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Sample records for regulates transport activity

  1. Regulators of Slc4 bicarbonate transporter activity

    PubMed Central

    Thornell, Ian M.; Bevensee, Mark O.

    2015-01-01

    The Slc4 family of transporters is comprised of anion exchangers (AE1-4), Na+-coupled bicarbonate transporters (NCBTs) including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2), electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2), and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE), as well as a borate transporter (BTR1). These transporters regulate intracellular pH (pHi) and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO−3 either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO−3 transporter contributes to a cell's ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s) (e.g., Na+ or Cl−). In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both well-known and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family. PMID:26124722

  2. Activation of ion transport systems during cell volume regulation

    SciTech Connect

    Eveloff, J.L.; Warnock, D.G.

    1987-01-01

    This review discusses the activation of transport pathways during volume regulation, including their characteristics, the possible biochemical pathways that may mediate the activation of transport pathways, and the relations between volume regulation and transepithelial transport in renal cells. Many cells regulate their volume when exposed to an anisotonic medium. The changes in cell volume are caused by activation of ion transport pathways, plus the accompanying osmotically driven water movement such that cell volume returns toward normal levels. The swelling of hypertonically shrunken cells is termed regulatory volume increase (RVI) and involves an influx of NaCl into the cell via either activation of Na-Cl, Na-K-2Cl cotransport systems, or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease (RVD) and involves an efflux of KCl and water from the cell by activation of either separate K/sup +/ and Cl/sup -/ conductances, a K-Cl cotransport system, or parallel K/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchangers. The biochemical mechanisms involved in the activation of transport systems are largely unknown, however, the phosphoinositide pathway may be implicated in RVI; phorbol esters, cGMP, and Ca/sup 2 +/ affect the process of volume regulation. Renal tubular cells, as well as the blood cells that transverse the medulla, are subjected to increasing osmotic gradients from the corticomedullary junction to the papillary tip, as well as changing interstitial and tubule fluid osmolarity, depending on the diuretic state of the animal. Medullary cells from the loop of Henle and the papilla can volume regulate by activating Na-K-2Cl cotransport or Na/sup +/-H/sup +/ and Cl/sup -/-HCO/sub 3//sup -/ exchange systems.

  3. Substrate regulation of ascorbate transport activity in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-10-01

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

  4. Serotonin transporter genotype modulates amygdala activity during mood regulation

    PubMed Central

    Rao, Hengyi; Wang, Jiongjiong; Detre, John A.; Breland, Jessica; Sankoorikal, Geena Mary V.; Brodkin, Edward S.; Farah, Martha J.

    2010-01-01

    Recent studies have implicated the short allele of the serotonin transporter-linked polymorphic region (5-HTTLPR) in depression vulnerability, particularly in the context of stress. Several neuroimaging studies have shown that 5-HTTLPR genotype predicts amygdala reactivity to negatively valenced stimuli, suggesting a mechanism whereby the short allele confers depression risk. The current study investigated whether 5-HTTLPR genotype similarly affects neural activity during an induced sad mood and during recovery from sad mood. Participants were 15 homozygous short (S) and 15 homozygous long (L) individuals. Regional cerebral blood flow was measured with perfusion functional magnetic resonance imaging during four scanning blocks: baseline, sad mood, mood recovery and following return to baseline. Comparing mood recovery to baseline, both whole brain analyses and template-based region-of-interest analyses revealed greater amygdala activity for the S vs the L-group. There were no significant amygdala differences found during the induced sad mood. These results demonstrate the effect of the S allele on amygdala activity during intentional mood regulation and suggest that amygdala hyperactivity during recovery from a sad mood may be one mechanism by which the S allele confers depression risk. PMID:19858108

  5. Regulation of airway surface liquid volume and mucus transport by active ion transport.

    PubMed

    Tarran, Robert

    2004-01-01

    Mucus clearance is an important component of the lung's innate defense against disease, and the ability of the airways to clear mucus is strongly dependent on the volume of liquid on airway surfaces. Whether airway surface liquid (ASL) volume is maintained by passive surface forces or by active ion transport is controversial yet crucial to the understanding of how this system operates in both health and disease. In support of active ion transport being the major determinant of ASL volume, we have demonstrated that normal airway epithelia sense and autoregulate ASL height (volume) by adjusting the rates of Na+ absorption and Cl- secretion to maintain mucus transport.

