterial homologue of GLUT members, XylE (an outward-facing, partly occluded conformation) and previously proposed GLUT1 homology model (an inward-facing conformation). A variety of native and mutant side chain interactions were modeled to highlight the potential roles of mutations in destabilizing protein-protein interaction hence triggering structural and functional defects. This study sets the stage for future studies of the structural properties that mediate GLUT1 dysfunction and further suggests that both SGLT and GLUT families share conserved domains that stabilize the transporter structure/function via a similar mechanism.

  1. Water transport through functionalized nanotubes with tunable hydrophobicity

    SciTech Connect

    Moskowitz, Ian; Snyder, Mark A.; Mittal, Jeetain

    2014-11-14

    Molecular dynamics simulations are used to study the occupancy and flow of water through nanotubes comprised of hydrophobic and hydrophilic atoms, which are arranged on a honeycomb lattice to mimic functionalized carbon nanotubes (CNTs). We consider single-file motion of TIP3P water through narrow channels of (6,6) CNTs with varying fractions (f) of hydrophilic atoms. Various arrangements of hydrophilic atoms are used to create heterogeneous nanotubes with separate hydrophobic/hydrophilic domains along the tube as well as random mixtures of the two types of atoms. The water occupancy inside the nanotube channel is found to vary nonlinearly as a function of f, and a small fraction of hydrophilic atoms (f ≈ 0.4) are sufficient to induce spontaneous and continuous filling of the nanotube. Interestingly, the average number of water molecules inside the channel and water flux through the nanotube are less sensitive to the specific arrangement of hydrophilic atoms than to the fraction, f. Two different regimes are observed for the water flux dependence on f – an approximately linear increase in flux as a function of f for f < 0.4, and almost no change in flux for higher f values, similar to the change in water occupancy. We are able to define an effective interaction strength between nanotube atoms and water's oxygen, based on a linear combination of interaction strengths between hydrophobic and hydrophilic nanotube atoms and water, that can quantitatively capture the observed behavior.

  2. Water transport through functionalized nanotubes with tunable hydrophobicity

    NASA Astrophysics Data System (ADS)

    Moskowitz, Ian; Snyder, Mark A.; Mittal, Jeetain

    2014-11-01

    Molecular dynamics simulations are used to study the occupancy and flow of water through nanotubes comprised of hydrophobic and hydrophilic atoms, which are arranged on a honeycomb lattice to mimic functionalized carbon nanotubes (CNTs). We consider single-file motion of TIP3P water through narrow channels of (6,6) CNTs with varying fractions (f) of hydrophilic atoms. Various arrangements of hydrophilic atoms are used to create heterogeneous nanotubes with separate hydrophobic/hydrophilic domains along the tube as well as random mixtures of the two types of atoms. The water occupancy inside the nanotube channel is found to vary nonlinearly as a function of f, and a small fraction of hydrophilic atoms (f ≈ 0.4) are sufficient to induce spontaneous and continuous filling of the nanotube. Interestingly, the average number of water molecules inside the channel and water flux through the nanotube are less sensitive to the specific arrangement of hydrophilic atoms than to the fraction, f. Two different regimes are observed for the water flux dependence on f - an approximately linear increase in flux as a function of f for f < 0.4, and almost no change in flux for higher f values, similar to the change in water occupancy. We are able to define an effective interaction strength between nanotube atoms and water's oxygen, based on a linear combination of interaction strengths between hydrophobic and hydrophilic nanotube atoms and water, that can quantitatively capture the observed behavior.

  3. Pyrethroid pesticide-induced alterations in dopamine transporter function

    PubMed Central

    Elwan, Mohamed A.; Richardson, Jason R.; Guillot, Thomas S.; Caudle, W. Michael; Miller, Gary W.

    2016-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative disease affecting the nigrostriatal dopaminergic pathway. Several epidemiological studies have demonstrated an association between pesticide exposure and the incidence of PD. Studies from our laboratory and others have demonstrated that certain pesticides increase levels of the dopamine transporter (DAT), an integral component of dopaminergic neurotransmission and a gateway for dopaminergic neurotoxins. Here, we report that repeated exposure (3 injections over 2 weeks) of mice to two commonly used pyrethroid pesticides, deltamethrin (3 mg/kg) and permethrin (0.8 mg/kg), increases DAT-mediated dopamine uptake by 31 and 28%, respectively. Using cells stably expressing DAT, we determined that exposure (10 min) to deltamethrin and permethrin (1 nM–100 μM) had no effect on DAT-mediated dopamine uptake. Extending exposures to both pesticides for 30 min (10 μM) or 24 h (1, 5, and 10 μM) resulted in significant decrease in dopamine uptake. This reduction was not the result of competitive inhibition, loss of DAT protein, or cytotoxicity. However, there was an increase in DNA fragmentation, an index of apoptosis, in cells exhibiting reduced uptake at 30 min and 24 h. These data suggest that up-regulation of DAT by in vivo pyrethroid exposure is an indirect effect and that longer-term exposure of cells results in apoptosis. Since DAT can greatly affect the vulnerability of dopamine neurons to neurotoxicants, up-regulation of DAT by deltamethrin and permethrin may increase the susceptibility of dopamine neurons to toxic insult, which may provide insight into the association between pesticide exposure and PD. PMID:16005927

  4. Pyrethroid pesticide-induced alterations in dopamine transporter function

    SciTech Connect

    Elwan, Mohamed A.; Richardson, Jason R.; Guillot, Thomas S.; Caudle, W. Michael; Miller, Gary W. . E-mail: gary.miller@emory.edu

    2006-03-15

    Parkinson's disease (PD) is a progressive neurodegenerative disease affecting the nigrostriatal dopaminergic pathway. Several epidemiological studies have demonstrated an association between pesticide exposure and the incidence of PD. Studies from our laboratory and others have demonstrated that certain pesticides increase levels of the dopamine transporter (DAT), an integral component of dopaminergic neurotransmission and a gateway for dopaminergic neurotoxins. Here, we report that repeated exposure (3 injections over 2 weeks) of mice to two commonly used pyrethroid pesticides, deltamethrin (3 mg/kg) and permethrin (0.8 mg/kg), increases DAT-mediated dopamine uptake by 31 and 28%, respectively. Using cells stably expressing DAT, we determined that exposure (10 min) to deltamethrin and permethrin (1 nM-100 {mu}M) had no effect on DAT-mediated dopamine uptake. Extending exposures to both pesticides for 30 min (10 {mu}M) or 24 h (1, 5, and 10 {mu}M) resulted in significant decrease in dopamine uptake. This reduction was not the result of competitive inhibition, loss of DAT protein, or cytotoxicity. However, there was an increase in DNA fragmentation, an index of apoptosis, in cells exhibiting reduced uptake at 30 min and 24 h. These data suggest that up-regulation of DAT by in vivo pyrethroid exposure is an indirect effect and that longer-term exposure of cells results in apoptosis. Since DAT can greatly affect the vulnerability of dopamine neurons to neurotoxicants, up-regulation of DAT by deltamethrin and permethrin may increase the susceptibility of dopamine neurons to toxic insult, which may provide insight into the association between pesticide exposure and PD.

  5. Proteoliposomes as Tool for Assaying Membrane Transporter Functions and Interactions with Xenobiotics

    PubMed Central

    Scalise, Mariafrancesca; Pochini, Lorena; Giangregorio, Nicola; Tonazzi, Annamaria; Indiveri, Cesare

    2013-01-01

    Proteoliposomes represent a suitable and up to date tool for studying membrane transporters which physiologically mediate absorption, excretion, trafficking and reabsorption of nutrients and metabolites. Using recently developed reconstitution strategies, transporters can be inserted in artificial bilayers with the same orientation as in the cell membranes and in the absence of other interfering molecular systems. These methodologies are very suitable for studying kinetic parameters and molecular mechanisms. After the first applications on mitochondrial transporters, in the last decade, proteoliposomes obtained with optimized methodologies have been used for studying plasma membrane transporters and defining their functional and kinetic properties and structure/function relationships. A lot of information has been obtained which has clarified and completed the knowledge on several transporters among which the OCTN sub-family members, transporters for neutral amino acid, B0AT1 and ASCT2, and others. Transporters can mediate absorption of substrate-like derivatives or drugs, improving their bioavailability or can interact with these compounds or other xenobiotics, leading to side/toxic effects. Therefore, proteoliposomes have recently been used for studying the interaction of some plasma membrane and mitochondrial transporters with toxic compounds, such as mercurials, H2O2 and some drugs. Several mechanisms have been defined and in some cases the amino acid residues responsible for the interaction have been identified. The data obtained indicate proteoliposomes as a novel and potentially important tool in drug discovery. PMID:24300519

  6. An Active Learning Exercise to Facilitate Understanding of Nephron Function: Anatomy and Physiology of Renal Transporters

    ERIC Educational Resources Information Center

    Dirks-Naylor, Amie J.

    2016-01-01

    Renal transport is a central mechanism underlying electrolyte homeostasis, acid base balance and other essential functions of the kidneys in human physiology. Thus, knowledge of the anatomy and physiology of the nephron is essential for the understanding of kidney function in health and disease. However, students find this content difficult to…

  7. Neurexin regulates visual function via mediating retinoid transport to promote rhodopsin maturation.

    PubMed

    Tian, Yao; Li, Tao; Sun, Mingkuan; Wan, Didi; Li, Qian; Li, Peipei; Zhang, Zi Chao; Han, Junhai; Xie, Wei

    2013-01-23

    Neurexins are cell adhesion molecules involved in synapse formation and synaptic regulation. Mutations in the neurexin genes are linked to a number of neurodevelopmental disorders such as autism. Here, we show that the Drosophila homolog of α-Neurexin is critical for fly visual function. Lack of Neurexin leads to significantly impaired visual function due to reduced rhodopsin levels. We show that the decreased chromophore levels cause deficits in rhodopsin maturation and that Neurexin is required for retinoid transport. Using yeast two-hybrid screening, we identify that Neurexin interacts with apolipoprotein I (ApoL I), a product generated by cleavage of retinoid- and fatty acid-binding glycoprotein (RFABG) that functions in retinoid transport. Finally, we demonstrate that Neurexin is essential for the apolipoproteins level. Our results reveal a role for Neurexin in mediating retinoid transport and subsequent rhodopsin maturation and suggest that Neurexin regulates lipoprotein function.

  8. Heterologous expression of a membrane-spanning auxin importer: implications for functional analyses of auxin transporters.

    PubMed

    Carrier, David John; Abu Bakar, Norliza Tendot; Lawler, Karen; Dorrian, James Matthew; Haider, Ameena; Bennett, Malcolm John; Kerr, Ian Derek

    2009-01-01

    Biochemical studies of plant auxin transporters in vivo are made difficult by the presence of multiple auxin transporters and auxin-interacting proteins. Furthermore, the expression level of most such transporters in plants is likely to be too low for purification and downstream functional analysis. Heterologous expression systems should address both of these issues. We have examined a number of such systems for their efficiency in expressing AUX1 from Arabidopsis thaliana. We find that a eukaryotic system based upon infection of insect cells with recombinant baculovirus provides a high level, easily scalable expression system capable of delivering a functional assay for AUX1. Furthermore, a transient transfection system in mammalian cells enables localization of AUX1 and AUX1-mediated transport of auxin to be investigated. In contrast, we were unable to utilise P. pastoris or L. lactis expression systems to reliably express AUX1.

  9. Functional profiles of orphan membrane transporters in the life cycle of the malaria parasite.

    PubMed

    Kenthirapalan, Sanketha; Waters, Andrew P; Matuschewski, Kai; Kooij, Taco W A

    2016-01-01

    Assigning function to orphan membrane transport proteins and prioritizing candidates for detailed biochemical characterization remain fundamental challenges and are particularly important for medically relevant pathogens, such as malaria parasites. Here we present a comprehensive genetic analysis of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes. Six genes, including four candidate aminophospholipid transporters, are refractory to gene deletion, indicative of essential functions. We generate and phenotypically characterize 29 mutant strains with deletions of individual transporter genes. Whereas seven genes appear to be dispensable under the experimental conditions tested, deletion of any of the 22 other genes leads to specific defects in life cycle progression in vivo and/or host transition. Our study provides growing support for a potential link between heavy metal homeostasis and host switching and reveals potential targets for rational design of new intervention strategies against malaria. PMID:26796412

  10. Functional profiles of orphan membrane transporters in the life cycle of the malaria parasite

    PubMed Central

    Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W. A.

    2016-01-01

    Assigning function to orphan membrane transport proteins and prioritizing candidates for detailed biochemical characterization remain fundamental challenges and are particularly important for medically relevant pathogens, such as malaria parasites. Here we present a comprehensive genetic analysis of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes. Six genes, including four candidate aminophospholipid transporters, are refractory to gene deletion, indicative of essential functions. We generate and phenotypically characterize 29 mutant strains with deletions of individual transporter genes. Whereas seven genes appear to be dispensable under the experimental conditions tested, deletion of any of the 22 other genes leads to specific defects in life cycle progression in vivo and/or host transition. Our study provides growing support for a potential link between heavy metal homeostasis and host switching and reveals potential targets for rational design of new intervention strategies against malaria. PMID:26796412

  11. Decreased vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function in knockout mice affects aging of dopaminergic systems

    PubMed Central

    Hall, F. S.; Itokawa, K.; Schmitt, A.; Moessner, R.; Sora, I.; Lesch, K. P.; Uhl, G. R.

    2013-01-01

    Dopamine (DA) is accumulated and compartmentalized by the dopamine transporter (DAT; SLC3A6) and the vesicular monoamine transporter 2 (VMAT2; SLC18A2). These transporters work at the plasma and vesicular membranes of dopaminergic neurons, respectively, and thus regulate levels of DA in neuronal compartments that include the extravesicular cytoplasmic compartment. DA in this compartment has been hypothesized to contribute to oxidative damage that can reduce the function of dopaminergic neurons in aging brains and may contribute to reductions in dopaminergic neurochemical markers, locomotor behavior and responses to dopaminergic drugs that are found in aged animals. The studies reported here examined aged mice with heterozygous deletions of VMAT2 or of DAT, which each reduce transporter expression to about 50% of levels found in wild-type (WT) mice. Aged mice displayed reduced locomotor responses under a variety of circumstances, including in response to locomotor stimulants, as well as changes in monoamine levels and metabolites in a regionally dependent manner. Several effects of aging were more pronounced in heterozygous VMAT2 knockout (KO) mice, including aging induced reductions in locomotion and reduced locomotor responses to cocaine. By contrast, some effects of aging were reduced or not observed in heterozygous DAT KO mice. These findings support the idea that altered DAT and VMAT2 expression affect age-related changes in dopaminergic function. These effects are most likely mediated by alterations in DA compartmentalization, and might be hypothesized to be more exacerbated by other factors that affect the metabolism of cytosolic DA. PMID:23978383

  12. The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11.

    PubMed

    Loganathan, Sampath K; Lukowski, Chris M; Casey, Joseph R

    2016-01-15

    Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function.

  13. Kymographic Analysis of Transport in an Individual Neuronal Sensory Cilium in Caenorhabditis elegans.

    PubMed

    O'Hagan, Robert; Barr, Maureen M

    2016-01-01

    Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules.Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM-3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2. PMID:27514919

  14. Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis

    PubMed Central

    Brandoni, Anabel; Hazelhoff, María Herminia; Bulacio, Romina Paula; Torres, Adriana Mónica

    2012-01-01

    Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions. PMID:23197884

  15. Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function.

    PubMed

    Tsigelny, Igor F; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K

    2008-10-01

    "rocker switch" may apply to certain MFS transporters, intermediate "tilted" states may exist under certain circumstances or as transitional structures. Although wet lab experimental confirmation is required, our results suggest that transport mechanisms in this transporter family should probably not be assumed to be conserved simply based on standard structural homology considerations. Furthermore, steered molecular dynamics elucidating energetic interactions of ligands with amino acid residues in an appropriately modeled transporter may have predictive value in understanding the impact of mutations and/or polymorphisms on transporter function. PMID:18942157

  16. Functional expression of two system A glutamine transporter isoforms in rat auditory brainstem neurons

    PubMed Central

    Blot, A.; Billups, D.; Bjørkmo, M.; Quazi, A.Z.; Uwechue, N.M.; Chaudhry, F.A.; Billups, B.

    2009-01-01

    Glutamine plays multiple roles in the CNS, including metabolic functions and production of the neurotransmitters glutamate and GABA. It has been proposed to be taken up into neurons via a variety of membrane transport systems, including system A, which is a sodium-dependent electrogenic amino acid transporter system. In this study, we investigate glutamine transport by application of amino acids to individual principal neurons of the medial nucleus of the trapezoid body (MNTB) in acutely isolated rat brain slices. A glutamine transport current was studied in patch-clamped neurons, which had the electrical and pharmacological properties of system A: it was sodium-dependent, had a non-reversing current-voltage relationship, was activated by proline, occluded by N-(methylamino)isobutyric acid (MeAIB), and was unaffected by 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH). Additionally, we examined the expression of different system A transporter isoforms using immunocytochemical staining with antibodies raised against system A transporter 1 and 2 (SAT1 and SAT2). Our results indicate that both isoforms are expressed in MNTB principal neurons, and demonstrate that functional system A transporters are present in the plasma membrane of neurons. Since system A transport is highly regulated by a number of cellular signaling mechanisms and glutamine then goes on to activate other pathways, the study of these transporters in situ gives an indication of the mechanisms of neuronal glutamine supply as well as points of regulation of neurotransmitter production, cellular signaling and metabolism in the native neuronal environment. PMID:19751803

  17. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    SciTech Connect

    Wang, Zhongshan; Xiang, Quanju; Zhu, Xiaofeng; Dong, Haohao; He, Chuan; Wang, Haiyan; Zhang, Yizheng; Wang, Wenjian; Dong, Changjiang

    2014-09-26

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

  18. Microscopic Characterization of Membrane Transporter Function by In Silico Modeling and Simulation.

    PubMed

    Vermaas, J V; Trebesch, N; Mayne, C G; Thangapandian, S; Shekhar, M; Mahinthichaichan, P; Baylon, J L; Jiang, T; Wang, Y; Muller, M P; Shinn, E; Zhao, Z; Wen, P-C; Tajkhorshid, E

    2016-01-01

    Membrane transporters mediate one of the most fundamental processes in biology. They are the main gatekeepers controlling active traffic of materials in a highly selective and regulated manner between different cellular compartments demarcated by biological membranes. At the heart of the mechanism of membrane transporters lie protein conformational changes of diverse forms and magnitudes, which closely mediate critical aspects of the transport process, most importantly the coordinated motions of remotely located gating elements and their tight coupling to chemical processes such as binding, unbinding and translocation of transported substrate and cotransported ions, ATP binding and hydrolysis, and other molecular events fueling uphill transport of the cargo. An increasing number of functional studies have established the active participation of lipids and other components of biological membranes in the function of transporters and other membrane proteins, often acting as major signaling and regulating elements. Understanding the mechanistic details of these molecular processes require methods that offer high spatial and temporal resolutions. Computational modeling and simulations technologies empowered by advanced sampling and free energy calculations have reached a sufficiently mature state to become an indispensable component of mechanistic studies of membrane transporters in their natural environment of the membrane. In this article, we provide an overview of a number of major computational protocols and techniques commonly used in membrane transporter modeling and simulation studies. The article also includes practical hints on effective use of these methods, critical perspectives on their strengths and weak points, and examples of their successful applications to membrane transporters, selected from the research performed in our own laboratory. PMID:27497175

  19. Expression and function of organic cation and anion transporters (SLC22 family) in the CNS.

    PubMed

    Farthing, Christine A; Sweet, Douglas H

    2014-01-01

    A major function of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier (BCSFB) is to exert selective control over the flux of organic cations and anions into and out of the CNS compartment. These barriers are dynamic tissues that accomplish this task by expressing dozens of transporter proteins representing numerous transporter families. One such family, belonging to the Solute Carrier (SLC) superfamily, is the organic cation/anion/zwitterion (SLC22) family of transporters, which includes the organic cation transporters (OCTs/OCTNs) and organic anion transporters (OATs). SLC22 transporters interact with a broad range of compounds that include drugs of abuse, environmental toxins/toxicants, opioid analgesics, antidepressant and anxiolytic agents and neurotransmitters and their metabolites. Defining the transport mechanisms controlling the CNS penetration, disposition and clearance of such compounds is fundamental to advancing our understanding of the underlying mechanisms that regulate CNS homeostasis and impact neuronal health. Such information might help direct efforts to improve the efficacy and clinical outcomes of current and future therapeutic agents used in the treatment of CNS disorders. This review focuses on highlighting the identification of the SLC22 transporter family, current knowledge of OCT and OAT expression within the CNS (including brain capillaries, choroid plexus and brain regions relevant to monoaminergic neuronal signaling), and recent data regarding behavioral changes related to mood and anxiety disorders and altered responses to stimulants and antidepressants in SLC22 loss of functions models (knockout/knockdown). In vitro and in vivo evidence of SLC22 localization and transport characteristics within the CNS compartment are summarized.

  20. Inflammatory Regulation of ATP Binding Cassette Efflux Transporter Expression and Function in Microglia

    PubMed Central

    Gibson, Christopher J.; Hossain, Muhammad M.; Richardson, Jason R.

    2012-01-01

    ATP-binding cassette (ABC) efflux transporters, including multidrug resistance protein 1 (Mdr1), breast cancer resistance protein (Bcrp), and multidrug resistance-associated proteins (Mrps) extrude chemicals from the brain. Although ABC transporters are critical for blood-brain barrier integrity, less attention has been placed on the regulation of these proteins in brain parenchymal cells such as microglia. Prior studies demonstrate that inflammation after lipopolysaccharide (LPS) treatment alters transporter expression in the livers of mice. Here, we sought to determine the effects of inflammation on the expression and function of transporters in microglia. To test this, the expression and function of ABC efflux transport proteins were quantified in mouse BV-2 microglial cells in response to activation with LPS. Intracellular retention of fluorescent rhodamine 123, Hoechst 33342, and calcein acetoxymethyl ester was increased in LPS-treated microglia, suggesting that the functions of Mdr1, Bcrp, and Mrps were decreased, respectively. LPS reduced Mdr1, Bcrp, and Mrp4 mRNA and protein expression between 40 and 70%. Conversely, LPS increased expression of Mrp1 and Mrp5 mRNA and protein. Immunofluorescent staining confirmed reduced Bcrp and Mrp4 and elevated Mrp1 and Mrp5 protein in activated microglia. Pharmacological inhibition of nuclear factor κB (NF-κB) transcriptional signaling attenuated down-regulation of Mdr1a mRNA and potentiated up-regulation of Mrp5 mRNA in LPS-treated cells. Together, these data suggest that LPS stimulates microglia and impairs efflux of prototypical ABC transporter substrates by altering mRNA and protein expression, in part through NF-κB signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation. PMID:22942241

  1. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT. PMID:22118932

  2. Decoupling catalytic activity from biological function of the ATPase that powers lipopolysaccharide transport.

    PubMed

    Sherman, David J; Lazarus, Michael B; Murphy, Lea; Liu, Charles; Walker, Suzanne; Ruiz, Natividad; Kahne, Daniel

    2014-04-01

    The cell surface of Gram-negative bacteria contains lipopolysaccharides (LPS), which provide a barrier against the entry of many antibiotics. LPS assembly involves a multiprotein LPS transport (Lpt) complex that spans from the cytoplasm to the outer membrane. In this complex, an unusual ATP-binding cassette transporter is thought to power the extraction of LPS from the outer leaflet of the cytoplasmic membrane and its transport across the cell envelope. We introduce changes into the nucleotide-binding domain, LptB, that inactivate transporter function in vivo. We characterize these residues using biochemical experiments combined with high-resolution crystal structures of LptB pre- and post-ATP hydrolysis and suggest a role for an active site residue in phosphate exit. We also identify a conserved residue that is not required for ATPase activity but is essential for interaction with the transmembrane components. Our studies establish the essentiality of ATP hydrolysis by LptB to power LPS transport in cells and suggest strategies to inhibit transporter function away from the LptB active site.

  3. Functional expression of pig renal organic anion transporter 3 (pOAT3).

    PubMed

    Hagos, Yohannes; Braun, Isabella M; Krick, Wolfgang; Burckhardt, Gerhard; Bahn, Andrew

    2005-05-01

    With the cloning of pig renal organic anion transporter 1 (pOAT1) (Biochimie 84 (2002) 1219) we set up a model system for comparative studies of cloned and natively isolated membrane located transport proteins. Meanwhile, another transport protein involved in p-aminohippurate (PAH) uptake on the basolateral side of the proximal tubule cells was identified, designated organic anion transporter 3 (OAT3). To explore the contribution of pOAT1 to the PAH clearance in comparison to OAT3, it was the aim of this study to extend our model by cloning of the pig ortholog of OAT3. Sequence comparisons of human organic anion transporter 3 (hOAT3) with the expressed sequence tag (EST) database revealed a clone and partial sequence of the pig renal organic anion transporter 3 (pOAT3) ortholog. Sequencing of the entire open reading frame resulted in a protein of 543 amino acid residues encoded by 1632 base pairs (EMBL Acc. No. AJ587003). It showed high homologies of 81%, 80%, 76%, and 77% to the human, rabbit, rat, and mouse OAT3, respectively. A functional characterization of pOAT3 in Xenopus laevis oocytes yielded an apparent Km (Kt) for [3H]estrone sulfate of 7.8 +/- 1.3 microM. Moreover, pOAT3 mediated [3H]estrone sulfate uptake was almost abolished by 0.5 mM of glutarate, dehydroepiandosterone sulfate, or probenecid consistent with the hallmarks of OAT3 function.

  4. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    SciTech Connect

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M. )

    1989-11-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K{sub m}, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

  5. Gravel threshold of motion: a state function of sediment transport disequilibrium?

    NASA Astrophysics Data System (ADS)

    Johnson, Joel P. L.

    2016-08-01

    In most sediment transport models, a threshold variable dictates the shear stress at which non-negligible bedload transport begins. Previous work has demonstrated that nondimensional transport thresholds (τc*) vary with many factors related not only to grain size and shape, but also with characteristics of the local bed surface and sediment transport rate (qs). I propose a new model in which qs-dependent τc*, notated as τc(qs)*, evolves as a power-law function of net erosion or deposition. In the model, net entrainment is assumed to progressively remove more mobile particles while leaving behind more stable grains, gradually increasing τc(qs)* and reducing transport rates. Net deposition tends to fill in topographic lows, progressively leading to less stable distributions of surface grains, decreasing τc(qs)* and increasing transport rates. Model parameters are calibrated based on laboratory flume experiments that explore transport disequilibrium. The τc(qs)* equation is then incorporated into a simple morphodynamic model. The evolution of τc(qs)* is a negative feedback on morphologic change, while also allowing reaches to equilibrate to sediment supply at different slopes. Finally, τc(qs)* is interpreted to be an important but nonunique state variable for morphodynamics, in a manner consistent with state variables such as temperature in thermodynamics.

  6. Soybean ureide transporters play a critical role in nodule development, function and nitrogen export.

    PubMed

    Collier, Ray; Tegeder, Mechthild

    2012-11-01

    Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen-fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long-distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins - GmUPS1-1 (XP_003516366) and GmUPS1-2 (XP_003518768) - in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1-1 and GmUPS1-2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.

  7. Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution.

    PubMed Central

    Saier, M H

    1994-01-01

    Three-dimensional structures have been elucidated for very few integral membrane proteins. Computer methods can be used as guides for estimation of solute transport protein structure, function, biogenesis, and evolution. In this paper the application of currently available computer programs to over a dozen distinct families of transport proteins is reviewed. The reliability of sequence-based topological and localization analyses and the importance of sequence and residue conservation to structure and function are evaluated. Evidence concerning the nature and frequency of occurrence of domain shuffling, splicing, fusion, deletion, and duplication during evolution of specific transport protein families is also evaluated. Channel proteins are proposed to be functionally related to carriers. It is argued that energy coupling to transport was a late occurrence, superimposed on preexisting mechanisms of solute facilitation. It is shown that several transport protein families have evolved independently of each other, employing different routes, at different times in evolutionary history, to give topologically similar transmembrane protein complexes. The possible significance of this apparent topological convergence is discussed. PMID:8177172

  8. OsABCB14 functions in auxin transport and iron homeostasis in rice (Oryza sativa L.).

    PubMed

    Xu, Yanxia; Zhang, Saina; Guo, Haipeng; Wang, Suikang; Xu, Ligen; Li, Chuanyou; Qian, Qian; Chen, Fan; Geisler, Markus; Qi, Yanhua; Jiang, De An

    2014-07-01

    Members of the ATP Binding Cassette B/Multidrug-Resistance/P-glyco-protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock-down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock-down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (-Fe). Their Fe concentration is higher and upregulation of Fe deficiency-responsive genes is lower in osabcb14 mutants than in wild-type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition.

  9. Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles

    SciTech Connect

    Gao Xiaoling; Wang Tao; Wu Bingxian; Chen Jun; Chen Jiyao; Yue Yang; Dai Ning; Chen Hongzhuan Jiang Xinguo

    2008-12-05

    Successful drug delivery by functionalized nanocarriers largely depends on their efficient intracellular transport which has not yet been fully understood. We developed a new tracking technique by encapsulating quantum dots into the core of wheat germ agglutinin-conjugated nanoparticles (WGA-NP) to track cellular transport of functionalized nanocarriers. The resulting nanoparticles showed no changes in particle size, zeta potential or biobinding activity, and the loaded probe presented excellent photostability and tracking ability. Taking advantage of these properties, cellular transport profiles of WGA-NP in Caco-2 cells was demonstrated. The cellular uptake begins with binding of WGA to its receptor at the cell surface. The subsequent endocytosis happened in a cytoskeleton-dependent manner and by means of clathrin and caveolae-mediated mechanisms. After endosome creating, transport occurs to both trans-Golgi and lysosome. Our study provides new evidences for quantum dots as a cellular tracking probe of nanocarriers and helps understand intracellular transport profile of lectin-functionalized nanoparticles.

  10. Insect glycerol transporters evolved by functional co-option and gene replacement

    PubMed Central

    Finn, Roderick Nigel; Chauvigné, François; Stavang, Jon Anders; Belles, Xavier; Cerdà, Joan

    2015-01-01

    Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects. PMID:26183829

  11. Insect glycerol transporters evolved by functional co-option and gene replacement.

    PubMed

    Finn, Roderick Nigel; Chauvigné, François; Stavang, Jon Anders; Belles, Xavier; Cerdà, Joan

    2015-01-01

    Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter converted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects. PMID:26183829

  12. Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics.

    SciTech Connect

    Bock KW; D Honys; JM. Ward; S Padmanaban; EP Nawrocki; KD Hirschi; D Twell; H Sze

    2006-01-01

    Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. About 1269 genes encoding classified transporters were collected from the Arabidopsis thaliana genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3 and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9); while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, were developmentally-regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter::GUS analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

  13. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation.

  14. Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

    PubMed

    Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

    2015-07-01

    Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. PMID:25883089

  15. Transportation.

    ERIC Educational Resources Information Center

    Crank, Ron

    This instructional unit is one of 10 developed by students on various energy-related areas that deals specifically with transportation and energy use. Its objective is for the student to be able to discuss the implication of energy usage as it applies to the area of transportation. Some topics covered are efficiencies of various transportation…

  16. Identification and functional characterization of the Caenorhabditis elegans riboflavin transporters rft-1 and rft-2.

    PubMed

    Biswas, Arundhati; Elmatari, Daniel; Rothman, Jason; LaMunyon, Craig W; Said, Hamid M

    2013-01-01

    Two potential orthologs of the human riboflavin transporter 3 (hRFVT3) were identified in the C. elegans genome, Y47D7A.16 and Y47D7A.14, which share 33.7 and 30.5% identity, respectively, with hRFVT3. The genes are tandemly arranged, and we assign them the names rft-1 (for Y47D7A.16) and rft-2 (for Y47D7A.14). Functional characterization of the coding sequences in a heterologous expression system demonstrated that both were specific riboflavin transporters, although the rft-1 encoded protein had greater transport activity. A more detailed examination of rft-1 showed its transport of riboflavin to have an acidic pH dependence, saturability (apparent Km = 1.4 ± 0.5 µM), inhibition by riboflavin analogues, and Na(+) independence. The expression of rft-1 mRNA was relatively higher in young larvae than in adults, and mRNA expression dropped in response to RF supplementation. Knocking down the two transporters individually via RNA interference resulted in a severe loss of fertility that was compounded in a double knockdown. Transcriptional fusions constructed with two fluorophores (rft-1::GFP, and rft-2::mCherry) indicated that rft-1 is expressed in the intestine and a small subset of neuronal support cells along the entire length of the animal. Expression of rft-2 is localized mainly to the intestine and pharynx. We also observed a drop in the expression of the two reporters in animals that were maintained in high riboflavin levels. These results report for the first time the identification of two riboflavin transporters in C. elegans and demonstrate their expression and importance to metabolic function in worms. Absence of transporter function renders worms sterile, making them useful in understanding human disease associated with mutations in hRFVT3.

  17. Identification and Functional Characterization of the Caenorhabditis elegans Riboflavin Transporters rft-1 and rft-2

    PubMed Central

    Biswas, Arundhati; Elmatari, Daniel; Rothman, Jason; LaMunyon, Craig W.; Said, Hamid M.

    2013-01-01

    Two potential orthologs of the human riboflavin transporter 3 (hRFVT3) were identified in the C. elegans genome, Y47D7A.16 and Y47D7A.14, which share 33.7 and 30.5% identity, respectively, with hRFVT3. The genes are tandemly arranged, and we assign them the names rft-1 (for Y47D7A.16) and rft-2 (for Y47D7A.14). Functional characterization of the coding sequences in a heterologous expression system demonstrated that both were specific riboflavin transporters, although the rft-1 encoded protein had greater transport activity. A more detailed examination of rft-1 showed its transport of riboflavin to have an acidic pH dependence, saturability (apparent Km = 1.4±0.5 µM), inhibition by riboflavin analogues, and Na+ independence. The expression of rft-1 mRNA was relatively higher in young larvae than in adults, and mRNA expression dropped in response to RF supplementation. Knocking down the two transporters individually via RNA interference resulted in a severe loss of fertility that was compounded in a double knockdown. Transcriptional fusions constructed with two fluorophores (rft-1::GFP, and rft-2::mCherry) indicated that rft-1 is expressed in the intestine and a small subset of neuronal support cells along the entire length of the animal. Expression of rft-2 is localized mainly to the intestine and pharynx. We also observed a drop in the expression of the two reporters in animals that were maintained in high riboflavin levels. These results report for the first time the identification of two riboflavin transporters in C. elegans and demonstrate their expression and importance to metabolic function in worms. Absence of transporter function renders worms sterile, making them useful in understanding human disease associated with mutations in hRFVT3. PMID:23483992

  18. Functional expression and cellular distribution of diastrophic dysplasia sulfate transporter (DTDST) gene mutations in HEK cells.

    PubMed

    Karniski, Lawrence P

    2004-10-01

    Defects in sulfate transport in chondrocytes lead to undersulfation of the cartilage extracellular matrix proteoglycans. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been linked to four chondrodysplasias of varying severity. To characterize disease-causing mutations of DTDST, we expressed DTDST-mediated sulfate transport in mammalian HEK-293 cells and determined that the wild-type protein is glycosylated and localized to the cell plasma membrane. Four mutations, A715V, C653S, Q454P and R279W, stimulated sulfate transport at rates only 39-62% of wild-type DTDST. These four mutations were expressed on the plasma membrane of the cell, but the amount of expressed protein was reduced when compared with wild-type DTDST. The Q454P mutant is unique in that it is not properly glycosylated in HEK cells. There was no difference in sulfate transport activity between cells transfected with either the DeltaV340 or the G678V mutations and control HEK cells. Furthermore, the G678V mutation is not expressed along the plasma membrane, but is trapped within the cytoplasm. When comparing the sulfate transport capacity of each DTDST mutation with the chondrodysplasia in which it has been identified, we find that individuals with severe achondrogenesis 1B phenotype have null mutations on both DTDST alleles. Heterozygotes for both a null mutation and a partial-function mutation result in either atelosteogenesis type 2 or DTD, whereas the milder, recessive multiple epiphyseal dysplasia phenotype is homozygous for partial-function mutations. In contrast to previous studies in Xenopus laevis oocytes, we find a strong correlation between the severity of the phenotype and the level of residual transport function in mammalian cells. PMID:15294877

  19. Evaluation of a functional model for simulating boron transport in soil

    SciTech Connect

    Corwin, D.L.; Goldberg, S.; David, A.

    1999-10-01

    There has been renewed interest in the application of functional models to the transport of nonpoint source pollutants at polypedon and watershed scales because of the ease of their coupling to a geographic information system and to the accepted organizational hierarchy of pedogenetic modeling approaches. However, very little work has been done to evaluate the performance of a functional transient-state model for the transport of a reactive solute over an extensive study period. Subsequently, the functional model TETrans (Trace Element Transport) was evaluated for model performance with boron (B) transport data collected from a meso-scale soil lysimeter column over a 1,000-day study period. Because the ability to simulate water flow has been evaluated previously for TETrans, the focus of this evaluation centered around the performance of various functional models of B adsorption used as subroutines within the TETrans model, including the (1) Freundlich, (2) kinetic Freundlich, (3) Langmuir, (4) temperature-dependent Langmuir, and (5) pH-dependent Keren adsorption isotherm equations. Model performance was evaluated with statistical functions, specifically the Average Absolute Prediction Error, the Root Mean Square Error, the Reduced Error Estimate and the Coefficient of Residual Mass, and graphic displays of observed and predicted B concentration profiles. Even though no single adsorption isotherm equation, when coupled to TETrans, could be considered poor in its performance, results indicated that the order of model performance was the pH-dependent Keren equation first, followed by the temperature-dependent Langmuir and kinetic Freundlich equations, the Freundlich equation, and, finally, the Langmuir equation. Overall, the TETrans model was able to simulate the transport of B with deviations because no functional adsorption equation incorporated all the influences of pH, ionic strength, temperature, and kinetic effects into a single equation. The inability to

  20. Functional Evidence of Multidrug Resistance Transporters (MDR) in Rodent Olfactory Epithelium

    PubMed Central

    Molinas, Adrien; Sicard, Gilles; Jakob, Ingrid

    2012-01-01

    Background P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated. Principal Findings Functional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM) accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of species-specific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG). In both animal species, MRPs inhibitors induced a marked reduction of the EOG magnitude, while Pgp inhibitors had only a minor or no measurable effect. Conclusions The findings suggest that both Pgp and MRP transporters are functional in the olfactory mucosa and in olfactory receptor neurons. Pgp and MRPs may be cellular constituents of olfactory receptor neurons and represent potential

  1. Limited transport of functionalized multi-walled carbon nanotubes in two natural soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Column experiments were conducted in undisturbed and in repacked soil columns at water contents close to saturation (85–96%) to investigate the transport and retention of functionalized 14C-labeled multi-walled carbon nanotubes (MWCNT) in two natural soils. Additionally, a field lysimeter experiment...

  2. Functional characterization in Xenopus oocytes of Na+ transport systems from durum wheat reveals diversity among two HKT1;4 transporters

    PubMed Central

    Masmoudi, Khaled; Véry, Anne-Aliénor

    2014-01-01

    Plant tolerance to salinity constraint involves complex and integrated functions including control of Na+ uptake, translocation, and compartmentalization. Several members of the high-affinity K+ transporter (HKT) family, which comprises plasma-membrane transporters permeable to K+ and Na+ or to Na+ only, have been shown to play major roles in plant Na+ and K+ homeostasis. Among them, HKT1;4 has been identified as corresponding to a quantitative trait locus (QTL) of salt tolerance in wheat but was not functionally characterized. Here, we isolated two HKT1;4-type cDNAs from a salt-tolerant durum wheat (Triticum turgidum L. subsp. durum) cultivar, Om Rabia3, and investigated the functional properties of the encoded transporters using a two-electrode voltage-clamp technique, after expression in Xenopus oocytes. Both transporters displayed high selectivity for Na+, their permeability to other monovalent cations (K+, Li+, Cs+, and Rb+) being ten times lower than that to Na+. Both TdHKT1;4-1 and TdHKT1;4-2 transported Na+ with low affinity, although the half-saturation of the conductance was observed at a Na+ concentration four times lower in TdHKT1;4-1 than in TdHKT1;4-2. External K+ did not inhibit Na+ transport through these transporters. Quinine slightly inhibited TdHKT1;4-2 but not TdHKT1;4-1. Overall, these data identified TdHKT1;4 transporters as new Na+-selective transporters within the HKT family, displaying their own functional features. Furthermore, they showed that important differences in affinity exist among durum wheat HKT1;4 transporters. This suggests that the salt tolerance QTL involving HKT1;4 may be at least in part explained by functional variability among wheat HKT1;4-type transporters. PMID:24192995

  3. Functional expression cloning and characterization of SFT, a stimulator of Fe transport.

    PubMed

    Gutierrez, J A; Yu, J; Rivera, S; Wessling-Resnick, M

    1997-11-17

    A stimulator of Fe transport (SFT) was identified by functional expression cloning in Xenopus oocytes. SFT-mediated transport has properties defined for transferrin-independent Fe uptake, but its cytolocalization in recycling endosomes and the observed stimulation of transferrin-bound Fe assimilation indicate a key role in intracellular Fe membrane transport as well. SFT has six predicted transmembranous domains and a functionally important RExxE motif that resembles domains involved in yeast Fe transport and Fe-binding by ferritin L-chains. The observation that SFT oligomerizes, along with other structural and mechanistic features, suggests it may be a member of either the ATP-binding cassette or cation diffusion facilitator families. The 3' untranslated region of SFT contains a translation inhibitory element and inhibition of SFT expression in Xenopus oocytes was found to be relieved by coinjection of transcripts from other defined cDNAs that are also described in this report. SFT is the first component of the mammalian Fe membrane transport machinery to be identified.

  4. N-Glycosylation is required for Na{sup +}-dependent vitamin C transporter functionality

    SciTech Connect

    Subramanian, Veedamali S. Marchant, Jonathan S.; Reidling, Jack C.; Said, Hamid M.

    2008-09-12

    The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased {sup 14}C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.

  5. Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2.

    PubMed

    Higashi, Kyohei; Imamura, Masataka; Fudo, Satoshi; Uemura, Takeshi; Saiki, Ryotaro; Hoshino, Tyuji; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2014-01-01

    Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.