  6. Active zone proteins are transported via distinct mechanisms regulated by Par-1 kinase

    PubMed Central

    Barber, Kara R.; Sherman, Michael

    2017-01-01

    Disruption of synapses underlies a plethora of neurodevelopmental and neurodegenerative disease. Presynaptic specialization called the active zone plays a critical role in the communication with postsynaptic neuron. While the role of many proteins at the active zones in synaptic communication is relatively well studied, very little is known about how these proteins are transported to the synapses. For example, are there distinct mechanisms for the transport of active zone components or are they all transported in the same transport vesicle? Is active zone protein transport regulated? In this report we show that overexpression of Par-1/MARK kinase, a protein whose misregulation has been implicated in Autism spectrum disorders (ASDs) and neurodegenerative disorders, lead to a specific block in the transport of an active zone protein component- Bruchpilot at Drosophila neuromuscular junctions. Consistent with a block in axonal transport, we find a decrease in number of active zones and reduced neurotransmission in flies overexpressing Par-1 kinase. Interestingly, we find that Par-1 acts independently of Tau-one of the most well studied substrates of Par-1, revealing a presynaptic function for Par-1 that is independent of Tau. Thus, our study strongly suggests that there are distinct mechanisms that transport components of active zones and that they are tightly regulated. PMID:28222093

  7. GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters.

    PubMed

    Ramesh, Sunita A; Tyerman, Stephen D; Xu, Bo; Bose, Jayakumar; Kaur, Satwinder; Conn, Vanessa; Domingos, Patricia; Ullah, Sana; Wege, Stefanie; Shabala, Sergey; Feijó, José A; Ryan, Peter R; Gilliham, Matthew; Gillham, Matthew

    2015-07-29

    The non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms.

  8. Regulation of ion channels and transporters by AMP-activated kinase (AMPK)

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

    The energy-sensing AMP-activated kinase AMPK ensures survival of energy-depleted cells by stimulating ATP production and limiting ATP utilization. Both energy production and energy consumption are profoundly influenced by transport processes across the cell membane including channels, carriers and pumps. Accordingly, AMPK is a powerful regulator of transport across the cell membrane. AMPK regulates diverse K+ channels, Na+ channels, Ca2+ release activated Ca2+ channels, Cl- channels, gap junctional channels, glucose carriers, Na+/H+-exchanger, monocarboxylate-, phosphate-, creatine-, amino acid-, peptide- and osmolyte-transporters, Na+/Ca2+-exchanger, H+-ATPase and Na+/K+-ATPase. AMPK activates ubiquitin ligase Nedd4–2, which labels several plasma membrane proteins for degradation. AMPK further regulates transport proteins by inhibition of Rab GTPase activating protein (GAP) TBC1D1. It stimulates phosphatidylinositol 3-phosphate 5-kinase PIKfyve and inhibits phosphatase and tensin homolog (PTEN) via glycogen synthase kinase 3β (GSK3β). Moreover, it stabilizes F-actin as well as downregulates transcription factor NF-κB. All those cellular effects serve to regulate transport proteins. PMID:24366036

  9. Regulation of hepatic drug transporter activity and expression by organochlorine pesticides.

    PubMed

    Bucher, Simon; Le Vee, Marc; Jouan, Elodie; Fardel, Olivier

    2014-03-01

    Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.

  10. Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation.