  6. Electronic transport properties of one dimensional lithium nanowire using density functional theory

    SciTech Connect

    Thakur, Anil; Kumar, Arun; Chandel, Surjeet; Ahluwalia, P. K.

    2015-05-15

    Single nanowire electrode devices are a unique platform for studying as energy storage devices. Lithium nanowire is of much importance in lithium ion batteries and therefore has received a great deal of attention in past few years. In this paper we investigated structural and electronic transport properties of Li nanowire using density functional theory (DFT) with SIESTA code. Electronic transport properties of Li nanowire are investigated theoretically. The calculations are performed in two steps: first an optimized geometry for Li nanowire is obtained using DFT calculations, and then the transport relations are obtained using NEGF approach. SIESTA and TranSIESTA simulation codes are used in the calculations correspondingly. The electrodes are chosen to be the same as the central region where transport is studied, eliminating current quantization effects due to contacts and focusing the electronic transport study to the intrinsic structure of the material. By varying chemical potential in the electrode regions, an I-V curve is traced which is in agreement with the predicted behavior. Agreement of bulk properties of Li with experimental values make the study of electronic and transport properties in lithium nanowires interesting because they are promising candidates as bridging pieces in nanoelectronics. Transmission coefficient and V-I characteristic of Li nano wire indicates that Li nanowire can be used as an electrode device.

  7. Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules

    PubMed Central

    Girard, Jean-Philippe; Baekkevold, Espen S.; Feliu, Jacques; Brandtzaeg, Per; Amalric, François

    1999-01-01

    High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na+-coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na+/sulfate cotransporter, NaSi-1, as well as with the human and rat Na+/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na+-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na+-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV. PMID:10535998

  8. Structural and functional analysis of SFT, a stimulator of Fe Transport.

    PubMed

    Yu, J; Wessling-Resnick, M

    1998-08-14

    Previous studies demonstrated that SFT (Stimulator of Fe Transport) facilitates both transferrin and nontransferrin-bound iron uptake in HeLa cells (Yu, J., and Wessling-Resnick, M. (1998) J. Biol. Chem. 273, 6909-6915). To further characterize the structure and function of SFT, we studied this human factor in rodent BHK cells. Kyte-Doolittle analysis suggests that SFT has six transmembrane-spanning segments. This transport protein also displays an REXXE motif resembling domains involved in iron binding by ferritin and in iron uptake mediated by the yeast transporter Ftr1. Using N- and C-terminal epitope tags, we have identified that modification of either protein terminus does not interfere with SFT function in nontransferrin-bound iron uptake. The N- and C-terminal domains are intracellularly disposed since antibodies against these epitopes fail to recognize expressed proteins unless BHK cells are solubilized with detergents. To define the topology of two large extramembranous loop domains, anti-peptide antibodies were employed; anti-loop 4 antibodies show no immunoreactivity unless cells are permeabilized but anti-loop 5 antibodies recognize and bind surface SFT. Thus, loop 4 must be intracellular while loop 5 is extracellular. These topological studies situate the putative iron-binding REXXE domain on the cytosolic face of the plasma membrane. However, 55Fe-binding studies reveal that the ability of SFT to bind and mediate transport of extracellular iron is defective in mutants with Glu --> Ala conversions in this motif. Curiously, we also find that depletion of intracellular iron by desferrioxamine impairs SFT transport and iron-binding functions. These observations lead to the speculation that the REXXE motif may play an important role in regulating SFT activity through interaction with intracellular iron and demonstrate that iron transport mediated by SFT is itself an iron-dependent process.

  9. Fabrication of self-supporting porous silicon membranes and tuning transport properties by surface functionalization

    NASA Astrophysics Data System (ADS)

    Velleman, Leonora; Shearer, Cameron James; Ellis, Amanda Vera; Losic, Dusan; Voelcker, Nicolas Hans; Shapter, Joseph George

    2010-09-01

    This study presents a simple approach to perform selective mass transport through freestanding porous silicon (pSi) membranes. pSi membranes were fabricated by the electrochemical etching of silicon to produce membranes with controlled structure and pore sizes close to molecular dimensions (~12 nm in diameter). While these membranes are capable of size-exclusion based separations, chemically specific filtration remains a great challenge especially in the biomedical field. Herein, we investigate the transport properties of chemically functionalized pSi membranes. The membranes were functionalized using silanes (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane (PFDS) and N-(triethoxysilylpropyl)-o-polyethylene oxide urethane (PEGS) to give membranes hydrophobic (PFDS) and hydrophilic (PEGS) properties. The transport of probe dyes tris(2,2'-bipyridyl)dichlororuthenium(ii) hexahydrate (Rubpy) and Rose Bengal (RB) through these functionalized membranes was examined to determine the effect surface functionalization has on the selectivity and separation ability of pSi membranes. This study provides the basis for further investigation into more sophisticated surface functionalization and coupled with the biocompatibility of pSi will lead to new advances in membrane based bio-separations.

  10. A DATABASE OF >20 keV ELECTRON GREEN'S FUNCTIONS OF INTERPLANETARY TRANSPORT AT 1 AU

    SciTech Connect

    Agueda, N.; Sanahuja, B.; Vainio, R.

    2012-10-15

    We use interplanetary transport simulations to compute a database of electron Green's functions, i.e., differential intensities resulting at the spacecraft position from an impulsive injection of energetic (>20 keV) electrons close to the Sun, for a large number of values of two standard interplanetary transport parameters: the scattering mean free path and the solar wind speed. The nominal energy channels of the ACE, STEREO, and Wind spacecraft have been used in the interplanetary transport simulations to conceive a unique tool for the study of near-relativistic electron events observed at 1 AU. In this paper, we quantify the characteristic times of the Green's functions (onset and peak time, rise and decay phase duration) as a function of the interplanetary transport conditions. We use the database to calculate the FWHM of the pitch-angle distributions at different times of the event and under different scattering conditions. This allows us to provide a first quantitative result that can be compared with observations, and to assess the validity of the frequently used term beam-like pitch-angle distribution.

  11. Recent Developments in Three Dimensional Radiation Transport Using the Green's Function Technique

    NASA Technical Reports Server (NTRS)

    Rockell, Candice; Tweed, John; Blattnig, Steve R.; Mertens, Christopher J.

    2010-01-01

    In the future, astronauts will be sent into space for longer durations of time compared to previous missions. The increased risk of exposure to dangerous radiation, such as Galactic Cosmic Rays and Solar Particle Events, is of great concern. Consequently, steps must be taken to ensure astronaut safety by providing adequate shielding. In order to better determine and verify shielding requirements, an accurate and efficient radiation transport code based on a fully three dimensional radiation transport model using the Green's function technique is being developed

  12. Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1

    PubMed Central

    Gui, Chunshan; Hagenbuch, Bruno

    2009-01-01

    The liver-specific organic anion transporting polypeptides OATP1B1 and OATP1B3 are highly homologous and share numerous substrates. However, at low concentrations OATP1B1 shows substrate selectivity for estrone-3-sulfate. In this study, we investigated the molecular mechanism for this substrate selectivity of OATP1B1 by constructing OATP1B1/1B3 chimeric transporters and by site-directed mutagenesis. Functional studies of chimeras showed that transmembrane domain 10 is critical for the function of OATP1B1. We further identified four amino acid residues, namely L545, F546, L550, and S554 in TM10, whose simultaneous mutation caused almost complete loss of OATP1B1-mediated estrone-3-sulfate transport. Comparison of the kinetics of estrone-3-sulfate transport confirmed a biphasic pattern for OATP1B1, but showed a monophasic pattern for the quadruple mutant L545S/F546L/L550T/S554T. This mutant also showed reduced transport for other OATP1B1 substrates such as bromosulfophthalein and [d-penicillamine2,5]enkephalin. Helical wheel analysis and molecular modeling suggest that L545 is facing the substrate translocation pathway, whereas F546, L550, and S554 are located inside the protein. These results indicate that L545 might contribute to OATP1B1 function by interacting with substrates, whereas F546, L550, and S554 seem important for protein structure. In conclusion, our results show that TM10 is critical for the function of OATP1B1. PMID:19760661

  13. The Expression and Function of Organic Anion Transporting Polypeptides in Normal Tissues and in Cancer

    PubMed Central

    Obaidat, Amanda; Roth, Megan; Hagenbuch, Bruno

    2011-01-01

    Organic anion transporting polypeptides (OATPs) are members of the SLCO gene superfamily of proteins. The 11 human OATPs are classified in 6 families and subfamilies on the basis of their amino acid sequence similarities. OATPs are expressed in several epithelial tissues throughout the body and transport mainly amphipathic molecules with molecular weights of more than 300 kDa. Members of the OATP1 and OATP2 families are functionally the best-characterized OATPs. Among these are the multispecific OATP1A2, OATP1B1, OATP1B3, and OATP2B1. They transport various endo- and xenobiotics, including hormones and their conjugates as well as numerous drugs such as several anticancer agents. Recent reports demonstrate that some OATPs are up- or downregulated in several cancers and that OATP expression might affect cancer development. On the basis of the findings summarized in this review, we propose that OATPs could be valuable targets for anticancer therapy. PMID:21854228

  14. Lipopolysaccharide (LPS) disrupts particle transport, cilia function and sperm motility in an ex vivo oviduct model

    PubMed Central

    O’Doherty, A. M.; Di Fenza, M.; Kölle, S.

    2016-01-01

    The oviduct functions in the transportation of gametes to the site of fertilization (the ampulla) and is the site of early embryonic development. Alterations of this early developmental environment, such as the presence of sexually transmitted pathogens, may affect oviduct function leading to reduced fertilization rates and contribute to compromised embryonic development. In this study, sperm interactions, particle transport speed (PTS) and cilia beat frequency (CBF) in the ampulla following exposure to lipopolysaccharide (LPS), a constituent of the sexually transmitted pathogens Chlamydia trachomatis and Chlamydia abortus, was investigated. Three complementary experiments were performed to analyse; (1) bound sperm motility and cilia function (2) transport velocity in the oviduct and (3) the expression of genes related to immune function and inflammatory response (CASP3, CD14, MYD88, TLR4 and TRAF6). The motility of bound sperm was significantly lower in ampullae that were exposed to LPS. CBF and PTS significantly increased after treatment with LPS for 2 hours. Finally, gene expression analysis revealed that CASP3 and CD14 were significantly upregulated and TLR4 trended towards increased expression following treatment with LPS. These findings provide an insight on the impact of LPS on the oviduct sperm interaction, and have implications for both male and female fertility. PMID:27079521

  15. Expression, solubilisation, and purification of a functional CMP-sialic acid transporter in Pichia pastoris.

    PubMed

    Maggioni, Andrea; Hadley, Barbara; von Itzstein, Mark; Tiralongo, Joe

    2014-09-01

    Membrane proteins, including solute transporters play crucial roles in cellular function and have been implicated in a variety of important diseases, and as such are considered important targets for drug development. Currently the drug discovery process is heavily reliant on the structural and functional information discerned from high-resolution crystal structures. However, membrane protein structure determination is notoriously difficult, due in part to challenges faced in their expression, solubilisation and purification. The CMP-sialic acid transporter (CST) is considered to be an attractive target for drug discovery. CST inhibition reduces cancer cell sialylation and decreases the metastatic potential of cancer cells and to date, no crystal structure of the CST, or any other nucleotide sugar transporter exists. Here we describe the optimised conditions for expression in Pichia pastoris, solubilisation using n-nonyl β-d-maltopyranoside (NM) and single step purification of a functional CST. Importantly we show that despite being able to solubilise and purify the CST using a number of different detergents, only NM was able to maintain CST functionality.

  16. Polymer nanoreactors with dual functionality: simultaneous detoxification of peroxynitrite and oxygen transport.

    PubMed

    Dobrunz, Dominik; Toma, Adriana C; Tanner, Pascal; Pfohl, Thomas; Palivan, Cornelia G

    2012-11-13

    The design of multifunctional systems is in focus today as a key strategy for coping with complex challenges in various domains that include chemistry, medicine, environmental sciences, and technology. Herein, we introduce protein-containing polymer nanoreactors with dual functionality: peroxynitrite degradation and oxygen transport. Vesicles made of poly-(2-methyloxazoline)-poly(dimethylsiloxane)-poly(2-methyloxazoline) successfully encapsulated hemoglobin (Hb), which serves as a model protein because of its dual function in oxygen transport and peroxynitrite degradation. By inserting channel proteins, the polymer membranes of vesicles permitted passage of various compounds that served for the assessment of in situ Hb activity. The requisite conformational changes in the protein structure and the change in oxidation states that took place within the confined space of the vesicle cavity demonstrated that Hb preserved its dual functionality: peroxynitrite degradation and oxygen transport. The functionality of our nanoreactor, combined with its simple procedure of production and extensive stability over several months, supports it as a promising system for further medical applications.

  17. Overview of Nucleotide Sugar Transporter Gene Family Functions Across Multiple Species.

    PubMed

    Orellana, Ariel; Moraga, Carol; Araya, Macarena; Moreno, Adrian

    2016-08-14

    Glycoproteins and glycolipids are crucial in a number of cellular processes, such as growth, development, and responses to external cues, among others. Polysaccharides, another class of sugar-containing molecules, also play important structural and signaling roles in the extracellular matrix. The additions of glycans to proteins and lipids, as well as polysaccharide synthesis, are processes that primarily occur in the Golgi apparatus, and the substrates used in this biosynthetic process are nucleotide sugars. These proteins, lipids, and polysaccharides are also modified by the addition of sulfate groups in the Golgi apparatus in a series of reactions where nucleotide sulfate is needed. The required nucleotide sugar substrates are mainly synthesized in the cytosol and transported into the Golgi apparatus by nucleotide sugar transporters (NSTs), which can additionally transport nucleotide sulfate. Due to the critical role of NSTs in eukaryotic organisms, any malfunction of these could change glycan and polysaccharide structures, thus affecting function and altering organism physiology. For example, mutations or deletion on NST genes lead to pathological conditions in humans or alter cell walls in plants. In recent years, many NSTs have been identified and functionally characterized, but several remain unanalyzed. This study examined existing information on functionally characterized NSTs and conducted a phylogenetic analysis of 257 NSTs predicted from nine animal and plant model species, as well as from protists and fungi. From this analysis, relationships between substrate specificity and the primary NST structure can be inferred, thereby advancing understandings of nucleotide sugar gene family functions across multiple species.

  18. Overview of Nucleotide Sugar Transporter Gene Family Functions Across Multiple Species.

    PubMed

    Orellana, Ariel; Moraga, Carol; Araya, Macarena; Moreno, Adrian

    2016-08-14

    Glycoproteins and glycolipids are crucial in a number of cellular processes, such as growth, development, and responses to external cues, among others. Polysaccharides, another class of sugar-containing molecules, also play important structural and signaling roles in the extracellular matrix. The additions of glycans to proteins and lipids, as well as polysaccharide synthesis, are processes that primarily occur in the Golgi apparatus, and the substrates used in this biosynthetic process are nucleotide sugars. These proteins, lipids, and polysaccharides are also modified by the addition of sulfate groups in the Golgi apparatus in a series of reactions where nucleotide sulfate is needed. The required nucleotide sugar substrates are mainly synthesized in the cytosol and transported into the Golgi apparatus by nucleotide sugar transporters (NSTs), which can additionally transport nucleotide sulfate. Due to the critical role of NSTs in eukaryotic organisms, any malfunction of these could change glycan and polysaccharide structures, thus affecting function and altering organism physiology. For example, mutations or deletion on NST genes lead to pathological conditions in humans or alter cell walls in plants. In recent years, many NSTs have been identified and functionally characterized, but several remain unanalyzed. This study examined existing information on functionally characterized NSTs and conducted a phylogenetic analysis of 257 NSTs predicted from nine animal and plant model species, as well as from protists and fungi. From this analysis, relationships between substrate specificity and the primary NST structure can be inferred, thereby advancing understandings of nucleotide sugar gene family functions across multiple species. PMID:27261257

  19. Aquatic models for the study of renal transport function and pollutant toxicity

    SciTech Connect

    Miller, D.S.

    1987-04-01

    Studies of renal cell transport mechanisms and their impairment by xenobiotics are often limited by technical difficulties related to renal tubule complexity. Problems include the juxtaposition of multiple tubule segments with different transport functions and severely limited access to the tubular lumen. Some limitations can be overcome by the careful selection of an appropriate aquatic experimental system. Two aquatic models for the vertebrate proximal segment are discussed here. The first is the kidney from certain marine flounder, which offers the following advantages: long-term viability, little tissue of nonproximal origin, and easy tubule isolation. Data are presented to demonstrate how studies with flounder kidney can be used to elucidate cellular mechanisms whereby different classes of toxic pollutants may interact. Results from these experiments indicate that the excretion of certain anionic xenobiotics can be delayed (1) by other anionic xenobiotics that compete for secretory transport sites and (2) by compounds that disrupt cellular ion gradients and energy metabolism needed to drive transport. The second system is the crustacean urinary bladder, a simple, flatsheet epithelium. Bladder morphology and transport physiology closely resemble those of vertebrate proximal segment. Electron micrographs show a brush border membrane at the luminal surface, numerous mitochondria, and an infolded serosal membrane, while in vivo and in vitro transport studies show reabsorption of NaCl, nutrients and water and secretion of organic cations; organic anions are secreted in bladders from some species and reabsorbed in others. Moreover, since bladders can be mounted as flat sheets in flux chambers, studies with this tissue avoid the problems of complex renal tubule geometry and tissue heterogeneity and tissue heterogeneity that limit transport studies in proximal tubule.

  20. Transporters for cationic amino acids in animal cells: discovery, structure, and function.

    PubMed

    Devés, R; Boyd, C A

    1998-04-01

    The structure and function of the four cationic amino acid transporters identified in animal cells are discussed. The systems differ in specificity, cation dependence, and physiological role. One of them, system y+, is selective for cationic amino acids, whereas the others (B[0,+], b[0,+], and y+ L) also accept neutral amino acids. In recent years, cDNA clones related to these activities have been isolated. Thus two families of proteins have been identified: 1) CAT or cationic amino acid transporters and 2) BAT or broad-scope transport proteins. In the CAT family, three genes encode for four different isoforms [CAT-1, CAT-2A, CAT-2(B) and CAT-3]; these are approximately 70-kDa proteins with multiple transmembrane segments (12-14), and despite their structural similarity, they differ in tissue distribution, kinetics, and regulatory properties. System y+ is the expression of the activity of CAT transporters. The BAT family includes two isoforms (rBAT and 4F2hc); these are 59- to 78-kDa proteins with one to four membrane-spanning segments, and it has been proposed that these proteins act as transport regulators. The expression of rBAT and 4F2hc induces system b[0,+] and system y+ L activity in Xenopus laevis oocytes, respectively. The roles of these transporters in nutrition, endocrinology, nitric oxide biology, and immunology, as well as in the genetic diseases cystinuria and lysinuric protein intolerance, are reviewed. Experimental strategies, which can be used in the kinetic characterization of coexpressed transporters, are also discussed.

  1. Purinergic P2Y receptors in airway epithelia: from ion transport to immune functions.

    PubMed

    Hao, Yuan; Ko, Wing-hung

    2014-02-25

    The regulated transport of salt and water is essential to the integrated function of many organ systems, including the respiratory, reproductive, and digestive tracts. Airway epithelial fluid secretion is a passive process that is driven by osmotic forces, which are generated by ion transport. The main determinant of a luminally-directed osmotic gradient is the mucosal transport of chloride ions (Cl(-)) into the lumen. As with many epithelial cells, a number of classic signal transduction cascades are involved in the regulation of ion transport. There are two well-known intracellular signaling systems: an increase in intracellular Ca(2+) concentration ([Ca(2+)]i) and an increase in the rate of synthesis of cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP). Therefore, Cl(-) secretion is primarily activated via the opening of apical Ca(2+)- or cAMP-dependent Cl(-) channels at the apical membrane. The opening of basolateral Ca(2+)- or cAMP-activated K(+) channels, which hyperpolarizes the cell to maintain the driving force for Cl(-) exit through apical Cl(-) channels that are constitutively open, is also important in regulating transepithelial ion transport. P2Y receptors are expressed in the apical and/or basolateral membranes of virtually all polarized epithelia to control the transport of fluid and electrolytes. Human airway epithelial cells express multiple nucleotide receptors. Extracellular nucleotides, such as UTP and ATP, are calcium-mobilizing secretagogues. They are released into the extracellular space from airway epithelial cells and act on the same cell in an autocrine fashion to stimulate transepithelial ion transport. In addition, recent data support the role of P2Y receptors in releasing inflammatory cytokines in the bronchial epithelium and other immune cells.

  2. Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum.

    PubMed Central

    Cohen, N R; Knecht, D A; Lodish, H F

    1996-01-01

    To facilitate expression of the rat GLUT 1 glucose transporter cDNA in Dictyostelium discoideum, we mutated the 5' end of the coding sequence such that the codons for the first ten amino acids conformed to preferred Dictyostelium codon usage. As determined by Western-blot analysis, a population of Dictyostelium transformed with the mutated cDNA expressed nonglycosylated GLUT 1 protein. Cell lines expressing GLUT 1 transport radiolabelled 2-deoxy-D-glucose at a rate 6-10 times that of cell lines transformed with vector alone. The initial rate of inward transport of 2-deoxy-D-glucose was stimulated several-fold by the presence of unlabelled glucose in the Dictyostelium cytoplasm, exemplifying the trans-activation of GLUT 1 transport characteristic of GLUT 1 present in erythrocyte membranes. The K(m) and Ki values for 2-deoxy-D-glucose, D-glucose, D-mannose and D-galactose were 3.7 mM, 2.6 mM, 11 mM and 30 mM respectively, similar to the values for GLUT 1 expressed in mammalian cells. L-Glucose and L-galactose, which are not transported by GLUT 1, do not inhibit uptake of 2-deoxy-D-glucose in Dictyostelium expressing GLUT 1. Thus, even though GLUT 1 expressed in Dictyostelium is not N-glycosylated, it transports hexoses normally; this is the first example of functional expression of a mammalian transport protein in this lower eukaryote. PMID:8645185

  3. Functional characterization of Citrus macrophylla BOR1 as a boron transporter.

    PubMed

    Cañon, Paola; Aquea, Felipe; Rodríguez-Hoces de la Guardia, Amparo; Arce-Johnson, Patricio

    2013-11-01

    Plants have evolved to develop an efficient system of boron uptake and transport using a range of efflux carriers named BOR proteins. In this work we isolated and characterized a boron transporter of citrus (Citrus macrophylla), which was named CmBOR1 for its high homology to AtBOR1. CmBOR1 has 4403 bp and 12 exons. Its coding region has 2145 bp and encodes for a protein of 714 amino acids. CmBOR1 possesses the molecular features of BORs such as an anion exchanger domain and the presence of 10 transmembrane domains. Functional analysis in yeast indicated that CmBOR1 has an efflux boron transporter activity, and transformants have increased tolerance to excess boron. CmBOR1 is expressed in leaves, stem and flowers and shows the greatest accumulation in roots. The transcript accumulation was significantly increased under boron deficiency conditions in shoots. In contrast, the accumulation of the transcript did not change in boron toxicity conditions. Finally, we observed that constitutive expression of CmBOR1 was able to increase tolerance to boron deficiency conditions in Arabidopsis thaliana, suggesting that CmBOR1 is a xylem loading boron transporter. Based on these results, it was determined that CmBOR1 encodes a boric acid/borate transporter involved in tolerance to boron deficiency in plants.

  4. Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics.

    PubMed

    Mullen, Anna; Hall, Jenny; Diegel, Janika; Hassan, Isa; Fey, Adam; MacMillan, Fraser

    2016-06-15

    During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated. PMID:27284059

  5. Function and repair of dental enamel - Potential role of epithelial transport processes of ameloblasts.

    PubMed

    Varga, Gábor; Kerémi, Beáta; Bori, Erzsébet; Földes, Anna

    2015-07-01

    The hardest mammalian tissue, dental enamel is produced by ameloblasts, which are electrolyte-transporting epithelial cells. Although the end product is very different, they show many similarities to transporting epithelia of the pancreas, salivary glands and kidney. Enamel is produced in a multi-step epithelial secretory process that features biomineralization which is an interplay of secreted ameloblast specific proteins and the time-specific transport of minerals, protons and bicarbonate. First, "secretory" ameloblasts form the entire thickness of the enamel layer, but with low mineral content. Then they differentiate into "maturation" ameloblasts, which remove organic matrix from the enamel and in turn further build up hydroxyapatite crystals. The protons generated by hydroxyapatite formation need to be buffered, otherwise enamel will not attain full mineralization. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. The whole process has been the focus of many immunohistochemical and gene knock-out studies, but, perhaps surprisingly, no functional data existed for mineral ion transport by ameloblasts. However, recent studies including ours provided a better insight for molecular mechanism of mineral formation. The secretory regulation is not completely known as yet, but its significance is crucial. Impairing regulation retards or prevents completion of enamel mineralization and results in the development of hypomineralized enamel that easily erodes after dental eruption. Factors that impair this function are fluoride and disruption of pH regulators. Revealing these factors may eventually lead to the treatment of enamel hypomineralization related to genetic or environmentally induced malformation.

  6. Water transport and functional dynamics of aquaporins in osmoregulatory organs of fishes.

    PubMed

    Madsen, Steffen S; Engelund, Morten B; Cutler, Christopher P

    2015-08-01

    Aquaporins play distinct roles for water transport in fishes as they do in mammals-both at the cellular, organ, and organismal levels. However, with over 32,000 known species of fishes inhabiting almost every aquatic environment, from tidal pools, small mountain streams, to the oceans and extreme salty desert lakes, the challenge to obtain consensus as well as specific knowledge about aquaporin physiology in these vertebrate clades is overwhelming. Because the integumental surfaces of these animals are in intimate contact with the surrounding milieu, passive water loss and uptake represent two of the major osmoregulatory challenges that need compensation. However, neither obligatory nor regulatory water transport nor their mechanisms have been elucidated to the same degree as, for example, ion transport in fishes. Currently fewer than 60 papers address fish aquaporins. Most of these papers identify "what is present" and describe tissue expression patterns in various teleosts. The agnathans, chondrichthyans, and functionality of fish aquaporins generally have received little attention. This review emphasizes the functional physiology of aquaporins in fishes, focusing on transepithelial water transport in osmoregulatory organs in euryhaline species - primarily teleosts, but covering other taxonomic groups as well. Most current knowledge comes from teleosts, and there is a strong need for related information on older fish clades. Our survey aims to stimulate new, original research in this area and to bring together new collaborations across disciplines.

  7. Functional mechanisms of neurotransmitter transporters regulated by lipid-protein interactions of their terminal loops

    PubMed Central

    Khelashvili, George; Weinstein, Harel

    2015-01-01

    The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP2) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP2 lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP2 lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins. PMID:25847498

  8. Organic cation transporter 3 modulates murine basophil functions by controlling intracellular histamine levels

    PubMed Central

    Schneider, Elke; Machavoine, François; Pléau, Jean-Marie; Bertron, Anne-France; Thurmond, Robin L.; Ohtsu, Hiroshi; Watanabe, Takehiko; Schinkel, Alfred H.; Dy, Michel

    2005-01-01

    In this study, we identify the bidirectional organic cation transporter 3 (OCT3/Slc22a3) as the molecule responsible for histamine uptake by murine basophils. We demonstrate that OCT3 participates in the control of basophil functions because exogenous histamine can inhibit its own synthesis—and that of interleukin (IL)-4, IL-6, and IL-13—through this means of transport. Furthermore, ligands of H3/H4 histamine receptors or OCT3 inhibit histamine uptake, and outward transport of newly synthesized histamine. By doing so, they increase the histamine content of basophils, which explains why they mimic the effect of exogenous histamine. These drugs were no longer effective in histamine-free histidine decarboxylase (HDC)-deficient mice, in contrast with histamine itself. Histamine was not taken up and lost its inhibitory effect in mice deficient for OCT3, which proved its specific involvement. Intracellular histamine levels were increased strongly in IL-3–induced OCT3−/− bone marrow basophils, and explained why they generated fewer cytokines than their wild-type counterpart. Their production was enhanced when histamine synthesis was blocked by the specific HDC inhibitor α-fluoro-methyl histidine, and underscored the determinant role of histamine in the inhibitory effect. We postulate that pharmacologic modulation of histamine transport might become instrumental in the control of basophil functions during allergic diseases. PMID:16061728

  9. Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design

    NASA Astrophysics Data System (ADS)

    Ishikawa, Toshihisa; Tamura, Ai; Saito, Hikaru; Wakabayashi, Kanako; Nakagawa, Hiroshi

    2005-10-01

    In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

  10. A structure-function analysis of ion transport in crustacean gills and excretory organs.

    PubMed

    Freire, Carolina A; Onken, Horst; McNamara, John C

    2008-11-01

    Osmotic and ionic regulation in the Crustacea is mostly accomplished by the multifunctional gills, together with the excretory organs. In addition to their role in gas exchange, the gills constitute organs of active, transepithelial, ion transport, an activity of major importance that underlies many essential physiological functions like osmoregulation, calcium homeostasis, ammonium excretion and extracellular pH regulation. This review focuses on structure-function relationships in crustacean gills and excretory effectors, from the organ to molecular levels of organization. We address the diversity of structural architectures encountered in different crustacean gill types, and in constituent cell types, before examining the physiological mechanisms of Na(+), Cl(-), Ca(2+) and NH(4)(+) transport, and of acid-base equivalents, based on findings obtained over the last two decades employing advanced techniques. The antennal and maxillary glands constitute the principal crustacean excretory organs, which have received less attention in functional studies. We examine the diversity present in antennal and maxillary gland architecture, highlighting the structural similarities between both organ types, and we analyze the functions ascribed to each glandular segment. Emphasis is given to volume and osmoregulatory functions, capacity to produce dilute urine in freshwater crustaceans, and the effect of acclimation salinity on urine volume and composition. The microanatomy and diversity of function ascribed to gills and excretory organs are appraised from an evolutionary perspective, and suggestions made as to future avenues of investigation that may elucidate evolutionary and adaptive trends underpinning the invasion and exploitation of novel habitats.

  11. The structure and function of the dopamine transporter and its role in CNS diseases.

    PubMed

    McHugh, Patrick C; Buckley, David A

    2015-01-01

    In this chapter, we explore the basic science of the dopamine transporter (DAT), an integral component of a system that regulates dopamine homeostasis. Dopamine is a key neurotransmitter for several brain functions including locomotor control and reward systems. The transporter structure, function, mechanism of action, localization, and distribution, in addition to gene regulation, are discussed. Over many years, a wealth of information concerning the DAT has been accrued and has led to increased interest in the role of the DAT in a plethora of central nervous system diseases. These DAT characteristics are explored in relation to a range of neurological and neuropsychiatric diseases, with a particular focus on the genetics of the DAT. In addition, we discuss the pharmacology of the DAT and how this relates to disease and addiction. PMID:25817874

  12. Using time-dependent density functional theory in real time for calculating electronic transport

    NASA Astrophysics Data System (ADS)

    Schaffhauser, Philipp; Kümmel, Stephan

    2016-01-01

    We present a scheme for calculating electronic transport within the propagation approach to time-dependent density functional theory. Our scheme is based on solving the time-dependent Kohn-Sham equations on grids in real space and real time for a finite system. We use absorbing and antiabsorbing boundaries for simulating the coupling to a source and a drain. The boundaries are designed to minimize the effects of quantum-mechanical reflections and electrical polarization build-up, which are the major obstacles when calculating transport by applying an external bias to a finite system. We show that the scheme can readily be applied to real molecules by calculating the current through a conjugated molecule as a function of time. By comparing to literature results for the conjugated molecule and to analytic results for a one-dimensional model system we demonstrate the reliability of the concept.

  13. Functional and transport analyses of CLCN5 genetic changes identified in Dent disease patients.

    PubMed

    Tang, Xiaojing; Brown, Matthew R; Cogal, Andrea G; Gauvin, Daniel; Harris, Peter C; Lieske, John C; Romero, Michael F; Chang, Min-Hwang

    2016-04-01

    Dent disease type 1, an X-linked inherited kidney disease is caused by mutations in electrogenic Cl(-)/H(+) exchanger, ClC-5. We functionally studied the most frequent mutation (S244L) and two mutations recently identified in RKSC patients, Q629X and R345W. We also studied T657S, which has a high minor-allele frequency (0.23%) in the African-American population, was published previously as pathogenic to cause Dent disease. The transport properties of CLC-5 were electrophysiologically characterized. WT and ClC-5 mutant currents were inhibited by pH 5.5, but not affected by an alkaline extracellular solution (pH 8.5). The T657S and R345W mutations showed the same anion selectivity sequence as WT ClC-5 (SCN(-)>NO3(-)≈Cl(-)>Br(-)>I(-)). However, the S244L and Q629X mutations abolished this anion conductance sequence. Cell surface CLC-5 expression was quantified using extracellular HA-tagged CLC-5 and a chemiluminescent immunoassay. Cellular localization of eGFP-tagged CLC-5 proteins was also examined in HEK293 cells with organelle-specific fluorescent probes. Functional defects of R345W and Q629X mutations were caused by the trafficking of the protein to the plasma membrane since proteins were mostly retained in the endoplasmic reticulum, and mutations showed positive correlations between surface expression and transport function. In contrast, although the S244L transport function was significantly lower than WT, cell surface, early endosome, and endoplasmic reticulum expression was equal to that of WT CLC-5. Function and trafficking of T657S was equivalent to the WT CLC-5 suggesting this is a benign variant rather than pathogenic. These studies demonstrate the useful information that can be gained by detailed functional studies of mutations predicted to be pathogenic. PMID:27117801

  14. Modulation of N-methyl-d-aspartate receptor function by glycine transport

    PubMed Central

    Bergeron, Richard; Meyer, Torsten M.; Coyle, Joseph T.; Greene, Robert W.

    1998-01-01

    The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-d-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch–clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 μM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR. PMID:9861038

  15. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    PubMed

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter. PMID:26907376

  16. Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation.

    PubMed

    Mendiondo, Guillermina M; Medhurst, Anne; van Roermund, Carlo W; Zhang, Xuebin; Devonshire, Jean; Scholefield, Duncan; Fernández, José; Axcell, Barry; Ramsay, Luke; Waterham, Hans R; Waugh, Robbie; Theodoulou, Frederica L; Holdsworth, Michael J

    2014-09-01

    In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ.

  17. Functional characterization of dopamine transporter in vivo using Drosophila melanogaster behavioral assays.

    PubMed

    Ueno, Taro; Kume, Kazuhiko

    2014-01-01

    Dopamine mediates diverse functions such as motivation, reward, attention, learning/memory and sleep/arousal. Recent studies using model organisms including the fruit fly, have elucidated various physiological functions of dopamine, and identified specific neural circuits for these functions. Flies with mutations in the Drosophila dopamine transporter (dDAT) gene show enhanced dopamine signaling, and short sleep and memory impairment phenotypes. However, understanding the mechanism by which dopamine signaling causes these phenotypes requires an understanding of the dynamics of dopamine release. Here we report the effects of dDAT expression on behavioral traits. We show that dDAT expression in a subset of dopaminergic neurons is sufficient for normal sleep. dDAT expression in other cell types such as Kenyon cells and glial cells can also rescue the short sleep phenotype of dDAT mutants. dDAT mutants also show a down-regulation of the D1-like dopamine receptor dDA1, and this phenotype is rescued when dDAT is expressed in the same cell types in which it rescues sleep. On the other hand, dDAT overexpression in mushroom bodies, which are the target of memory forming dopamine neurons, abolishes olfactory aversive memory. Our data demonstrate that expression of extrasynaptic dopamine transporters can rescue some aspects of dopamine signaling in dopamine transporter mutants. These results provide novel insights into regulatory systems that modulate dopamine signaling. PMID:25232310

  18. Transport and detection of unlabeled nucleotide targets by microtubules functionalized with molecular beacons.

    PubMed

    Raab, Matthew; Hancock, William O

    2008-03-01

    Shrinking biosensors down to microscale dimensions enables increases in sensitivity and the ability to analyze minute samples such as the contents of individual cells. The goal of the present study is to create mobile microscale biosensors by attaching molecular beacons to microtubules and using kinesin molecular motors to transport these functionalized microtubules across two-dimensional surfaces. Previous work has shown that microfluidic channels can be functionalized with kinesin motors such that microtubules can be transported and directed through these channels without the need for external power or pressure-driven pumping. In this work, we show that molecular beacons can be attached to microtubules such that both the fluorescence reporting capability of the beacon and the motility of the microtubules are retained. These molecular beacon-functionalized microtubules were able to bind ssDNA target sequences, transport them across surfaces, and report their presence by an increase in fluorescence that was detected by fluorescence microscopy. This work is an important step toward creating hybrid microdevices for sensitive virus detection or analyzing mRNA profiles of individual cells.

  19. Multi-term approximation to the Boltzmann transport equation for electron energy distribution functions in nitrogen

    NASA Astrophysics Data System (ADS)

    Feng, Yue

    Plasma is currently a hot topic and it has many significant applications due to its composition of both positively and negatively charged particles. The energy distribution function is important in plasma science since it characterizes the ability of the plasma to affect chemical reactions, affect physical outcomes, and drive various applications. The Boltzmann Transport Equation is an important kinetic equation that provides an accurate basis for characterizing the distribution function---both in energy and space. This dissertation research proposes a multi-term approximation to solve the Boltzmann Transport Equation by treating the relaxation process using an expansion of the electron distribution function in Legendre polynomials. The elastic and 29 inelastic cross sections for electron collisions with nitrogen molecules (N2) and singly ionized nitrogen molecules ( N+2 ) have been used in this application of the Boltzmann Transport Equation. Different numerical methods have been considered to compare the results. The numerical methods discussed in this thesis are the implicit time-independent method, the time-dependent Euler method, the time-dependent Runge-Kutta method, and finally the implicit time-dependent relaxation method by generating the 4-way grid with a matrix solver. The results show that the implicit time-dependent relaxation method is the most accurate and stable method for obtaining reliable results. The results were observed to match with the published experimental data rather well.

  20. Study of Transport Properties in Armchair Graphyne Nanoribbons: A Density Functional Approach

    NASA Astrophysics Data System (ADS)

    Golafrooz Shahri, S.; Roknabadi, M. R.; Shahtahmasebi, N.; Behdani, M.

    2016-07-01

    In present paper, the non-equilibrium Green function (NEGF) method along with the density functional theory (DFT) are used to investigate the effect of width on transport and electronic properties of armchair graphyne (γ-graphyne) nanoribbons. The results show that all the studied nanoribbons are semiconductor and their band gaps decrease as the widths of nanoribbons increase, which will result in increasing current at a certain voltage. Also our results show the promising application of armchair graphyne nanoribbons in nano-electrical devices.

  1. Numerical Methods for a Kohn-Sham Density Functional Model Based on Optimal Transport.

    PubMed

    Chen, Huajie; Friesecke, Gero; Mendl, Christian B

    2014-10-14

    In this paper, we study numerical discretizations to solve density functional models in the "strictly correlated electrons" (SCE) framework. Unlike previous studies, our work is not restricted to radially symmetric densities. In the SCE framework, the exchange-correlation functional encodes the effects of the strong correlation regime by minimizing the pairwise Coulomb repulsion, resulting in an optimal transport problem. We give a mathematical derivation of the self-consistent Kohn-Sham-SCE equations, construct an efficient numerical discretization for this type of problem for N = 2 electrons, and apply it to the H2 molecule in its dissociating limit. PMID:26588133

  2. Workshop on Functional Requirements for the Modeling of Fate and Transport of Waterborne CBRN Materials

    SciTech Connect

    Giles, GE

    2005-02-03

    The purpose of this Workshop on ''Functional Requirements for the Modeling of Fate and Transport of Waterborne CBRN Materials'' was to solicit functional requirements for tools that help Incident Managers plan for and deal with the consequences of industrial or terrorist releases of materials into the nation's waterways and public water utilities. Twenty representatives attended and several made presentations. Several hours of discussions elicited a set of requirements. These requirements were summarized in a form for the attendees to vote on their highest priority requirements. These votes were used to determine the prioritized requirements that are reported in this paper and can be used to direct future developments.