    PubMed

    Mason, J N; Farmer, H; Tomlinson, I D; Schwartz, J W; Savchenko, V; DeFelice, L J; Rosenthal, S J; Blakely, R D

    2005-04-15

    Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common approaches for studying these transporters involve use of radiolabeled substrates or antagonists, methods possessing limited spatial resolution and that bear limited opportunities for repeated monitoring of living preparations. To circumvent these issues, we have explored two novel assay platforms that permit temporally resolved quantitation of transport activity and transporter protein localization. To monitor the binding and transport function of NET and DAT in real-time, we have investigated the uptake of the fluorescent organic compound 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). We have extended our previous single cell level application of this substrate to monitor transport activity via high-throughput assay platforms. Compared to radiotracer uptake methods, acquisition of ASP+ fluorescence is non-isotopic and allows for continuous, repeated transport measurements on both transfected and native preparations. Secondly, we have extended our application of small-molecule-conjugated fluorescent CdSe/ZnS nanocrystals, or quantum dots (Qdots), to utilize antibody and peptide ligands that can identify surface expressed transporters, receptors and other membrane proteins in living cell systems. Unlike typical organic fluorophores, Qdots are highly resistant to bleaching and can be conjugated to multiple ligands. They can also be illuminated by conventional light sources, yet produce narrow, gaussian emission spectra compatible with multiple target visualization (multiplexing). Together, these approaches offer novel opportunities to investigate changes in transporter function and distribution in real-time with superior spatial and temporal resolution.

  11. Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

    PubMed Central

    Di Giovanni, Jerome; Sheng, Zu-Hang

    2015-01-01

    Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca2+ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca2+ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting. PMID:26108535

  12. Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting.

    PubMed

    Di Giovanni, Jerome; Sheng, Zu-Hang

    2015-08-04

    Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin(-/-) neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin(-/-) phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca(2+) sensor, we demonstrate that snapin-dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca(2+) sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting.

  13. 5-HT1B autoreceptor regulation of serotonin transporter activity in synaptosomes

    PubMed Central

    Hagan, Catherine E.; McDevitt, Ross A.; Liu, Yusha; Furay, Amy R.; Neumaier, John F.

    2012-01-01

    Serotonin-1B (5-HT1B) autoreceptors are located in serotonin (5-HT) terminals along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission, and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high-affinity reuptake of 5-HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT Km and Vmax, and previous work suggests that 5-HT1B autoreceptors may regulate 5-HT reuptake, in addition to modulating 5-HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5-HT1B autoreceptor regulation of SERT-mediated 5-HT uptake into synaptosomes. The selective 5-HT1B antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild-type but not 5-HT1B knockout mice, whereas SERT uptake was enhanced after pre-treatment with the selective 5-HT1B agonist CP94253. Furthermore, SERT activity varies as a function of 5-HT1B receptor expression—specifically, genetic deletion of 5-HT1B decreased SERT function, while viral-mediated overexpression of 5-HT1B autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5-HT1B autoreceptors regulate SERT activity. Since SERT clearance rate varies as a function of 5-HT1B autoreceptor expression levels and is modulated by both activation and inhibition of 5-HT1B autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications. PMID:22961814

  14. Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

    PubMed Central

    Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Shin, Misun; Nam, Yeon-Ju; Choi, Dong-Hwa; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

    2015-01-01

    The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH. PMID:26057890

  15. Coordinative modulation of human zinc transporter 2 gene expression through active and suppressive regulators.

    PubMed

    Lu, Yu-Ju; Liu, Ya-Chuan; Lin, Meng-Chieh; Chen, Yi-Ting; Lin, Lih-Yuan

    2015-04-01

    Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5' to 3') shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.

  16. Structural basis for the metal-selective activation of the manganese transport regulator of Bacillus subtilis.

    PubMed

    Kliegman, Joseph I; Griner, Sarah L; Helmann, John D; Brennan, Richard G; Glasfeld, Arthur

    2006-03-21

    The manganese transport regulator (MntR) of Bacillus subtilis is activated by Mn(2+) to repress transcription of genes encoding transporters involved in the uptake of manganese. MntR is also strongly activated by cadmium, both in vivo and in vitro, but it is poorly activated by other metal cations, including calcium and zinc. The previously published MntR.Mn(2+) structure revealed a binuclear complex of manganese ions with a metal-metal separation of 3.3 A (herein designated the AB conformer). Analysis of four additional crystal forms of MntR.Mn(2+) reveals that the AB conformer is only observed in monoclinic crystals at 100 K, suggesting that this conformation may be stabilized by crystal packing forces. In contrast, monoclinic crystals analyzed at room temperature (at either pH 6.5 or pH 8.5), and a second hexagonal crystal form (analyzed at 100 K), all reveal the shift of one manganese ion by 2.5 A, thereby leading to a newly identified conformation (the AC conformer) with an internuclear distance of 4.4 A. Significantly, the cadmium and calcium complexes of MntR also contain binuclear complexes with a 4.4 A internuclear separation. In contrast, the zinc complex of MntR contains only one metal ion per subunit, in the A site. Isothermal titration calorimetry confirms the stoichiometry of Mn(2+), Cd(2+), and Zn(2+) binding to MntR. We propose that the specificity of MntR activation is tied to productive binding of metal ions at two sites; the A site appears to act as a selectivity filter, determining whether the B or C site will be occupied and thereby fully activate MntR.

  17. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  18. Activation of Big Grain1 significantly improves grain size by regulating auxin transport in rice.

    PubMed

    Liu, Linchuan; Tong, Hongning; Xiao, Yunhua; Che, Ronghui; Xu, Fan; Hu, Bin; Liang, Chengzhen; Chu, Jinfang; Li, Jiayang; Chu, Chengcai

    2015-09-01

    Grain size is one of the key factors determining grain yield. However, it remains largely unknown how grain size is regulated by developmental signals. Here, we report the identification and characterization of a dominant mutant big grain1 (Bg1-D) that shows an extra-large grain phenotype from our rice T-DNA insertion population. Overexpression of BG1 leads to significantly increased grain size, and the severe lines exhibit obviously perturbed gravitropism. In addition, the mutant has increased sensitivities to both auxin and N-1-naphthylphthalamic acid, an auxin transport inhibitor, whereas knockdown of BG1 results in decreased sensitivities and smaller grains. Moreover, BG1 is specifically induced by auxin treatment, preferentially expresses in the vascular tissue of culms and young panicles, and encodes a novel membrane-localized protein, strongly suggesting its role in regulating auxin transport. Consistent with this finding, the mutant has increased auxin basipetal transport and altered auxin distribution, whereas the knockdown plants have decreased auxin transport. Manipulation of BG1 in both rice and Arabidopsis can enhance plant biomass, seed weight, and yield. Taking these data together, we identify a novel positive regulator of auxin response and transport in a crop plant and demonstrate its role in regulating grain size, thus illuminating a new strategy to improve plant productivity.

  19. Dietary P regulates phosphate transporter expression, phosphatase activity, and effluent P partitioning in trout culture.

    PubMed

    Coloso, R M; King, K; Fletcher, J W; Weis, P; Werner, A; Ferraris, R P

    2003-08-01

    Phosphate utilization by fish is an important issue because of its critical roles in fish growth and aquatic environmental pollution. High dietary phosphorus (P) levels typically decrease the efficiency of P utilization, thereby increasing the amount of P excreted as metabolic waste in effluents emanating from rainbow trout aquaculture. In mammals, vitamin D3 is a known regulator of P utilization but in fish, its regulatory role is unclear. Moreover, the effects of dietary P and vitamin D3 on expression of enzymatic and transport systems potentially involved in phosphate utilization are little known. We therefore monitored production of effluent P, levels of plasma vitamin D3 metabolites, as well as expression of phosphatases and the sodium phosphate cotransporter (NaPi2) in trout fed semipu diets that varied in dietary P and vitamin D3 levels. Mean soluble P concentrations varied markedly with dietary P but not with vitamin D3, and constituted 40-70% of total effluent P production by trout. Particulate P concentrations accounted for 25-50% of effluent P production, but did not vary with dietary P or vitamin D3. P in settleable wastes accounted for <10% of effluent P. The stronger effect of dietary P on effluent P levels is paralleled by its striking effects on phosphatases and NaPi2. The mRNA abundance of the intestinal and renal sodium phosphate transporters increased in fish fed low dietary P; vitamin D3 had no effect. Low-P diets reduced plasma phosphate concentrations. Intracellular phytase activity increased but brushborder alkaline phosphatase activity decreased in the intestine, pyloric caeca, and gills of trout fed diets containing low dietary P. Vitamin D3 had no effect on enzyme activities. Moreover, plasma concentrations of 25-hydroxyvitamin D3 and of 1,25-dihydroxyvitamin D3 were unaffected by dietary P and vitamin D3 levels. The major regulator of P metabolism, and ultimately of levels of P in the effluent from trout culture, is dietary P.