  3. Loss of divalent metal transporter 1 function promotes brain copper accumulation and increases impulsivity.

    PubMed

    Han, Murui; Chang, JuOae; Kim, Jonghan

    2016-09-01

    The divalent metal transporter 1 (DMT1) is a major iron transporter required for iron absorption and erythropoiesis. Loss of DMT1 function results in microcytic anemia. While iron plays an important role in neural function, the behavioral consequences of DMT1 deficiency are largely unexplored. The goal of this study was to define the neurobehavioral and neurochemical phenotypes of homozygous Belgrade (b/b) rats that carry DMT1 mutation and explore potential mechanisms of these phenotypes. The b/b rats (11-12 weeks old) and their healthy littermate heterozygous (+/b) Belgrade rats were subject to elevated plus maze tasks. The b/b rats spent more time in open arms, entered open arms more frequently and traveled more distance in the maze than +/b controls, suggesting increased impulsivity. Impaired emotional behavior was associated with down-regulation of GABA in the hippocampus in b/b rats. Also, b/b rats showed increased GABAA receptor α1 and GABA transporter, indicating altered GABAergic function. Furthermore, metal analysis revealed that b/b rats have decreased total iron, but normal non-heme iron, in the brain. Interestingly, b/b rats exhibited unusually high copper levels in most brain regions, including striatum and hippocampus. Quantitative PCR analysis showed that both copper importer copper transporter 1 and exporter copper-transporting ATPase 1 were up-regulated in the hippocampus from b/b rats. Finally, b/b rats exhibited increased 8-isoprostane levels and decreased glutathione/glutathione disulfide ratio in the hippocampus, reflecting elevated oxidative stress. Combined, our results suggest that copper loading in DMT1 deficiency could induce oxidative stress and impair GABA metabolism, which promote impulsivity-like behavior. Iron-copper model: Mutations in the divalent metal transporter 1 (DMT1) decrease body iron status and up-regulate copper absorption, which leads to copper loading in the brain and consequently increases metal-induced oxidative

  4. Loss of divalent metal transporter 1 function promotes brain copper accumulation and increases impulsivity.

    PubMed

    Han, Murui; Chang, JuOae; Kim, Jonghan

    2016-09-01

    The divalent metal transporter 1 (DMT1) is a major iron transporter required for iron absorption and erythropoiesis. Loss of DMT1 function results in microcytic anemia. While iron plays an important role in neural function, the behavioral consequences of DMT1 deficiency are largely unexplored. The goal of this study was to define the neurobehavioral and neurochemical phenotypes of homozygous Belgrade (b/b) rats that carry DMT1 mutation and explore potential mechanisms of these phenotypes. The b/b rats (11-12 weeks old) and their healthy littermate heterozygous (+/b) Belgrade rats were subject to elevated plus maze tasks. The b/b rats spent more time in open arms, entered open arms more frequently and traveled more distance in the maze than +/b controls, suggesting increased impulsivity. Impaired emotional behavior was associated with down-regulation of GABA in the hippocampus in b/b rats. Also, b/b rats showed increased GABAA receptor α1 and GABA transporter, indicating altered GABAergic function. Furthermore, metal analysis revealed that b/b rats have decreased total iron, but normal non-heme iron, in the brain. Interestingly, b/b rats exhibited unusually high copper levels in most brain regions, including striatum and hippocampus. Quantitative PCR analysis showed that both copper importer copper transporter 1 and exporter copper-transporting ATPase 1 were up-regulated in the hippocampus from b/b rats. Finally, b/b rats exhibited increased 8-isoprostane levels and decreased glutathione/glutathione disulfide ratio in the hippocampus, reflecting elevated oxidative stress. Combined, our results suggest that copper loading in DMT1 deficiency could induce oxidative stress and impair GABA metabolism, which promote impulsivity-like behavior. Iron-copper model: Mutations in the divalent metal transporter 1 (DMT1) decrease body iron status and up-regulate copper absorption, which leads to copper loading in the brain and consequently increases metal-induced oxidative

  5. Molecular changes in hepatic metabolism and transport in cirrhosis and their functional importance

    PubMed Central

    Dietrich, Christoph G; Götze, Oliver; Geier, Andreas

    2016-01-01

    Liver cirrhosis is the common endpoint of many hepatic diseases and represents a relevant risk for liver failure and hepatocellular carcinoma. The progress of liver fibrosis and cirrhosis is accompanied by deteriorating liver function. This review summarizes the regulatory and functional changes in phase I and phase II metabolic enzymes as well as transport proteins and provides an overview regarding lipid and glucose metabolism in cirrhotic patients. Interestingly, phase I enzymes are generally downregulated transcriptionally, while phase II enzymes are mostly preserved transcriptionally but are reduced in their function. Transport proteins are regulated in a specific way that resembles the molecular changes observed in obstructive cholestasis. Lipid and glucose metabolism are characterized by insulin resistance and catabolism, leading to the disturbance of energy expenditure and wasting. Possible non-invasive tests, especially breath tests, for components of liver metabolism are discussed. The heterogeneity and complexity of changes in hepatic metabolism complicate the assessment of liver function in individual patients. Additionally, studies in humans are rare, and species differences preclude the transferability of data from rodents to humans. In clinical practice, some established global scores or criteria form the basis for the functional evaluation of patients with liver cirrhosis, but difficult treatment decisions such as selection for transplantation or resection require further research regarding the application of existing non-invasive tests and the development of more specific tests. PMID:26755861

  6. Functional Impact of Corticotropin-Releasing Factor Exposure on Tau Phosphorylation and Axon Transport

    PubMed Central

    Le, Michelle H.; Weissmiller, April M.; Monte, Louise; Lin, Po Han; Hexom, Tia C.; Natera, Orlangie; Wu, Chengbiao; Rissman, Robert A.

    2016-01-01

    Stress exposure or increased levels of corticotropin-releasing factor (CRF) induce hippocampal tau phosphorylation (tau-P) in rodent models, a process that is dependent on the type-1 CRF receptor (CRFR1). Although these preclinical studies on stress-induced tau-P provide mechanistic insight for epidemiological work that identifies stress as a risk factor for Alzheimer’s disease (AD), the actual impact of stress-induced tau-P on neuronal function remains unclear. To determine the functional consequences of stress-induced tau-P, we developed a novel mouse neuronal cell culture system to explore the impact of acute (0.5hr) and chronic (2hr) CRF treatment on tau-P and integral cell processes such as axon transport. Consistent with in vivo reports, we found that chronic CRF treatment increased tau-P levels and caused globular accumulations of phosphorylated tau in dendritic and axonal processes. Furthermore, while both acute and chronic CRF treatment led to significant reduction in CREB activation and axon transport of brain-derived neurotrophic factor (BDNF), this was not the case with mitochondrial transport. Acute CRF treatment caused increased mitochondrial velocity and distance traveled in neurons, while chronic CRF treatment modestly decreased mitochondrial velocity and greatly increased distance traveled. These results suggest that transport of cellular energetics may take priority over growth factors during stress. Tau-P was required for these changes, as co-treatment of CRF with a GSK kinase inhibitor prevented CRF-induced tau-P and all axon transport changes. Collectively, our results provide mechanistic insight into the consequences of stress peptide-induced tau-P and provide an explanation for how chronic stress via CRF may lead to neuronal vulnerability in AD. PMID:26790099

  7. Functional Characterization and Metal Ion Specificity of the Metal-Citrate Complex Transporter from Streptomyces coelicolor▿

    PubMed Central

    Lensbouer, Joshua J.; Patel, Ami; Sirianni, Joseph P.; Doyle, Robert P.

    2008-01-01

    Secondary transporters of citrate in complex with metal ions belong to the bacterial CitMHS family, about which little is known. The transport of metal-citrate complexes in Streptomyces coelicolor has been investigated. The best cofactor for citrate uptake in Streptomyces coelicolor is Fe3+, but uptake was also noted for Ca2+, Pb2+, Ba2+, and Mn2+. Uptake was not observed with the Mg2+, Ni2+, or Co2+ cofactor. The transportation of iron- and calcium-citrate makes these systems unique among the CitMHS family members reported to date. No complementary uptake akin to that observed for the CitH (Ca2+, Ba2+, Sr2+) and CitM (Mg2+, Ni2+, Mn2+, Co2+, Zn2+) systems of Bacillus subtilis was noted. Competitive experiments using EGTA confirmed that metal-citrate complex formation promoted citrate uptake. Uptake of free citrate was not observed. The open reading frame postulated as being responsible for the metal-citrate transport observed in Streptomyces coelicolor was cloned and overexpressed in Escherichia coli strains with the primary Fe3+-citrate transport system (fecABCDE) removed. Functional expression was successful, with uptake of Ca2+-citrate, Fe3+-citrate, and Pb2+-citrate observed. No free-citrate transport was observed in IPTG (isopropyl-β-d-thiogalactopyranoside)-induced or -uninduced E. coli. Metabolism of the Fe3+-citrate and Ca2+-citrate complexes, but not the Pb2+-citrate complex, was observed. Rationalization is based on the difference in metal-complex coordination upon binding of the metal by citrate. PMID:18556792

  8. Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells.

    PubMed

    Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

    2012-08-15

    Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration.

  9. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    PubMed

    Polireddy, Kishore; Khan, Mohiuddin Md Taimur; Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J; Haynes, Mark K; Bologa, Cristian G; Oprea, Tudor I; Tegos, George P; Sklar, Larry A; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity. PMID:22808084

  10. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

    PubMed Central

    Reck, Jaimee; Schauer, Alexandria M.; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A.; Porter, Mary E.

    2016-01-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  11. The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas.

    PubMed

    Reck, Jaimee; Schauer, Alexandria M; VanderWaal Mills, Kristyn; Bower, Raqual; Tritschler, Douglas; Perrone, Catherine A; Porter, Mary E

    2016-08-01

    The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms. PMID:27251063

  12. Loss of GSNOR1 Function Leads to Compromised Auxin Signaling and Polar Auxin Transport.

    PubMed

    Shi, Ya-Fei; Wang, Da-Li; Wang, Chao; Culler, Angela Hendrickson; Kreiser, Molly A; Suresh, Jayanti; Cohen, Jerry D; Pan, Jianwei; Baker, Barbara; Liu, Jian-Zhong

    2015-09-01

    Cross talk between phytohormones, nitric oxide (NO), and auxin has been implicated in the control of plant growth and development. Two recent reports indicate that NO promoted auxin signaling but inhibited auxin transport probably through S-nitrosylation. However, genetic evidence for the effect of S-nitrosylation on auxin physiology has been lacking. In this study, we used a genetic approach to understand the broader role of S-nitrosylation in auxin physiology in Arabidopsis. We compared auxin signaling and transport in Col-0 and gsnor1-3, a loss-of-function GSNOR1 mutant defective in protein de-nitrosylation. Our results showed that auxin signaling was impaired in the gsnor1-3 mutant as revealed by significantly reduced DR5-GUS/DR5-GFP accumulation and compromised degradation of AXR3NT-GUS, a useful reporter in interrogating auxin-mediated degradation of Aux/IAA by auxin receptors. In addition, polar auxin transport was compromised in gsnor1-3, which was correlated with universally reduced levels of PIN or GFP-PIN proteins in the roots of the mutant in a manner independent of transcription and 26S proteasome degradation. Our results suggest that S-nitrosylation and GSNOR1-mediated de-nitrosylation contribute to auxin physiology, and impaired auxin signaling and compromised auxin transport are responsible for the auxin-related morphological phenotypes displayed by the gsnor1-3 mutant.

  13. Loss of GSNOR1 Function Leads to Compromised Auxin Signaling and Polar Auxin Transport.

    PubMed

    Shi, Ya-Fei; Wang, Da-Li; Wang, Chao; Culler, Angela Hendrickson; Kreiser, Molly A; Suresh, Jayanti; Cohen, Jerry D; Pan, Jianwei; Baker, Barbara; Liu, Jian-Zhong

    2015-09-01

    Cross talk between phytohormones, nitric oxide (NO), and auxin has been implicated in the control of plant growth and development. Two recent reports indicate that NO promoted auxin signaling but inhibited auxin transport probably through S-nitrosylation. However, genetic evidence for the effect of S-nitrosylation on auxin physiology has been lacking. In this study, we used a genetic approach to understand the broader role of S-nitrosylation in auxin physiology in Arabidopsis. We compared auxin signaling and transport in Col-0 and gsnor1-3, a loss-of-function GSNOR1 mutant defective in protein de-nitrosylation. Our results showed that auxin signaling was impaired in the gsnor1-3 mutant as revealed by significantly reduced DR5-GUS/DR5-GFP accumulation and compromised degradation of AXR3NT-GUS, a useful reporter in interrogating auxin-mediated degradation of Aux/IAA by auxin receptors. In addition, polar auxin transport was compromised in gsnor1-3, which was correlated with universally reduced levels of PIN or GFP-PIN proteins in the roots of the mutant in a manner independent of transcription and 26S proteasome degradation. Our results suggest that S-nitrosylation and GSNOR1-mediated de-nitrosylation contribute to auxin physiology, and impaired auxin signaling and compromised auxin transport are responsible for the auxin-related morphological phenotypes displayed by the gsnor1-3 mutant. PMID:25917173

  14. Vitamin C Function in the Brain: Vital Role of the Ascorbate Transporter (SVCT2)

    PubMed Central

    Harrison, Fiona E.; May, James M.

    2009-01-01

    Ascorbate (vitamin C) is a vital antioxidant molecule in the brain. However, it also has a number of other important functions, participating as a co-factor in several enzyme reactions including catecholamine synthesis, collagen production and regulation of HIF-1α. Ascorbate is transported into the brain and neurons via the Sodium-dependent Vitamin C Transporter-2 (SVCT2), which causes accumulation of ascorbate within cells against a concentration gradient. Dehydroascorbic acid, the oxidized form of ascorbate, is transported via glucose transporters of the GLUT family. Once in cells, it is rapidly reduced to ascorbate. The highest concentrations of ascorbate in the body are found in the brain and neuroendocrine tissues such as adrenal, although the brain is the most difficult organ to deplete of ascorbate. Combined with regional asymmetry in ascorbate distribution within different brain areas, these facts suggest an important role for ascorbate in the brain. Ascorbate is proposed as a neuromodulator of glutamatergic, dopaminergic, cholinergic and GABAergic transmission and related behaviors. Neurodegenerative diseases typically involve high levels of oxidative stress and thus ascorbate has been posited to have potential therapeutic roles against ischemic stroke, Alzheimer's disease, Parkinson's disease and Huntingdon's disease. PMID:19162177

  15. Consideration of allosterism and interacting proteins in the physiological functions of the serotonin transporter.

    PubMed

    Zhong, Huailing; Sánchez, Connie; Caron, Marc G

    2012-02-15

    The serotonin transporter (SERT) functions to transport serotonin (5-HT) from the extracellular space into neurons to maintain homeostatic control of 5-HT. It is the molecular target for selective serotonin reuptake inhibitor (SSRI) antidepressants. Preclinical research has shown that some SERT inhibitors can bind to two distinct binding sites on the SERT, a primary high affinity binding site and a low affinity allosteric binding site. Mutational studies of the SERT and computational modeling methods with escitalopram resulted in the identification of key amino acid residues important for the function of the allosteric binding site. While this allosteric binding site appears to influence the clinical efficacy of escitalopram under physiological conditions, the molecular mechanism of this effect is still poorly understood and may involve a large network of protein-protein interactions with the SERT. Dynamic interfaces between the SERT and the SERT interacting proteins (SIPs) potentially influence not only the SERT on its uptake function, its regulation, and trafficking, but also on known as well as yet to be identified non-canonical signaling pathways through SIPs. In this commentary, we outline approaches in the areas of selective small-molecule allosteric compound discovery, biochemistry, in vivo genetic knock-in mouse models, as well as computational and structural biology. These studies of the intra-molecular allosteric modulation of the SERT in the context of the myriad of potential inter-molecular signaling interactions with SIPs may help uncover unknown physiological functions of the SERT.

  16. A parametric transfer function methodology for analyzing reactive transport in nonuniform flow.

    PubMed

    Luo, Jian; Cirpka, Olaf A; Fienen, Michael N; Wu, Wei-min; Mehlhorn, Tonia L; Carley, Jack; Jardine, Philip M; Criddle, Craig S; Kitanidis, Peter K

    2006-02-01

    We analyze reactive transport during in-situ bioremediation in a nonuniform flow field, involving multiple extraction and injection wells, by the method of transfer functions. Gamma distributions are used as parametric models of the transfer functions. Apparent parameters of classical transport models may be estimated from those of the gamma distributions by matching temporal moments. We demonstrate the method by application to measured data taken at a field experiment on bioremediation conducted in a multiple-well system in Oak Ridge, TN. Breakthrough curves (BTCs) of a conservative tracer (bromide) and a reactive compound (ethanol) are measured at multi-level sampling (MLS) wells and in extraction wells. The BTCs of both compounds are jointly analyzed to estimate the first-order degradation rate of ethanol. To quantify the tracer loss, we compare the approaches of using a scaling factor and a first-order decay term. Results show that by including a scaling factor both gamma distributions and inverse-Gaussian distributions (transfer functions according to the advection-dispersion equation) are suitable to approximate the transfer functions and estimate the reactive rate coefficients for both MLS and extraction wells. However, using a first-order decay term for tracer loss fails to describe the BTCs at the extraction well, which is affected by the nonuniform distribution of travel paths.

  17. Functional assembly of thylakoid deltapH-dependent/Tat protein transport pathway components in vitro.

    PubMed

    Fincher, Vivian; Dabney-Smith, Carole; Cline, Kenneth

    2003-12-01

    Assembly of the components of the thylakoid deltapH-dependent/Tat protein transport machinery was analyzed in vitro. Upon incubation with intact chloroplasts, precursors to all three components, Hcf106, cpTatC and Tha4, were imported into the organelle and assembled into characteristic endogenous complexes. In particular, all of the imported cpTatC and approximately two-thirds of the imported Hcf106 functionally assembled into 700 kDa complexes capable of binding Tat pathway precursor proteins. The amounts assembled into thylakoids by this procedure were moderate. However, physiological quantities of mature forms of Tha4 and Hcf106 were integrated into isolated thylakoids and a significant percentage of the Hcf106 so integrated was assembled into the 700 kDa complex. Interestingly, a mutant form of Hcf106 in which an invariant transmembrane glutamate was changed to glutamine integrated into the membrane but did not assemble into the receptor complex. Analysis of energy and known pathway component requirements indicated that Hcf106 and Tha4 integrate by an unassisted or 'spontaneous' mechanism. The functionality of in vitro integrated Tha4 was verified by its ability to restore transport to thylakoid membranes from the maize tha4 mutant, which lacks the Tha4 protein. Development of this functional in vitro assembly assay will facilitate structure-function studies of the thylakoid Tat pathway translocation machinery.

  18. Lactate Transport and Receptor Actions in Retina: Potential Roles in Retinal Function and Disease.

    PubMed

    Kolko, Miriam; Vosborg, Fia; Henriksen, Ulrik L; Hasan-Olive, Md Mahdi; Diget, Elisabeth Holm; Vohra, Rupali; Gurubaran, Iswariya Raja Sridevi; Gjedde, Albert; Mariga, Shelton Tendai; Skytt, Dorte M; Utheim, Tor Paaske; Storm-Mathisen, Jon; Bergersen, Linda H

    2016-06-01

    In retina, like in brain, lactate equilibrates across cell membranes via monocarboxylate transporters and in the extracellular space by diffusion, forming a basis for the action of lactate as a transmitter of metabolic signals. In the present paper, we argue that the lactate receptor GPR81, also known as HCAR1, may contribute importantly to the control of retinal cell functions in health and disease. GPR81, a G-protein coupled receptor, is known to downregulate cAMP both in adipose and nervous tissue. The receptor also acts through other down-stream mechanisms to control functions, such as excitability, metabolism and inflammation. Recent publications predict effects of the lactate receptor on neurodegeneration. Neurodegenerative diseases in retina, where the retinal ganglion cells die, notably glaucoma and diabetic retinopathy, may be linked to disturbed lactate homeostasis. Pilot studies reveal high GPR81 mRNA in retina and indicate GPR81 localization in Müller cells and retinal ganglion cells. Moreover, monocarboxylate transporters are expressed in retinal cells. We envision that lactate receptors and transporters could be useful future targets of novel therapeutic strategies to protect neurons and prevent or counteract glaucoma as well as other retinal diseases. PMID:26677077

  19. Expression and functional studies of genes involved in transport and metabolism of glycerol in Pachysolen tannophilus

    PubMed Central

    2013-01-01

    Background Pachysolen tannophilus is a non-conventional yeast, which can metabolize many of the carbon sources found in low cost feedstocks including glycerol and xylose. The xylose utilisation pathways have been extensively studied in this organism. However, the mechanism behind glycerol metabolism is poorly understood. Using the recently published genome sequence of P. tannophilus CBS4044, we searched for genes with functions in glycerol transport and metabolism by performing a BLAST search using the sequences of the relevant genes from Saccharomyces cerevisiae as queries. Results Quantitative real-time PCR was performed to unveil the expression patterns of these genes during growth of P. tannophilus on glycerol and glucose as sole carbon sources. The genes predicted to be involved in glycerol transport in P. tannophilus were expressed in S. cerevisiae to validate their function. The S. cerevisiae strains transformed with heterologous genes showed improved growth and glycerol consumption rates with glycerol as the sole carbon source. Conclusions P. tannophilus has characteristics relevant for a microbial cell factory to be applied in a biorefinery setting, i.e. its ability to utilise the carbon sources such as xylose and glycerol. However, the strain is not currently amenable to genetic modification and transformation. Heterologous expression of the glycerol transporters from P. tannophilus, which has a relatively high growth rate on glycerol, could be used as an approach for improving the efficiency of glycerol assimilation in other well characterized and applied cell factories such as S. cerevisiae. PMID:23514356

  20. Oxygen transport and the function of myoglobin. Theoretical model and experiments in chicken gizzard smooth muscle.

    PubMed

    de Koning, J; Hoofd, L J; Kreuzer, F

    1981-03-01

    We studied the steady-state oxygen transfer across thin layers of respiring chicken gizzard smooth muscle and compared three models for oxygen consumption with respect to their influence on the facilitation of oxygen diffusion by myoglobin. These models assumed zero-order, Michaelis-Menten or exponential kinetics. The transport equation was solved for these models with simultaneous oxygen facilitation assuming chemical equilibrium between oxygen and myoglobin. Experimental flux data were obtained in two situations: a) high oxygen pressure throughout the layer of tissue providing maximum oxygen consumption and oxygen permeability, and b) anoxic conditions in part of the layer and with submaximal oxygen consumption and desaturation of myoglobin. Measurements in the presence of functional myoglobin were compared with data obtained after abolishing the transport function of myoglobin by application of 1 kPa carbon monoxide. It was found that oxygen consumption interferes with the facilitation effect. The oxygen pressure at half maximum oxygen consumption in the Michaelis-Menten model was 0.3 +/- 0.1 (S.E) kPa. The facilitation of the oxygen transport by myoglobin was 50 to 100% of the maximum value to be expected on the basis of the prevailing myoglobin concentration. PMID:7195007

  1. Lactate Transport and Receptor Actions in Retina: Potential Roles in Retinal Function and Disease.

    PubMed

    Kolko, Miriam; Vosborg, Fia; Henriksen, Ulrik L; Hasan-Olive, Md Mahdi; Diget, Elisabeth Holm; Vohra, Rupali; Gurubaran, Iswariya Raja Sridevi; Gjedde, Albert; Mariga, Shelton Tendai; Skytt, Dorte M; Utheim, Tor Paaske; Storm-Mathisen, Jon; Bergersen, Linda H

    2016-06-01

    In retina, like in brain, lactate equilibrates across cell membranes via monocarboxylate transporters and in the extracellular space by diffusion, forming a basis for the action of lactate as a transmitter of metabolic signals. In the present paper, we argue that the lactate receptor GPR81, also known as HCAR1, may contribute importantly to the control of retinal cell functions in health and disease. GPR81, a G-protein coupled receptor, is known to downregulate cAMP both in adipose and nervous tissue. The receptor also acts through other down-stream mechanisms to control functions, such as excitability, metabolism and inflammation. Recent publications predict effects of the lactate receptor on neurodegeneration. Neurodegenerative diseases in retina, where the retinal ganglion cells die, notably glaucoma and diabetic retinopathy, may be linked to disturbed lactate homeostasis. Pilot studies reveal high GPR81 mRNA in retina and indicate GPR81 localization in Müller cells and retinal ganglion cells. Moreover, monocarboxylate transporters are expressed in retinal cells. We envision that lactate receptors and transporters could be useful future targets of novel therapeutic strategies to protect neurons and prevent or counteract glaucoma as well as other retinal diseases.

  2. Divergent metabolome and proteome suggest functional independence of dual phloem transport systems in cucurbits

    PubMed Central

    Zhang, Baichen; Tolstikov, Vladimir; Turnbull, Colin; Hicks, Leslie M.; Fiehn, Oliver

    2010-01-01

    Cucurbitaceous plants (cucurbits) have long been preferred models for studying phloem physiology. However, these species are unusual in that they possess two different phloem systems, one within the main vascular bundles [fascicular phloem (FP)] and another peripheral to the vascular bundles and scattered through stem and petiole cortex tissues [extrafascicular phloem (EFP)]. We have revisited the assumption that the sap released after shoot incision originates from the FP, and also investigated the long-standing question of why the sugar content of this sap is ~30-fold less than predicted for requirements of photosynthate delivery. Video microscopy and phloem labeling experiments unexpectedly reveal that FP very quickly becomes blocked upon cutting, whereas the extrafascicular phloem bleeds for extended periods. Thus, all cucurbit phloem sap studies to date have reported metabolite, protein, and RNA composition and transport in the relatively minor extrafascicular sieve tubes. Using tissue dissection and direct sampling of sieve tube contents, we show that FP in fact does contain up to 1 M sugars, in contrast to low-millimolar levels in the EFP. Moreover, major phloem proteins in sieve tubes of FP differ from those that predominate in the extrafascicular sap, and include several previously uncharacterized proteins with little or no homology to databases. The overall compositional differences of the two phloem systems strongly indicate functional isolation. On this basis, we propose that the fascicular phloem is largely responsible for sugar transport, whereas the extrafascicular phloem may function in signaling, defense, and transport of other metabolites. PMID:20566864

  3. Linking suspended sediment transport metrics with fish functional traits in the Northwestern Great Plains (Invited)

    NASA Astrophysics Data System (ADS)

    Schwartz, J. S.; Simon, A.; Klimetz, L.

    2009-12-01

    Loss of ecological integrity due to excessive suspended sediment in rivers and streams is a major cause of water quality impairment in the United States. Although 32 states have developed numeric criteria for turbidity or suspended solids, or both according to the USEPA (2006), criteria is typically written as a percent exceedance above background and what constitutes background is not well defined. Defining a background level is problematic considering suspended sediments and related turbidity levels change with flow stage and season, and limited scientific data exists on relationships between sediment exposure and biotic response. Current assessment protocols for development of sediment total maximum daily loads (TMDLs) lack a means to link temporally-variable sediment transport rates with specific losses of ecological functions as loads increase. This study, within the in Northwestern Great Plains Ecoregion, co-located 58 USGS gauging stations with existing flow and suspended sediment data, and fish data from federal and state agencies. Suspended sediment concentration (SSC) transport metrics were quantified into exceedance frequencies of a given magnitude, duration as the number of consecutive days a given concentration was equaled or exceeded, dosage as concentration x duration, and mean annual suspended sediment yields. A functional traits-based approach was used to correlate SSC transport metrics with site occurrences of 20 fish traits organized into four main groups: preferred rearing mesohabitat, trophic structure, feeding habits, and spawning behavior. Negative correlations between SSC metrics and trait occurrences were assumed to represent potential conditions for impairment, specifically identifying an ecological loss by functional trait. Potential impairment conditions were linked with presence of the following traits: habitat preferences for stream pool and river shallow waters; feeding generalists, omnivores, piscivores; and several spawning

  4. Classification of transporters using efficient radial basis function networks with position-specific scoring matrices and biochemical properties.

    PubMed

    Ou, Yu-Yen; Chen, Shu-An; Gromiha, M Michael

    2010-05-15

    Transporters are proteins that are involved in the movement of ions or molecules across biological membranes. Transporters are generally classified into channels/pores, electrochemical transporters, and active transporters. Discriminating the specific class of transporters and their subfamilies are essential tasks in computational biology for the advancement of structural and functional genomics. We have systematically analyzed the amino acid composition, residue pair preference and amino acid properties in six different families of transporters. Utilizing the information, we have developed a radial basis function (RBF) network method based on profiles obtained with position specific scoring matrices for discriminating transporters belonging to three different classes and six families. Our method showed a fivefold cross validation accuracy of 76%, 73%, and 69% for discriminating transporters and nontransporters, three different classes and six different families of transporters, respectively. Further, the method was tested with independent datasets, which showed similar level of accuracy. A web server has been developed for discriminating transporters based on three classes and six families, and it is available at http://rbf.bioinfo.tw/ approximately sachen/tcrbf.html. We suggest that our method could be effectively used to identify transporters and discriminating them into different classes and families.

  5. Function and expression of ATP-binding cassette transporters in cultured human Y79 retinoblastoma cells.

    PubMed

    Ishikawa, Yuka; Nagai, Junya; Okada, Yumi; Sato, Koya; Yumoto, Ryoko; Takano, Mikihisa

    2010-01-01

    The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet. PMID:20190417

  6. Green function solution of the Boltzmann transport equation for semiconducting thin film with rough boundaries

    NASA Astrophysics Data System (ADS)

    Ketenoğlu, D.; Ünal, B.

    2012-08-01

    In this study the Green function solution of the Boltzmann transport equation on semiconducting thin film with irregular walls has been applied for the first time. The effects of electron scattering caused by these irregularities on the electrical conductivity have been investigated. First of all by using coordinate transformations, the irregularities on the walls have been transferred into the volume and in this way the both surfaces have been brought into flat forms. By taking two models, Gaussian and exponential, for random potential energy term contained in the transformed Hamiltonian as the perturbation, the resistivity results have been calculated and compared with the ones obtained from the methods widely known in the literature. The Boltzmann transport equation has been solved in relaxation time approximation for the irregular walled system in the case of no magnetic field.

  7. [Sauna effect on blood oxygen transport function and proxidant/antioxidant balance in youths].

    PubMed

    Zinchuk, V V; Zhad'ko, D D

    2012-01-01

    There was investigated sauna effect on blood oxygen transport function and proxidant/antioxidant balance in 18 to 22 years old males. Subjects being tested underwent thermal exposure once per week over a period of 5 months (20 procedures). There were two exposure over the course of sauna bathing (temperature 85-90 degrees C, humidity 10-15%): the first exposure lasted for 5 minutes and the second one for 10 minutes. Dry air sauna in youth's leads to respiratory alkalosis, increases pO2, decreases haemoglobin binding capacity to venous blood oxygen thus facilitating oxygen transport into body tissues. Single sauna visit results in oxidative stress (augmentation of free radical processes and deterioration of antioxidant defence mechanisms), while its manifestations being diminished after multiple thermal exposures. Increase in nitrogen monoxide formation being observed might matter for the modification of the oxygen dependent processes of the human body.

  8. Functional expression of the glycine transporter 1 on bullfrog retinal cones.

    PubMed

    Zhang, Xin-Jun; Ge, Li-Hao; Liu, Jian; Yang, Xiong-Li

    2008-11-19

    Using patch clamp techniques, we characterized glycine-induced currents from cones in bullfrog retinal slices. Application of glycine to cone terminals induced an inward current, which was in part suppressed by strychnine. The remaining strychnine-resistant current component, which did not show polarity reversion in a range of -120 mV to +40 mV, was blocked by N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl] sarcosine, an antagonist of glycine transporter 1 (GlyT1), but not affected by amoxapine, an inhibitor of glycine transporter 2. Application of sarcosine, an agonist of GlyT1, to cone terminals induced an inward current that was completely suppressed by N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl] sarcosine or when external Na in Ringer's was replaced by choline. All these results show for the first time the functional expression of GlyT1 on bullfrog cones. PMID:18841092

  9. Limited transport of functionalized multi-walled carbon nanotubes in two natural soils.

    PubMed

    Kasel, Daniela; Bradford, Scott A; Simůnek, Jiří; Pütz, Thomas; Vereecken, Harry; Klumpp, Erwin

    2013-09-01

    Column experiments were conducted in undisturbed and in repacked soil columns at water contents close to saturation (85-96%) to investigate the transport and retention of functionalized (14)C-labeled multi-walled carbon nanotubes (MWCNT) in two natural soils. Additionally, a field lysimeter experiment was performed to provide long-term information at a larger scale. In all experiments, no breakthrough of MWCNTs was detectable and more than 85% of the applied radioactivity was recovered in the soil profiles. The retention profiles exhibited a hyper-exponential shape with greater retention near the column or lysimeter inlet and were successfully simulated using a numerical model that accounted for depth-dependent retention. In conclusion, results indicated that the soils acted as a strong sink for MWCNTs. Little transport of MWCNTs is therefore likely to occur in the vadose zone, and this implies limited potential for groundwater contamination in the investigated soils.

  10. Transport characteristics of nanoscale functional zerovalent iron/silica composites for in situ remediation of trichloroethylene.

    PubMed

    Zhan, Jingjing; Zheng, Tonghua; Piringer, Gerhard; Day, Christopher; McPherson, Gary L; Lu, Yunfeng; Papadopoulos, Kyriakos; John, Vijay T

    2008-12-01

    Effective in situ remediation of groundwater requires the successful delivery of reactive iron particles through soil. In this paper we report the transport characteristics of nanoscale zerovalent iron entrapped in porous silica particles and prepared through an aerosol-assisted process. The entrapment of iron nanoparticles into the silica matrix prevents their aggregation while maintaining the particles' reactivity. Furthermore, the silica particles are functionalized with alkyl groups and are extremely efficient in adsorbing dissolved trichloroethylene (TCE). Because of synthesis through the aerosol route, the particles are of the optimal size range (0.1-1 microm) for mobility through sediments. Column and capillary transport experiments confirm that the particles move far more effectivelythrough model soils than commercially available uncoated nanoscale reactive iron particles. Microcapillary experiments indicate that the particles partition to the interface of TCE droplets, further enhancing their potential for dense non-aqueous-phase liquid source-zone remediation.

  11. TCTEX1D4 Interactome in Human Testis: Unraveling the Function of Dynein Light Chain in Spermatozoa

    PubMed Central

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; da Cruz e Silva, Edgar

    2014-01-01

    Abstract Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function. PMID:24606217

  12. TCTEX1D4 interactome in human testis: unraveling the function of dynein light chain in spermatozoa.

    PubMed

    Freitas, Maria João; Korrodi-Gregório, Luís; Morais-Santos, Filipa; Cruz e Silva, Edgar da; Fardilha, Margarida

    2014-04-01

    Studies were designed to identify the TCTEX1D4 interactome in human testis, with the purpose of unraveling putative protein complexes essential to male reproduction and thus novel TCTEX1D4 functions. TCTEX1D4 is a dynein light chain that belongs to the DYNT1/TCTEX1 family. In spermatozoa, it appears to be important to sperm motility, intraflagellar transport, and acrosome reaction. To contribute to the knowledge on TCTEX1D4 function in testis and spermatozoa, a yeast two-hybrid assay was performed in testis, which allowed the identification of 40 novel TCTEX1D4 interactors. Curiously, another dynein light chain, TCTEX1D2, was identified and its existence demonstrated for the first time in human spermatozoa. Immunofluorescence studies proved that TCTEX1D2 is an intra-acrosomal protein also present in the midpiece, suggesting a role in cargo movement in human spermatozoa. Further, an in silico profile of TCTEX1D4 revealed that most TCTEX1D4 interacting proteins were not previously characterized and the ones described present a very broad nature. This reinforces TCTEX1D4 as a dynein light chain that is capable of interacting with a variety of functionally different proteins. These observations collectively contribute to a deeper molecular understanding of the human spermatozoa function.

  13. Chloroplast Iron Transport Proteins – Function and Impact on Plant Physiology

    PubMed Central

    López-Millán, Ana F.; Duy, Daniela; Philippar, Katrin

    2016-01-01

    Chloroplasts originated about three billion years ago by endosymbiosis of an ancestor of today’s cyanobacteria with a mitochondria-containing host cell. During evolution chloroplasts of higher plants established as the site for photosynthesis and thus became the basis for all life dependent on oxygen and carbohydrate supply. To fulfill this task, plastid organelles are loaded with the transition metals iron, copper, and manganese, which due to their redox properties are essential for photosynthetic electron transport. In consequence, chloroplasts for example represent the iron-richest system in plant cells. However, improvement of oxygenic photosynthesis in turn required adaptation of metal transport and homeostasis since metal-catalyzed generation of reactive oxygen species (ROS) causes oxidative damage. This is most acute in chloroplasts, where radicals and transition metals are side by side and ROS-production is a usual feature of photosynthetic electron transport. Thus, on the one hand when bound by proteins, chloroplast-intrinsic metals are a prerequisite for photoautotrophic life, but on the other hand become toxic when present in their highly reactive, radical generating, free ionic forms. In consequence, transport, storage and cofactor-assembly of metal ions in plastids have to be tightly controlled and are crucial throughout plant growth and development. In the recent years, proteins for iron transport have been isolated from chloroplast envelope membranes. Here, we discuss their putative functions and impact on cellular metal homeostasis as well as photosynthetic performance and plant metabolism. We further consider the potential of proteomic analyses to identify new players in the field. PMID:27014281

  14. A Nonradioactive Uptake Assay for Rapid Analysis of Thyroid Hormone Transporter Function.

    PubMed

    Jayarama-Naidu, Roopa; Johannes, Jörg; Meyer, Franziska; Wirth, Eva Katrin; Schomburg, Lutz; Köhrle, Josef; Renko, Kostja

    2015-07-01

    Thyroid hormones (TH) are actively taken up into target cells via TH-transmembrane transporters (THTT). Their activity and expression patterns define a layer of endocrine regulation that is poorly understood. Therefore, THTT are potential targets for interfering agents (endocrine disruptors) as well as for pharmacological interventions. Inactivating mutations have been identified as the underlying cause of heritable diseases (monocarboxylate transporter 8-associated Allan-Herndon-Dudley syndrome) and might also define a class of subclinical TH insensitivity. As a basic tool to solve questions regarding THTT substrate specificity, activation or inactivation by compounds and functional changes from mutations, uptake assays with radiolabeled tracers are standard. Due to the need for radioactive isotopes, this technique is limited to screening of labelled substrates and disadvantageous regarding handling, setup, and regulatory issues. To overcome these hurdles, we developed an uptake assay protocol using nonradioactive ligands. In brief, uptake of nonradioactive iodine-containing substrate molecules was monitored via Sandell-Kolthoff reaction. The novel assay was designed to the common microtiter plate layout. As a prove-of-principle, we measured TH uptake by monocarboxylate transporter 8-transfected MDCK1 cells. Titrations with bromosulphthalein as an example for inhibitor screening setups and a side-by-side comparison with the radioactive method prove this assay to be reliable, sensitive, and convenient. Furthermore, the method was applicable on primary murine astrocytes, which enables high-throughput screening studies on in vitro model systems with physiological transporter regulation. Due to its design, it is applicable for high-throughput screening of modulatory compounds, but it is also a safe, inexpensive and an easily accessible method for functional testing of THTT in basic science. PMID:25910050

  15. Cloning, functional characterization and heterologous expression of TaLsi1, a wheat silicon transporter gene.

    PubMed

    Montpetit, Jonatan; Vivancos, Julien; Mitani-Ueno, Namiki; Yamaji, Naoki; Rémus-Borel, Wilfried; Belzile, François; Ma, Jian Feng; Bélanger, Richard R

    2012-05-01

    Silicon (Si) is known to be beneficial to plants, namely in alleviating biotic and abiotic stresses. The magnitude of such positive effects is associated with a plant's natural ability to absorb Si. Many grasses can accumulate as much as 10% on a dry weight basis while most dicots, including Arabidopsis, will accumulate less than 0.1%. In this report, we describe the cloning and functional characterization of TaLsi1, a wheat Si transporter gene. In addition, we developed a heterologous system for the study of Si uptake in plants by introducing TaLsi1 and OsLsi1, its ortholog in rice, into Arabidopsis, a species with a very low innate Si uptake capacity. When expressed constitutively under the control of the CaMV 35S promoter, both TaLsi1 and OsLsi1 were expressed in cells of roots and shoots. Such constitutive expression of TaLsi1 or OsLsi1 resulted in a fourfold to fivefold increase in Si accumulation in transformed plants compared to WT. However, this Si absorption caused deleterious symptoms. When the wheat transporter was expressed under the control of a root-specific promoter (a boron transporter gene (AtNIP5;1) promoter), a similar increase in Si absorption was noted but the plants did not exhibit symptoms and grew normally. These results demonstrate that TaLsi1 is indeed a functional Si transporter as its expression in Arabidopsis leads to increased Si uptake, but that this expression must be confined to root cells for healthy plant development. The availability of this heterologous expression system will facilitate further studies into the mechanisms and benefits of Si uptake. PMID:22351076

  16. Functional characterization of Prickle2 and BBS7 identify overlapping phenotypes yet distinct mechanisms

    PubMed Central

    Mei, Xue; Westfall, Trudi A.; Zhang, Qihong; Sheffield, Val C.; Bassuk, Alexander G.; Slusarski, Diane C.

    2014-01-01

    Ciliopathies are genetic disorders that are caused by dysfunctional cilia and affect multiple organs. One type of ciliopathy, Bardet-Biedl Syndrome, is a rare disorder characterized by obesity, retinitis pigmentosa, polydactyly, mental retardation and susceptibility to cardiovascular diseases. The Wnt/Planar Cell Polarity (PCP) has been associated with cilia function and ciliogenesis in directing the orientation of cilia and basal bodies. Yet the exact relationship between PCP and ciliopathy is not well understood. Here, we examine interactions between a core PCP component, Prickle2 (Pk2), and a central BBS gene, Bbs7, using gene knockdown in the zebrafish. pk2 and bbs7 knockdown both disrupt the formation of a ciliated organ, the Kupffer’s Vesicle (KV), but do not display a synergistic interaction. By measuring cell polarity in the neural tube, we find that bbs7 activity is not required for Pk asymmetric localization. Moreover, BBS protein complex formation is preserved in the Pk2-deficient (Pk2−/−) mouse. Previously we reported an intracellular melanosome transport delay as a cardinal feature of reduced bbs gene activity. We find that pk2 knockdown suppresses bbs7-related retrograde transport delay. Similarly, knockdown of ift22, an anterograde intraflagellar transport component, also suppresses the bbs7-related retrograde delay. Notably, we find that pk2 knockdown larvae show a delay in anterograde transport. These data suggest a novel role for Pk2 in directional intracellular transport and our analyses show that PCP and BBS function independently, yet result in overlapping phenotypes when knocked down in zebrafish. PMID:24938409

  17. The kinesin superfamily protein KIF17: one protein with many functions

    PubMed Central

    Wong-Riley, Margaret T.T.; Besharse, Joseph C.

    2012-01-01

    Kinesins are ATP-dependent molecular motors that carry cargos along microtubules, generally in an anterograde direction. They are classified into 14 distinct families with varying structural and functional characteristics. KIF17 is a member of the kinesin-2 family that is plus end-directed. It is a homodimer with a pair of head motor domains that bind microtubules, a coiled-coil stalk, and a tail domain that binds cargos. In neurons, KIF17 transports N-methyl-D-aspartate receptor NR2B subunit, kainate receptor GluR5, and potassium Kv4.2 channels from cell bodies exclusively to dendrites. These cargos are necessary for synaptic transmission, learning, memory, and other functions. KIF17’s interaction with NXF2 enables the transport of mRNA bidirectionally in dendrites. KIF17 or its homolog OSM-3 also mediates intraflagellar transport of cargos to the distal tips of flagella or cilia, thereby aiding in ciliogenesis. In many invertebrate and vertebrate sensory cells, KIF17 delivers cargos that contribute to chemosensory perception and signal transduction. In vertebrate photoreceptors, KIF17 is necessary for outer segment development and disc morphogenesis. In the testis, KIF17 (KIF17b) mediates microtubule-independent delivery of ACT from the nucleus to the cytoplasm and microtubule-dependent transport of Spatial-ε, both are presumably involved in spermatogenesis. KIF17 is also implicated in epithelial polarity and morphogenesis, placental transport and development, and the development of specific brain regions. The transcriptional regulation of KIF17 has recently been found to be mediated by nuclear respiratory factor 1 (NRF-1), which also regulates NR2B as well as energy metabolism in neurons. Dysfunctions of KIF17 are linked to a number of pathologies. PMID:23762210

  18. The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and Perspective

    PubMed Central

    Kimura, Tomoki; Kambe, Taiho

    2016-01-01

    Around 3000 proteins are thought to bind zinc in vivo, which corresponds to ~10% of the human proteome. Zinc plays a pivotal role as a structural, catalytic, and signaling component that functions in numerous physiological processes. It is more widely used as a structural element in proteins than any other transition metal ion, is a catalytic component of many enzymes, and acts as a cellular signaling mediator. Thus, it is expected that zinc metabolism and homeostasis have sophisticated regulation, and elucidating the underlying molecular basis of this is essential to understanding zinc functions in cellular physiology and pathogenesis. In recent decades, an increasing amount of evidence has uncovered critical roles of a number of proteins in zinc metabolism and homeostasis through influxing, chelating, sequestrating, coordinating, releasing, and effluxing zinc. Metallothioneins (MT) and Zrt- and Irt-like proteins (ZIP) and Zn transporters (ZnT) are the proteins primarily involved in these processes, and their malfunction has been implicated in a number of inherited diseases such as acrodermatitis enteropathica. The present review updates our current understanding of the biological functions of MTs and ZIP and ZnT transporters from several new perspectives. PMID:26959009

  19. Ion Binding Energies Determining Functional Transport of ClC Proteins

    NASA Astrophysics Data System (ADS)

    Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

    2014-06-01

    The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

  20. Restored glial glutamate transporter EAAT2 function as a potential therapeutic approach for Alzheimer's disease.

    PubMed

    Takahashi, Kou; Kong, Qiongman; Lin, Yuchen; Stouffer, Nathan; Schulte, Delanie A; Lai, Liching; Liu, Qibing; Chang, Ling-Chu; Dominguez, Sky; Xing, Xuechao; Cuny, Gregory D; Hodgetts, Kevin J; Glicksman, Marcie A; Lin, Chien-Liang Glenn

    2015-03-01

    Glutamatergic systems play a critical role in cognitive functions and are known to be defective in Alzheimer's disease (AD) patients. Previous literature has indicated that glial glutamate transporter EAAT2 plays an essential role in cognitive functions and that loss of EAAT2 protein is a common phenomenon observed in AD patients and animal models. In the current study, we investigated whether restored EAAT2 protein and function could benefit cognitive functions and pathology in APPSw,Ind mice, an animal model of AD. A transgenic mouse approach via crossing EAAT2 transgenic mice with APPSw,Ind. mice and a pharmacological approach using a novel EAAT2 translational activator, LDN/OSU-0212320, were conducted. Findings from both approaches demonstrated that restored EAAT2 protein function significantly improved cognitive functions, restored synaptic integrity, and reduced amyloid plaques. Importantly, the observed benefits were sustained one month after compound treatment cessation, suggesting that EAAT2 is a potential disease modifier with therapeutic potential for AD. PMID:25711212

  1. Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation

    PubMed Central

    Mendiondo, Guillermina M.; Medhurst, Anne; van Roermund, Carlo W.; Zhang, Xuebin; Devonshire, Jean; Scholefield, Duncan; Fernández, José; Axcell, Barry; Ramsay, Luke; Waterham, Hans R.; Waugh, Robbie; Theodoulou, Frederica L.; Holdsworth, Michael J.

    2014-01-01

    In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ. PMID:24913629

  2. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health.

    NASA Astrophysics Data System (ADS)

    Pochini, Lorena; Scalise, Mariafrancesca; Galluccio, Michele; Indiveri, Cesare

    2014-08-01

    Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

  3. A New Endogenous Overexpression System of Multidrug Transporters of Candida albicans Suitable for Structural and Functional Studies

    PubMed Central

    Banerjee, Atanu; Khandelwal, Nitesh K.; Sanglard, Dominique; Prasad, Rajendra

    2016-01-01

    Fungal pathogens have a robust array of multidrug transporters which aid in active expulsion of drugs and xenobiotics to help them evade toxic effects of drugs. Thus, these transporters impose a major impediment to effective chemotherapy. Although the Saccharomyces cerevisiae strain AD1-8u− has catered well to the need of an overexpression system to study drug transport by multidrug transporters of Candida albicans, artifacts associated with a heterologous system could not be excluded. To avoid the issue, we exploited a azole-resistant clinical isolate of C. albicans to develop a new system devoid of three major multidrug transporters (Cdr1p, Cdr2p, and Mdr1p) for the overexpression of multidrug transporters under native hyperactive CDR1 promoter due to gain of function (GOF) mutation in TAC1. The study deals with overexpression and functional characterization of representatives of two major classes of multidrug transporters, Cdr1p and Mdr1p, to prove the functionality of this newly developed endogenous expression system. Expression of native Cdr1 and Mdr1 protein in C. albicans cells was confirmed by confocal microscopy and immunodetection and resulted in increased resistance to the putative substrates as compared to control. The system was further validated by overexpressing a few key mutant variants of Cdr1p and Mdr1p. Together, our data confirms the utility of new endogenous overexpression system which is devoid of artifactual factors as most suited for functional characterization of multidrug transporter proteins of C. albicans. PMID:26973635

  4. Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers*

    PubMed Central

    Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; Van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

    2014-01-01

    ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters. PMID:25043761

  5. High pressure modulated transport and signaling functions of membrane proteins in models and in vivo

    NASA Astrophysics Data System (ADS)

    Vogel, R. F.; Linke, K.; Teichert, H.; Ehrmann, M. A.

    2008-07-01

    Cellular membranes serve in the separation of compartments, recognition of the environment, selective transport and signal transduction. Membrane lipids and membrane proteins play distinct roles in these processes, which are affected by environmental chemical (e. g. pH) or physical (e. g. pressure and temperature) changes. High hydrostatic pressure (HHP) affects fluidity and integrity of bacterial membranes instantly during the ramp, resulting in a loss of membrane potential and vital membrane protein functions. We have used the multiple drug transporter LmrA from Lactococcus lactis and ToxR, a membrane protein sensor from Photobacterium profundum, a deep-sea bacterium, and Vibrio cholerae to study membrane protein interaction and functionality in proteolioposomes and by the use of in vivo reporter systems, respectively. Both proteins require dimerization in the phospholipid bilayer for their functionality, which was favoured in the liquid crystalline lipid phase with ToxR and LmrA. Whereas LmrA, which resides in liposomes consisting of DMPC, DMPC/cholesterol or natural lipids, lost its ATPase activity above 20 or 40 MPa, it maintained its active dimeric structure in DOPC/DPPC/cholesterol liposomes up to 120 MPa. By using a specific indicator strain in which the dimerisation of ToxR initiates the transcription of lacZ it was demonstrated, that the amino acid sequence of the transmembrane domain influences HHP stability of ToxR dimerization in vivo. Thus, both the lipid structure and the nature of the protein affect membrane protein interaction. It is suggested that the protein structure determines basic functionality, e.g. principle ability or kinetics to dimerize to a functional complex, while the lipid environment modulates this property.

  6. Kinesin family 17 (osmotic avoidance abnormal-3) is dispensable for photoreceptor morphology and function.

    PubMed

    Jiang, Li; Tam, Beatrice M; Ying, Guoxing; Wu, Sen; Hauswirth, William W; Frederick, Jeanne M; Moritz, Orson L; Baehr, Wolfgang

    2015-12-01

    In Caenorhabditis elegans, homodimeric [kinesin family (KIF) 17, osmotic avoidance abnormal-3 (OSM-3)] and heterotrimeric (KIF3) kinesin-2 motors are required to establish sensory cilia by intraflagellar transport (IFT) where KIF3 and KIF17 cooperate to build the axoneme core and KIF17 builds the distal segments. However, the function of KIF17 in vertebrates is unresolved. We expressed full-length and motorless KIF17 constructs in mouse rod photoreceptors using adeno-associated virus in Xenopus laevis rod photoreceptors using a transgene and in ciliated IMCD3 cells. We found that tagged KIF17 localized along the rod outer segment axoneme when expressed in mouse and X. laevis photoreceptors, whereas KIF3A was restricted to the proximal axoneme. Motorless KIF3A and KIF17 mutants caused photoreceptor degeneration, likely through dominant negative effects on IFT. KIF17 mutant lacking the motor domain translocated to nuclei after exposure of a C-terminal nuclear localization signal. Germ-line deletion of Kif17 in mouse did not affect photoreceptor function. A rod-specific Kif3/Kif17 double knockout mouse demonstrated that KIF17 and KIF3 do not act synergistically and did not prevent rhodopsin trafficking to rod outer segments. In summary, the nematode model of KIF3/KIF17 cooperation apparently does not apply to mouse photoreceptors in which the photosensory cilium is built exclusively by KIF3. PMID:26229057

  7. Mutations in DYNC2LI1 disrupt cilia function and cause short rib polydactyly syndrome

    PubMed Central

    Taylor, S. Paige; Dantas, Tiago J.; Duran, Ivan; Wu, Sulin; Lachman, Ralph S.; Nelson, Stanley F.; Cohn, Daniel H.; Vallee, Richard B.; Krakow, Deborah

    2015-01-01

    The short rib polydactyly syndromes (SRPS) are a heterogeneous group of autosomal recessive, perinatal-lethal skeletal disorders characterized primarily by short, horizontal ribs, short limbs, and poly-dactyly. Mutations in several genes affecting intraflagellar transport (IFT) cause SRPS but they do not account for all cases. Here we identify additional SRPS genes and further unravel the functional basis for IFT. We perform whole exome sequencing and identify mutations in a new disease-producing gene, cytoplasmic dynein-2 light intermediate chain 1, DYNC2LI1, segregating with disease in three families. Using primary fibroblasts, we show that DYNC2LI1 is essential for dynein-2 complex stability and that mutations in DYNC2LI1 result in variable-length, including hyperelongated, cilia, Hedgehog pathway impairment, and ciliary IFT accumulations. The findings in this study expand our understanding of SRPS locus heterogeneity and demonstrate the importance of DYNC2LI1 in dynein-2 complex stability, cilium function, Hedgehog regulation, and skeletogenesis. PMID:26077881

  8. Characterization of Tetratricopeptide Repeat-Containing Proteins Critical for Cilia Formation and Function

    PubMed Central

    Xu, Yanan; Cao, Jingli; Huang, Shan; Feng, Di; Zhang, Wei; Zhu, Xueliang; Yan, Xiumin

    2015-01-01

    Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT), consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. Tetratricopeptide repeat-containing (TTC) proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs) into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, ttc4, -9c, -36, and -39c, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of ttc4 and ttc25, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia. PMID:25860617

  9. Insights into the Structure and Function of Ciliary and Flagellar Doublet Microtubules

    PubMed Central

    Linck, Richard; Fu, Xiaofeng; Lin, Jianfeng; Ouch, Christna; Schefter, Alexandra; Steffen, Walter; Warren, Peter; Nicastro, Daniela

    2014-01-01

    Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. PMID:24794867

  10. [Physiological functions of carnitine and carnitine transporters in the central nervous system].

    PubMed

    Inazu, Masato; Matsumiya, Teruhiko

    2008-06-01

    L-Carnitine is an essential co-factor in the metabolism of lipids and consequently in the production of cellular energy. This molecule has important physiological roles, including its involvement in the beta-oxidation of fatty acids by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane as acylcarnitine esters. In the brain, L-carnitine and acetyl-L-carnitine have important roles in cerebral bioenergetics and in neuroprotection through a variety of mechanisms including their antioxidant properties and in the modulation and promotion of synaptic neurotransmission, most notably cholinergic neurotransmission. Acetyl-L-carnitine was successfully applied as pharmacological agents for treatment of chronic degenerative diseases of the senile brain and for slowing down the progression of mental deterioration in Alzheimer's disease, and they may involve both the cholinergic neuronal transmission activity of acetyl-L-carnitine and its ability to enhance neuronal metabolism in mitochondria. Astrocytes are able to produce large amounts of ketone bodies, which are thought to supply adjacent neurons with easily transferable substrates for generation of energy. Thus, the L-carnitine uptake mechanism becomes the rate-limiting step for astrocyte ketogenesis. Several carnitine transporters have been known to be present in peripheral tissues. In this review, the functional expression and physiological role of carnitine transporters in central nervous system is further discussed. PMID:18646596

  11. Sulfate transporters in the plant’s response to drought and salinity: regulation and possible functions

    PubMed Central

    Gallardo, Karine; Courty, Pierre-Emmanuel; Le Signor, Christine; Wipf, Daniel; Vernoud, Vanessa

    2014-01-01

    Drought and salinity are two frequently combined abiotic stresses that affect plant growth, development, and crop productivity. Sulfate, and molecules derived from this anion such as glutathione, play important roles in the intrinsic responses of plants to such abiotic stresses. Therefore, understanding how plants facing environmental constraints re-equilibrate the flux of sulfate between and within different tissues might uncover perspectives for improving tolerance against abiotic stresses. In this review, we took advantage of genomics and post-genomics resources available in Arabidopsis thaliana and in the model legume species Medicago truncatula to highlight and compare the regulation of sulfate transporter genes under drought and salt stress. We also discuss their possible function in the plant’s response and adaptation to abiotic stresses and present prospects about the potential benefits of mycorrhizal associations, which by facilitating sulfate uptake may assist plants to cope with abiotic stresses. Several transporters are highlighted in this review that appear promising targets for improving sulfate transport capacities of crops under fluctuating environmental conditions. PMID:25400648

  12. Functional Analysis of Sterol Transporter Orthologues in the Filamentous Fungus Aspergillus nidulans

    PubMed Central

    Bühler, Nicole; Hagiwara, Daisuke

    2015-01-01

    Polarized growth in filamentous fungi needs a continuous supply of proteins and lipids to the growing hyphal tip. One of the important membrane compounds in fungi is ergosterol. At the apical plasma membrane ergosterol accumulations, which are called sterol-rich plasma membrane domains (SRDs). The exact roles and formation mechanism of the SRDs remained unclear, although the importance has been recognized for hyphal growth. Transport of ergosterol to hyphal tips is thought to be important for the organization of the SRDs. Oxysterol binding proteins, which are conserved from yeast to human, are involved in nonvesicular sterol transport. In Saccharomyces cerevisiae seven oxysterol-binding protein homologues (OSH1 to -7) play a role in ergosterol distribution between closely located membranes independent of vesicle transport. We found five homologous genes (oshA to oshE) in the filamentous fungi Aspergillus nidulans. The functions of OshA-E were characterized by gene deletion and subcellular localization. Each gene-deletion strain showed characteristic phenotypes and different sensitivities to ergosterol-associated drugs. Green fluorescent protein-tagged Osh proteins showed specific localization in the late Golgi compartments, puncta associated with the endoplasmic reticulum, or diffusely in the cytoplasm. The genes expression and regulation were investigated in a medically important species Aspergillus fumigatus, as well as A. nidulans. Our results suggest that each Osh protein plays a role in ergosterol distribution at distinct sites and contributes to proper fungal growth. PMID:26116213

  13. Different functional roles of arginine residues 39 and 61 and tyrosine residue 98 in transport and channel mode of the glutamate transporter EAAC1.

    PubMed

    Zhu, Yani; Vasilets, Larisa A; Fei, Jian; Guo, Lihe; Schwarz, Wolfgang

    2004-10-11

    The excitatory amino acid transporter EAAC1 is an electrogenic Na+ - and K+ -gradient-driven transporter. In addition, the transporter mediates in the presence of Na+ and glutamate an anion conductance uncoupled from the transport of the glutamate. The first two N-terminal domains, important for forming the conductance mode, are extracellularly bordered by positively charged arginine residues, R39 and R61, being completely conserved throughout the transporter family. Also the conserved tyrosine residue Y98 could be important for Cl- conductance. We have investigated, by measurements of glutamate uptake and glutamate-induced currents, the effects of mutation of the arginines and the tyrosine to alanine. The mutation R39A hardly affects transport and channel mode. The mutation R61A, on the other hand, reduces the activity of transport but stimulates the channel conductance. In addition, the apparent Km values for glutamate uptake and for the glutamate-activated current are reduced. Glutamate stimulation of current seems to be associated with a voltage-dependent step, and the apparent valence of charge moved during binding is reduced in the R61A mutant. The mutation Y98A leads to reduced function with reduced apparent Km value for glutamate, and with strong reduction of the selectivity ration between NO3- and Cl- of the conductance mode.

  14. Identification of high-level functional/system requirements for future civil transports

    NASA Technical Reports Server (NTRS)

    Swink, Jay R.; Goins, Richard T.

    1992-01-01

    In order to accommodate the rapid growth in commercial aviation throughout the remainder of this century, the Federal Aviation Administration (FAA) is faced with a formidable challenge to upgrade and/or modernize the National Airspace System (NAS) without compromising safety or efficiency. A recurring theme in both the Aviation System Capital Investment Plan (CIP), which has replaced the NAS Plan, and the new FAA Plan for Research, Engineering, and Development (RE&D) rely on the application of new technologies and a greater use of automation. Identifying the high-level functional and system impacts of such modernization efforts on future civil transport operational requirements, particularly in terms of cockpit functionality and information transfer, was the primary objective of this project. The FAA planning documents for the NAS of the 2005 era and beyond were surveyed; major aircraft functional capabilities and system components required for such an operating environment were identified. A hierarchical structured analysis of the information processing and flows emanating from such functional/system components were conducted and the results documented in graphical form depicting the relationships between functions and systems.

  15. Functional Implications and Ubiquitin-Dependent Degradation of the Peptide Transporter Ptr2 in Saccharomyces cerevisiae

    PubMed Central

    Kawai, Ken; Moriya, Atsuto; Uemura, Satoshi

    2014-01-01

    The peptide transporter Ptr2 plays a central role in di- or tripeptide import in Saccharomyces cerevisiae. Although PTR2 transcription has been extensively analyzed in terms of upregulation by the Ubr1-Cup9 circuit, the structural and functional information for this transporter is limited. Here we identified 14 amino acid residues required for peptide import through Ptr2 based on the crystallographic information of Streptococcus thermophilus peptide transporter PepTst and based on the conservation of primary sequences among the proton-dependent oligopeptide transporters (POTs). Expression of Ptr2 carrying one of the 14 mutations of which the corresponding residues of PepTst are involved in peptide recognition, salt bridge interaction, or peptide translocation failed to enable ptr2Δtrp1 cell growth in alanyl-tryptophan (Ala-Trp) medium. We observed that Ptr2 underwent rapid degradation after cycloheximide treatment (half-life, approximately 1 h), and this degradation depended on Rsp5 ubiquitin ligase. The ubiquitination of Ptr2 most likely occurs at the N-terminal lysines 16, 27, and 34. Simultaneous substitution of arginine for the three lysines fully prevented Ptr2 degradation. Ptr2 mutants of the presumed peptide-binding site (E92Q, R93K, K205R, W362L, and E480D) exhibited severe defects in peptide import and were subjected to Rsp5-dependent degradation when cells were moved to Ala-Trp medium, whereas, similar to what occurs in the wild-type Ptr2, mutant proteins of the intracellular gate were upregulated. These results suggest that Ptr2 undergoes quality control and the defects in peptide binding and the concomitant conformational change render Ptr2 subject to efficient ubiquitination and subsequent degradation. PMID:25172766

  16. The varied functions of aluminium-activated malate transporters-much more than aluminium resistance.

    PubMed

    Palmer, Antony J; Baker, Alison; Muench, Stephen P

    2016-06-15

    The ALMT (aluminium-activated malate transporter) family comprises a functionally diverse but structurally similar group of ion channels. They are found ubiquitously in plant species, expressed throughout different tissues, and located in either the plasma membrane or tonoplast. The first family member identified was TaALMT1, discovered in wheat root tips, which was found to be involved in aluminium resistance by means of malate exudation into the soil. However, since this discovery other family members have been shown to have many other functions such as roles in stomatal opening, general anionic homoeostasis, and in economically valuable traits such as fruit flavour. Recent evidence has also shown that ALMT proteins can act as key molecular actors in GABA (γ-aminobutyric acid) signalling, the first evidence that GABA can act as a signal transducer in plants. PMID:27284052

  17. Insights into the Modulation of Dopamine Transporter Function by Amphetamine, Orphenadrine, and Cocaine Binding.

    PubMed

    Cheng, Mary Hongying; Block, Ethan; Hu, Feizhuo; Cobanoglu, Murat Can; Sorkin, Alexander; Bahar, Ivet

    2015-01-01

    Human dopamine (DA) transporter (hDAT) regulates dopaminergic signaling in the central nervous system by maintaining the synaptic concentration of DA at physiological levels, upon reuptake of DA into presynaptic terminals. DA translocation involves the co-transport of two sodium ions and the channeling of a chloride ion, and it is achieved via alternating access between outward-facing (OF) and inward-facing states of DAT. hDAT is a target for addictive drugs, such as cocaine, amphetamine (AMPH), and therapeutic antidepressants. Our recent quantitative systems pharmacology study suggested that orphenadrine (ORPH), an anticholinergic agent and anti-Parkinson drug, might be repurposable as a DAT drug. Previous studies have shown that DAT-substrates like AMPH or -blockers like cocaine modulate the function of DAT in different ways. However, the molecular mechanisms of modulation remained elusive due to the lack of structural data on DAT. The newly resolved DAT structure from Drosophila melanogaster opens the way to a deeper understanding of the mechanism and time evolution of DAT-drug/ligand interactions. Using a combination of homology modeling, docking analysis, molecular dynamics simulations, and molecular biology experiments, we performed a comparative study of the binding properties of DA, AMPH, ORPH, and cocaine and their modulation of hDAT function. Simulations demonstrate that binding DA or AMPH drives a structural transition toward a functional form predisposed to translocate the ligand. In contrast, ORPH appears to inhibit DAT function by arresting it in the OF open conformation. The analysis shows that cocaine and ORPH competitively bind DAT, with the binding pose and affinity dependent on the conformational state of DAT. Further assays show that the effect of ORPH on DAT uptake and endocytosis is comparable to that of cocaine. PMID:26106364

  18. Insights into the Modulation of Dopamine Transporter Function by Amphetamine, Orphenadrine, and Cocaine Binding

    PubMed Central

    Cheng, Mary Hongying; Block, Ethan; Hu, Feizhuo; Cobanoglu, Murat Can; Sorkin, Alexander; Bahar, Ivet

    2015-01-01

    Human dopamine (DA) transporter (hDAT) regulates dopaminergic signaling in the central nervous system by maintaining the synaptic concentration of DA at physiological levels, upon reuptake of DA into presynaptic terminals. DA translocation involves the co-transport of two sodium ions and the channeling of a chloride ion, and it is achieved via alternating access between outward-facing (OF) and inward-facing states of DAT. hDAT is a target for addictive drugs, such as cocaine, amphetamine (AMPH), and therapeutic antidepressants. Our recent quantitative systems pharmacology study suggested that orphenadrine (ORPH), an anticholinergic agent and anti-Parkinson drug, might be repurposable as a DAT drug. Previous studies have shown that DAT-substrates like AMPH or -blockers like cocaine modulate the function of DAT in different ways. However, the molecular mechanisms of modulation remained elusive due to the lack of structural data on DAT. The newly resolved DAT structure from Drosophila melanogaster opens the way to a deeper understanding of the mechanism and time evolution of DAT–drug/ligand interactions. Using a combination of homology modeling, docking analysis, molecular dynamics simulations, and molecular biology experiments, we performed a comparative study of the binding properties of DA, AMPH, ORPH, and cocaine and their modulation of hDAT function. Simulations demonstrate that binding DA or AMPH drives a structural transition toward a functional form predisposed to translocate the ligand. In contrast, ORPH appears to inhibit DAT function by arresting it in the OF open conformation. The analysis shows that cocaine and ORPH competitively bind DAT, with the binding pose and affinity dependent on the conformational state of DAT. Further assays show that the effect of ORPH on DAT uptake and endocytosis is comparable to that of cocaine. PMID:26106364

  19. Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

    PubMed

    Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

    2015-12-01

    The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses.

  20. Discovering thiamine transporters as targets of chloroquine using a novel functional genomics strategy.

    PubMed

    Huang, Zhiwei; Srinivasan, Sankaranarayanan; Zhang, Jianhuai; Chen, Kaifu; Li, Yongxiang; Li, Wei; Quiocho, Florante A; Pan, Xuewen

    2012-01-01

    Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7(T287N)) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets.

  1. Multitasking by Exploitation of Intracellular Transport Functions: The Many Faces of FcRn

    PubMed Central

    Ward, E. Sally; Ober, Raimund J.

    2015-01-01

    The MHC Class I-related receptor, FcRn, transports antibodies of the immunoglobulin G (IgG) class within and across a diverse array of different cell types. Through this transport, FcRn serves multiple roles throughout adult life that extend well beyond its earlier defined function of transcytosing IgGs from mother to offspring. These roles include the maintenance of IgG levels and the delivery of antigen in the form of immune complexes to degradative compartments within cells. Recent studies have led to significant advances in knowledge of the intracellular trafficking of FcRn and (engineered) IgGs at both the molecular and cellular levels. The engineering of FcRn–IgG (or Fc) interactions to generate antibodies of increased longevity represents an area of active interest, particularly in the light of the expanding use of antibodies in therapy. The strict pH dependence of FcRn–IgG interactions, with binding at pH 6 that becomes essentially undetectable as near neutral pH is approached, is essential for efficient transport. The requirement for retention of low affinity at near neutral pH increases the complexity of engineering antibodies for increased half-life. Conversely, engineered IgGs that have gained significant binding for FcRn at this pH can be potent inhibitors of FcRn that lower endogenous IgG levels and have multiple potential uses as therapeutics. In addition, molecular studies of FcRn–IgG interactions indicate that mice have limitations as preclinical models for FcRn function, primarily due to cross-species differences in FcRn-binding specificity. PMID:19755184

  2. Discovering Thiamine Transporters as Targets of Chloroquine Using a Novel Functional Genomics Strategy

    PubMed Central

    Huang, Zhiwei; Srinivasan, Sankaranarayanan; Zhang, Jianhuai; Chen, Kaifu; Li, Yongxiang; Li, Wei; Quiocho, Florante A.; Pan, Xuewen

    2012-01-01

    Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7T287N) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets. PMID:23209439

  3. Dissociable Roles of Dopamine and Serotonin Transporter Function in a Rat Model of Negative Urgency

    PubMed Central

    Yates, Justin R.; Darna, Mahesh; Gipson, Cassandra D.; Dwoskin, Linda P.; Bardo, Michael T.

    2015-01-01

    Negative urgency is a facet of impulsivity that reflects mood-based rash action and is associated with various maladaptive behaviors in humans. However, the underlying neural mechanisms of negative urgency are not fully understood. Several brain regions within the mesocorticolimbic pathway, as well as the neurotransmitters dopamine (DA) and serotonin (5-HT), have been implicated in impulsivity. Extracellular DA and 5-HT concentrations are regulated by DA transporters (DAT) and 5-HT transporters (SERT); thus, these transporters may be important molecular mechanisms underlying individual differences in negative urgency. The current study employed a reward omission task to model negative urgency in rats. During reward trials, a cue light signaled the non-contingent delivery of one sucrose pellet; immediately following the non-contingent reward, rats responded on a lever to earn sucrose pellets (operant phase). Omission trials were similar to reward trials, except that non-contingent sucrose was omitted following the cue light prior to the operant phase. As expected, contingent responding was higher following omission of expected reward than following delivery of expected reward, thus reflecting negative urgency. Upon completion of behavioral training, Vmax and Km were obtained from kinetic analysis of [3H]DA and [3H]5-HT uptake using synaptosomes prepared from nucleus accumbens (NAc), dorsal striatum (Str), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC) isolated from individual rats. Vmax for DAT in NAc and for SERT in OFC were positively correlated with negative urgency scores. The current findings suggest that mood-based impulsivity (negative urgency) is associated with enhanced DAT function in NAc and SERT function in OFC. PMID:26005123

  4. Memory Functions to Represent Transport Through Heterogeneous Media: Can They Make Physical Sense?

    NASA Astrophysics Data System (ADS)

    Carrera, J.; Gouze, P.; Willmann, M.; Méléan, Y.; Dentz, M.; Le Borgne, T.; Alcolea, A.; Sanchez-Vila, X.

    2006-12-01

    Adding a memory sink-source term to the Advection Dispersion Equation (ADE) helps in alleviating many of the discrepancies between ADE predictions and field observations. Specifically, such a term can explain the scale dependence of apparent dispersivity, the time dependence of cinematic porosity, asymmetry in the spatial distribution of concentrations and, specially, tailing in breakthrough curves. This memory sink-source term can be quite easily incorporated in conventional ADE simulators as the convolution of a memory function times the concentration history. The resulting approach is equivalent to the Multi Rate Mass Transfer models and can be viewed as a special case of Continuous Time Random Walk. The big question is whether the memory function should be viewed as just a toolbox full of additional fitting parameters or one can assign it a physical meaning. And, in the latter case, whether could one predict the evolution of solutes on the sole basis of flow information (e.g., statistics of hydraulic conductivity, and the like). Here, we summarize the efforts we have made in developing a positive response to those questions. We find that neither simple matrix diffusion nor transport through stationary random log conductivity fields lead to satisfactory results. These can be obtained either when diffusion into immobile zones is treated as spatially variable or when transport is simulated over fields resulting from the superposition of an evolving range of scales (i.e., Neuman's Universal Scaling approach).

  5. Expression, purification and functional reconstitution of FeoB, the ferrous iron transporter from Pseudomonas aeruginosa.

    PubMed

    Seyedmohammad, Saeed; Born, Diana; Venter, Henrietta

    2014-09-01

    The FeoB Fe(II) transporter from the drug resistant pathogen, Pseudomonas aeruginosa is essential for ferrous iron transport and is implicated in virulence and biofilm development. Hence it is an attractive target for the development of new anti-infective drugs. FeoB is an intriguing protein that consists of a cytosolic N-terminal GTPase domain and an integral membrane domain which most likely acts as ferrous iron permease. Characterisation of FeoB is critical for developing therapeutics aimed at inhibiting this protein. However, structural and functional analysis of FeoB is hampered by the lack of high yield homogenously pure protein which is monodisperse, stable and active in solution. Here we describe the optimised procedure for the recombinant expression of FeoB from P. aeruginosa and provide an evaluation of the most favourable purification, pH and detergent conditions. The functional reconstitution of FeoB in liposomes is also described. This represents the first detailed procedure for obtaining a pure, active and stable FeoB solution in milligram quantities which would be amenable to biochemical, biophysical and structural studies.

  6. Cellular effect evaluation of micropollutants using transporter functions of renal proximal tubule cells.

    PubMed

    Ren, Xianghao; Lee, Yu Jin; Han, Ho Jae; Kim, In S

    2009-11-01

    Issues pertaining to the effects of micropollutants in reclaimed water are arising in terms of their effect on human health. However, current cellular methodologies face some difficulties to detect subtle effects of waterborne micropollutants at environmental concentrations (ngL(-1)-microgL(-1)) on human and animal cells. In this study, an appropriate cellular model capable of detecting the subtle effects of aquatic micropollutants at environmental concentrations using the functions of primary cultured rabbit renal proximal tubule cells (PTCs) is proposed. Tris-(2-chloroethyl)-phosphate (TCEP) was chosen as the representative micropollutant from eight typical micropollutants via lactate dehydrogenase assay. TCEP significantly decreased not only ion (sodium, calcium, and phosphate) uptake from 10(-2) mg L(-1) (64.8-82.5%, 60.4-68.8%, and 91.9-93.8% of the control, respectively), but also the expression of ion transporters (NHE-3 and L-type Ca channel) from 10(-2) mg L(-1) (53.9-87.4% and 38.6-63.6% of the control, respectively). Moreover, TCEP significantly decreased both the non-ion (glucose, fructose, and l-arginine) uptake and the expression of non-ion transporters (SGLT 1, GLUT 5, and rBAT) from 10(-2) mg L(-1). Therefore, the results demonstrated that the function of PTCs as a cellular model can be used to determine subtle effects of environmental micropollutants at low concentrations. PMID:19729184

  7. Function of a p24 Heterodimer in Morphogenesis and Protein Transport in Penicillium oxalicum

    PubMed Central

    Wang, Fangzhong; Liu, Kuimei; Han, Lijuan; Jiang, Baojie; Wang, Mingyu; Fang, Xu

    2015-01-01

    The lignocellulose degradation capacity of filamentous fungi has been widely studied because of their cellulase hypersecretion. The p24 proteins in eukaryotes serve important functions in this secretory pathway. However, little is known about the functions of the p24 proteins in filamentous fungi. In this study, four p24 proteins were identified in Penicillium oxalicum. Six p24 double-deletion strains were constructed, and further studies were carried out with the ΔerpΔpδ strain. The experimental results suggested that Erp and Pδ form a p24 heterodimer in vivo. This p24 heterodimer participates in important morphogenetic events, including sporulation, hyphal growth, and lateral branching. The results suggested that the p24 heterodimer mediates protein transport, particularly that of cellobiohydrolase. Analysis of the intracellular proteome revealed that the ΔerpΔpδ double mutant is under secretion stress due to attempts to remove proteins that are jammed in the endomembrane system. These results suggest that the p24 heterodimer participates in morphogenesis and protein transport. Compared with P. oxalicum Δerp, a greater number of cellular physiological pathways were impaired in ΔerpΔpδ. This finding may provide new insights into the secretory pathways of filamentous fungi. PMID:26149342

  8. Function of a p24 Heterodimer in Morphogenesis and Protein Transport in Penicillium oxalicum.

    PubMed

    Wang, Fangzhong; Liu, Kuimei; Han, Lijuan; Jiang, Baojie; Wang, Mingyu; Fang, Xu

    2015-01-01

    The lignocellulose degradation capacity of filamentous fungi has been widely studied because of their cellulase hypersecretion. The p24 proteins in eukaryotes serve important functions in this secretory pathway. However, little is known about the functions of the p24 proteins in filamentous fungi. In this study, four p24 proteins were identified in Penicillium oxalicum. Six p24 double-deletion strains were constructed, and further studies were carried out with the ΔerpΔpδ strain. The experimental results suggested that Erp and Pδ form a p24 heterodimer in vivo. This p24 heterodimer participates in important morphogenetic events, including sporulation, hyphal growth, and lateral branching. The results suggested that the p24 heterodimer mediates protein transport, particularly that of cellobiohydrolase. Analysis of the intracellular proteome revealed that the ΔerpΔpδ double mutant is under secretion stress due to attempts to remove proteins that are jammed in the endomembrane system. These results suggest that the p24 heterodimer participates in morphogenesis and protein transport. Compared with P. oxalicum Δerp, a greater number of cellular physiological pathways were impaired in ΔerpΔpδ. This finding may provide new insights into the secretory pathways of filamentous fungi. PMID:26149342

  9. Functional Traits and Water Transport Strategies in Lowland Tropical Rainforest Trees.

    PubMed

    Apgaua, Deborah M G; Ishida, Françoise Y; Tng, David Y P; Laidlaw, Melinda J; Santos, Rubens M; Rumman, Rizwana; Eamus, Derek; Holtum, Joseph A M; Laurance, Susan G W

    2015-01-01

    Understanding how tropical rainforest trees may respond to the precipitation extremes predicted in future climate change scenarios is paramount for their conservation and management. Tree species clearly differ in drought susceptibility, suggesting that variable water transport strategies exist. Using a multi-disciplinary approach, we examined the hydraulic variability in trees in a lowland tropical rainforest in north-eastern Australia. We studied eight tree species representing broad plant functional groups (one palm and seven eudicot mature-phase, and early-successional trees). We characterised the species' hydraulic system through maximum rates of volumetric sap flow and velocities using the heat ratio method, and measured rates of tree growth and several stem, vessel, and leaf traits. Sap flow measures exhibited limited variability across species, although early-successional species and palms had high mean sap velocities relative to most mature-phase species. Stem, vessel, and leaf traits were poor predictors of sap flow measures. However, these traits exhibited different associations in multivariate analysis, revealing gradients in some traits across species and alternative hydraulic strategies in others. Trait differences across and within tree functional groups reflect variation in water transport and drought resistance strategies. These varying strategies will help in our understanding of changing species distributions under predicted drought scenarios. PMID:26087009

  10. Expression, purification and functional reconstitution of FeoB, the ferrous iron transporter from Pseudomonas aeruginosa.

    PubMed

    Seyedmohammad, Saeed; Born, Diana; Venter, Henrietta

    2014-09-01

    The FeoB Fe(II) transporter from the drug resistant pathogen, Pseudomonas aeruginosa is essential for ferrous iron transport and is implicated in virulence and biofilm development. Hence it is an attractive target for the development of new anti-infective drugs. FeoB is an intriguing protein that consists of a cytosolic N-terminal GTPase domain and an integral membrane domain which most likely acts as ferrous iron permease. Characterisation of FeoB is critical for developing therapeutics aimed at inhibiting this protein. However, structural and functional analysis of FeoB is hampered by the lack of high yield homogenously pure protein which is monodisperse, stable and active in solution. Here we describe the optimised procedure for the recombinant expression of FeoB from P. aeruginosa and provide an evaluation of the most favourable purification, pH and detergent conditions. The functional reconstitution of FeoB in liposomes is also described. This represents the first detailed procedure for obtaining a pure, active and stable FeoB solution in milligram quantities which would be amenable to biochemical, biophysical and structural studies. PMID:24993789

  11. Functional Traits and Water Transport Strategies in Lowland Tropical Rainforest Trees

    PubMed Central

    Apgaua, Deborah M. G.; Ishida, Françoise Y.; Tng, David Y. P.; Laidlaw, Melinda J.; Santos, Rubens M.; Rumman, Rizwana; Eamus, Derek; Holtum, Joseph A. M.; Laurance, Susan G. W.

    2015-01-01

    Understanding how tropical rainforest trees may respond to the precipitation extremes predicted in future climate change scenarios is paramount for their conservation and management. Tree species clearly differ in drought susceptibility, suggesting that variable water transport strategies exist. Using a multi-disciplinary approach, we examined the hydraulic variability in trees in a lowland tropical rainforest in north-eastern Australia. We studied eight tree species representing broad plant functional groups (one palm and seven eudicot mature-phase, and early-successional trees). We characterised the species’ hydraulic system through maximum rates of volumetric sap flow and velocities using the heat ratio method, and measured rates of tree growth and several stem, vessel, and leaf traits. Sap flow measures exhibited limited variability across species, although early-successional species and palms had high mean sap velocities relative to most mature-phase species. Stem, vessel, and leaf traits were poor predictors of sap flow measures. However, these traits exhibited different associations in multivariate analysis, revealing gradients in some traits across species and alternative hydraulic strategies in others. Trait differences across and within tree functional groups reflect variation in water transport and drought resistance strategies. These varying strategies will help in our understanding of changing species distributions under predicted drought scenarios. PMID:26087009

  12. Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function.

    PubMed

    Chen, Gin-Fu; Sudhahar, Varadarajan; Youn, Seock-Won; Das, Archita; Cho, Jaehyung; Kamiya, Tetsuro; Urao, Norifumi; McKinney, Ronald D; Surenkhuu, Bayasgalan; Hamakubo, Takao; Iwanari, Hiroko; Li, Senlin; Christman, John W; Shantikumar, Saran; Angelini, Gianni D; Emanueli, Costanza; Ushio-Fukai, Masuko; Fukai, Tohru

    2015-10-06

    Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1(-/-) mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.

  13. Functional Traits and Water Transport Strategies in Lowland Tropical Rainforest Trees.

    PubMed

    Apgaua, Deborah M G; Ishida, Françoise Y; Tng, David Y P; Laidlaw, Melinda J; Santos, Rubens M; Rumman, Rizwana; Eamus, Derek; Holtum, Joseph A M; Laurance, Susan G W

    2015-01-01

    Understanding how tropical rainforest trees may respond to the precipitation extremes predicted in future climate change scenarios is paramount for their conservation and management. Tree species clearly differ in drought susceptibility, suggesting that variable water transport strategies exist. Using a multi-disciplinary approach, we examined the hydraulic variability in trees in a lowland tropical rainforest in north-eastern Australia. We studied eight tree species representing broad plant functional groups (one palm and seven eudicot mature-phase, and early-successional trees). We characterised the species' hydraulic system through maximum rates of volumetric sap flow and velocities using the heat ratio method, and measured rates of tree growth and several stem, vessel, and leaf traits. Sap flow measures exhibited limited variability across species, although early-successional species and palms had high mean sap velocities relative to most mature-phase species. Stem, vessel, and leaf traits were poor predictors of sap flow measures. However, these traits exhibited different associations in multivariate analysis, revealing gradients in some traits across species and alternative hydraulic strategies in others. Trait differences across and within tree functional groups reflect variation in water transport and drought resistance strategies. These varying strategies will help in our understanding of changing species distributions under predicted drought scenarios.

  14. Copper Transport Protein Antioxidant-1 Promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function

    PubMed Central

    Chen, Gin-Fu; Sudhahar, Varadarajan; Youn, Seock-Won; Das, Archita; Cho, Jaehyung; Kamiya, Tetsuro; Urao, Norifumi; McKinney, Ronald D.; Surenkhuu, Bayasgalan; Hamakubo, Takao; Iwanari, Hiroko; Li, Senlin; Christman, John W.; Shantikumar, Saran; Angelini, Gianni D.; Emanueli, Costanza; Ushio-Fukai, Masuko; Fukai, Tohru

    2015-01-01

    Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1−/− mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease. PMID:26437801

  15. Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi

    PubMed Central

    Gallo-Ebert, Christina; McCourt, Paula C.; Donigan, Melissa; Villasmil, Michelle L.; Chen, WeiWei; Pandya, Devanshi; Franco, Judith; Romano, Desiree; Chadwick, Sean; Gygax, Scott; Nickels, Joseph T.

    2011-01-01

    The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicans arv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1. PMID:22142782

  16. Arv1 lipid transporter function is conserved between pathogenic and nonpathogenic fungi.

    PubMed

    Gallo-Ebert, Christina; McCourt, Paula C; Donigan, Melissa; Villasmil, Michelle L; Chen, WeiWei; Pandya, Devanshi; Franco, Judith; Romano, Desiree; Chadwick, Sean G; Gygax, Scott E; Nickels, Joseph T

    2012-02-01

    The lipid transporter Arv1 regulates sterol trafficking, and glycosylphosphatidylinositol and sphingolipid biosyntheses in Saccharomyces cerevisiae. ScArv1 contains an Arv1 homology domain (AHD) that is conserved at the amino acid level in the pathogenic fungal species, Candida albicans and Candida glabrata. Here we show S. cerevisiae cells lacking Arv1 are highly susceptible to antifungal drugs. In the presence of drug, Scarv1 cells are unable to induce ERG gene expression, have an altered pleiotrophic drug response, and are defective in multi-drug resistance efflux pump expression. All phenotypes are remediated by ectopic expression of CaARV1 or CgARV1. The AHDs of these pathogenic fungi are required for specific drug tolerance, demonstrating conservation of function. In order to understand how Arv1 regulates antifungal susceptibility, we examined sterol trafficking. CaARV1/CgARV1 expression suppressed the sterol trafficking defect of Scarv1 cells. Finally, we show that C. albicansarv1/arv1 cells are avirulent using a BALB/c disseminated mouse model. We suggest that overall cell survival in response to antifungal treatment requires the lipid transporter function of Arv1.

  17. Sequence- and Structure-Based Functional Annotation and Assessment of Metabolic Transporters in Aspergillus oryzae: A Representative Case Study

    PubMed Central

    Raethong, Nachon; Wong-ekkabut, Jirasak; Laoteng, Kobkul; Vongsangnak, Wanwipa

    2016-01-01

    Aspergillus oryzae is widely used for the industrial production of enzymes. In A. oryzae metabolism, transporters appear to play crucial roles in controlling the flux of molecules for energy generation, nutrients delivery, and waste elimination in the cell. While the A. oryzae genome sequence is available, transporter annotation remains limited and thus the connectivity of metabolic networks is incomplete. In this study, we developed a metabolic annotation strategy to understand the relationship between the sequence, structure, and function for annotation of A. oryzae metabolic transporters. Sequence-based analysis with manual curation showed that 58 genes of 12,096 total genes in the A. oryzae genome encoded metabolic transporters. Under consensus integrative databases, 55 unambiguous metabolic transporter genes were distributed into channels and pores (7 genes), electrochemical potential-driven transporters (33 genes), and primary active transporters (15 genes). To reveal the transporter functional role, a combination of homology modeling and molecular dynamics simulation was implemented to assess the relationship between sequence to structure and structure to function. As in the energy metabolism of A. oryzae, the H+-ATPase encoded by the AO090005000842 gene was selected as a representative case study of multilevel linkage annotation. Our developed strategy can be used for enhancing metabolic network reconstruction. PMID:27274991

  18. Clinico-Pathologic Function of Cerebral ABC Transporters – Implications for the Pathogenesis of Alzheimer’s Disease

    PubMed Central

    Pahnke, Jens; Wolkenhauer, Olaf; Krohn, Markus; Walker, Lary C.

    2009-01-01

    In recent years it has become evident that ABC transporters fulfill important barrier functions in normal organs and during disease processes. Most importantly, resistance to drugs in cancer cells led to intense oncological and pharmacological investigations in which researchers were able to highlight important pharmacological interactions of chemotherapeuticals with ABC transporter function. Recently, the development of neurodegenerative diseases and the maintenance of neuronal stem cells have been linked to the activity of ABC transporters. Here, we summarize findings from cell culture experiments, animal models and studies of patients with Alzheimer’s disease. Furthermore, we discuss pharmacological interactions and computational methods for risk assessment. PMID:18690837

  19. Functional characterisation of human SGLT-5 as a novel kidney-specific sodium-dependent sugar transporter.

    PubMed

    Grempler, Rolf; Augustin, Robert; Froehner, Stefanie; Hildebrandt, Tobias; Simon, Eric; Mark, Michael; Eickelmann, Peter

    2012-02-01

    Sodium glucose cotransporters (SGLT) actively catalyse carbohydrate transport across cellular membranes. Six of the 12 known SGLT family members have the capacity to bind and/or transport monosaccharides (SGLT-1 to 6); of these, all but SGLT-5 have been characterised. Here we demonstrate that human SGLT-5 is exclusively expressed in the kidney. Four splice variants were detected and the most abundant SGLT-5-mRNA was functionally characterised. SGLT-5 mediates sodium-dependent [(14)C]-α-methyl-D-glucose (AMG) transport that can be inhibited by mannose, fructose, glucose, and galactose. Uptake studies using demonstrated high capacity transport for mannose and fructose and, to a lesser extent, glucose, AMG, and galactose. SGLT-5 mediated mannose, fructose and AMG transport was weakly (μM potency) inhibited by SGLT-2 inhibitors. In summary, we have characterised SGLT-5 as a kidney mannose transporter. Further studies are warranted to explore the physiological role of SGLT-5.

  20. New advances in the pathophysiology of intestinal ion transport and barrier function in diarrhea and the impact on therapy.

    PubMed

    Hoque, Kazi Mirajul; Chakraborty, Subhra; Sheikh, Irshad Ali; Woodward, Owen M

    2012-06-01

    Diarrhea remains a continuous threat to human health worldwide. Scaling up the best practices for diarrhea prevention requires improved therapies. Diarrhea results from dysregulation of normal intestinal ion transport functions. Host-microbe contact is a key determinant of this response. Underlying mechanisms in the disease state are regulated by intracellular signals that modulate the activity of individual transport proteins responsible for ion transport and barrier function. Similarly, virulence factors of pathogens and their complex interaction with the host has shed light on the mechanism of enteric infection. Great advances in our understanding of the pathophysiologic mechanisms of epithelial transport, and host-microbe interaction have been made in recent years. Application of these new advances may represent strategies to decrease pathogen attachment, enhance intestinal cation absorption, decrease anion secretion and repair barrier function. This review highlights the new advances and better understanding in the pathophysiology of diarrheal diseases and their impact on therapy.

  1. What is Hidden in Hiding Functions? The Influence of Packing on Size-Selective Transport

    NASA Astrophysics Data System (ADS)

    Yager, E.; Buxton, T. H.; Buffington, J. M.; Fremier, A. K.; Hassan, M. A.

    2015-12-01

    Studying landscape evolution and developing defensible land management plans require predictions of sediment flux in rivers. Bed load transport equations often use hiding functions to represent the relative mobility of different grain sizes, which is assumed to be caused by hiding effects. Hiding effects describe the tendency for small particles to have lower projection into the flow and higher friction angles than large grains, decreasing their mobility. Hiding function exponents (b) are fit using reach-scale measurements, whereas hiding effects operate at the grain scale. Therefore, the relation between b values and hiding effects has not been explicitly demonstrated. Hiding effects are also commonly measured for grains placed on bed surfaces rather than for in situ grains that experience the packing and burial that are typical of natural streambeds. To examine this issue, we measured resisting forces and projection for in situ and surface grains in a set of laboratory flume experiments. We used these values in a force balance equation to calculate critical shear stresses and then fit hiding functions to the predicted stresses. Lower projection and higher resistance of in situ grains caused their critical shear stresses to be much higher than those for surface grains. In situ grains also had much lower hiding effects because fine and coarse in situ sediment exhibited relatively similar projection values. We compared b values calculated for in situ and surface grains to those obtained from bedload transport measurements in our experiments. Only in situ sediment with relatively weak hiding effects predicted the observed b value from bedload observations. Our results demonstrate that the relative mobility of fine and coarse sediment was largely controlled by packing resistance and that hiding effects are less significant than previously thought.

  2. Analysis of cargo transport by IFT and GFP imaging of IFT in Chlamydomonas.

    PubMed

    Diener, Dennis

    2009-01-01

    Chlamydomonas reinhardtii is the organism in which intraflagellar transport (IFT) was first visualized and in which the composition of IFT particles was originally elucidated. As the universality of IFT among ciliated/flagellated cells was uncovered, the diversity of organisms used to study IFT has grown. Still, because of the ease of isolation of flagella from Chlamydomonas and the battery of temperature-sensitive mutants affecting IFT proteins and motors, this unicellular alga remains the principal model for biochemical studies of IFT motors and cargo; furthermore, the long, exposed flagella of this cell are ideally suited for observing IFT in real time with GFP-tagged components of IFT.

  3. Molecular Basis of Tubulin Transport Within the Cilium by IFT74 and IFT81

    PubMed Central

    Bhogaraju, Sagar; Cajanek, Lukas; Fort, Cécile; Blisnick, Thierry; Weber, Kristina; Taschner, Michael; Mizuno, Naoko; Lamla, Stefan; Bastin, Philippe; Nigg, Erich A.; Lorentzen, Esben

    2015-01-01

    Intraflagellar transport (IFT) of ciliary precursors such as tubulin from the cytoplasm to the ciliary tip is involved in the construction of the cilium, a hairlike organelle found on most eukaryotic cells. However, the molecular mechanisms of IFT are poorly understood. Here, we found that the two core IFT proteins IFT74 and IFT81 form a tubulin-binding module and mapped the interaction to a calponin homology domain of IFT81 and a highly basic domain in IFT74. Knockdown of IFT81 and rescue experiments with point mutants showed that tubulin binding by IFT81 was required for ciliogenesis in human cells. PMID:23990561

  4. Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

    PubMed Central

    Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

    2002-01-01

    In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

  5. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells

    PubMed Central

    Raghunayakula, Sarita; Subramonian, Divya; Dasso, Mary; Kumar, Rita; Zhang, Xiang-Dong

    2015-01-01

    Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the

  6. Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells.

    PubMed

    Raghunayakula, Sarita; Subramonian, Divya; Dasso, Mary; Kumar, Rita; Zhang, Xiang-Dong

    2015-01-01

    Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/β and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/β-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the

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

    PubMed

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

    2010-01-01

    Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5min decreased [(3)H]NE uptake capacity, an effect characterized by a robust decrease in the V(max) of the transport of [(3)H]NE. As expected, reserpine did not displace the binding of [(3)H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [(3)H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [(3)H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca(2+)/Ca(2+)-calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [(3)H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, alpha-methyl-p-tyrosine, increased [(3)H]NE uptake and eliminated the inhibitory effects of reserpine on [(3)H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca(2+)-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors. PMID:20176067

  8. "Sticky"-Ends-Guided Creation of Functional Hollow Nanopores for Guest Encapsulation and Water Transport.

    PubMed

    Huo, Yanping; Zeng, Huaqiang

    2016-05-17

    Commercial uses of water-transporting aquaporins for seawater desalination and wastewater reclamation/reuse are being investigated in both academia and the industry. Presently, structural complexity, stability, scalability, and activity reconstitution of these costly channel proteins still present substantial challenges to scientists and engineers. An attractive strategy is to develop robust synthetic water channels able to mimic the water-transporting function of aquaporins for utility in the making of next generation of water channel-based biomimetic porous membranes for various water purification applications. In sharp contrast to burgeoning development in constructing synthetic ion channels over the past four decades, very limited progress has been made in the area of synthetic water channels. A handful of such examples include the first report by Percec in 2007 (Percec et al. J. Am. Chem. Soc. 2007, 129, 11698-11699), which was followed by Barboiu in 2011 (Barboiu et al. Angew. Chem., Int. Ed. 2011, 50, 11366-11372), Gong and Hou in 2012 (Gong et al. Nat. Commun. 2012, 3, 949; Hou et al. J. Am. Chem. Soc. 2012, 134, 8384-8387), and Zeng in 2014 (Zeng et al. J. Am. Chem. Soc. 2014, 136, 14270-14276). Radically deviating from the fact that the discovery of novel synthetic channel systems with desired transport selectivity is most often empirical and very often serendipitous, we have instead adopted a more rational designer approach whereby molecular building blocks have been carefully designed from scratch to perform their intended built-in functions. Our designer journey started in 2008, two years after I started leading a group at the National University of Singapore. Since then, we have been actively investigating the use of designed water-binding "aquafoldamers" to construct synthetic water channels for the rapid and selective transport of water molecules ideally with the exclusion of all other nonproton molecular species. Toward this goal, we designed and

  9. "Sticky"-Ends-Guided Creation of Functional Hollow Nanopores for Guest Encapsulation and Water Transport.

    PubMed

    Huo, Yanping; Zeng, Huaqiang

    2016-05-17

    Commercial uses of water-transporting aquaporins for seawater desalination and wastewater reclamation/reuse are being investigated in both academia and the industry. Presently, structural complexity, stability, scalability, and activity reconstitution of these costly channel proteins still present substantial challenges to scientists and engineers. An attractive strategy is to develop robust synthetic water channels able to mimic the water-transporting function of aquaporins for utility in the making of next generation of water channel-based biomimetic porous membranes for various water purification applications. In sharp contrast to burgeoning development in constructing synthetic ion channels over the past four decades, very limited progress has been made in the area of synthetic water channels. A handful of such examples include the first report by Percec in 2007 (Percec et al. J. Am. Chem. Soc. 2007, 129, 11698-11699), which was followed by Barboiu in 2011 (Barboiu et al. Angew. Chem., Int. Ed. 2011, 50, 11366-11372), Gong and Hou in 2012 (Gong et al. Nat. Commun. 2012, 3, 949; Hou et al. J. Am. Chem. Soc. 2012, 134, 8384-8387), and Zeng in 2014 (Zeng et al. J. Am. Chem. Soc. 2014, 136, 14270-14276). Radically deviating from the fact that the discovery of novel synthetic channel systems with desired transport selectivity is most often empirical and very often serendipitous, we have instead adopted a more rational designer approach whereby molecular building blocks have been carefully designed from scratch to perform their intended built-in functions. Our designer journey started in 2008, two years after I started leading a group at the National University of Singapore. Since then, we have been actively investigating the use of designed water-binding "aquafoldamers" to construct synthetic water channels for the rapid and selective transport of water molecules ideally with the exclusion of all other nonproton molecular species. Toward this goal, we designed and

  10. Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome.

    PubMed Central

    Webb, G C; Zhang, J; Garlow, S J; Wesp, A; Riezman, H; Jones, E W

    1997-01-01

    Saccharomyces cerevisiae pep7 mutants are defective in transport of soluble vacuolar hydrolases to the lysosome-like vacuole. PEP7 is a nonessential gene that encodes a hydrophilic protein of 515 amino acids. A cysteine-rich tripartite motif in the N-terminal half of the polypeptide shows striking similarity to sequences found in many other eukaryotic proteins. Several of these proteins are thought to function in the vacuolar/lysosomal pathway. Mutations that change highly conserved cysteine residues in this motif lead to a loss of Pep7p function. Kinetic studies demonstrate that Pep7p function is required for the transport of the Golgi-precursors of the soluble hydrolases carboxypeptidase Y, proteinase A, and proteinase B to the endosome. Integral membrane hydrolase alkaline phosphatase is transported to the vacuole by a parallel intracellular pathway that does not require Pep7p function. pep7 mutants accumulate a 40-60-nm vesicle population, suggesting that Pep7p functions in a vesicle consumption step in vesicle-mediated transport of soluble hydrolases to the endosome. Whereas pep7 mutants demonstrate no defects in endocytic uptake at the plasma membrane, the mutants demonstrate defects in transport of receptor-mediated macromolecules through the endocytic pathway. Localization studies indicate that Pep7p is found both as a soluble cytoplasmic protein and associated with particulate fractions. We conclude that Pep7p functions as a novel regulator of vesicle docking and/or fusion at the endosome. Images PMID:9168472

  11. Application of the multigrid amplitude function method for time-dependent transport equation using MOC

    SciTech Connect

    Tsujita, K.; Endo, T.; Yamamoto, A.

    2013-07-01

    An efficient numerical method for time-dependent transport equation, the mutigrid amplitude function (MAF) method, is proposed. The method of characteristics (MOC) is being widely used for reactor analysis thanks to the advances of numerical algorithms and computer hardware. However, efficient kinetic calculation method for MOC is still desirable since it requires significant computation time. Various efficient numerical methods for solving the space-dependent kinetic equation, e.g., the improved quasi-static (IQS) and the frequency transform methods, have been developed so far mainly for diffusion calculation. These calculation methods are known as effective numerical methods and they offer a way for faster computation. However, they have not been applied to the kinetic calculation method using MOC as the authors' knowledge. Thus, the MAF method is applied to the kinetic calculation using MOC aiming to reduce computation time. The MAF method is a unified numerical framework of conventional kinetic calculation methods, e.g., the IQS, the frequency transform, and the theta methods. Although the MAF method is originally developed for the space-dependent kinetic calculation based on the diffusion theory, it is extended to transport theory in the present study. The accuracy and computational time are evaluated though the TWIGL benchmark problem. The calculation results show the effectiveness of the MAF method. (authors)

  12. Ovarian steroid regulation of serotonin reuptake transporter (SERT) binding, distribution, and function in female macaques.

    PubMed

    Lu, N Z; Eshleman, A J; Janowsky, A; Bethea, C L

    2003-03-01

    The serotonin reuptake transporter (SERT) plays an important role in serotonin neurotransmission and in several psychopathological disorders such as depression and anxiety disorders. In this study, we investigated whether the ovarian steroids, estrogen (E) and progesterone (P) regulate SERT binding, intracellular distribution, and function using [(3)H]citalopram ligand binding with quantitative autoradiography, immunofluorescence histochemistry with confocal microscopy and [(3)H]serotonin uptake, respectively. Ovariectomized macaques received either placebo, E alone, P alone or E plus P for 28 days. In the raphe, E, P, and E+P treatments did not change SERT binding density. In several hypothalamic nuclei, [(3)H]citalopram binding was increased by E, P, and E+P. Immunofluorescent SERT in serotonin soma was intracellular and similar among treatments. In the hypothalamus, immunofluorescent SERT was located along the serotonergic axons and there was a significant proliferation of immunofluorescent fibers in hormone-treated animals. In addition, E and E+P treatment increased serotonin uptake in the basal ganglia. These findings suggest that ovarian hormones regulate SERT protein expression and distribution, perhaps via extracellular serotonin or mRNA stability, but not solely at the level of gene transcription. Further investigation on the possible action of ovarian steroids on the directionality of SERT transport is indicated. PMID:12660809

  13. Presenilin influences glycogen synthase kinase-3 β (GSK-3β) for kinesin-1 and dynein function during axonal transport.

    PubMed

    Dolma, Kunsang; Iacobucci, Gary J; Hong Zheng, Kan; Shandilya, Jayasha; Toska, Eneda; White, Joseph A; Spina, Elizabeth; Gunawardena, Shermali

    2014-03-01

    Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3β (GSK-3β). PS genetically interacts with GSK-3β in an activity-dependent manner. Excess of active GSK-3β perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3β-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3β did not affect axonal transport. Strikingly, GSK-3β-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3β are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3β activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.

  14. EVALUATION OF DMSO TRANSPORT IN HUMAN ARTICULAR CARTILAGE: VEHICLE SOLUTIONS AND EFFECTS ON CELL FUNCTION.

    PubMed

    Kay, A G; Rooney, P; Kearney, J; Pegg, D E

    2015-01-01

    Osteochondral allografting techniques are limited by the availability of suitable donor tissue; there is an urgent need for effective cryopreservation. A fundamental requirement is the need to establish initial conditions of exposure to cryoprotectant that the chondrocytes will tolerate and that load the tissue with an adequate concentration of cryoprotectant. Three vehicle solutions to transport DMSO into the tissue were studied. Knee joints were obtained from deceased donors with appropriate consent. Whole condyles were treated with 20% w/w DMSO in each of three vehicle solutions and chondrocyte function and tissue CPA content measured. The results showed that exposure to 20% DMSO in each vehicle solution for 2 hours at 0 degrees C was tolerated without loss of GAG synthetic activity. It was observed that penetration of DMSO increased little after 1 hour of CPA exposure at 0 degrees C but the final tissue concentration of CPA was markedly lower than that in the medium. PMID:26510337

  15. Role of serotonin transporter function in rat orbitofrontal cortex in impulsive choice

    PubMed Central

    Darna, Mahesh; Chow, Jonathan J.; Yates, Justin R.; Charnigo, Richard J.; Beckmann, Joshua S.; Bardo, Michael T.; Dwoskin, Linda P.

    2016-01-01

    Impulsivity is a multi-faceted personality construct that plays a prominent role in drug abuse vulnerability. Dysregulation of 5-hydroxytryptamine (serotonin, 5-HT) systems in subregions of the prefrontal cortex has been implicated in impulsivity. Extracellular 5-HT concentrations are regulated by 5-HT transporters (SERTs), indicating that these transporters may be important molecular targets underlying individual differences in impulsivity and drug abuse vulnerability. The present study evaluated the role of SERT in mediating individual differences in impulsivity. Rats were tested for both impulsive action using the cued go/no-go task and for impulsive choice using a delay discounting task in a counterbalanced design. Following behavioral evaluation, Km and Vmax were obtained from kinetic analysis of [3H]5-HT uptake by SERT using synaptosomes prepared from both orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC) obtained from each individual rat. Vmax for SERT in OFC, but not mPFC, was negatively correlated with mean adjusted delay scores in the delay discounting task. In contrast, Vmax for SERT in OFC and mPFC was not correlated with performance in the cued go/no-go task. To further evaluate the relationship between SERT function and impulsive choice, a selective SERT inhibitor, fluoxetine (0, 15, 50 and 150 pmol/side) was microinjected bilaterally into OFC and effects on the delay discounting task determined. Following stabilization of behavior, fluoxetine increased mean adjusted delay scores (decreased impulsivity) in high impulsive rats compared to saline microinjection, but had no effect in low impulsive rats. These ex vivo and in vivo results suggest that enhanced SERT function in OFC underlies high impulsive choice behavior. PMID:26183652

  16. The Arabidopsis COPT6 transport protein functions in copper distribution under copper-deficient conditions.

    PubMed

    Garcia-Molina, Antoni; Andrés-Colás, Nuria; Perea-García, Ana; Neumann, Ulla; Dodani, Sheel C; Huijser, Peter; Peñarrubia, Lola; Puig, Sergi

    2013-08-01

    Copper (Cu), an essential redox active cofactor, participates in fundamental biological processes, but it becomes highly cytotoxic when present in excess. Therefore, living organisms have established suitable mechanisms to balance cellular and systemic Cu levels. An important strategy to maintain Cu homeostasis consists of regulating uptake and mobilization via the conserved family of CTR/COPT Cu transport proteins. In the model plant Arabidopsis thaliana, COPT1 protein mediates root Cu acquisition, whereas COPT5 protein functions in Cu mobilization from intracellular storage organelles. The function of these transporters becomes critical when environmental Cu bioavailability diminishes. However, little is know about the mechanisms that mediate plant Cu distribution. In this report, we present evidence supporting an important role for COPT6 in Arabidopsis Cu distribution. Similarly to COPT1 and COPT2, COPT6 fully complements yeast mutants defective in high-affinity Cu uptake and localizes to the plasma membrane of Arabidopsis cells. Whereas COPT2 mRNA is only up-regulated upon severe Cu deficiency, COPT6 transcript is expressed under Cu excess conditions and displays a more gradual increase in response to decreases in environmental Cu levels. Consistent with COPT6 expression in aerial vascular tissues and reproductive organs, copt6 mutant plants exhibit altered Cu distribution under Cu-deficient conditions, including increased Cu in rosette leaves but reduced Cu levels in seeds. This altered Cu distribution is fully rescued when the wild-type COPT6 gene is reintroduced into the copt6 mutant line. Taken together, these findings highlight the relevance of COPT6 in shoot Cu redistribution when environmental Cu is limited.

  17. Endosomal transport function in yeast requires a novel AAA-type ATPase, Vps4p.

    PubMed Central

    Babst, M; Sato, T K; Banta, L M; Emr, S D

    1997-01-01

    In a late-Golgi compartment of the yeast Saccharomyces cerevisiae, vacuolar proteins such as carboxypeptidase Y (CPY) are actively sorted away from the secretory pathway and transported to the vacuole via a pre-vacuolar, endosome-like intermediate. The vacuolar protein sorting (vps) mutant vps4 accumulates vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar pre-vacuolar compartment. The VPS4 gene has been cloned and found to encode a 48 kDa protein which belongs to the protein family of AAA-type ATPases. The Vps4 protein was purified and shown to exhibit an N-ethylmaleimide-sensitive ATPase activity. A single amino acid change within the AAA motif of Vps4p yielded a protein that lacked ATPase activity and did not complement the protein sorting or morphological defects of the vps4 delta1 mutant. Indeed, when expressed at normal levels in wild-type cells, the mutant vps4 gene acted as a dominant-negative allele. The phenotypic characterization of a temperature-sensitive vps4 allele showed that the immediate consequence of loss of Vps4p function is a defect in vacuolar protein delivery. In this mutant, precursor CPY was not secreted but instead accumulated in an intracellular compartment, presumably the pre-vacuolar endosome. Electron microscopy revealed that upon temperature shift, exaggerated stacks of curved cisternal membranes (aberrant endosome) also accumulated in the vps4ts mutant. Based on these and other observations, we propose that Vps4p function is required for efficient transport out of the pre-vacuolar endosome. PMID:9155008

  18. Channel-mediated lactic acid transport: a novel function for aquaglyceroporins in bacteria.

    PubMed

    Bienert, Gerd P; Desguin, Benoît; Chaumont, François; Hols, Pascal

    2013-09-15

    MIPs (major intrinsic proteins), also known as aquaporins, are membrane proteins that channel water and/or uncharged solutes across membranes in all kingdoms of life. Considering the enormous number of different bacteria on earth, functional information on bacterial MIPs is scarce. In the present study, six MIPs [glpF1 (glycerol facilitator 1)-glpF6] were identified in the genome of the Gram-positive lactic acid bacterium Lactobacillus plantarum. Heterologous expression in Xenopus laevis oocytes revealed that GlpF2, GlpF3 and GlpF4 each facilitated the transmembrane diffusion of water, dihydroxyacetone and glycerol. As several lactic acid bacteria have GlpFs in their lactate racemization operon (GlpF1/F4 phylogenetic group), their ability to transport this organic acid was tested. Both GlpF1 and GlpF4 facilitated the diffusion of D/L-lactic acid. Deletion of glpF1 and/or glpF4 in Lb. plantarum showed that both genes were involved in the racemization of lactic acid and, in addition, the double glpF1 glpF4 mutant showed a growth delay under conditions of mild lactic acid stress. This provides further evidence that GlpFs contribute to lactic acid metabolism in this species. This lactic acid transport capacity was shown to be conserved in the GlpF1/F4 group of Lactobacillales. In conclusion, we have functionally analysed the largest set of bacterial MIPs and demonstrated that the lactic acid membrane permeability of bacteria can be regulated by aquaglyceroporins.

  19. Revealing the Molecular Structure and the Transport Mechanism at the Base of Primary Cilia Using Superresolution STED Microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Tung-Lin

    The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227+/-18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205+/-20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165+/-8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186+/-21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to

  20. Towards a filtered density function approach for reactive transport in groundwater

    NASA Astrophysics Data System (ADS)

    Suciu, N.; Schüler, L.; Attinger, S.; Knabner, P.

    2016-04-01

    Evolution equations for probability density functions (PDFs) and filtered density functions (FDFs) of random species concentrations weighted by conserved scalars are formulated as Fokker-Planck equations describing stochastically equivalent processes in concentration-position spaces. This approach provides consistent numerical PDF/FDF solutions, given by the density in the concentration-position space of an ensemble of computational particles governed by the associated Itô equations. The solutions are obtained by a global random walk (GRW) algorithm, which is stable, free of numerical diffusion, and practically insensitive to the increase of the number of particles. The general FDF approach and the GRW numerical solution are illustrated for a reduced complexity problem consisting of the transport of a single scalar in groundwater. Randomness is induced by the stochastic parameterization of the hydraulic conductivity, characterized by short range correlations and small variance. The objective is to infer the statistics of the random concentration sampled at the plume center of mass, integrated over the transverse dimension of a two-dimensional spatial domain. The PDF/FDF problem can therefore be formulated in a two-dimensional domain as well, a spatial dimension and one in the concentration space. The upscaled drift and diffusion coefficients describing the PDF transport in the physical space are estimated on single-trajectories of diffusion in velocity fields with short-range correlations, owing to their self-averaging property. The mixing coefficients describing the PDF transport in concentration spaces are parameterized by the trend and the noise inferred from the statistical analysis of an ensemble of simulated concentration time series, as well as by classical mixing models. A Gaussian spatial filter applied to a Kraichnan velocity field generator is used to construct coarse-grained simulations (CGS) for FDF problems. The purposes of the CGS simulations are

  1. Theoretical investigations into the electronic structures and electron transport properties of fluorine and carbonyl end-functionalized quarterthiophenes.

    PubMed

    Li, Qian; Duan, Yuai; Gao, Hong-Ze; Su, Zhong-Мin; Geng, Yun

    2015-06-01

    In this work, we concentrate on systematic investigation on the fluorination and carbonylation effect on electron transport properties of thiophene-based materials with the aim of seeking and designing electron transport materials. Some relative factors, namely, frontier molecular orbital (FMO), vertical electron affinity (VEA), electron reorganization energy (λele), electron transfer integral (tele), electron drift mobility (μele) and band structures have been calculated and discussed based on density functional theory. The results show that the introduction of fluorine atoms and carbonyl group especially for the latter could effectively increase EA and reduce λele, which is beneficial to the improvement of electron transport performance. Furthermore, these introductions could also affect the tele by changing molecular packing manner and distribution of FMO. Finally, according to our calculation, the 3d system is considered to be a promising electron transport material with small λele, high electron transport ability and good ambient stability.

  2. Theoretical investigations into the electronic structures and electron transport properties of fluorine and carbonyl end-functionalized quarterthiophenes.

    PubMed

    Li, Qian; Duan, Yuai; Gao, Hong-Ze; Su, Zhong-Мin; Geng, Yun

    2015-06-01

    In this work, we concentrate on systematic investigation on the fluorination and carbonylation effect on electron transport properties of thiophene-based materials with the aim of seeking and designing electron transport materials. Some relative factors, namely, frontier molecular orbital (FMO), vertical electron affinity (VEA), electron reorganization energy (λele), electron transfer integral (tele), electron drift mobility (μele) and band structures have been calculated and discussed based on density functional theory. The results show that the introduction of fluorine atoms and carbonyl group especially for the latter could effectively increase EA and reduce λele, which is beneficial to the improvement of electron transport performance. Furthermore, these introductions could also affect the tele by changing molecular packing manner and distribution of FMO. Finally, according to our calculation, the 3d system is considered to be a promising electron transport material with small λele, high electron transport ability and good ambient stability. PMID:25909689

  3. Functional interactions between the ciliopathy-associated Meckel syndrome 1 (MKS1) protein and two novel MKS1-related (MKSR) proteins.

    PubMed

    Bialas, Nathan J; Inglis, Peter N; Li, Chunmei; Robinson, Jon F; Parker, Jeremy D K; Healey, Michael P; Davis, Erica E; Inglis, Chrystal D; Toivonen, Tiina; Cottell, David C; Blacque, Oliver E; Quarmby, Lynne M; Katsanis, Nicholas; Leroux, Michel R

    2009-03-01

    Meckel syndrome (MKS) is a ciliopathy characterized by encephalocele, cystic renal disease, liver fibrosis and polydactyly. An identifying feature of MKS1, one of six MKS-associated proteins, is the presence of a B9 domain of unknown function. Using phylogenetic analyses, we show that this domain occurs exclusively within a family of three proteins distributed widely in ciliated organisms. Consistent with a ciliary role, all Caenorhabditis elegans B9-domain-containing proteins, MKS-1 and MKS-1-related proteins 1 and 2 (MKSR-1, MKSR-2), localize to transition zones/basal bodies of sensory cilia. Their subcellular localization is largely co-dependent, pointing to a functional relationship between the proteins. This localization is evolutionarily conserved, because the human orthologues also localize to basal bodies, as well as cilia. As reported for MKS1, disrupting human MKSR1 or MKSR2 causes ciliogenesis defects. By contrast, single, double and triple C. elegans mks/mksr mutants do not display overt defects in ciliary structure, intraflagellar transport or chemosensation. However, we find genetic interactions between all double mks/mksr mutant combinations, manifesting as an increased lifespan phenotype, which is due to abnormal insulin-IGF-I signaling. Our findings therefore demonstrate functional interactions between a novel family of proteins associated with basal bodies or cilia, providing new insights into the molecular etiology of a pleiotropic human disorder. PMID:19208769

  4. Proximal renal tubular acidosis mediated by mutations in NBCe1-A: unraveling the transporter's structure-functional properties

    PubMed Central

    Kurtz, Ira; Zhu, Quansheng

    2013-01-01

    NBCe1 belongs to the SLC4 family of base transporting membrane proteins that plays a significant role in renal, extrarenal, and systemic acid-base homeostasis. Recent progress has been made in characterizing the structure-function properties of NBCe1 (encoded by the SLC4A4 gene), and those factors that regulate its function. In the kidney, the NBCe1-A variant that is expressed on the basolateral membrane of proximal tubule is the key transporter responsible for overall transepithelial bicarbonate absorption in this nephron segment. NBCe1 mutations impair transepithelial bicarbonate absorption causing the syndrome of proximal renal tubular acidosis (pRTA). Studies of naturally occurring NBCe1 mutant proteins in heterologous expression systems have been very helpful in elucidation the structure-functional properties of the transporter. NBCe1 mutations are now known to cause pRTA by various mechanisms including the alteration of the transporter function (substrate ion interaction, electrogenicity), abnormal processing to the plasma membrane, and a perturbation in its structural properties. The elucidation of how NBCe1 mutations cause pRTA in addition to the recent studies which have provided further insight into the topology of the transporter have played an important role in uncovering its critically important structural-function properties. PMID:24391589

  5. ATP-binding cassette transporter A7 (ABCA7) loss of function alters Alzheimer amyloid processing.

    PubMed

    Satoh, Kanayo; Abe-Dohmae, Sumiko; Yokoyama, Shinji; St George-Hyslop, Peter; Fraser, Paul E

    2015-10-01

    The ATP-binding cassette transporter A7 (ABCA7) has been identified as a susceptibility factor of late onset Alzheimer disease in genome-wide association studies. ABCA7 has been shown to mediate phagocytosis and affect membrane trafficking. The current study examined the impact of ABCA7 loss of function on amyloid precursor protein (APP) processing and generation of amyloid-β (Aβ). Suppression of endogenous ABCA7 in several different cell lines resulted in increased β-secretase cleavage and elevated Aβ. ABCA7 knock-out mice displayed an increased production of endogenous murine amyloid Aβ42 species. Crossing ABCA7-deficient animals to an APP transgenic model resulted in significant increases in the soluble Aβ as compared with mice expressing normal levels of ABCA7. Only modest changes in the amount of insoluble Aβ and amyloid plaque densities were observed once the amyloid pathology was well developed, whereas Aβ deposition was enhanced in younger animals. In vitro studies indicated a more rapid endocytosis of APP in ABCA7 knock-out cells that is mechanistically consistent with the increased Aβ production. These in vitro and in vivo findings indicate a direct role of ABCA7 in amyloid processing that may be associated with its primary biological function to regulate endocytic pathways. Several potential loss-of-function ABCA7 mutations and deletions linked to Alzheimer disease that in some instances have a greater impact than apoE allelic variants have recently been identified. A reduction in ABCA7 expression or loss of function would be predicted to increase amyloid production and that may be a contributing factor in the associated Alzheimer disease susceptibility. PMID:26260791

  6. Reduced dopamine transporter functioning induces high-reward risk-preference consistent with bipolar disorder.

    PubMed

    van Enkhuizen, Jordy; Henry, Brook L; Minassian, Arpi; Perry, William; Milienne-Petiot, Morgane; Higa, Kerin K; Geyer, Mark A; Young, Jared W

    2014-12-01

    Individuals with bipolar disorder (BD) exhibit deleterious decision making, negatively impacting their lives. Such aberrant decision making can be quantified using the Iowa Gambling Task (IGT), which requires choosing between advantageous and disadvantageous options based on different reward/punishment schedules. The mechanisms underlying this behavioral deficit are unknown, but may include the reduced dopamine transporter (DAT) functioning reported in BD patients. Using both human and mouse IGTs, we tested whether reduced DAT functioning would recreate patterns of deficient decision making of BD patients. We assessed the IGT performance of 16 BD subjects (7 female) and 17 healthy control (HC) subjects (12 female). We recorded standard IGT performance measures and novel post-reward and post-punishment decision-making strategies. We characterized a novel single-session mouse IGT using C57BL/6J mice (n = 44). The BD and HC IGT performances were compared with the effects of chronic (genetic knockdown (KD; n = 31) and wild-type (n = 28) mice) and acute (C57BL/6J mice (n = 89) treated with the DAT inhibitor GBR12909) reductions of DAT functioning in mice performing this novel IGT. BD patients exhibited impaired decision making compared with HC subjects. Both the good-performing DAT KD and GBR12909-treated mice exhibited poor decision making in the mouse IGT. The deficit of each population was driven by high-reward sensitivity. The single-session mouse IGT measures dynamic risk-based decision making similar to humans. Chronic and acute reductions of DAT functioning in mice impaired decision-making consistent with poor IGT performance of BD patients. Hyperdopaminergia caused by reduced DAT may impact poor decision making in BD patients, which should be confirmed in future studies.

  7. Expression, regulation, and function of drug transporters in cervicovaginal tissues of a mouse model used for microbicide testing.

    PubMed

    Zhou, Tian; Hu, Minlu; Pearlman, Andrew; Rohan, Lisa C

    2016-09-15

    P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4) are three efflux transporters that play key roles in the pharmacokinetics of antiretroviral drugs used in the pre-exposure prophylaxis of HIV sexual transmission. In this study, we investigated the expression, regulation, and function of these transporters in cervicovaginal tissues of a mouse model. Expression and regulation were examined using real-time RT-PCR and immunohistochemical staining, in the mouse tissues harvested at estrus and diestrus stages under natural cycling or after hormone synchronization. The three transporters were expressed at moderate to high levels compared to the liver. Transporter proteins were localized in various cell types in different tissue segments. Estrous cycle and exogenous hormone treatment affected transporter mRNA and protein expression, in a tissue- and transporter-dependent manner. Depo-Provera-synchronized mice were dosed vaginally or intraperitoneally with (3)H-TFV, with or without MK571 co-administration, to delineate the function of cervicovaginal Mrp4. Co-administration of MK571 significantly increased the concentration of vaginally-administered TFV in endocervix and vagina. MK571 increased the concentration of intraperitoneally-administered TFV in the cervicovaginal lavage and vagina by several fold. Overall, P-gp, Bcrp, and Mrp4 were positively expressed in mouse cervicovaginal tissues, and their expression can be regulated by the estrous cycle or by exogenous hormones. In this model, the Mrp4 transporter impacted TFV distribution in cervicovaginal tissues. PMID:27453435

  8. Transplantation of Glial Progenitors That Overexpress Glutamate Transporter GLT1 Preserves Diaphragm Function Following Cervical SCI

    PubMed Central

    Li, Ke; Javed, Elham; Hala, Tamara J; Sannie, Daniel; Regan, Kathleen A; Maragakis, Nicholas J; Wright, Megan C; Poulsen, David J; Lepore, Angelo C

    2015-01-01

    Approximately half of traumatic spinal cord injury (SCI) cases affect cervical regions, resulting in chronic respiratory compromise. The majority of these injuries affect midcervical levels, the location of phrenic motor neurons (PMNs) that innervate the diaphragm. A valuable opportunity exists following SCI for preventing PMN loss that occurs during secondary degeneration. One of the primary causes of secondary injury is excitotoxicity due to dysregulation of extracellular glutamate homeostasis. Astrocytes express glutamate transporter 1 (GLT1), which is responsible for the majority of CNS glutamate clearance. Given our observations of GLT1 dysfunction post-SCI, we evaluated intraspinal transplantation of Glial-Restricted Precursors (GRPs)—a class of lineage-restricted astrocyte progenitors—into ventral horn following cervical hemicontusion as a novel strategy for reconstituting GLT1 function, preventing excitotoxicity and protecting PMNs in the acutely injured spinal cord. We find that unmodified transplants express low levels of GLT1 in the injured spinal cord. To enhance their therapeutic properties, we engineered GRPs with AAV8 to overexpress GLT1 only in astrocytes using the GFA2 promoter, resulting in significantly increased GLT1 protein expression and functional glutamate uptake following astrocyte differentiation in vitro and after transplantation into C4 hemicontusion. Compared to medium-only control and unmodified GRPs, GLT1-overexpressing transplants reduced lesion size, diaphragm denervation and diaphragm dysfunction. Our findings demonstrate transplantation-based replacement of astrocyte GLT1 is a promising approach for SCI. PMID:25492561

  9. A computational model of oxygen transport in the cerebrocapillary levels for normal and pathologic brain function.

    PubMed

    Safaeian, Navid; David, Tim

    2013-10-01

    The oxygen exchange and correlation between the cerebral blood flow (CBF) and cerebral metabolic rate of oxygen consumption (CMRO2) in the cortical capillary levels for normal and pathologic brain functions remain the subject of debate. A 3D realistic mesoscale model of the cortical capillary network (non-tree like) is constructed using a random Voronoi tessellation in which each edge represents a capillary segment. The hemodynamics and oxygen transport are numerically simulated in the model, which involves rheological laws in the capillaries, oxygen diffusion, and non-linear binding of oxygen to hemoglobin, respectively. The findings show that the cerebral hypoxia due to a significant decreased perfusion (as can occur in stroke) can be avoided by a moderate reduction in oxygen demand. Oxygen extraction fraction (OEF) can be an important indicator for the brain oxygen metabolism under normal perfusion and misery-perfusion syndrome (leading to ischemia). The results demonstrated that a disproportionately large increase in blood supply is required for a small increase in the oxygen demand, which, in turn, is strongly dependent on the resting OEF. The predicted flow-metabolism coupling in the model supports the experimental studies of spatiotemporal stimulations in humans by positron emission tomography and functional magnetic resonance imaging.

  10. Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p.

    PubMed

    Wysocki, Robert; Clemens, Stephan; Augustyniak, Daria; Golik, Pawel; Maciaszczyk, Ewa; Tamás, Markus J; Dziadkowiec, Dorota

    2003-05-01

    Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.

  11. Loss of synaptic Zn2+ transporter function increases risk of febrile seizures

    PubMed Central

    Hildebrand, Michael S.; Phillips, A. Marie; Mullen, Saul A.; Adlard, Paul A.; Hardies, Katia; Damiano, John A.; Wimmer, Verena; Bellows, Susannah T.; McMahon, Jacinta M.; Burgess, Rosemary; Hendrickx, Rik; Weckhuysen, Sarah; Suls, Arvid; De Jonghe, Peter; Scheffer, Ingrid E.; Petrou, Steven; Berkovic, Samuel F.; Reid, Christopher A.

    2015-01-01

    Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn2+) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn2+ homeostasis contributes to susceptibility is unknown. Synaptic Zn2+ is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn2+ into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn2+ increases the risk of FS and more broadly support the idea that impaired synaptic Zn2+ homeostasis can contribute to neuronal hyperexcitability. PMID:26647834

  12. Quantum maximum-entropy principle for closed quantum hydrodynamic transport within a Wigner function formalism

    SciTech Connect

    Trovato, M.; Reggiani, L.

    2011-12-15

    By introducing a quantum entropy functional of the reduced density matrix, the principle of quantum maximum entropy is asserted as fundamental principle of quantum statistical mechanics. Accordingly, we develop a comprehensive theoretical formalism to construct rigorously a closed quantum hydrodynamic transport within a Wigner function approach. The theoretical formalism is formulated in both thermodynamic equilibrium and nonequilibrium conditions, and the quantum contributions are obtained by only assuming that the Lagrange multipliers can be expanded in powers of ({h_bar}/2{pi}){sup 2}. In particular, by using an arbitrary number of moments, we prove that (1) on a macroscopic scale all nonlocal effects, compatible with the uncertainty principle, are imputable to high-order spatial derivatives, both of the numerical density n and of the effective temperature T; (2) the results available from the literature in the framework of both a quantum Boltzmann gas and a degenerate quantum Fermi gas are recovered as a particular case; (3) the statistics for the quantum Fermi and Bose gases at different levels of degeneracy are explicitly incorporated; (4) a set of relevant applications admitting exact analytical equations are explicitly given and discussed; (5) the quantum maximum entropy principle keeps full validity in the classical limit, when ({h_bar}/2{pi}){yields}0.

  13. Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

    PubMed Central

    Boll, M; Herget, M; Wagener, M; Weber, W M; Markovich, D; Biber, J; Clauss, W; Murer, H; Daniel, H

    1996-01-01

    The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences. Images Fig. 7 PMID:8552623

  14. Novel riboflavin transporter family RFVT/SLC52: identification, nomenclature, functional characterization and genetic diseases of RFVT/SLC52.

    PubMed

    Yonezawa, Atsushi; Inui, Ken-ichi

    2013-01-01

    Riboflavin, a water-soluble vitamin also known as vitamin B2, is essential for normal cellular functions. Riboflavin transporters play important roles in its homeostasis. Recently, three novel riboflavin transporters were identified, and designated as RFT1, RFT2 and RFT3. Because the RFTs did not show similarity to other SLC transporters, and RFT1 and RFT3 are similar in sequence and function, they were assigned into a new SLC family, SLC52. Subsequently, RFT1/GPR172B, RFT3/GPR172A and RFT2/C20orf54 were renamed as RFVT1/SLC52A1, RFVT2/SLC52A2 and RFVT3/SLC52A3, respectively. In this review, we summarize recent findings on the cloning, nomenclature, functional characterization and genetic diseases of RFVT1/SLC52A1, RFVT2/SLC52A2 and RFVT3/SLC52A3.

  15. Functional Coding Variation in Recombinant Inbred Mouse Lines Reveals Novel Serotonin Transporter-Associated Phenotypes

    SciTech Connect

    Carneiro, Ana; Airey, David; Thompson, Brent; Zhu, C; Rinchik, Eugene M; Lu, Lu; Chesler, Elissa J; Erikson, Keith; Blakely, Randy

    2009-01-01

    The human serotonin (5-hydroxytryptamine, 5-HT) transporter (hSERT, SLC6A4) figures prominently in the etiology or treatment of many prevalent neurobehavioral disorders including anxiety, alcoholism, depression, autism and obsessive-compulsive disorder (OCD). Here we utilize naturally occurring polymorphisms in recombinant inbred (RI) lines to identify novel phenotypes associated with altered SERT function. The widely used mouse strain C57BL/6J, harbors a SERT haplotype defined by two nonsynonymous coding variants (Gly39 and Lys152 (GK)). At these positions, many other mouse lines, including DBA/2J, encode Glu39 and Arg152 (ER haplotype), assignments found also in hSERT. Synaptosomal 5-HT transport studies revealed reduced uptake associated with the GK variant. Heterologous expression studies confirmed a reduced SERT turnover rate for the GK variant. Experimental and in silico approaches using RI lines (C57Bl/6J X DBA/2J=BXD) identifies multiple anatomical, biochemical and behavioral phenotypes specifically impacted by GK/ER variation. Among our findings are multiple traits associated with anxiety and alcohol consumption, as well as of the control of dopamine (DA) signaling. Further bioinformatic analysis of BXD phenotypes, combined with biochemical evaluation of SERT knockout mice, nominates SERT-dependent 5-HT signaling as a major determinant of midbrain iron homeostasis that, in turn, dictates ironregulated DA phenotypes. Our studies provide a novel example of the power of coordinated in vitro, in vivo and in silico approaches using murine RI lines to elucidate and quantify the system-level impact of gene variation.

  16. Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum.

    PubMed

    Watanabe, Akira; Hiraga, Kazumi; Suda, Masako; Yukawa, Hideaki; Inui, Masayuki

    2015-06-15

    The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s(-1)), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s(-1)), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. PMID:25862223

  17. Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

    PubMed Central

    Watanabe, Akira; Hiraga, Kazumi; Suda, Masako; Yukawa, Hideaki

    2015-01-01

    The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. PMID:25862223

  18. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence

    PubMed Central

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W.

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes. PMID:26741360

  19. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

    PubMed

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes. PMID:26741360

  20. Role of Annular Lipids in the Functional Properties of Leucine Transporter LeuT Proteomicelles

    PubMed Central

    2016-01-01

    Recent work has shown that the choice of the type and concentration of detergent used for the solubilization of membrane proteins can strongly influence the results of functional experiments. In particular, the amino acid transporter LeuT can bind two substrate molecules in low concentrations of n-dodecyl β-d-maltopyranoside (DDM), whereas high concentrations reduce the molar binding stoichiometry to 1:1. Subsequent molecular dynamics (MD) simulations of LeuT in DDM proteomicelles revealed that DDM can penetrate to the extracellular vestibule and make stable contacts in the functionally important secondary substrate binding site (S2), suggesting a potential competitive mechanism for the reduction in binding stoichiometry. Because annular lipids can be retained during solubilization, we performed MD simulations of LeuT proteomicelles at various stages of the solubilization process. We find that at low DDM concentrations, lipids are retained around the protein and penetration of detergent into the S2 site does not occur, whereas at high concentrations, lipids are displaced and the probability of DDM binding in the S2 site is increased. This behavior is dependent on the type of detergent, however, as we find in the simulations that the detergent lauryl maltose-neopentyl glycol, which is approximately twice the size of DDM and structurally more closely resembles lipids, does not penetrate the protein even at very high concentrations. We present functional studies that confirm the computational findings, emphasizing the need for careful consideration of experimental conditions, and for cautious interpretation of data in gathering mechanistic information about membrane proteins. PMID:26811944

  1. A Trypanosomatid Iron Transporter that Regulates Mitochondrial Function Is Required for Leishmania amazonensis Virulence.

    PubMed

    Mittra, Bidyottam; Laranjeira-Silva, Maria Fernanda; Perrone Bezerra de Menezes, Juliana; Jensen, Jennifer; Michailowsky, Vladimir; Andrews, Norma W

    2016-01-01

    Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.

  2. Transport properties of proton-exchange membranes: Effect of supercritical-fluid processing and chemical functionality

    NASA Astrophysics Data System (ADS)

    Pulido Ayazo

    NafionRTM membranes commonly used in direct methanol fuel cells (DMFC), are tipically limited by high methanol permeability (also known as the cross-over limitation). These membranes have phase segregated sulfonated ionic domains in a perfluorinated backbone, which makes processing challenging and limited by phase equilibria considerations. This study used supercritical fluids (SCFs) as a processing alternative, since the gas-like mass transport properties of SCFs allow a better penetration into the membranes and the use of polar co-solvents influenced their morphology, fine-tuning the physical and transport properties in the membrane. Measurements of methanol permeability and proton conductivity were performed to the NafionRTM membranes processed with SCFs at 40ºC and 200 bar and the co-solvents as: acetone, tetrahydrofuran (THF), isopropyl alcohol, HPLC-grade water, acetic acid, cyclohexanone. The results obtained for the permeability data were of the order of 10 -8-10-9 cm2/s, two orders of magnitude lower than unprocessed Nafion. Proton conductivity results obtained using AC impedance electrochemical spectroscopy was between 0.02 and 0.09 S/cm, very similar to the unprocessed Nafion. SCF processing with ethanol as co-solvent reduced the methanol permeability by two orders of magnitude, while the proton conductivity was only reduced by 4%. XRD analysis made to the treated samples exhibited a decreasing pattern in the crystallinity, which affects the transport properties of the membrane. Also, SAXS profiles of the Nafion membranes processed were obtained with the goal of determining changes produced by the SCF processing in the hydrophilic domains of the polymer. With the goal of searching for new alternatives in proton exchange membranes (PEMs) triblock copolymer of poly(styrene-isobutylene-styrene) (SIBS) and poly(styrene-isobutylene-styrene) SEBS were studied. These sulfonated tri-block copolymers had lower methanol permeabilities, but also lower proton

  3. Coupled particle-fluid transport and magnetic separation in microfluidic systems with passive magnetic functionality

    NASA Astrophysics Data System (ADS)

    Khashan, Saud A.; Furlani, Edward P.

    2013-03-01

    A study is presented of coupled particle-fluid transport and field-directed particle capture in microfluidic systems with passive magnetic functionality. These systems consist of a microfluidic flow cell on a substrate that contains embedded magnetic elements. Two systems are considered that utilize soft- and hard-magnetic elements, respectively. In the former, an external field is applied to magnetize the elements, and in the latter, they are permanently magnetized. The field produced by the magnetized elements permeates into the flow cell giving rise to an attractive force on magnetic particles that flow through it. The systems are studied using a novel numerical/closed-form modelling approach that combines numerical transport analysis with closed-form field analysis. Particle-fluid transport is computed using computational fluid dynamics (CFD), while the magnetic force that governs particle capture is obtained in closed form. The CFD analysis takes into account dominant particle forces and two-way momentum transfer between the particles and the fluid. The two-way particle-fluid coupling capability is an important feature of the model that distinguishes it from more commonly used and simplified one-way coupling analysis. The model is used to quantify the impact of two-way particle-fluid coupling on both the capture efficiency and the flow pattern in the systems considered. Many effects such as particle-induced flow-enhanced capture efficiency and flow circulation are studied that cannot be predicted using one-way coupling analysis. In addition, dilute particle dispersions are shown to exhibit significant localized particle-fluid coupling near the capture regions, which contradicts the commonly held view that two-way coupling can be ignored when analysing high-gradient magnetic separation involving such particle systems. Overall, the model demonstrates that two-way coupling needs to be taken into account for rigorous predictions of capture efficiency, especially

  4. A functional-phylogenetic system for the classification of transport proteins.

    PubMed

    Saier, M H

    1999-01-01

    Twenty completely sequenced genomes of bacteria, archaea, and eukaryotes have been surveyed for the presence of genes encoding homologues of known solute transport proteins. These analyses and others have demonstrated the presence of nearly 250 families of sequence-related transporters. All such proteins have been classified according to the system we call the transporter classification system of the transport commission (TC). This short summary article describes the main features of this system; the families are presented in tabular form. Detailed information concerning these families and their constituent transporters is available on our web sites. J. Cell. Biochem. Suppls. 32/33:84-94, 1999.

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

    NASA Astrophysics Data System (ADS)

    Payne, Christine

    2014-03-01

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

  6. Functional Comparison of 45 Naturally Occurring Isoforms of the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT).

    PubMed

    Callaghan, Paul S; Hassett, Matthew R; Roepe, Paul D

    2015-08-18

    At least 53 distinct isoforms of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein are expressed in strains or isolates of P. falciparum malarial parasites from around the globe. These parasites exhibit a range of sensitivities to chloroquine (CQ) and other drugs. Mutant PfCRT is believed to confer cytostatic CQ resistance (CQR(CS)) by transporting CQ away from its DV target (free heme released upon hemoglobin digestion). One theory is that variable CQ transport catalyzed by these different PfCRT isoforms is responsible for the range of CQ sensitivities now found for P. falciparum. Alternatively, additional mutations in drug-selected parasites, or additional functions of PfCRT, might complement PfCRT-mediated CQ transport in conferring the range of observed resistance phenotypes. To distinguish between these possibilities, we recently optimized a convenient method for measuring PfCRT-mediated CQ transport, involving heterologous expression in Saccharomyces cerevisiae. Here, we use this method to quantify drug transport activity for 45 of 53 of the naturally occurring PfCRT isoforms. Data show that variable levels of CQR likely depend upon either additional PfCRT functions or additional genetic events, including perhaps changes that influence DV membrane potential. The data also suggest that the common K76T PfCRT mutation that is often used to distinguish a P. falciparum CQR phenotype is not, in and of itself, a fully reliable indicator of CQR status.

  7. Functional characterization of a transition metal ion transporter, OsZIP6 from rice (Oryza sativa L.).

    PubMed

    P G, Kavitha; Kuruvilla, Sam; Mathew, M K

    2015-12-01

    Micronutrients are important for the growth and development of plants, which deploy families of transporters for their uptake and distribution. We have functionally characterized a novel transition metal ion transporter from rice, OsZIP6 (Oryza sativa zinc regulated transporter, iron regulated transporter-like protein 6). The transporter was found to be transcriptionally activated in shoot and root tissues in response to deficiency in Fe(2+), Zn(2+) and Mn(2+). OsZIP6 was expressed in Xenopus laevis oocytes, where currents were observed on addition of Co(2+), Fe(2+) and Cd(2+) but not Zn(2+), Mn(2+) and Ni(2+). This substrate range for OsZIP6, identified using two-electrode voltage clamp electrophysiology was confirmed by atomic absorption spectroscopy. Ion transport by OsZIP6 was found to be pH dependent and enhanced transport was observed at acidic pH. Radioisotope uptake suggested that Co(2+) competitively inhibits Fe(2+) uptake by OsZIP6. Identification and characterization of ZIP family members from crop plants will contribute to an understanding of nutrient mineral homeostasis in these plants.

  8. Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

    PubMed

    Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

    2016-09-01

    ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. PMID:27602881

  9. A systematic approach to the amplified expression, functional characterization and purification of inositol transporters from Bacillus subtilis.

    PubMed

    Bettaney, Kim E; Sukumar, Preethi; Hussain, Rohanah; Siligardi, Giuliano; Henderson, Peter J F; Patching, Simon G

    2013-02-01

    Abstract A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ∼ 10-15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of (3)H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for (3)H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.

  10. Identification and functional characterization of silicon transporters in soybean using comparative genomics of major intrinsic proteins in Arabidopsis and rice.

    PubMed

    Deshmukh, Rupesh K; Vivancos, Julien; Guérin, Valérie; Sonah, Humira; Labbé, Caroline; Belzile, François; Bélanger, Richard R

    2013-11-01

    Silicon (Si) confers several benefits to many plant species when absorbed as silicic acid through nodulin 26-like intrinsic proteins (NIPs). The NIPs belong to major intrinsic protein (MIP) family, members of which form channels with high selectivity to control transport of water and different solutes. Here, comparative genomic analysis of the MIPs was performed to investigate the presence of Si transporter MIPs in soybean. Thorough analysis of phylogeny, gene organization, transcriptome profiling and protein modeling was performed to characterize MIPs in rice, Arabidopsis and soybean. Based on several attributes, two putative Si transporter genes, GmNIP2-1 and GmNIP2-2, were identified, characterized and cloned from soybean. Expression of both genes was detected in shoot and root tissues, and decreased as Si increased. The protein encoded by GmNIP2-2 showed functionality for Si transport when expressed in Xenopus oocytes, thus confirming the genetic capability of soybean to absorb the element. Comparative analysis of MIPs in plants provides opportunities to decipher gene evolution, functionality and selectivity of nutrient uptake mechanisms. Exploitation of this strategy has helped to uncover unique features of MIPs in soybean. The identification and functional characterization of Si transporters can be exploited to optimize the benefits that plants can derive from Si absorption. PMID:23771580

  11. Functional test of pedotransfer functions to predict water flow and solute transport with the dual-permeability model MACRO

    NASA Astrophysics Data System (ADS)

    Moeys, J.; Larsbo, M.; Bergström, L.; Brown, C. D.; Coquet, Y.; Jarvis, N. J.

    2012-02-01

    Estimating pesticide leaching risks at the regional scale requires the ability to completely parameterise a pesticide fate model using only survey data, such as soil and land-use maps. Such parameterisation usually rely on a set of lookup tables and (pedo)transfer functions, relating elementary soil and site properties to model parameters. The aim of this paper is to describe and test a complete set of parameter estimation algorithms developed for the pesticide fate model MACRO, which accounts for preferential flow in soil macropores. We used tracer monitoring data from 16 lysimeter studies, carried out in three European countries, to evaluate the ability of MACRO and this "blind parameterisation" scheme to reproduce measured solute leaching at the base of each lysimeter. We focused on the prediction of early tracer breakthrough due to preferential flow, because this is critical for pesticide leaching. We then calibrated a selected number of parameters in order to assess to what extent the prediction of water and solute leaching could be improved. Our results show that water flow was generally reasonably well predicted (median model efficiency, ME, of 0.42). Although the general pattern of solute leaching was reproduced well by the model, the overall model efficiency was low (median ME = -0.26) due to errors in the timing and magnitude of some peaks. Preferential solute leaching at early pore volumes was also systematically underestimated. Nonetheless, the ranking of soils according to solute loads at early pore volumes was reasonably well estimated (concordance correlation coefficient, CCC, between 0.54 and 0.72). Moreover, we also found that ignoring macropore flow leads to a significant deterioration in the ability of the model to reproduce the observed leaching pattern, and especially the early breakthrough in some soils. Finally, the calibration procedure showed that improving the estimation of solute transport parameters is probably more important than the

  12. Functional test of pedotransfer functions to predict water flow and solute transport with the dual-permeability model MACRO

    NASA Astrophysics Data System (ADS)

    Moeys, J.; Larsbo, M.; Bergström, L.; Brown, C. D.; Coquet, Y.; Jarvis, N. J.

    2012-07-01

    Estimating pesticide leaching risks at the regional scale requires the ability to completely parameterise a pesticide fate model using only survey data, such as soil and land-use maps. Such parameterisations usually rely on a set of lookup tables and (pedo)transfer functions, relating elementary soil and site properties to model parameters. The aim of this paper is to describe and test a complete set of parameter estimation algorithms developed for the pesticide fate model MACRO, which accounts for preferential flow in soil macropores. We used tracer monitoring data from 16 lysimeter studies, carried out in three European countries, to evaluate the ability of MACRO and this "blind parameterisation" scheme to reproduce measured solute leaching at the base of each lysimeter. We focused on the prediction of early tracer breakthrough due to preferential flow, because this is critical for pesticide leaching. We then calibrated a selected number of parameters in order to assess to what extent the prediction of water and solute leaching could be improved. Our results show that water flow was generally reasonably well predicted (median model efficiency, ME, of 0.42). Although the general pattern of solute leaching was reproduced well by the model, the overall model efficiency was low (median ME = -0.26) due to errors in the timing and magnitude of some peaks. Preferential solute leaching at early pore volumes was also systematically underestimated. Nonetheless, the ranking of soils according to solute loads at early pore volumes was reasonably well estimated (concordance correlation coefficient, CCC, between 0.54 and 0.72). Moreover, we also found that ignoring macropore flow leads to a significant deterioration in the ability of the model to reproduce the observed leaching pattern, and especially the early breakthrough in some soils. Finally, the calibration procedure showed that improving the estimation of solute transport parameters is probably more important than the

  13. Individual variation in incentive salience attribution and accumbens dopamine transporter expression and function.

    PubMed

    Singer, Bryan F; Guptaroy, Bipasha; Austin, Curtis J; Wohl, Isabella; Lovic, Vedran; Seiler, Jillian L; Vaughan, Roxanne A; Gnegy, Margaret E; Robinson, Terry E; Aragona, Brandon J

    2016-03-01

    Cues (conditioned stimuli; CSs) associated with rewards can come to motivate behavior, but there is considerable individual variation in their ability to do so. For example, a lever-CS that predicts food reward becomes attractive and wanted, and elicits reward-seeking behavior, to a greater extent in some rats ('sign-trackers'; STs) than others ('goal-trackers'; GTs). Variation in dopamine (DA) neurotransmission in the nucleus accumbens (NAc) core is thought to contribute to such individual variation. Given that the DA transporter (DAT) exerts powerful regulation over DA signaling, we characterized the expression and function of the DAT in the accumbens of STs and GTs. STs showed greater DAT surface expression in ventral striatal synaptosomes than GTs, and ex vivo fast-scan cyclic voltammetry recordings of electrically evoked DA release confirmed enhanced DAT function in STs, as indicated by faster DA uptake, specifically in the NAc core. Consistent with this, systemic amphetamine (AMPH) produced greater inhibition of DA uptake in STs than in GTs. Furthermore, injection of AMPH directly into the NAc core enhanced lever-directed approach in STs, presumably by amplifying the incentive value of the CS, but had no effect on goal-tracking behavior. On the other hand, there were no differences between STs and GTs in electrically-evoked DA release in slices, or in total ventral striatal DA content. We conclude that greater DAT surface expression may facilitate the attribution of incentive salience to discrete reward cues. Investigating this variability in animal sub-populations may help explain why some people abuse drugs while others do not.

  14. Physical Interactions and Functional Relationships of Neuroligin 2 and Midbrain Serotonin Transporters.

    PubMed

    Ye, Ran; Quinlan, Meagan A; Iwamoto, Hideki; Wu, Hsiao-Huei; Green, Noah H; Jetter, Christopher S; McMahon, Douglas G; Veestra-VanderWeele, Jeremy; Levitt, Pat; Blakely, Randy D

    2015-01-01

    The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] modulates many key brain functions including those subserving sensation, emotion, reward, and cognition. Efficient clearance of 5-HT after release is achieved by the antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). To identify novel SERT regulators, we pursued a proteomic analysis of mouse midbrain SERT complexes, evaluating findings in the context of prior studies that established a SERT-linked transcriptome. Remarkably, both efforts converged on a relationship of SERT with the synaptic adhesion protein neuroligin 2 (NLGN2), a post-synaptic partner for presynaptic neurexins, and a protein well-known to organize inhibitory GABAergic synapses. Western blots of midbrain reciprocal immunoprecipitations confirmed SERT/NLGN2 associations, and also extended to other NLGN2 associated proteins [e.g., α-neurexin (NRXN), gephyrin]. Midbrain SERT/NLGN2 interactions were found to be Ca(2+)-independent, supporting cis vs. trans-synaptic interactions, and were absent in hippocampal preparations, consistent with interactions arising in somatodendritic compartments. Dual color in situ hybridization confirmed co-expression of Tph2 and Nlgn2 mRNA in the dorsal raphe, with immunocytochemical studies confirming SERT:NLGN2 co-localization in raphe cell bodies but not axons. Consistent with correlative mRNA expression studies, loss of NLGN2 expression in Nlgn2 null mice produced significant reductions in midbrain and hippocampal SERT expression and function. Additionally, dorsal raphe 5-HT neurons from Nlgn2 null mice exhibit reduced excitability, a loss of GABAA receptor-mediated IPSCs, and increased 5-HT1A autoreceptor sensitivity. Finally, Nlgn2 null mice display significant changes in behaviors known to be responsive to SERT and/or 5-HT receptor manipulations. We discuss our findings in relation to the possible coordination of intrinsic and extrinsic regulation afforded by somatodendritic SERT:NLGN2 complexes

  15. Membrane Lipid Changes During Formation of a Functional Electron Transport System in Staphylococcus aureus

    PubMed Central

    Frerman, Frank E.; White, David C.

    1967-01-01

    Addition of oxygen to a culture of anaerobically growing Staphylococcus aureus results in the formation of a membrane-bound, functional electron transport system. With the shift to aerobic growth, there is at least a 15-fold increase in cytochrome a and at least a 55-fold increase in cytochrome oxidase o. At the completion of the shift to aerobic growth, the cytochrome levels equal those found in bacteria grown with aeration throughout the entire growth cycle. Cytochromes b1 and o are formed first. Their synthesis slows when cytochrome a becomes detectable. Concentrations of cytochromes b1 and sometimes cytochrome a increase late in the adaptive period. Concomitant with this is a decrease in the oxygen tension at which the rate of oxygen utilization becomes dependent on the oxygen concentration. During the shift to aerobic growth, the protoheme content increases ninefold, and all the protoheme can be accounted for in enzymatically reducible cytochrome b1 and cytochrome oxidase o. Protoheme, but not a functional cytochrome system, is synthesized by anaerobically growing S. aureus. Heme a appears only after a period of aerobic growth. During the shift to aerobic growth, there is a 1.6-fold increase in the vitamin K2 content, with an alteration in the ratios of the 35 and 45 carbon side chain isoprenologues. A twofold increase in phosphatidyl glycerol and a 1.6-fold increase in cardiolipin occur with the shift to aerobic growth. Lysyl-phosphtidyl glycerol remains essentially constant in this period. Concentrations of mono- and diglucosyl diglycerides increase coordinately 1.3-fold during the shift to aerobic growth at a 2.5 to 1 m ratio. PMID:4294593

  16. Individual variation in incentive salience attribution and accumbens dopamine transporter expression and function.

    PubMed

    Singer, Bryan F; Guptaroy, Bipasha; Austin, Curtis J; Wohl, Isabella; Lovic, Vedran; Seiler, Jillian L; Vaughan, Roxanne A; Gnegy, Margaret E; Robinson, Terry E; Aragona, Brandon J

    2016-03-01

    Cues (conditioned stimuli; CSs) associated with rewards can come to motivate behavior, but there is considerable individual variation in their ability to do so. For example, a lever-CS that predicts food reward becomes attractive and wanted, and elicits reward-seeking behavior, to a greater extent in some rats ('sign-trackers'; STs) than others ('goal-trackers'; GTs). Variation in dopamine (DA) neurotransmission in the nucleus accumbens (NAc) core is thought to contribute to such individual variation. Given that the DA transporter (DAT) exerts powerful regulation over DA signaling, we characterized the expression and function of the DAT in the accumbens of STs and GTs. STs showed greater DAT surface expression in ventral striatal synaptosomes than GTs, and ex vivo fast-scan cyclic voltammetry recordings of electrically evoked DA release confirmed enhanced DAT function in STs, as indicated by faster DA uptake, specifically in the NAc core. Consistent with this, systemic amphetamine (AMPH) produced greater inhibition of DA uptake in STs than in GTs. Furthermore, injection of AMPH directly into the NAc core enhanced lever-directed approach in STs, presumably by amplifying the incentive value of the CS, but had no effect on goal-tracking behavior. On the other hand, there were no differences between STs and GTs in electrically-evoked DA release in slices, or in total ventral striatal DA content. We conclude that greater DAT surface expression may facilitate the attribution of incentive salience to discrete reward cues. Investigating this variability in animal sub-populations may help explain why some people abuse drugs while others do not. PMID:26613374

  17. Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

    PubMed Central

    Reverter, Meritxell; Rentero, Carles; de Muga, Sandra Vilà; Alvarez-Guaita, Anna; Mulay, Vishwaroop; Cairns, Rose; Wood, Peta; Monastyrskaya, Katia; Pol, Albert; Tebar, Francesc; Blasi, Joan; Grewal, Thomas; Enrich, Carlos

    2011-01-01

    Cholesterol regulates plasma membrane (PM) association and functioning of syntaxin-4 and soluble N-ethylmaleimide-sensitive fusion protein 23 (SNAP23) in the secretory pathway. However, the molecular mechanism and cellular cholesterol pools that determine the localization and assembly of these target membrane SNAP receptors (t-SNAREs) are largely unknown. We recently demonstrated that high levels of annexin A6 (AnxA6) induce accumulation of cholesterol in late endosomes, thereby reducing cholesterol in the Golgi and PM. This leads to an impaired supply of cholesterol needed for cytosolic phospholipase A2 (cPLA2) to drive Golgi vesiculation and caveolin transport to the cell surface. Using AnxA6-overexpressing cells as a model for cellular cholesterol imbalance, we identify impaired cholesterol egress from late endosomes and diminution of Golgi cholesterol as correlating with the sequestration of SNAP23/syntaxin-4 in Golgi membranes. Pharmacological accumulation of late endosomal cholesterol and cPLA2 inhibition induces a similar phenotype in control cells with low AnxA6 levels. Ectopic expression of Niemann-Pick C1 (NPC1) or exogenous cholesterol restores the location of SNAP23 and syntaxin-4 within the PM. Importantly, AnxA6-mediated mislocalization of these t-SNAREs correlates with reduced secretion of cargo via the SNAP23/syntaxin-4–dependent constitutive exocytic pathway. We thus conclude that inhibition of late endosomal export and Golgi cholesterol depletion modulate t-SNARE localization and functioning along the exocytic pathway. PMID:22039070

  18. HIV protease inhibitors disrupt astrocytic glutamate transporter function and neurobehavioral performance

    PubMed Central

    Vivithanaporn, Pornpun; Asahchop, Eugene L.; Acharjee, Shaona; Baker, Glen B.; Power, Christopher

    2016-01-01

    Objective: The neurotoxic actions of the HIV protease inhibitors, amprenavir (APV) and lopinavir (LPV) were investigated. Design: With combination antiretroviral therapy (cART), HIV-infected persons exhibit neurocognitive impairments, raising the possibility that cART might exert adverse central nervous system (CNS) effects. We examined the effects of LPV and APV using in-vitro and in-vivo assays of CNS function. Methods: Gene expression, cell viability and amino-acid levels were measured in human astrocytes, following exposure to APV or LPV. Neurobehavioral performance, amino-acid levels and neuropathology were examined in HIV-1 Vpr transgenic mice after treatment with APV or LPV. Results: Excitatory amino-acid transporter-2 (EAAT2) expression was reduced in astrocytes treated with LPV or APV, especially LPV (P < 0.05), which was accompanied by reduced intracellular l-glutamate levels in LPV-treated cells (P < 0.05). Treatment of astrocytes with APV or LPV reduced the expression of proliferating cell nuclear antigen (PCNA) and Ki-67 (P < 0.05) although cell survival was unaffected. Exposure of LPV to astrocytes augmented glutamate-evoked transient rises in [Cai] (P < 0.05). Vpr mice treated with LPV showed lower concentrations of l-glutamate, l-aspartate and l-serine in cortex compared with vehicle-treated mice (P < 0.05). Total errors in T-maze assessment were increased in LPV and APV-treated animals (P < 0.05). EAAT2 expression was reduced in the brains of protease inhibitor-treated animals, which was associated with gliosis (P < 0.05). Conclusion: These results indicated that contemporary protease inhibitors disrupt astrocyte functions at therapeutic concentrations with enhanced sensitivity to glutamate, which can lead to neurobehavioral impairments. ART neurotoxicity should be considered in future therapeutic regimens for HIV/AIDS. PMID:26558720

  19. Serotonin transporter genotype modulates functional connectivity between amygdala and PCC/PCu during mood recovery

    PubMed Central

    Fang, Zhuo; Zhu, Senhua; Gillihan, Seth J.; Korczykowski, Marc; Detre, John A.; Rao, Hengyi

    2013-01-01

    The short (S) allele of the serotonin transporter-linked polymorphic region (5-HTTLPR) has been associated with increased susceptibility to depression. Previous neuroimaging studies have consistently showed increased amygdala activity during the presentation of negative stimuli or regulation of negative emotion in the homozygous short allele carriers, suggesting the key role of amygdala response in mediating increased risk for depression. The brain default mode network (DMN) has also been shown to modulate amygdala activity. However, it remains unclear whether 5-HTTLPR genetic variation modulates functional connectivity (FC) between the amygdala and regions of DMN. In this study, we re-analyzed our previous imaging dataset and examined the effects of 5-HTTLPR genetic variation on amygdala connectivity. A total of 15 homozygous short (S/S) and 15 homozygous long individuals (L/L) were scanned in functional magnetic resonance imaging (fMRI) during four blocks: baseline, sad mood, mood recovery, and return to baseline. The S/S and L/L groups showed a similar pattern of FC and no differences were found between the two groups during baseline and sad mood scans. However, during mood recovery, the S/S group showed significantly reduced anti-correlation between amygdala and posterior cingulate cortex/precuneus (PCC/PCu) compared to the L/L group. Moreover, PCC/PCu-amygdala connectivity correlated with amygdala activity in the S/S group but not the L/L group. These results suggest that 5-HTTLPR genetic variation modulates amygdala connectivity which subsequently affects its activity during mood regulation, providing an additional mechanism by which the S allele confers depression risk. PMID:24198772

  20. Effects of cation and anion solvation on ion transport in functionalized perfluoropolyethers electrolytes

    NASA Astrophysics Data System (ADS)

    Timachova, Ksenia; Chintapalli, Mahati; Olsen, Kevin; Desimone, Joseph; Balsara, Nitash

    Advances in polymer electrolytes for use in lithium batteries have been limited by the incorporation of selective lithium binding groups that provide necessary solvation for the lithium but ultimately restrict the mobility of the lithium ions relative to anions. Perfluoropolyether electrolytes (PFPE) are a new class of nonflammable liquid polymer electrolytes that have been functionalized with solvating groups for both lithium ions and fluorinated anions. PFPEs with different endgroups mixed with LiN(SO2CF3)2 salt have shown substantial differences in conductivity and allows us to investigate the effects of varying solvating environments on ion transport. To study the independent motion of cations and anions in these systems, the individual diffusion coefficients of the Li + and (SO2CF3)2 - ions were measured using pulsed-field gradient nuclear magnetic resonance (PFG-NMR). Comparing conductivity calculated using these diffusion coefficients with electrochemical measurements yields an estimation for the number of charge carrier in the system. The amount of salt dissociation, not the mobility of the salt, is the primary driver of differences in electrochemical conductivities between PFPEs with different solvating groups.

  1. Membrane topology and functional importance of the periplasmic region of ABC transporter LolCDE.

    PubMed

    Yasuda, Masaki; Iguchi-Yokoyama, Asako; Matsuyama, Shin-ichi; Tokuda, Hajime; Narita, Shin-ichiro

    2009-10-01

    The LolCDE complex is an ATP-binding cassette transporter that mediates the release of newly synthesized lipoproteins from the cytoplasmic membrane of gram-negative bacteria, which results in the initiation of outer-membrane sorting of lipoproteins through the Lol pathway. LolCDE is composed of one copy each of membrane subunits LolC and LolE, and two copies of nucleotide-binding subunit LolD. In this study, we examined the membrane topology of LolC and LolE by PhoA fusion analysis. Both LolC and LolE were found to have four transmembrane segments with a large periplasmic loop exposed to the periplasm. Despite similarities in sequence and topology, the accessibility of a sulfhydryl reagent to Cys introduced into the periplasmic loop suggested that the structure of the periplasmic region differs between LolC and LolE. Inhibition of the release of lipoproteins by the sulfhydryl reagent supported a previous proposal that LolC and LolE have distinct functions. PMID:19809197

  2. Aly/ REF, a factor for mRNA transport, activates RH gene promoter function.

    PubMed

    Suganuma, Hiroshi; Kumada, Maki; Omi, Toshinori; Gotoh, Takaya; Lkhagvasuren, Munkhtulga; Okuda, Hiroshi; Kamesaki, Toyomi; Kajii, Eiji; Iwamoto, Sadahiko

    2005-06-01

    The rhesus (Rh) blood group antigens are of considerable importance in transfusion medicine as well as in newborn or autoimmune hemolytic diseases due to their high antigenicity. We identified a major DNaseI hypersensitive site at the 5' flanking regions of both RHD and RHCE exon 1. A 34 bp fragment located at -191 to -158 from a translation start position, and containing the TCCCCTCCC sequence, was involved in enhancing promoter activity, which was assessed by luciferase reporter gene assay. A biotin-labelled 34 bp probe isolated an mRNA transporter protein, Aly/REF. The specific binding of Aly/REF to RH promoter in erythroid was confirmed by chromatin immunoprecipitation assay. The silencing of Aly/REF by siRNA reduced not only the RH promoter activity of the reporter gene but also transcription from the native genome. These facts provide second proof of Aly/REF as a transcription coactivator, initially identified as a coactivator for the TCRalpha enhancer function. Aly/REF might be a novel transcription cofactor for erythroid-specific genes.

  3. A quality function deployment method applied to highly reusable space transportation

    SciTech Connect

    Zapata, E.

    1997-01-01

    This paper will describe a Quality Function Deployment (QFD) currently in work the goal of which is to add definition and insight to the development of long term Highly Reusable Space Transportation (HRST). The objective here is twofold. First, to describe the process, the actual QFD experience as applies to the HRST study. Second, to describe the preliminary results of this process, in particular the assessment of possible directions for future pursuit such as promising candidate technologies or approaches that may finally open the space frontier. The iterative and synergistic nature of QFD provides opportunities in the process for the discovery of what is key in so far as it is useful, what is not, and what is merely true. Key observations on the QFD process will be presented. The importance of a customer definition as well as the similarity of the process of developing a technology portfolio to product development will be shown. Also, the relation of identified cost and operating drivers to future space vehicle designs that are robust to an uncertain future will be discussed. The results in particular of this HRST evaluation will be preliminary given the somewhat long term (or perhaps not?) nature of the task being considered. {copyright} {ital 1997 American Institute of Physics.}

  4. Modeling structure-function relationships for diffusive drug transport in inert porous geopolymer matrices.

    PubMed

    Jämstorp, Erik; Strømme, Maria; Frenning, Göran

    2011-10-01

    A unique structure-function relationship investigation of mechanically strong geopolymer drug delivery vehicles for sustained release of potent substances is presented. The effect of in-synthesis water content on geopolymer pore structure and diffusive drug transport is investigated. Scanning electron microscopy, N2 gas adsorption, mercury intrusion porosimetry, compression strength test, drug permeation, and release experiments are performed. Effective diffusion coefficients are measured and compared with corresponding theoretical values as derived from pore size distribution and connectivity via pore-network modeling. By solely varying the in-synthesis water content, mesoporous and mechanically strong geopolymers with porosities of 8%-45% are obtained. Effective diffusion coefficients of the model drugs Saccharin and Zolpidem are observed to span two orders of magnitude (∼1.6-120 × 10(-8) cm(2) /s), comparing very well to theoretical estimations. The ability to predict drug permeation and release from geopolymers, and materials alike, allows future formulations to be tailored on a structural and chemical level for specific applications such as controlled drug delivery of highly potent substances.

  5. Oxygen transport and cardiovascular function at extreme altitude: lessons from Operation Everest II

    NASA Technical Reports Server (NTRS)

    Sutton, J. R.; Reeves, J. T.; Groves, B. M.; Wagner, P. D.; Alexander, J. K.; Hultgren, H. N.; Cymerman, A.; Houston, C. S.

    1992-01-01

    Operation Everest II was designed to examine the physiological responses to gradual decompression simulating an ascent of Mt Everest (8,848 m) to an inspired PO2 of 43 mmHg. The principal studies conducted were cardiovascular, respiratory, muscular-skeletal and metabolic responses to exercise. Eight healthy males aged 21-31 years began the "ascent" and six successfully reached the "summit", where their resting arterial blood gases were PO2 = 30 mmHg and PCO2 = 11 mmHg, pH = 7.56. Their maximal oxygen uptake decreased from 3.98 +/- 0.2 L/min at sea level to 1.17 +/- 0.08 L/min at PIO2 43 mmHg. The principal factors responsible for oxygen transport from the atmosphere to tissues were (1) Alveolar ventilation--a four fold increase. (2) Diffusion from the alveolus to end capillary blood--unchanged. (3) Cardiac function (assessed by hemodynamics, echocardiography and electrocardiography)--normal--although maximum cardiac output and heart rate were reduced. (4) Oxygen extraction--maximal with PvO2 14.8 +/- 1 mmHg. With increasing altitude maximal blood and muscle lactate progressively declined although at any submaximal intensity blood and muscle lactate was higher at higher altitudes.

  6. Open Quantum Transport and Non-Hermitian Real-Time Time-Dependent Density Functional Theory

    NASA Astrophysics Data System (ADS)

    Elenewski, Justin; Zhao, Yanxiang; Chen, Hanning

    Sub-nanometer electronic devices are notoriously difficult to simulate, with the most widely adopted transport schemes predicting currents that diverge from experiment by several orders of magnitude. This deviation arises from numerous factors, including the inability of these methods to accommodate dynamic processes such as charge reorganization. A promising alternative entails the direct propagation of an electronic structure calculation, as exemplified by real-time time-dependent density functional theory (RT-TDDFT). Unfortunately this framework is inherently that of a closed system, and modifications must be made to handle incoming and outgoing particle fluxes. To this end, we establish a formal correspondence between the quantum master equation for an open, many-particle system and its description in terms of RT-TDDFT and non-Hermitian boundary potentials. By dynamically constraining the particle density within the boundary regions corresponding to the device leads, a simulation may be selectively converged to the non-equilibrium steady state associated with a given electrostatic bias. Our numerical tests demonstrate that this algorithm is both highly stable and readily integrated into existing electronic structure frameworks

  7. Functional expression of electron transport chain complexes in mouse rod outer segments.

    PubMed

    Calzia, Daniela; Garbarino, Greta; Caicci, Federico; Manni, Lucia; Candiani, Simona; Ravera, Silvia; Morelli, Alessandro; Traverso, Carlo Enrico; Panfoli, Isabella

    2014-07-01

    Rod photoreceptors efficiently carry out phototransduction cascade, an energetically costly process. Our recent data in bovine rod outer segment (OS) demonstrated that ATP for phototransduction is produced by an extramitochondrial oxidative phosphorylation, thanks to the expression of the Electron Transport Chain (ETC) complexes and of F1Fo ATP synthase in disks. Here we have focused on mouse retinas, reporting the activity of ETC complexes I, II, IV assayed directly on unfixed mouse eye sections, as well as immunogold TEM analysis of fixed mouse eye sections to verify the presence of ND4L subunit of ETC complex I and subunit IV of ETC complex IV in rod OS. Data suggest the presence of functional ETC in mouse rod OS, like their bovine counterpart. The protocol here developed for in situ assay of the ETC complexes activity represents a reliable method for the detection of ETC dysfunction in mice models of retinal pathologies. In fact, the ETC is a major source of reactive oxygen intermediates, and oxidative stress, especially when ectopically expressed in the OS. In turn, oxidative stress contributes to many retinal pathologies, such as diabetic retinopathy, age related macular degeneration, photoreceptor death after retinal detachment and some forms of retinitis pigmentosa.

  8. [Effect of simvastatin on the oxygen transport function and prooxidant - antioxidant balance in blood].

    PubMed

    Glutkina, N V

    2013-01-01

    The effects of simvastatin on the blood oxygen transport function and indices of prooxidant - antioxidant balance at incubation have been studied. Simvastatin at a concentration of 100 ng/ml increases p50 (the blood pO2 corresponding to its 50% oxygen saturation) at real values of pH and pCO2 from 39.53 + 2.41 (p <0.05) to 36.60 (36, 40, 37, 60) (p <0.05) mm Hg. An increase in the drug concentration led to a decrease in the level of this parameter, but in a dose-independent manner. In addition, the level of nitrates/nitrites in the blood plasma was also increased, which was evidence of increasing activity of the L-arginine-NO system. The indices of prooxidant - antioxidant balance exhibited no significant changes. The results demonstrate a new pleiotropic effect of simvastatin, which is realized via a change in the hemoglobin - oxygen affinity through modification of NO production. This effect must be taken into account in the treatment of pathology in the blood circulation.

  9. U and Xe transport in UO2±x: Density functional theory calculations

    NASA Astrophysics Data System (ADS)

    Andersson, D. A.; Uberuaga, B. P.; Nerikar, P. V.; Unal, C.; Stanek, C. R.

    2011-08-01

    The detrimental effects of the fission gas Xe on the performance of oxide nuclear fuels are well known. However, less well known are the mechanisms that govern fission gas evolution. Here, to better understand bulk Xe behavior (diffusion mechanisms) in UO2±x we calculate the relevant activation energies using density functional theory techniques. By analyzing a combination of Xe solution thermodynamics, migration barriers, and the interaction of dissolved Xe atoms with U, we demonstrate that Xe diffusion predominantly occurs via a vacancy-mediated mechanism. Since Xe transport is closely related to the diffusion of U vacancies, we have also studied the activation energy for this process. To best reproduce experimental data for the Xe and U activation energies, it is critical to consider the active charge-compensation mechanism for intrinsic defects in UO2±x. Due to the high thermodynamic cost of reducing U4+ ions, any defect formation occurring at a fixed composition, i.e., no change in UO2±x stoichiometry, always avoids such reactions, which, for example, implies that the ground-state configuration of an O Frenkel pair in UO2 does not involve any explicit local reduction (oxidation) of U ions at the O vacancy (interstitial).

  10. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function

    PubMed Central

    Margolis, Kara Gross; Li, Zhishan; Stevanovic, Korey; Saurman, Virginia; Anderson, George M.; Snyder, Isaac; Blakely, Randy D.; Gershon, Michael D.

    2016-01-01

    Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT4 agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT4–mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation. PMID:27111230

  11. Validation of a multi-layer Green's function code for ion beam transport

    NASA Astrophysics Data System (ADS)

    Walker, Steven; Tweed, John; Tripathi, Ram; Badavi, Francis F.; Miller, Jack; Zeitlin, Cary; Heilbronn, Lawrence

    To meet the challenge of future deep space programs, an accurate and efficient engineering code for analyzing the shielding requirements against high-energy galactic heavy radiations is needed. In consequence, a new version of the HZETRN code capable of simulating high charge and energy (HZE) ions with either laboratory or space boundary conditions is currently under development. The new code, GRNTRN, is based on a Green's function approach to the solution of Boltzmann's transport equation and like its predecessor is deterministic in nature. The computational model consists of the lowest order asymptotic approximation followed by a Neumann series expansion with non-perturbative corrections. The physical description includes energy loss with straggling, nuclear attenuation, nuclear fragmentation with energy dispersion and down shift. Code validation in the laboratory environment is addressed by showing that GRNTRN accurately predicts energy loss spectra as measured by solid-state detectors in ion beam experiments with multi-layer targets. In order to validate the code with space boundary conditions, measured particle fluences are propagated through several thicknesses of shielding using both GRNTRN and the current version of HZETRN. The excellent agreement obtained indicates that GRNTRN accurately models the propagation of HZE ions in the space environment as well as in laboratory settings and also provides verification of the HZETRN propagator.

  12. A quality function deployment method applied to highly reusable space transportation

    NASA Astrophysics Data System (ADS)

    Zapata, Edgar

    1997-01-01

    This paper will describe a Quality Function Deployment (QFD) currently in work the goal of which is to add definition and insight to the development of long term Highly Reusable Space Transportation (HRST). The objective here is twofold. First, to describe the process, the actual QFD experience as applies to the HRST study. Second, to describe the preliminary results of this process, in particular the assessment of possible directions for future pursuit such as promising candidate technologies or approaches that may finally open the space frontier. The iterative and synergistic nature of QFD provides opportunities in the process for the discovery of what is key in so far as it is useful, what is not, and what is merely true. Key observations on the QFD process will be presented. The importance of a customer definition as well as the similarity of the process of developing a technology portfolio to product development will be shown. Also, the relation of identified cost and operating drivers to future space vehicle designs that are robust to an uncertain future will be discussed. The results in particular of this HRST evaluation will be preliminary given the somewhat long term (or perhaps not?) nature of the task being considered.

  13. Serotonin transporter variant drives preventable gastrointestinal abnormalities in development and function.

    PubMed

    Margolis, Kara Gross; Li, Zhishan; Stevanovic, Korey; Saurman, Virginia; Israelyan, Narek; Anderson, George M; Snyder, Isaac; Veenstra-VanderWeele, Jeremy; Blakely, Randy D; Gershon, Michael D

    2016-06-01

    Autism spectrum disorder (ASD) is an increasingly common behavioral condition that frequently presents with gastrointestinal (GI) disturbances. It is not clear, however, how gut dysfunction relates to core ASD features. Multiple, rare hyperfunctional coding variants of the serotonin (5-HT) transporter (SERT, encoded by SLC6A4) have been identified in ASD. Expression of the most common SERT variant (Ala56) in mice increases 5-HT clearance and causes ASD-like behaviors. Here, we demonstrated that Ala56-expressing mice display GI defects that resemble those seen in mice lacking neuronal 5-HT. These defects included enteric nervous system hypoplasia, slow GI transit, diminished peristaltic reflex activity, and proliferation of crypt epithelial cells. An opposite phenotype was seen in SERT-deficient mice and in progeny of WT dams given the SERT antagonist fluoxetine. The reciprocal phenotypes that resulted from increased or decreased SERT activity support the idea that 5-HT signaling regulates enteric neuronal development and can, when disturbed, cause long-lasting abnormalities of GI function. Administration of a 5-HT4 agonist to Ala56 mice during development prevented Ala56-associated GI perturbations, suggesting that excessive SERT activity leads to inadequate 5-HT4-mediated neurogenesis. We propose that deficient 5-HT signaling during development may contribute to GI and behavioral features of ASD. The consequences of therapies targeting SERT during pregnancy warrant further evaluation. PMID:27111230

  14. First-principles transport calculation method based on real-space finite-difference nonequilibrium Green's function scheme

    NASA Astrophysics Data System (ADS)

    Ono, Tomoya; Egami, Yoshiyuki; Hirose, Kikuji

    2012-11-01

    We demonstrate an efficient nonequilibrium Green's function transport calculation procedure based on the real-space finite-difference method. The direct inversion of matrices for obtaining the self-energy terms of electrodes is computationally demanding in the real-space method because the matrix dimension corresponds to the number of grid points in the unit cell of electrodes, which is much larger than that of sites in the tight-binding approach. The procedure using the ratio matrices of the overbridging boundary-matching technique [Y. Fujimoto and K. Hirose, Phys. Rev. BPRBMDO1098-012110.1103/PhysRevB.67.195315 67, 195315 (2003)], which is related to the wave functions of a couple of grid planes in the matching regions, greatly reduces the computational effort to calculate self-energy terms without losing mathematical strictness. In addition, the present procedure saves computational time to obtain the Green's function of the semi-infinite system required in the Landauer-Büttiker formula. Moreover, the compact expression to relate Green's functions and scattering wave functions, which provide a real-space picture of the scattering process, is introduced. An example of the calculated results is given for the transport property of the BN ring connected to (9,0) carbon nanotubes. The wave-function matching at the interface reveals that the rotational symmetry of wave functions with respect to the tube axis plays an important role in electron transport. Since the states coming from and going to electrodes show threefold rotational symmetry, the states in the vicinity of the Fermi level, the wave function of which exhibits fivefold symmetry, do not contribute to the electron transport through the BN ring.

  15. Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells.

    PubMed

    Tega, Yuma; Yuzurihara, Chihiro; Kubo, Yoshiyuki; Akanuma, Shin-ichi; Ehrhardt, Carsten; Hosoya, Ken-ichi

    2016-02-01

    Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 μM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and β2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with β2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation. PMID:26830082

  16. HKT transporters mediate salt stress resistance in plants: from structure and function to the field.

    PubMed

    Hamamoto, Shin; Horie, Tomoaki; Hauser, Felix; Deinlein, Ulrich; Schroeder, Julian I; Uozumi, Nobuyuki

    2015-04-01

    Plant cells are sensitive to salinity stress and do not require sodium as an essential element for their growth and development. Saline soils reduce crop yields and limit available land. Research shows that HKT transporters provide a potent mechanism for mediating salt tolerance in plants. Knowledge of the molecular ion transport and regulation mechanisms and the control of HKT gene expression are crucial for understanding the mechanisms by which HKT transporters enhance crop performance under salinity stress. This review focuses on HKT transporters in monocot plants and in Arabidopsis as a dicot plant, as a guide to efforts toward improving salt tolerance of plants for increasing the production of crops and bioenergy feedstocks.

  17. Functional requirements for the Automated Transportation Management System: TTP number: RL 439002

    SciTech Connect

    Portsmouth, J.H.

    1992-12-31

    This requirements analysis, documents Department of Energy (DOE) transportation management procedures for the purpose of providing a clear and mutual understanding between users and designers of the proposed Automated Transportation Management System (ATMS). It is imperative that one understand precisely how DOE currently performs traffic management tasks; only then can an integrated system be proposed that successfully satisfies the major requirements of transportation managers and other system users. Accordingly, this report describes the current workings of DOE transportation organizations and then proposes a new system which represents a synthesis of procedures (both current and desired) which forms the basis for further systems development activities.

  18. Modulation and Functional Role of the Orientations of the N- and P-Domains of Cu+ -Transporting ATPase along the Ion Transport Cycle.

    PubMed

    Meng, Dan; Bruschweiler-Li, Lei; Zhang, Fengli; Brüschweiler, Rafael

    2015-08-18

    Ion transport of different P-type ATPases is regulated similarly through the interplay of multiple protein domains. In the presence of ATP, binding of a cation to the ion binding site in the transmembrane helices leads to the phosphorylation of the P-domain, allowing ion transfer across the membrane. The details of the mechanism, however, are not clear. Here, we report the modulation of the orientation between the N- and P-domains of Cu(+)-transporting ATPase along the ion transport cycle using high-resolution nuclear magnetic resonance spectroscopy in solution. On the basis of residual dipolar coupling measurements, it is found that the interdomain orientation (relative openness) of the N- and P-domains is distinctly modulated depending on the specific state of the N- and P-domains along the ion translocation cycle. The two domains' relative position in the apo state is semiopen, whereas it becomes closed upon binding of ATP to the N-domain. After phosphorylation of the P-domain and the release of ADP, the opening, however, becomes the widest among all the states. We reason such wide opening resulting from the departure of ADP prepares the N- and P-domains to accommodate the A-domain for interaction and, hence, promote ion transport and allow dephosphorylation of the P-domain. Such wide interdomain opening is abolished when an Asn to Asp mutation is introduced into the conserved DXXK motif located in the hinge region of the N- and P-domains of Cu(+)-ATPase, suggesting the indispensible role of the N- and P-interdomain orientation during ion transportation. Our results shed new light on the structural and mechanistic details of P-type ATPase function at large.

  19. A Novel Member of the Trehalose Transporter Family Functions as an H+-Dependent Trehalose Transporter in the Reabsorption of Trehalose in Malpighian Tubules

    PubMed Central

    Kikuta, Shingo; Hagiwara-Komoda, Yuka; Noda, Hiroaki; Kikawada, Takahiro

    2012-01-01

    In insects, Malpighian tubules are functionally analogous to mammalian kidneys in that they not only are essential to excrete waste molecules into the lumen but also are responsible for the reabsorption of indispensable molecules, such as sugars, from the lumen to the principal cells. Among sugars, the disaccharide trehalose is highly important to insects because it is the main hemolymph sugar to serve as a source of energy and carbon. The trehalose transporter TRET1 participates in the transfer of newly synthesized trehalose from the fat body across the cellular membrane into the hemolymph. Although transport proteins must play a pivotal role in the reabsorption of trehalose in Malpighian tubules, the molecular context underlying this process remains obscure. Previously, we identified a Tret1 homolog (Nlst8) that is expressed principally in the Malpighian tubules of the brown planthopper (BPH). Here, we used the Xenopus oocyte expression system to show that NlST8 exerts trehalose transport activity that is elevated under low pH conditions. These functional assays indicate that Nlst8 encodes a proton-dependent trehalose transporter (H-TRET1). To examine the involvement of Nlst8 in trehalose reabsorption, we analyzed the sugar composition of honeydew by using BPH with RNAi gene silencing. Trehalose was detected in the honeydew as waste excreted from Nlst8-dsRNA-injected BPH under hyperglycemic conditions. However, trehalose was not expelled from GFP-dsRNA-injected BPH even under hyperglycemic conditions. We conclude that NlST8 could participate in trehalose reabsorption driven by a H+ gradient from the lumen to the principal cells of the Malpighian tubules. PMID:22934042

  20. Transport and deposition of functionalized CdTe nanoparticles in saturated porous media

    NASA Astrophysics Data System (ADS)

    Torkzaban, Saeed; Kim, Yongman; Mulvihill, Martin; Wan, Jiamin; Tokunaga, Tetsu K.

    2010-11-01

    Comprehensive understanding of the transport and deposition of engineered nanoparticles (NPs) in subsurface is required to assess their potential negative impact on the environment. We studied the deposition behavior of functionalized quantum dot (QD) NPs (CdTe) in different types of sands (Accusand, ultrapure quartz, and iron-coated sand) at various solution ionic strengths (IS). The observed transport behavior in ultrapure quartz and iron-coated sand was consistent with conventional colloid deposition theories. However, our results from the Accusand column showed that deposition was minimal at the lowest IS (1 mM) and increased significantly as the IS increased. The effluent breakthrough occurred with a delay, followed by a rapid rise to the maximum normalized concentration of unity. Negligible deposition in the column packed with ultrapure quartz sand (100 mM) and Accusand (1 mM) rules out the effect of straining and suggests the importance of surface charge heterogeneity in QD deposition in Accusand at higher IS. Data analyses further show that only a small fraction of sand surface area contributed in QD deposition even at the highest IS (100 mM) tested. The observed delay in breakthrough curves of QDs was attributed to the fast diffusive mass transfer rate of QDs from bulk solution to the sand surface and QD mass transfer on the solid phase. Scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis were used to examine the morphology and elemental composition of sand grains. It was observed that there were regions on the sand covered with layers of clay particles. EDX spectra collected from these regions revealed that Si and Al were the major elements suggesting that the clay particles were kaolinite. Additional batch experiments using gold NPs and SEM analysis were performed and it was observed that the gold NPs were only deposited on clay particles originally on the Accusand surface. After removing the clays from the sand surface, we

  1. Casein kinase 1δ functions at the centrosome and Golgi to promote ciliogenesis

    PubMed Central

    Greer, Yoshimi Endo; Westlake, Christopher J.; Gao, Bo; Bharti, Kapil; Shiba, Yoko; Xavier, Charles P.; Pazour, Gregory J.; Yang, Yingzi; Rubin, Jeffrey S.

    2014-01-01

    Inhibition of casein kinase 1 delta (CK1δ) blocks primary ciliogenesis in human telomerase reverse transcriptase immortalized retinal pigmented epithelial and mouse inner medullary collecting duct cells-3. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1δ)-null mice also exhibit ciliogenesis defects. CK1δ catalytic activity and centrosomal localization signal (CLS) are required to rescue cilia formation in MEFsCsnk1d null. Furthermore, expression of a truncated derivative containing the CLS displaces full-length CK1δ from the centrosome and decreases ciliary length in control MEFs, suggesting that centrosomal CK1δ has a role in ciliogenesis. CK1δ inhibition also alters pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Ras-like in rat brain-11A, Ras-like in rat brain-8A, centrosomal protein of 290 kDa, pericentriolar material protein 1, and polycystin-2, as well as the Golgi distribution of its binding partner, A-kinase anchor protein 450 (AKAP450). As reported for AKAP450, CK1δ was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1δ-binding site inhibits Golgi-derived microtubule nucleation, Golgi distribution of intraflagellar transport protein 20 homologue, and ciliogenesis. Our results suggest that CK1δ mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi, where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport. PMID:24648492

  2. Functional and transcript analysis of a novel metal transporter gene EpNramp from a dark septate endophyte (Exophiala pisciphila).

    PubMed

    Wei, Yun-Fang; Li, Tao; Li, Ling-Fei; Wang, Jun-Ling; Cao, Guan-Hua; Zhao, Zhi-Wei

    2016-02-01

    Various metal transporters mediate sub-cellular sequestration of diverse metal ions, contribute to cellular metal tolerance, and control metal partitioning, particularly under conditions of high rates of metal influx into organisms. In the current study, a ubiquitous and evolutionary conserved metal transporter gene, homology to natural resistance associated macrophage protein (Nramp), was cloned from a metal-tolerant isolate of dark septate endophyte (DSE, Exophiala pisciphila), and its functional and transcript characterization were analyzed. The full-length Nramp gene from E. pisciphila (named EpNramp) was 1716 bp and expected to encode a polypeptide of 571 amino acid residues. EpNramp fused to green fluorescent protein suggested that EpNramp was a plasma membrane metal transporter, which was consistent with the results of bioinformatics analysis with 11 transmembrane domains. Yeast functional complementation revealed that EpNramp could complement the growth defect of Fe-uptake yeast mutant (fet3fet4 double mutant) by mediating the transport of Fe(2+). Expression of EpNramp increased Cd(2+) sensitivity and Cd(2+) accumulation in yeast. In addition, qPCR data revealed that E. pisciphila significantly down-regulated EpNramp expression with elevated Cd(2+) exposure. Altogether, EpNramp is a bivalent cation transporter localized in cell membrane, which is necessary for efficient translocation of both Fe and Cd, and its activities partly attributed to the tolerance of DSE to toxic and excessive Cd(2+) supplements. PMID:26595509

  3. Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals

    PubMed Central

    Furukawa, Junji; Inoue, Katsuhisa; Maeda, Junya; Yasujima, Tomoya; Ohta, Kinya; Kanai, Yoshikatsu; Takada, Tappei; Matsuo, Hirotaka; Yuasa, Hiroaki

    2015-01-01

    The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na+ and H+, but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes. PMID:26455426

  4. Functional analysis of diastrophic dysplasia sulfate transporter. Its involvement in growth regulation of chondrocytes mediated by sulfated proteoglycans.

    PubMed

    Satoh, H; Susaki, M; Shukunami, C; Iyama, K; Negoro, T; Hiraki, Y

    1998-05-15

    Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene constitute a family of recessively inherited osteochondrodysplasias including achondrogenesis type 1B, atelosteogenesis type II, and diastrophic dysplasia. However, the functional properties of the gene product have yet to be elucidated. We cloned rat DTDST cDNA from rat UMR-106 osteoblastic cells. Northern blot analysis suggested that cartilage and intestine were the major expression sites for DTDST mRNA. Analysis of the genomic sequence revealed that the rat DTDST gene was composed of at least five exons. Two distinct transcripts were expressed in chondrocytes due to alternative utilization of the third exon, corresponding to an internal portion of the 5'-untranslated region of the cDNA. Injection of rat and human DTDST cRNA into Xenopus laevis oocytes induced Na+-independent sulfate transport. Transport activity of the expressed DTDST was markedly inhibited by extracellular chloride and bicarbonate. In contrast, canalicular Na+-independent sulfate transporter Sat-1 required the presence of extracellular chloride in the cRNA-injected oocytes. The activity profile of sulfate transport in growth plate chondrocytes was studied in the extracellular presence of various anions and found substantially identical to DTDST expressed in oocytes. Thus, sulfate transport of chondrocytes is dominantly dependent on the DTDST system. Finally, we demonstrate that undersulfation of proteoglycans by the chlorate treatment of chondrocytes significantly impaired growth response of the cells to fibroblast growth factor, suggesting a role for DTDST in endochondral bone formation. PMID:9575183

  5. Functional changes in glutamate transporters and astrocyte biophysical properties in a rodent model of focal cortical dysplasia

    PubMed Central

    Campbell, Susan L.; Hablitz, John J.; Olsen, Michelle L.

    2014-01-01

    Cortical dysplasia is associated with intractable epilepsy and developmental delay in young children. Recent work with the rat freeze-induced focal cortical dysplasia (FCD) model has demonstrated that hyperexcitability in the dysplastic cortex is due in part to higher levels of extracellular glutamate. Astrocyte glutamate transporters play a pivotal role in cortical maintaining extracellular glutamate concentrations. Here we examined the function of astrocytic glutamate transporters in a FCD model in rats. Neocortical freeze lesions were made in postnatal day (PN) 1 rat pups and whole cell electrophysiological recordings and biochemical studies were performed at PN 21–28. Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls. Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate. Reduced astrocytic glutamate transport clearance contributed to increased NMDA receptor-mediated current decay kinetics in lesioned animals. The electrophysiological profile of astrocytes in the lesion group was also markedly changed compared to sham operated animals. Control astrocytes demonstrate large-amplitude linear leak currents in response to voltage-steps whereas astrocytes in lesioned animals demonstrated significantly smaller voltage-activated inward and outward currents. Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals. However, Western blotting, immunohistochemistry and quantitative PCR demonstrated no differences in the expression of the astrocytic glutamate transporter GLT-1 in lesioned animals relative to controls. These data suggest that, in the absence of changes in protein or mRNA expression levels, functional changes in astrocytic glutamate transporters contribute to neuronal hyperexcitability in the FCD model

  6. Tubulin transport by IFT is upregulated during ciliary growth by a cilium-autonomous mechanism

    PubMed Central

    Craft, Julie M.; Harris, J. Aaron; Hyman, Sebastian; Kner, Peter

    2015-01-01

    The assembly of the axoneme, the structural scaffold of cilia and flagella, requires translocation of a vast quantity of tubulin into the growing cilium, but the mechanisms that regulate the targeting, quantity, and timing of tubulin transport are largely unknown. In Chlamydomonas, GFP-tagged α-tubulin enters cilia as an intraflagellar transport (IFT) cargo and by diffusion. IFT-based transport of GFP-tubulin is elevated in growing cilia and IFT trains carry more tubulin. Cells possessing both nongrowing and growing cilia selectively target GFP-tubulin into the latter. The preferential delivery of tubulin boosts the concentration of soluble tubulin in the matrix of growing versus steady-state cilia. Cilia length mutants show abnormal kinetics of tubulin transport. We propose that cells regulate the extent of occupancy of IFT trains by tubulin cargoes. During ciliary growth, IFT concentrates soluble tubulin in cilia and thereby promotes elongation of the axonemal microtubules. PMID:25583998

  7. Functional characterisation of an intron retaining K(+) transporter of barley reveals intron-mediated alternate splicing.

    PubMed

    Shahzad, K; Rauf, M; Ahmed, M; Malik, Z A; Habib, I; Ahmed, Z; Mahmood, K; Ali, R; Masmoudi, K; Lemtiri-Chlieh, F; Gehring, C; Berkowitz, G A; Saeed, N A

    2015-07-01

    Intron retention in transcripts and the presence of 5' and 3' splice sites within these introns mediate alternate splicing, which is widely observed in animals and plants. Here, functional characterisation of the K(+) transporter, HvHKT2;1, with stably retained introns from barley (Hordeum vulgare) in yeast (Saccharomyces cerevisiae), and transcript profiling in yeast and transgenic tobacco (Nicotiana tabacum) is presented. Expression of intron-retaining HvHKT2;1 cDNA (HvHKT2;1-i) in trk1, trk2 yeast strain defective in K(+) uptake restored growth in medium containing hygromycin in the presence of different concentrations of K(+) and mediated hypersensitivity to Na(+) . HvHKT2;1-i produces multiple transcripts via alternate splicing of two regular introns and three exons in different compositions. HKT isoforms with retained introns and exon skipping variants were detected in relative expression analysis of (i) HvHKT2;1-i in barley under native conditions, (ii) in transgenic tobacco plants constitutively expressing HvHKT2;1-i, and (iii) in trk1, trk2 yeast expressing HvHKT2;1-i under control of an inducible promoter. Mixed proportions of three HKT transcripts: HvHKT2;1-e (first exon region), HvHKT2;1-i1 (first intron) and HvHKT2;1-i2 (second intron) were observed. The variation in transcript accumulation in response to changing K(+) and Na(+) concentrations was observed in both heterologous and plant systems. These findings suggest a link between intron-retaining transcripts and different splice variants to ion homeostasis, and their possible role in salt stress.

  8. Identification and functional characterization of three novel alleles for the serotonin transporter-linked polymorphic region.

    PubMed

    Ehli, E A; Hu, Y; Lengyel-Nelson, T; Hudziak, J J; Davies, G E

    2012-02-01

    A promoter polymorphism in the serotonin transporter gene (5-HTTLPR) has been reported to confer relative risk for phenotypes (depression/anxiety) and endophenotypes (amygdala reactivity). In this report, we identify and characterize three rare 5-HTTLPR alleles not previously described in the human literature. The three novel alleles were identified while genotyping 5-HTTLPR in a family-based attention deficit hyperactivity disorder clinical population. Two of the novel alleles are longer than the common 16-repeat long (L) allele (17 and 18 repeats) and the third is significantly smaller than the 14-repeat short (S) allele (11 repeats). The sequence and genetic architecture of each novel allele is described in detail. We report a significant decrease in the expression between the XL₁₇ (17r) allele and the L(A) (16r) allele. The XS₁₁ (11r) allele showed similar expression with the S (14r) allele. A 1.8-fold increase in expression was observed with the L(A)(16r) allele compared with the L(G) (16r) allele, which replicates results from earlier 5-HTTLPR expression experiments. In addition, transcription factor binding site (TFBS) analysis was performed using MatInspector (Genomatix) that showed the presence or absence of different putative TFBSs between the novel alleles and the common L (16r) and S (14r) alleles. The identification of rare variants and elucidation of their functional impact could potentially lead to understanding the contribution that the rare variant may have on the inheritance/susceptibility of multifactorial common diseases.

  9. Identification of functionally distinct Na-HCO3 co-transporters in colon.

    PubMed

    Barmeyer, Christian; Ye, Jeff Huaqing; Soroka, Carol; Geibel, Peter; Hingsammer, Lukas M; Weitgasser, Laurence; Atway, Danny; Geibel, John P; Binder, Henry J; Rajendran, Vazhaikkurichi M

    2013-01-01

    Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5'-diisothiocyanato-2-2'-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H(+)]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in p

  10. Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms.

    PubMed Central

    Elluru, R G; Bloom, G S; Brady, S T

    1995-01-01

    The mechanochemical ATPase kinesin is thought to move membrane-bounded organelles along microtubules in fast axonal transport. However, fast transport includes several classes of organelles moving at rates that differ by an order of magnitude. Further, the fact that cytoplasmic forms of kinesin exist suggests that kinesins might move cytoplasmic structures such as the cytoskeleton. To define cellular roles for kinesin, the axonal transport of kinesin was characterized. Retinal proteins were pulse-labeled, and movement of radiolabeled kinesin through optic nerve and tract into the terminals was monitored by immunoprecipitation. Heavy and light chains of kinesin appeared in nerve and tract at times consistent with fast transport. Little or no kinesin moved with slow axonal transport indicating that effectively all axonal kinesin is associated with membranous organelles. Both kinesin heavy chain molecular weight variants of 130,000 and 124,000 M(r) (KHC-A and KHC-B) moved in fast anterograde transport, but KHC-A moved at 5-6 times the rate of KHC-B. KHC-A cotransported with the synaptic vesicle marker synaptophysin, while a portion of KHC-B cotransported with the mitochondrial marker hexokinase. These results suggest that KHC-A is enriched on small tubulovesicular structures like synaptic vesicles and that at least one form of KHC-B is predominantly on mitochondria. Biochemical specialization may target kinesins to appropriate organelles and facilitate differential regulation of transport. Images PMID:7538359

  11. Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

    PubMed Central

    Covarrubias, Alejandra A.; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I.

    2014-01-01

    Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility. PMID:25402186

  12. Role of membrane-associated thiol groups in the functional regulation of gastric microsomal (H+ + K+)-transporting ATPase system.

    PubMed Central

    Nandi, J; Meng-Ai, Z; Ray, T K

    1983-01-01

    The distribution of free thiol groups associated with the membrane proteins of the purified pig gastric microsomal vesicles was quantified, and the relation of thiol groups to the function of the gastric (H+ + K+)-transporting ATPase system was investigated. Two different thiol-specific agents, carboxypyridine disulphide (CPDS) and N-(1-naphthyl)maleimide (NNM) were used for the study. The structure-function relationship of the membrane thiol groups was studied after modification by the probes under various conditions, relating the inhibition of the (H+ + K+)-transporting ATPase to the ATP-dependent H+ accumulation by the gastric microsomal vesicles. On the basis of the extent of stimulation of the microsomal (H+ + K+)-transporting ATPase in the presence and absence of valinomycin (val) about 85% of the vesicles were found to be intact. CPDS at 1 mM completely inhibits the valinomycin-stimulated ATPase and the associated p-nitrophenyl phosphatase with a concomitant inhibition of vesicular H+ uptake. Both the enzyme and dye-uptake activities were fully protected against CPDS inhibition when the treatment with CPDS was carried out in the presence of ATP. ATP also offered protection (about 65%) against NNM inhibition of the (H+ + K+)-transporting ATPase system and vesicular H+ uptake. Under similar conditions ATP also protected about 10 and 6 nmol of thiol groups/mg of protein respectively from CPDS and NNM reaction. Our data suggest that the thiol groups on the outer surface of the vesicles are primarily involved in gastric (H+ + K+)-transporting ATPase function. Furthermore, at least about 15% of the total microsomal thiol groups appear to be associated with the ATPase system. The data have been discussed in terms of the structure-function relationship of gastric microsomes. PMID:6311168

  13. Functional expression and regulation of drug transporters in monolayer- and sandwich-cultured mouse hepatocytes.

    PubMed

    Noel, Gregory; Le Vee, Marc; Moreau, Amélie; Stieger, Bruno; Parmentier, Yannick; Fardel, Olivier

    2013-04-11

    Primary hepatocyte cultures are now considered as convenient models for in vitro analyzing liver drug transport. However, if primary human and rat hepatocytes have been well-characterized with respect to drug transporter expression and regulation, much less is known for primary mouse hepatocytes. The present study was therefore designed to gain insights about this point. The profile of sinusoidal and canalicular drug transporter mRNA expression in short time (4h)-cultured mouse hepatocytes was found to be highly correlated with that of freshly isolated hepatocytes; by contrast, those of counterparts cultured for a longer time (until 4 days) either in monolayer configurations on plastic or collagen or in sandwich configuration with matrigel were profoundly altered: uptake drug transporters such as Oct1, Oatps and Oat2 were thus down-regulated, whereas most of efflux transporters such as Mdr1a/b, Mrp3, Mrp4 and Bcrp were induced. Moreover, short time-cultured hepatocytes exhibited the highest levels of sinusoidal influx transporter activities. Transporter-mediated drug secretion into canalicular networks was however only observed in sandwich-cultured hepatocytes. Mouse hepatocytes cultured either in monolayer or sandwich configurations were finally shown to exhibit up-regulation of referent transporters in response to exposure to prototypical activators of the drug sensing receptors pregnane X receptor, aryl hydrocarbon receptor or constitutive androstane receptor. Taken together, these data demonstrate the feasibility of using primary mouse hepatocytes for investigating potential interactions of xenobiotics with hepatic transporter activity or regulation, provided that adequate culture conditions are retained. PMID:23396053

  14. Combinatorial theory of the semiclassical evaluation of transport moments II: Algorithmic approach for moment generating functions

    SciTech Connect

    Berkolaiko, G.; Kuipers, J.

    2013-12-15

    Electronic transport through chaotic quantum dots exhibits universal behaviour which can be understood through the semiclassical approximation. Within the approximation, calculation of transport moments reduces to codifying classical correlations between scattering trajectories. These can be represented as ribbon graphs and we develop an algorithmic combinatorial method to generate all such graphs with a given genus. This provides an expansion of the linear transport moments for systems both with and without time reversal symmetry. The computational implementation is then able to progress several orders further than previous semiclassical formulae as well as those derived from an asymptotic expansion of random matrix results. The patterns observed also suggest a general form for the higher orders.

  15. Getting the tail to wag the dog: Incorporating groundwater transport into catchment solute transport models using rank StorAge Selection (rSAS) functions

    NASA Astrophysics Data System (ADS)

    Harman, C. J.

    2015-12-01

    Surface water hydrologic models are increasingly used to analyze the transport of solutes through the landscape, such as nitrate. However, many of these models cannot adequately capture the effect of groundwater flow paths, which can have long travel times and accumulate legacy contaminants, releasing them to streams over decades. If these long lag times are not accounted for, the short-term efficacy of management activities to reduce nitrogen loads may be overestimated. Models that adopt a simple 'well-mixed' assumption, leading to an exponential transit time distribution at steady state, cannot adequately capture the broadly skewed nature of groundwater transit times in typical watersheds. Here I will demonstrate how StorAge Selection functions can be used to capture the long lag times of groundwater in a typical subwatershed-based hydrologic model framework typical of models like SWAT, HSPF, HBV, PRMS and others. These functions can be selected and calibrated to reproduce historical data where available, but can also be fitted to the results of a steady-state groundwater transport model like MODFLOW/MODPATH, allowing those results to directly inform the parameterization of an unsteady surface water model. The long tails of the transit time distribution predicted by the groundwater model can then be completely captured by the surface water model. Examples of this application in the Chesapeake Bay watersheds and elsewhere will be given.

  16. A three dimensional Green's function solution technique for the transport of heavy ions in laboratory and space

    NASA Astrophysics Data System (ADS)

    Gerstner, Candice Rockell

    In the future, astronauts will be sent into space for longer durations of time compared to previous missions. The increased risk of exposure to ionizing radiation, such as Galactic Cosmic Rays and Solar Particle Events, is of great concern. Consequently, steps must be taken to ensure astronaut safety by providing adequate shielding. The shielding and exposure of space travelers is controlled by the transport properties of the radiation through the spacecraft, its onboard systems and the bodies of the individuals themselves. Meeting the challenge of future space programs will therefore require accurate and efficient methods for performing radiation transport calculations to analyze and predict shielding requirements. One such method, which is developed in this dissertation, is based on a three dimensional Green's function solution technique for the transport of heavy ions in both laboratory and space.

  17. Functional Linkage between Genes That Regulate Osmotic Stress Responses and Multidrug Resistance Transporters: Challenges and Opportunities for Antibiotic Discovery

    PubMed Central

    2014-01-01

    All cells need to protect themselves against the osmotic challenges of their environment by maintaining low permeability to ions across their cell membranes. This is a basic principle of cellular function, which is reflected in the interactions among ion transport and drug efflux genes that have arisen during cellular evolution. Thus, upon exposure to pore-forming antibiotics such as amphotericin B (AmB) or daptomycin (Dap), sensitive cells overexpress common resistance genes to protect themselves from added osmotic challenges. These genes share pathway interactions with the various types of multidrug resistance (MDR) transporter genes, which both preserve the native lipid membrane composition and at the same time eliminate disruptive hydrophobic molecules that partition excessively within the lipid bilayer. An increased understanding of the relationships between the genes (and their products) that regulate osmotic stress responses and MDR transporters will help to identify novel strategies and targets to overcome the current stalemate in drug discovery. PMID:24295980

  18. Impact of disruption of secondary binding site S2 on dopamine transporter function.

    PubMed

    Zhen, Juan; Reith, Maarten E A

    2016-09-01

    The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm. PMID

  19. A mitochondrial monocarboxylate transporter in rat liver and heart and its possible function in cell control.

    PubMed Central

    Mowbray, J

    1975-01-01

    Several hydroxy- and keto-substituted monocarboxylates were found to undergo co- as well as counter-exchange across the mitochondrial membrane. The results argue against a simple Donnan system and may be explained by the existence of a transporter for monocarboxylates. In support of this explanation it was apparently possible to 'pump' pyruvate to the sucrose-inaccessible space by using the dicarboxylate transporter. Further, several aromatic and aliphatic analogues of pyruvate, but not of di- or tri-carboxylate transport inhibitors, have been shown to prevent pyruvate-exchange reactions. Palmitoylcarnitine was found to have a much stronger affinity for the carrier than either carnitine or pyruvate and the possible consequences of this for carnitine-palmitoylcarnitine exchange and on the control of the pyruvate dehydrogenase complex are explored. In view of the range of transport inhibitors and substrates it is suggested that the carrier has a fairly broad specificity. 'Inhibitor-stop' kinetic studies show that the speed of translocation of pyruvate at 1 degrees C is of the same order as malate. The possible correlation between the role of a hydroxy-keto acid transporter in substrate exchange and some whole animal experiments is briefly discussed. It is proposed that for reasons of control the cell will require membrane monocarboxylate transporters no less than di- or tri-carboxylate carriers. PMID:1156399

  20. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

  1. The coiled-coil domain containing protein CCDC151 is required for the function of IFT-dependent motile cilia in animals.

    PubMed

    Jerber, Julie; Baas, Dominique; Soulavie, Fabien; Chhin, Brigitte; Cortier, Elisabeth; Vesque, Christine; Thomas, Joëlle; Durand, Bénédicte

    2014-02-01

    Cilia are evolutionarily conserved organelles endowed with essential physiological and developmental functions. In humans, disruption of cilia motility or signaling leads to complex pleiotropic genetic disorders called ciliopathies. Cilia motility requires the assembly of multi-subunit motile components such as dynein arms, but mechanisms underlying their assembly pathway and transport into the axoneme are still largely unknown. We identified a previously uncharacterized coiled-coil domain containing protein CCDC151, which is evolutionarily conserved in motile ciliated species and shares ancient features with the outer dynein arm-docking complex 2 of Chlamydomonas. In Drosophila, we show that CG14127/CCDC151 is associated with motile intraflagellar transport (IFT)-dependent cilia and required for geotaxis behavior of adult flies. In zebrafish, Ccdc151 is expressed in tissues with motile cilia, and morpholino-induced depletion of Ccdc151 leads to left-right asymmetry defects and kidney cysts. We demonstrate that Ccdc151 is required for proper motile function of cilia in the Kupffer's vesicle and in the pronephros by controlling dynein arm assembly, showing that Ccdc151 is a novel player in the control of IFT-dependent dynein arm assembly in animals. However, we observed that CCDC151 is also implicated in other cellular functions in vertebrates. In zebrafish, ccdc151 is involved in proper orientation of cell divisions in the pronephros and genetically interacts with prickle1 in this process. Furthermore, knockdown experiments in mammalian cells demonstrate that CCDC151 is implicated in the regulation of primary cilium length. Hence, CCDC151 is required for motile cilia function in animals but has acquired additional non-motile functions in vertebrates.

  2. Functional Properties of the Arabidopsis Peptide Transporters AtPTR1 and AtPTR5*

    PubMed Central

    Hammes, Ulrich Z.; Meier, Stefan; Dietrich, Daniela; Ward, John M.; Rentsch, Doris

    2010-01-01

    The Arabidopsis di- and tripeptide transporters AtPTR1 and AtPTR5 were expressed in Xenopus laevis oocytes, and their selectivity and kinetic properties were determined by voltage clamping and by radioactive uptake. Dipeptide transport by AtPTR1 and AtPTR5 was found to be electrogenic and dependent on protons but not sodium. In the absence of dipeptides, both transporters showed proton-dependent leak currents that were inhibited by Phe-Ala (AtPTR5) and Phe-Ala, Trp-Ala, and Phe-Phe (AtPTR1). Phe-Ala was shown to reduce leak currents by binding to the substrate-binding site with a high apparent affinity. Inhibition of leak currents was only observed when the aromatic amino acids were present at the N-terminal position. AtPTR1 and AtPTR5 transport activity was voltage-dependent, and currents increased supralinearly with more negative membrane potentials and did not saturate. The voltage dependence of the apparent affinities differed between Ala-Ala, Ala-Lys, and Ala-Asp and was not conserved between the two transporters. The apparent affinity of AtPTR1 for these dipeptides was pH-dependent and decreased with decreasing proton concentration. In contrast to most proton-coupled transporters characterized so far, −Imax increased at high pH, indicating that regulation of the transporter by pH overrides the importance of protons as co-substrate. PMID:20937801

  3. STRUCTURE AND FUNCTION OF SUBSURFACE MICROBIAL COMMUNITIES AFFECTING RADIONUCLIDE TRANSPORT AND BIOIMMOBILIZATION

    SciTech Connect

    Joel E. Kostka; Lee Kerkhof; Kuk-Jeong Chin; Martin Keller; Joseph W. Stucki

    2011-06-15

    The objectives of this project were to: (1) isolate and characterize novel anaerobic prokaryotes from subsurface environments exposed to high levels of mixed contaminants (U(VI), nitrate, sulfate), (2) elucidate the diversity and distribution of metabolically active metal- and nitrate-reducing prokaryotes in subsurface sediments, and (3) determine the biotic and abiotic mechanisms linking electron transport processes (nitrate, Fe(III), and sulfate reduction) to radionuclide reduction and immobilization. Mechanisms of electron transport and U(VI) transformation were examined under near in situ conditions in sediment microcosms and in field investigations at the Oak Ridge Field Research Center (ORFRC), in Oak Ridge, Tennessee, where the subsurface is exposed to mixed contamination predominated by uranium and nitrate. A total of 20 publications (16 published or 'in press' and 4 in review), 10 invited talks, and 43 contributed seminars/ meeting presentations were completed during the past four years of the project. PI Kostka served on one proposal review panel each year for the U.S. DOE Office of Science during the four year project period. The PI leveraged funds from the state of Florida to purchase new instrumentation that aided the project. Support was also leveraged by the PI from the Joint Genome Institute in the form of two successful proposals for genome sequencing. Draft genomes are now available for two novel species isolated during our studies and 5 more genomes are in the pipeline. We effectively addressed each of the three project objectives and research highlights are provided. Task I - Isolation and characterization of novel anaerobes: (1) A wide range of pure cultures of metal-reducing bacteria, sulfate-reducing bacteria, and denitrifying bacteria (32 strains) were isolated from subsurface sediments of the Oak Ridge Field Research Center (ORFRC), where the subsurface is exposed to mixed contamination of uranium and nitrate. These isolates which are new

  4. Transport proteins.

    PubMed

    Thatcher, Jack D

    2013-04-16

    This Teaching Resource provides and describes two animated lessons that illustrate general properties of transport proteins. The lesson called "transport protein classes" depicts major classes and subclasses of transport proteins. The "transporters, mechanism of action" lesson explains how transporters and P class ATPase (adenosine triphosphatase) pumps function. These animations serve as valuable resources for any collegiate-level course that describes these important factors. Courses that might use them include introductory biology, biochemistry, cell biology, physiology, and biophysics.

  5. [Oxygen-transporting function of the blood circulation system in sevoflurane anesthesia during myocardial revascularization under extracorporeal circulation].

    PubMed

    Skopets, A A; Lomivorotov, V V; Karakhalis, N B; Makarov, A A; Duman'ian, E S; Lomivorotova, L V

    2009-01-01

    The purpose of the study was to evaluate the efficiency of oxygen-transporting function of the circulatory system under sevoflurane anesthesia during myocardial revascularization operations under extracorporeal circulation. Twenty-five patients with coronary heart disease were examined. Mean blood pressure, heart rate, cardiac index, total peripheral vascular resistance index, pulmonary pressure, pulmonary wedge pressure, and central venous pressure were measured. Arterial and mixed venous blood oxygen levels, oxygen delivery and consumption index, arteriovenous oxygen difference, and glucose and lactate concentrations were calculated. The study has demonstrated that sevoflurane is an effective and safe anesthetic for myocardial revascularization operations in patients with coronary heart disease. The use of sevoflurane contributes to steady-state oxygen-transporting function of the circulatory system at all surgical stages.

  6. Functional Characterization of Water Transport and Cellular Localization of Three Aquaporin Paralogs in the Salmonid Intestine

    PubMed Central

    Madsen, Steffen S.; Olesen, Jesper H.; Bedal, Konstanze; Engelund, Morten Buch; Velasco-Santamaría, Yohana M.; Tipsmark, Christian K.

    2011-01-01

    Intestinal water absorption is greatly enhanced in salmonids upon acclimation from freshwater (FW) to seawater (SW); however, the molecular mechanism for water transport is unknown. We conducted a pharmacological characterization of water absorption in the rainbow trout intestine along with an investigation of the distribution and cellular localization of three aquaporins (Aqp1aa, -1ab, and -8ab) in pyloric caeca, middle (M), and posterior (P) intestine of the Atlantic salmon. In vitro iso-osmotic water absorption (Jv) was higher in SW than FW-trout and was inhibited by (mmol L−1): 0.1 KCN (41%), 0.1 ouabain (72%), and 0.1 bumetanide (82%) suggesting that active transport, Na+, K+-ATPase and Na+, K+, 2Cl−-co-transport are involved in establishing the driving gradient for water transport. Jv was also inhibited by 1 mmol L−1 HgCl2, serosally (23% in M and 44% in P), mucosally (27% in M), or both (61% in M and 58% in P), suggesting involvement of both apical and basolateral aquaporins in water transport. The inhibition was antagonized by 5 mmol L−1 mercaptoethanol. By comparison, 10 mmol L−1 mucosal tetraethylammonium, an inhibitor of certain aquaporins, inhibited Jv by 20%. In the presence of glucose, mucosal addition of phloridzin inhibited water transport by 20%, suggesting that water transport is partially linked to the Na+-glucose co-transporter. Using polyclonal antibodies against salmon Aqp1aa, -1ab, and -8ab, we detected Aqp1aa, and -1ab immunoreactivity in the brush border and sub-apical region of enterocytes in all intestinal segments. The Aqp8ab antibody showed a particularly strong immunoreaction in the brush border and sub-apical region of enterocytes throughout the intestine and also stained lateral membranes and peri-nuclear regions though at lower intensity. The present localization of three aquaporins in both apical and lateral membranes of salmonid enterocytes facilitates a model for transcellular water transport in the

  7. Functional characterization of a Na(+)-coupled dicarboxylate transporter from Bacillus licheniformis.

    PubMed

    Strickler, Melodie A; Hall, Jason A; Gaiko, Olga; Pajor, Ana M

    2009-12-01

    The Na(+)-coupled dicarboxylate transporter, SdcL, from Bacillus licheniformis is a member of the divalent anion/Na(+) symporter (DASS) family that includes the bacterial Na(+)/dicarboxylate cotransporter SdcS (from Staphyloccocus aureus) and the mammalian Na(+)/dicarboxylate cotransporters, NaDC1 and NaDC3. The transport properties of SdcL produced in Escherichia coli are similar to those of its prokaryotic and eukaryotic counterparts, involving the Na(+)-dependent transport of dicarboxylates such as succinate or malate across the cytoplasmic membrane with a K(m) of approximately 6 microM. SdcL may also transport aspartate, alpha-ketoglutarate and oxaloacetate with low affinity. The cotransport of Na(+) and dicarboxylate by SdcL has an apparent stoichiometry of 2:1, and a K(0.5) for Na(+) of 0.9 mM. Our findings represent the characterization of another prokaryotic protein of the DASS family with transport properties similar to its eukaryotic counterparts, but with a broader substrate specificity than other prokaryotic DASS family members. The broader range of substrates carried by SdcL may provide insight into domains of the protein that allow a more flexible or larger substrate binding pocket.

  8. Cyclosporin A reduces canalicular membrane fluidity and regulates transporter function in rats.

    PubMed

    Yasumiba, S; Tazuma, S; Ochi, H; Chayama, K; Kajiyama, G

    2001-03-15

    Changes of the biliary canalicular membrane lipid content can affect membrane fluidity and biliary lipid secretion in rats. The immunosuppressant cyclosporin A is known to cause intrahepatic cholestasis. This study investigated whether cyclosporin A influenced canalicular membrane fluidity by altering membrane phospholipids or transporter expression. In male Sprague-Dawley rats, a bile-duct cannula was inserted to collect bile, and sodium taurocholate was infused (100 nmol/min per 100 g) for 60 min. During steady-state taurocholate infusion, cyclosporin A (20 mg/kg) or vehicle was injected intravenously and then bile was collected for 80 min. After killing the rats, canalicular membrane vesicles were prepared. Expression of canalicular membrane transporters was assessed by Western blotting and canalicular membrane vesicle fluidity was estimated by fluorescence polarization. Cyclosporin A reduced biliary lipid secretion along with a disproportionate reduction of lipids relative to bile acids. Cyclosporin A significantly decreased canalicular membrane fluidity along with an increase of the cholesterol/phospholipid molar ratio. Only expression of the transporter P-glycoprotein was increased by cyclosporin A. Because canalicular membrane transporter expression was largely unchanged by cyclosporin A despite a marked decrease of biliary lipid secretion, transporter activity may partly depend upon canalicular membrane fluidity.

  9. A small synthetic molecule functions as a chloride-bicarbonate dual-transporter and induces chloride secretion in cells.

    PubMed

    Liu, Peng-Yun; Li, Shing-To; Shen, Fang-Fang; Ko, Wing-Hung; Yao, Xiao-Qiang; Yang, Dan

    2016-05-31

    A C2 symmetric small molecule composed of l-phenylalanine and isophthalamide was found to function as a Cl(-)/HCO3(-) dual transporter and self-assemble into chloride channels. In Ussing-chamber based short-circuit current measurements, this molecule elicited chloride-dependent short-circuit current (Isc) increase in both Calu-3 cell and CFBE41o-cell (with F508del mutant CFTR) monolayers. PMID:27188496

  10. Modifying Surface Energy of Graphene via Plasma-Based Chemical Functionalization to Tune Thermal and Electrical Transport at Metal Interfaces.

    PubMed

    Foley, Brian M; Hernández, Sandra C; Duda, John C; Robinson, Jeremy T; Walton, Scott G; Hopkins, Patrick E

    2015-08-12

    The high mobility exhibited by both supported and suspended graphene, as well as its large in-plane thermal conductivity, has generated much excitement across a variety of applications. As exciting as these properties are, one of the principal issues inhibiting the development of graphene technologies pertains to difficulties in engineering high-quality metal contacts on graphene. As device dimensions decrease, the thermal and electrical resistance at the metal/graphene interface plays a dominant role in degrading overall performance. Here we demonstrate the use of a low energy, electron-beam plasma to functionalize graphene with oxygen, fluorine, and nitrogen groups, as a method to tune the thermal and electrical transport properties across gold-single layer graphene (Au/SLG) interfaces. We find that while oxygen and nitrogen groups improve the thermal boundary conductance (hK) at the interface, their presence impairs electrical transport leading to increased contact resistance (ρC). Conversely, functionalization with fluorine has no impact on hK, yet ρC decreases with increasing coverage densities. These findings indicate exciting possibilities using plasma-based chemical functionalization to tailor the thermal and electrical transport properties of metal/2D material contacts. PMID:26125524

  11. Modifying Surface Energy of Graphene via Plasma-Based Chemical Functionalization to Tune Thermal and Electrical Transport at Metal Interfaces.

    PubMed

    Foley, Brian M; Hernández, Sandra C; Duda, John C; Robinson, Jeremy T; Walton, Scott G; Hopkins, Patrick E

    2015-08-12

    The high mobility exhibited by both supported and suspended graphene, as well as its large in-plane thermal conductivity, has generated much excitement across a variety of applications. As exciting as these properties are, one of the principal issues inhibiting the development of graphene technologies pertains to difficulties in engineering high-quality metal contacts on graphene. As device dimensions decrease, the thermal and electrical resistance at the metal/graphene interface plays a dominant role in degrading overall performance. Here we demonstrate the use of a low energy, electron-beam plasma to functionalize graphene with oxygen, fluorine, and nitrogen groups, as a method to tune the thermal and electrical transport properties across gold-single layer graphene (Au/SLG) interfaces. We find that while oxygen and nitrogen groups improve the thermal boundary conductance (hK) at the interface, their presence impairs electrical transport leading to increased contact resistance (ρC). Conversely, functionalization with fluorine has no impact on hK, yet ρC decreases with increasing coverage densities. These findings indicate exciting possibilities using plasma-based chemical functionalization to tailor the thermal and electrical transport properties of metal/2D material contacts.

  12. Nanoparticle-assisted optical tethering of endosomes reveals the cooperative function of dyneins in retrograde axonal transport.

    PubMed

    Chowdary, Praveen D; Che, Daphne L; Kaplan, Luke; Chen, Ou; Pu, Kanyi; Bawendi, Moungi; Cui, Bianxiao

    2015-12-10

    Dynein-dependent transport of organelles from the axon terminals to the cell bodies is essential to the survival and function of neurons. However, quantitative knowledge of dyneins on axonal organelles and their collective function during this long-distance transport is lacking because current technologies to do such measurements are not applicable to neurons. Here, we report a new method termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the cooperative mechanics of dyneins on retrograde axonal endosomes in live neurons. In this method, the opposing force from an elastic tether causes the endosomes to gradually stall under load and detach with a recoil velocity proportional to the dynein forces. These recoil velocities reveal that the axonal endosomes, despite their small size, can recruit up to 7 dyneins that function as independent mechanical units stochastically sharing load, which is vital for robust retrograde axonal transport. This study shows that NOTE, which relies on controlled generation of reactive oxygen species, is a viable method to manipulate small cellular cargos that are beyond the reach of current technology.

  13. Use of fish functional traits to associate in-stream suspended sediment transport metrics with biological impairment.

    PubMed

    Schwartz, John S; Simon, Andrew; Klimetz, Lauren

    2011-08-01

    Loss of ecological integrity due to excessive suspended sediment in rivers and streams is a major cause of water quality impairment in the USA. Current assessment protocols for development of sediment total maximum daily loads (TMDLs) lack a means to link temporally variable sediment transport rates with specific losses of ecological functions as loads increase. In order to accomplish this linkage assessment, a functional traits-based approach was used to correlate site occurrences of 17 fish species traits in three main groups (preferred rearing habitat, trophic feeding guild, and spawning behavior) with suspended sediment transport metrics. The sediment transport metrics included concentrations, durations, and dosages for a range of exceedance frequencies; and mean annual suspended sediment yields (SSY). In addition, this study in the Northwestern Great Plains Ecoregion examined trait relationships with three environmental gradients: channel stability, drainage area, and elevation. Potential stressor responses due to elevated suspended sediment concentration (SSC) levels were correlated with occurrences of five traits: preferred pool habitat; feeding generalists, omnivores, piscivores, and nest-building spawners; and development of ecologically based TMDL targets were demonstrated for specific SSC exceedance frequencies. In addition, reduced site occurrences for preferred pool habitat and nest-building spawners traits were associated with unstable channels and higher SSY. At an ecoregion scale, a functional traits assessment approach provided a means to quantify relations between biological impairment and episodically elevated levels of suspended sediment, supporting efforts to develop ecologically based sediment TMDLs.

  14. Interacting fermions in one-dimensional disordered lattices: Exploring localization and transport properties with lattice density-functional theories

    NASA Astrophysics Data System (ADS)

    Vettchinkina, V.; Kartsev, A.; Karlsson, D.; Verdozzi, C.

    2013-03-01

    We investigate the static and dynamical behavior of one-dimensional interacting fermions in disordered Hubbard chains contacted to semi-infinite leads. The chains are described via the repulsive Anderson-Hubbard Hamiltonian, using static and time-dependent lattice density-functional theory. The dynamical behavior of our quantum transport system is studied using an integration scheme available in the literature, which we modify via the recursive Lanczos method to increase its efficiency. To quantify the degree of localization due to disorder and interactions, we adapt the definition of the inverse participation ratio to obtain an indicator which is suitable for quantum transport geometries and can be obtained within density-functional theory. Lattice density-functional theories are reviewed and, for contacted chains, we analyze the merits and limits of the coherent-potential approximation in describing the spectral properties, with interactions included via lattice density-functional theory. Our approach appears to be able to capture complex features due to the competition between disorder and interactions. Specifically, we find a dynamical enhancement of delocalization in the presence of a finite bias and an increase of the steady-state current induced by interparticle interactions. This behavior is corroborated by results for the time-dependent densities and for the inverse participation ratio. Using short isolated chains with interaction and disorder, a brief comparative analysis between time-dependent density-functional theory and exact results is then given, followed by general concluding remarks.

  15. Nonequilibrium fluctuation-dissipation relations for one- and two-particle correlation functions in steady-state quantum transport

    SciTech Connect

    Ness, H.; Dash, L. K.

    2014-04-14

    We study the non-equilibrium (NE) fluctuation-dissipation (FD) relations in the context of quantum thermoelectric transport through a two-terminal nanodevice in the steady-state. The FD relations for the one- and two-particle correlation functions are derived for a model of the central region consisting of a single electron level. Explicit expressions for the FD relations of the Green's functions (one-particle correlations) are provided. The FD relations for the current-current and charge-charge (two-particle) correlations are calculated numerically. We use self-consistent NE Green's functions calculations to treat the system in the absence and in the presence of interaction (electron-phonon) in the central region. We show that, for this model, there is no single universal FD theorem for the NE steady state. There are different FD relations for each different class of problems. We find that the FD relations for the one-particle correlation function are strongly dependent on both the NE conditions and the interactions, while the FD relations of the current-current correlation function are much less dependent on the interaction. The latter property suggests interesting applications for single-molecule and other nanoscale transport experiments.

  16. Functional characterization of common protein variants in the efflux transporter ABCC11 and identification of T546M as functionally damaging variant.

    PubMed

    Arlanov, R; Lang, T; Jedlitschky, G; Schaeffeler, E; Ishikawa, T; Schwab, M; Nies, A T

    2016-04-01

    Multidrug resistance protein 8 (ABCC11) is an efflux transporter for anionic lipophilic compounds, conferring resistance to antiviral and anticancer agents like 5-fluorouracil (5-FU). ABCC11 missense variants may contribute to variability in drug response but functional consequences, except for the 'earwax variant' c.538G>A, are unknown. Using the 'Screen and Insert' technology, we generated human embryonic kidney 293 cells stably expressing ABCC11 missense variants frequently occurring in different ethnic populations: c.57G>A, c.538G>A, c.950C>A, c.1637C>T, c.1942G>A, c.4032A>G. A series of in silico prediction analyses and in vitro plasma membrane vesicle uptake, immunoblotting and immunolocalization experiments were undertaken to investigate functional consequences. We identified c.1637C>T (T546M), previously associated with 5-FU-related toxicity, as a novel functionally damaging ABCC11 variant exhibiting markedly reduced transport function of 5-FdUMP, the active cytotoxic metabolite of 5-FU. Detailed analysis of 14 subpopulations revealed highest allele frequencies of c.1637C>T in Europeans and Americans (up to 11%) compared with Africans and Asians (up to 3%).

  17. Structure-function analysis of HKE4, a member of the new LIV-1 subfamily of zinc transporters.

    PubMed Central

    Taylor, Kathryn M; Morgan, Helen E; Johnson, Andrea; Nicholson, Robert I

    2004-01-01

    The KE4 proteins are an emerging group of proteins with little known functional data. In the present study, we report the first characterization of the recombinant human KE4 protein in mammalian cells. The KE4 sequences are included in the subfamily of ZIP (Zrt-, Irt-like Proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc Transporters). All these LZT sequences contain similarities to ZIP transporters, including the consensus sequence in transmembrane domain IV, which is essential for zinc transport. However, the new LZT subfamily can be separated from other ZIP transporters by the presence of a highly conserved potential metalloprotease motif (HEXPHEXGD) in transmembrane domain V. Here we report the location of HKE4 on intracellular membranes, including the endoplasmic reticulum, and its ability to increase the intracellular free zinc as measured with the zinc-specific fluorescent dye, Newport Green, in a time-, temperature- and concentration-dependent manner. This is in contrast with the zinc influx ability of another LZT protein, LIV-1, which was due to its plasma membrane location. Therefore we have added to the functionality of LZT proteins by reporting their ability to increase intracellular-free zinc, whether they are located on the plasma membrane or on intracellular membranes. This result, in combination with the crucial role that zinc plays in cell growth, emphasizes the importance of this new LZT subfamily, including the KE4 sequences, in the control of intracellular zinc homoeostasis, aberrations of which can lead to diseases such as cancer, immunological disorders and neurological dysfunction. PMID:14525538

  18. Immuno-detection of OCTN1 (SLC22A4) in HeLa cells and characterization of transport function.

    PubMed

    Pochini, Lorena; Scalise, Mariafrancesca; Indiveri, Cesare

    2015-11-01

    OCTN1 was immuno-detected in the cervical cancer cell HeLa, in which the complete pattern of acetylcholine metabolizing enzymes is expressed. Comparison of immuno-staining intensity of HeLa OCTN1 with the purified recombinant human OCTN1 allowed measuring the specific OCTN1 concentration in the HeLa cell extract and, hence calculating the HeLa OCTN1 specific transport activity that was about 10 nmol×min(-1)×mg protein(-1), measured as uptake of [(3)H]acetylcholine in proteoliposomes reconstituted with HeLa extract. This value was very similar to the specific activity of the recombinant protein. Acetylcholine transport was suppressed by incubation of the protein or proteoliposomes with the anti-OCTN1 antibody and was strongly inhibited by PLP and MTSEA, known inhibitors of OCTN1. The absence of ATP in the internal side of proteoliposomes strongly impaired transport function of both the HeLa and, as expected, the recombinant OCTN1. HeLa OCTN1 was inhibited by spermine, NaCl (Na(+)), TEA, γ-butyrobetaine, choline, acetylcarnitine and ipratropium but not by neostigmine. Besides acetylcholine, choline was taken up by HeLa OCTN1 proteoliposomes. The transporter catalyzed also acetylcholine and choline efflux which, differently from uptake, was not inhibited by MTSEA. Time course of [(3)H]acetylcholine uptake in intact HeLa cells was measured. As in proteoliposomes, acetylcholine transport in intact cells was inhibited by TEA and NaCl. Efflux of [(3)H]acetylcholine occurred in intact cells, as well. The experimental data concur in demonstrating a role of OCTN1 in transporting acetylcholine and choline in HeLa cells.

  19. Taurine transporter in fetal T lymphocytes and platelets: differential expression and functional activity.

    PubMed

    Iruloh, C G; D'Souza, S W; Speake, P F; Crocker, I; Fergusson, W; Baker, P N; Sibley, C P; Glazier, J D

    2007-01-01

    Transplacental transfer of taurine, a beta-amino acid essential for fetal and neonatal development, constitutes the primary source of taurine for the fetus. Placental transport of taurine is compromised in pregnancies complicated by intrauterine growth restriction, resulting in a reduced concentration of taurine in cord plasma. This could impact on fetal cellular metabolism as taurine represents the most abundant intracellular amino acid in many fetal cell types. In the present study, we have used pure isolates of fetal platelets and T lymphocytes from cord blood of placentas, from normal, term pregnancies, as fetal cell types to examine the cellular uptake mechanisms for taurine by the system beta transporter and have compared gene and protein expression for the taurine transporter protein (TAUT) in these two cell types. System beta activity in fetal platelets was 15-fold higher compared with fetal T lymphocytes (P < 0.005), mirroring greater TAUT mRNA expression in platelets than T lymphocytes (P < 0.005). Cell-specific differences in TAUT protein moieties were detected with a doublet of 75 and 80 kDa in fetal platelets compared with 114 and 120 kDa in fetal T lymphocytes, with relatively higher expression in platelets. We conclude that greater system beta activity in fetal platelets compared with T lymphocytes is the result of relatively greater TAUT mRNA and protein expression. This study represents the first characterization of amino acid transporters in fetal T lymphocytes.

  20. Clinical Efficacy of Fluvoxamine and Functional Polymorphism in a Serotonin Transporter Gene on Childhood Autism

    ERIC Educational Resources Information Center

    Sugie, Yoko; Sugie, Hideo; Fukuda,Tokiko; Ito, Masataka; Sasada, Yumiko; Nakabayashi, Mutsumi; Fukashiro, Kazunobu; Ohzeki, Takehiko

    2005-01-01

    We studied the correlation between response to fluvoxamine and serotonin transporter gene promoter region polymorphism (5-HTTLPR). Eighteen children with autistic disorder completed a 12-week double-blind, placebo-controlled, randomized crossover study of fluvoxamine. Behavioral assessments were obtained before and at 12 weeks of treatment.…

  1. Characterization of the osmoprotectant transporter OpuC from Pseudomonas syringae and demonstration that cystathionine-beta-synthase domains are required for its osmoregulatory function.

    PubMed

    Chen, Chiliang; Beattie, Gwyn A

    2007-10-01

    The plant pathogen Pseudomonas syringae may cope with osmotic stress on plants, in part, by importing osmoprotective compounds. In this study, we found that P. syringae pv. tomato strain DC3000 was distinct from most bacterial species in deriving greater osmoprotection from exogenous choline than from glycine betaine. This superior osmoprotection was correlated with a higher capacity for uptake of choline than for uptake of glycine betaine. Of four putative osmoregulatory ABC transporters in DC3000, one, designated OpuC, functioned as the primary or sole transporter for glycine betaine and as one of multiple transporters for choline under high osmolarity. Surprisingly, the homolog of the well-characterized ProU transporter from Escherichia coli and Salmonella enterica serovar Typhimurium did not function in osmoprotection. The P. syringae pv. tomato OpuC transporter was more closely related to the Bacillus subtilis and Listeria monocytogenes OpuC transporters than to known osmoprotectant transporters in gram-negative bacteria based on sequence similarity and genetic arrangement. The P. syringae pv. tomato OpuC transporter had a high affinity for glycine betaine, a low affinity for choline, and a broad substrate specificity that included acetylcholine, carnitine, and proline betaine. Tandem cystathionine-beta-synthase (CBS) domains in the ATP-binding component of OpuC were required for transporter function. The presence of these CBS domains was correlated with osmoregulatory function among the putative transporters examined in DC3000 and was found to be predictive of functional osmoregulatory transporters in other pseudomonads. These results provide the first functional evaluation of an osmoprotectant transporter in a Pseudomonas species and demonstrate the usefulness of the CBS domains as predictors of osmoregulatory activity. PMID:17660277

  2. Transmembrane domain V plays a stabilizing role in the function of human bile acid transporter SLC10A2.

    PubMed

    Moore, Robyn H; Chothe, Paresh; Swaan, Peter W

    2013-07-30

    The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2), primarily expressed in the ileum, is involved in both the recycling of bile acids and cholesterol homeostasis. In this study, the structure-function relationship of transmembrane domain 5 (TM5) residues involved in transport is elucidated. Cysteine scanning mutagenesis of each consecutive residue on TM5 resulted in 96% of mutants having a significantly decreased transport activity, although each was expressed at the cell surface. Specifically, G197 and I208 were no longer functional, and G201 and G212 functioned at a level of <10% upon cysteine mutation. Interestingly, each of these exists along one face of the helix. Studies suggest that neither G201 nor G212 is on the substrate pathway. Conservative alanine mutations of the four residues displayed a higher activity in all but G197A, indicating its functional importance. G197 and G201 form a GxxxG motif, which has been found to be important in helix-helix interactions. According to our model, G197 and G201 face transmembrane domain 4 (TM4) residues G179 and P175, respectively. Similarly, G212 faces G237, which forms part of a GxxxG domain in transmembrane domain 6 (TM6). It is possible that these GxxxG domains and their interacting partners are responsible for maintaining the structure of the helices and their interactions with one another. I205 and I208 are both in positions to anchor the GxxxG domains and direct the change in interaction of TM5 from TM4 to TM6. Combined, the results suggest that residues along TM5 are critical for ASBT function but are not directly involved in substrate translocation.

  3. Functional reconstitution of the. gamma. -aminobutyric acid transporter from synaptic vesicles using artificial ion gradients

    SciTech Connect

    Hell, J.W.; Edelmann, L.; Hartinger, J.; Jahn, R. )

    1991-12-24

    The {gamma}-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3{prime}-diisopropylthiodicarbocyanine iodide, and changes of the H{sup +} gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K{sup +} gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of ({sup 3}H)GABA which was saturable. Similarly, ({sup 3}H)glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K{sup +}-loaded proteoliposomes in a buffer free of K{sup +} or Na{sup +} ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H{sup +} ATPase by incubation of K{sup +}-loaded proteoliposomes in equimolar K{sup +} buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and {beta}-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, these data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

  4. Function of the Caenorhabditis elegans ABC Transporter PGP-2 in the Biogenesis of a Lysosome-related Fat Storage Organelle

    PubMed Central

    Schroeder, Lena K.; Kremer, Susan; Kramer, Maxwell J.; Currie, Erin; Kwan, Elizabeth; Watts, Jennifer L.; Lawrenson, Andrea L.

    2007-01-01

    Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(−) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase–containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(−) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red–stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red–stained fat in pgp-2(−) animals as well as insight into the specialized function of this lysosome-related organelle. PMID:17202409

  5. Quantitative Targeted Absolute Proteomics of Transporters and Pharmacoproteomics-Based Reconstruction of P-Glycoprotein Function in Mouse Small Intestine.

    PubMed

    Akazawa, Takanori; Uchida, Yasuo; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2016-07-01

    The purpose of this study was to investigate whether a pharmacokinetic model integrating in vitro mdr1a efflux activity (which we previously reported) with in vitro/in vivo differences in protein expression level can reconstruct intestinal mdr1a function. In situ intestinal permeability-surface area product ratio between wild-type and mdr1a/1b (-/-) mice is one of the parameters used to describe intestinal mdr1a function. The reconstructed ratios of six mdr1a substrates (dexamethasone, digoxin, loperamide, quinidine, verapamil, vinblastine) and one nonsubstrate (diazepam) were consistent with the observed values reported by Adachi et al. within 2.1-fold difference. Thus, intestinal mdr1a function can be reconstructed by our pharmacoproteomic modeling approach. Furthermore, we evaluated regional differences in protein expression levels of mouse intestinal transporters. Sixteen (mdr1a, mrp4, bcrp, abcg5, abcg8, glut1, 4f2hc, sglt1, lat2, pept1, mct1, slc22a18, ostβ, villin1, Na(+)/K(+)-ATPase, γ-gtp) out of 46 target molecules were detected by employing our established quantitative targeted absolute proteomics technique. The protein expression amounts of mdr1a and bcrp increased progressively from duodenum to ileum. Sglt1, lat2, and 4f2hc were highly expressed in jejunum and ileum. Mct1 and ostβ were highly expressed in ileum. The quantitative expression profiles established here should be helpful to understand and predict intestinal transporter functions. PMID:27276518

  6. Conserved Functions of the MATE Transporter BIG EMBRYO1 in Regulation of Lateral Organ Size and Initiation Rate.

    PubMed

    Suzuki, Masaharu; Sato, Yutaka; Wu, Shan; Kang, Byung-Ho; McCarty, Donald R

    2015-08-01

    Genetic networks that determine rates of organ initiation and organ size are key regulators of plant architecture. Whereas several genes that influence the timing of lateral organ initiation have been identified, the regulatory pathways in which these genes operate are poorly understood. Here, we identify a class of genes implicated in regulation of the lateral organ initiation rate. Loss-of-function mutations in the MATE transporter encoded by maize (Zea mays) Big embryo 1 (Bige1) cause accelerated leaf and root initiation as well as enlargement of the embryo scutellum. BIGE1 is localized to trans-Golgi, indicating a possible role in secretion of a signaling molecule. Interestingly, phenotypes of bige1 bear striking similarity to cyp78a mutants identified in diverse plant species. We show that a CYP78A gene is upregulated in bige1 mutant embryos, suggesting a role for BIGE1 in feedback regulation of a CYP78A pathway. We demonstrate that accelerated leaf formation and early flowering phenotypes conditioned by mutants of Arabidopsis thaliana BIGE1 orthologs are complemented by maize Bige1, showing that the BIGE1 transporter has a conserved function in regulation of lateral organ initiation in plants. We propose that BIGE1 is required for transport of an intermediate or product associated with the CYP78A pathway. PMID:26276834

  7. Conserved Functions of the MATE Transporter BIG EMBRYO1 in Regulation of Lateral Organ Size and Initiation Rate.

    PubMed

    Suzuki, Masaharu; Sato, Yutaka; Wu, Shan; Kang, Byung-Ho; McCarty, Donald R

    2015-08-01

    Genetic networks that determine rates of organ initiation and organ size are key regulators of plant architecture. Whereas several genes that influence the timing of lateral organ initiation have been identified, the regulatory pathways in which these genes operate are poorly understood. Here, we identify a class of genes implicated in regulation of the lateral organ initiation rate. Loss-of-function mutations in the MATE transporter encoded by maize (Zea mays) Big embryo 1 (Bige1) cause accelerated leaf and root initiation as well as enlargement of the embryo scutellum. BIGE1 is localized to trans-Golgi, indicating a possible role in secretion of a signaling molecule. Interestingly, phenotypes of bige1 bear striking similarity to cyp78a mutants identified in diverse plant species. We show that a CYP78A gene is upregulated in bige1 mutant embryos, suggesting a role for BIGE1 in feedback regulation of a CYP78A pathway. We demonstrate that accelerated leaf formation and early flowering phenotypes conditioned by mutants of Arabidopsis thaliana BIGE1 orthologs are complemented by maize Bige1, showing that the BIGE1 transporter has a conserved function in regulation of lateral organ initiation in plants. We propose that BIGE1 is required for transport of an intermediate or product associated with the CYP78A pathway.

  8. Using the plant vacuole as a biological system to investigate the functional properties of exogenous channels and transporters.

    PubMed

    Festa, M; Lagostena, L; Carpaneto, A

    2016-03-01

    Plant cells possess a large intracellular compartment that animal cells do not, the central vacuole, which has been investigated for a long time. The central vacuole can occupy up to 90% of the cellular volume and, differently from intracellular organelles from animal cells such as lysosomes or endosomes, it is easy to isolate. Because of its large dimension (up to 40 μm diameter) it can be successfully studied using the classical patch-clamp technique. Following the idea that the vacuolar membrane could be used as a convenient model to characterize the functional properties of channel-forming peptides, we verified that the phytotoxic lipodepsipeptide Syringopeptin 25A from Pseudomonas syringae pv syringae was able to form ionic pores in sugar beet vacuoles and we performed a detailed biophysical analysis. Recently, we extended the use of plant vacuoles to the expression and functional characterization of animal intracellular transporters, namely rat CLC-7, and channels, i.e. human TPC2. Since endo-lysosomal transporters and channels are still largely unexplored, principally because their intracellular localization renders them difficult to study, we believe that this novel approach will prove to be a powerful system for the investigation of the molecular mechanisms of exogenous transporters and channels. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  9. Homoepitaxial tunnel barriers with functionalized graphene-on-graphene for charge and spin transport.

    PubMed

    Friedman, Adam L; van 't Erve, Olaf M J; Li, Connie H; Robinson, Jeremy T; Jonker, Berend T

    2014-01-01

    The coupled imperatives for reduced heat dissipation and power consumption in high-density electronics have rekindled interest in devices based on tunnelling. Such devices require mating dissimilar materials, raising issues of heteroepitaxy, layer uniformity, interface stability and electronic states that severely complicate fabrication and compromise performance. Two-dimensional materials such as graphene obviate these issues and offer a new paradigm for tunnel barriers. Here we demonstrate a homoepitaxial tunnel barrier structure in which graphene serves as both the tunnel barrier and the high-mobility transport channel. We fluorinate the top layer of a graphene bilayer to decouple it from the bottom layer, so that it serves as a single-monolayer tunnel barrier for both charge and spin injection into the lower graphene channel. We demonstrate high spin injection efficiency with a tunnelling spin polarization >60%, lateral transport of spin currents in non-local spin-valve structures and determine spin lifetimes with the Hanle effect.

  10. Homoepitaxial tunnel barriers with functionalized graphene-on-graphene for charge and spin transport.

    PubMed

    Friedman, Adam L; van 't Erve, Olaf M J; Li, Connie H; Robinson, Jeremy T; Jonker, Berend T

    2014-01-01

    The coupled imperatives for reduced heat dissipation and power consumption in high-density electronics have rekindled interest in devices based on tunnelling. Such devices require mating dissimilar materials, raising issues of heteroepitaxy, layer uniformity, interface stability and electronic states that severely complicate fabrication and compromise performance. Two-dimensional materials such as graphene obviate these issues and offer a new paradigm for tunnel barriers. Here we demonstrate a homoepitaxial tunnel barrier structure in which graphene serves as both the tunnel barrier and the high-mobility transport channel. We fluorinate the top layer of a graphene bilayer to decouple it from the bottom layer, so that it serves as a single-monolayer tunnel barrier for both charge and spin injection into the lower graphene channel. We demonstrate high spin injection efficiency with a tunnelling spin polarization >60%, lateral transport of spin currents in non-local spin-valve structures and determine spin lifetimes with the Hanle effect. PMID:24445349

  11. Wave-front sensing by use of a Green's function solution to the intensity transport equation.

    PubMed

    Woods, Simon C; Greenaway, Alan H

    2003-03-01

    A method for reconstructing an unknown wave front from measurements of its intensity distribution on two planes along the direction of propagation is described. The method solves the intensity transport equation by use of Neumann boundary conditions, leading to a solution that requires only matrix multiplication. The method provides real-time wave-front reconstruction with high accuracy and is easily reposed to permit reconstruction of the wave front in any orthonormal basis set. PMID:12630836

  12. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function.

    PubMed

    Reynafarje, B; Lehninger, A L

    1978-10-01

    This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP(4-) (out) for ADP(3-) (in) + 0.5 phosphate(2-) (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated.

  13. The BBSome Controls Energy Homeostasis by Mediating the Transport of the Leptin Receptor to the Plasma Membrane

    PubMed Central

    Guo, Deng-Fu; Cui, Huxing; Zhang, Qihong; Morgan, Donald A.; Thedens, Daniel R.; Nishimura, Darryl; Grobe, Justin L.; Sheffield, Val C.; Rahmouni, Kamal

    2016-01-01

    Bardet-Biedl syndrome (BBS) is a highly pleiotropic autosomal recessive disorder associated with a wide range of phenotypes including obesity. However, the underlying mechanism remains unclear. Here, we show that neuronal BBSome is a critical determinant of energy balance through its role in the regulation of the trafficking of the long signaling form of the leptin receptor (LRb). Targeted disruption of the BBSome by deleting the Bbs1 gene from the nervous system causes obesity in mice, and this phenotype is reproduced by ablation of the Bbs1 gene selectively in the LRb-expressing cells, but not from adipocytes. Obesity developed as a consequence of both increased food intake and decreased energy expenditure in mice lacking the Bbs1 gene in LRb-expressing cells. Strikingly, the well-known role of BBS proteins in the regulation of ciliary formation and function is unlikely to account for the obesogenic effect of BBS1 loss as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb-expressing cells caused a marginal increase in body weight and adiposity. Instead, we demonstrate that silencing BBS proteins, but not IFT88, impair the trafficking of the LRb to the plasma membrane leading to central leptin resistance in a manner independent of obesity. Our data also demonstrate that postnatal deletion of the Bbs1 gene in the mediobasal hypothalamus can cause obesity in mice, arguing against an early neurodevelopmental origin of obesity in BBS. Our results depict a novel mechanism underlying energy imbalance and obesity in BBS with potential implications in common forms of human obesity. PMID:26926121

  14. The mitochondrial ornithine transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution of two human isoforms.

    PubMed

    Fiermonte, Giuseppe; Dolce, Vincenza; David, Laura; Santorelli, Filippo Maria; Dionisi-Vici, Carlo; Palmieri, Ferdinando; Walker, John E

    2003-08-29

    Two isoforms of the human ornithine carrier, ORC1 and ORC2, have been identified by overexpression of the proteins in bacteria and by study of the transport properties of the purified proteins reconstituted into liposomes. Both transport L-isomers of ornithine, lysine, arginine, and citrulline by exchange and by unidirectional mechanisms, and they are inactivated by the same inhibitors. ORC2 has a broader specificity than ORC1, and L- and D-histidine, L-homoarginine, and D-isomers of ornithine, lysine, and ornithine are all substrates. Both proteins are expressed in a wide range of human tissues, but ORC1 is the predominant form. The highest levels of expression of both isoforms are in the liver. Five mutant forms of ORC1 associated with the human disease hyperornithinemia-hyperammonemia-homocitrullinuria were also made. The mutations abolish the transport properties of the protein. In patients with hyperornithinemia-hyperammonemia-homocitrullinuria, isoform ORC2 is unmodified, and its presence compensates partially for defective ORC1. PMID:12807890

  15. Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation

    SciTech Connect

    Piwnica-Worms, D.; Vallabhaneni, V.R.; Kronauge, J.F.

    1995-09-26

    Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an {approximately}170 integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis(2-methoxyisobutyl isonitrile) technetium(I) (Tc-SESTAMIBI), a {gamma}-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (T{sub 1/2} {approx} 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. 70 refs., 7 figs., 2 tabs.

  16. Transport Behavior of Functionalized Multi-Wall Carbon Nanotubes in Water-Saturated Quartz Sand as a Function of Tube Length

    PubMed Central

    Wang, Yonggang; Kim, Jae-Hong; Baek, Jong-Beom; Miller, Gary W.; Pennell, Kurt D.

    2012-01-01

    A series of one-dimensional column experiments was conducted to examine the effects of tube length on the transport and deposition of 4-ethoxybenzoic acid functionalized multi-wall carbon nanotubes (MWCNTs) in water-saturated porous media. Aqueous MWCNTs suspensions were prepared to yield three distributions of tube lengths; 0.02–1.3 μm (short), 0.2–7.5 μm (medium), and 0.2–21.4 μm (long). Results of the column studies showed that MWCNT retention increased with increasing tube length. Nevertheless, more than 76% of the MWCNT mass delivered to the columns was detected in effluent samples under all experimental conditions, indicating that the functionalized MWCNTs were readily transported through 40–50 mesh Ottawa sand. Examination of MWCNT length distributions in the effluent samples revealed that nanotubes with lengths greater than 8 μm were preferentially deposited. In addition, measured retention profiles exhibited the greatest MWCNT deposition near the column inlet, which was most pronounced for the long MWCNTs, and decreased sharply with travel distance. Scanning electron microscope (SEM) images showed that MWCNTs were deposited on sand surfaces over the entire column length, while larger MWCNT bundles were retained at grain intersections and near the column inlet. A mathematical model based on clean bed filtration theory (CBFT) was unable to accurately simulate the measured retention profile data, even after varying the weighting function and incorporating a nonuniform attachment rate coefficient expression. Modification of the mathematical model to account for physical straining greatly improved predictions of MWCNT retention, yielding straining rate coefficients that were four orders-of-magnitude greater than corresponding attachment rate coefficients. Taken in concert, these experimental and modeling results demonstrate the potential importance of, and need to consider, particle straining and tube length distribution when describing MWCNT

  17. Impaired mitochondrial energy production and ABC transporter function-A crucial interconnection in dementing proteopathies of the brain.

    PubMed

    Pahnke, Jens; Fröhlich, Christina; Krohn, Markus; Schumacher, Toni; Paarmann, Kristin

    2013-10-01

    Ageing is the main risk factor for the development of dementing neurodegenerative diseases (NDs) and it is accompanied by the accumulation of variations in mitochondrial DNA. The resulting tissue-specific alterations in ATP production and availability cause deteriorations of cerebral clearance mechanisms that are important for the removal of toxic peptides and its aggregates. ABC transporters were shown to be the most important exporter superfamily for toxic peptides, e.g. β-amyloid and α-synuclein. Their activity is highly dependent on the availability of ATP and forms a directed energy-exporter network, linking decreased mitochondrial function with highly impaired ABC transporter activity and disease progression. In this paper, we describe a network based on interactions between ageing, energy metabolism, regeneration, accumulation of toxic peptides and the development of proteopathies of the brain with a focus on Alzheimer's disease (AD). Additionally, we provide new experimental evidence for interactions within this network in regenerative processes in AD.

  18. Electronic and thermoelectric properties of InN studied using ab initio density functional theory and Boltzmann transport calculations

    NASA Astrophysics Data System (ADS)

    Borges, P. D.; Scolfaro, L.

    2014-12-01

    The thermoelectric properties of indium nitride in the most stable wurtzite phase (w-InN) as a function of electron and hole concentrations and temperature were studied by solving the semiclassical Boltzmann transport equations in conjunction with ab initio electronic structure calculations, within Density Functional Theory. Based on maximally localized Wannier function basis set and the ab initio band energies, results for the Seebeck coefficient are presented and compared with available experimental data for n-type as well as p-type systems. Also, theoretical results for electric conductivity and power factor are presented. Most cases showed good agreemen