  20. Resveratrol Prevents Retinal Dysfunction by Regulating Glutamate Transporters, Glutamine Synthetase Expression and Activity in Diabetic Retina.

    PubMed

    Zeng, Kaihong; Yang, Na; Wang, Duozi; Li, Suping; Ming, Jian; Wang, Jing; Yu, Xuemei; Song, Yi; Zhou, Xue; Yang, Yongtao

    2016-05-01

    This study investigated the effects of resveratrol (RSV) on retinal functions, glutamate transporters (GLAST) and glutamine synthetase (GS) expression in diabetic rats retina, and on glutamate uptake, GS activity, GLAST and GS expression in high glucose-cultured Müller cells. The electroretinogram was used to evaluate retinal functions. Müller cells cultures were prepared from 5- to 7-day-old Sprague-Dawley rats. The expression of GLAST and GS was examined by qRT-PCR, ELISA and western-blotting. Glutamate uptake was measured as (3)H-glutamate contents of the lysates. GS activity was assessed by a spectrophotometric assay. 1- to 7-month RSV administrations (5 and 10 mg/kg/day) significantly alleviated hyperglycemia and weight loss in diabetic rats. RSV administrations also significantly attenuated diabetes-induced decreases in amplitude of a-wave in rod response, decreases in amplitude of a-, and b-wave in cone and rod response and decreases in amplitude of OP2 in oscillatory potentials. 1- to 7-month RSV treatments also significantly inhibited diabetes-induced delay in OP2 implicit times in scotopic 3.0 OPS test. The down-regulated mRNA and protein expression of GLAST and GS in diabetic rats retina was prevented by RSV administrations. In high glucose-treated cultures, Müller cells' glutamate uptake, GS activity, GLAST and GS expression were decreased significantly compared with normal control cultures. RSV (10, 20, and 30 mmol/l) significantly inhibited the HG-induced decreases in glutamate uptake, GS activity, GLAST and GS expression (at least P < 0.05). These beneficial results suggest that RSV may be considered as a therapeutic option to prevent from diabetic retinopathy.

  1. Regulation of Human Hepatic Drug Transporter Activity and Expression by Diesel Exhaust Particle Extract

    PubMed Central

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2015-01-01

    Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their

  2. Genetic Complementation Screen Identifies a Mitogen-activated Protein Kinase Phosphatase, MKP3, as a Regulator of Dopamine Transporter Trafficking

    PubMed Central

    Larsen, Mads Breum; Prasad, Balakrishna M.; Amara, Susan G.

    2008-01-01

    The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C–induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis. PMID:18434601

  3. Regulation of vitamin C transport.

    PubMed

    Wilson, John X

    2005-01-01

    Ascorbic acid and dehydroascorbic acid (DHAA, oxidized vitamin C) are dietary sources of vitamin C in humans. Both nutrients are absorbed from the lumen of the intestine and renal tubules by, respectively, enterocytes and renal epithelial cells. Subsequently vitamin C circulates in the blood and enters all of the other cells of the body. Concerning flux across the plasma membrane, simple diffusion of ascorbic acid plays only a small or negligible role. More important are specific mechanisms of transport and metabolism that concentrate vitamin C intracellularly to enhance its function as an enzyme cofactor and antioxidant. The known transport mechanisms are facilitated diffusion of DHAA through glucose-sensitive and -insensitive transporters, facilitated diffusion of ascorbate through channels, exocytosis of ascorbate in secretory vesicles, and secondary active transport of ascorbate through the sodium-dependent vitamin C transporters SVCT1 and SVCT2 proteins that are encoded by the genes Slc23a1 and Slc23a2, respectively. Evidence is reviewed indicating that these transport pathways are regulated under physiological conditions and altered by aging and disease.

  4. Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

    PubMed Central

    Al Awabdh, Sana; Gupta‐Agarwal, Swati; Sheehan, David F.; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E.; Griffin, Lewis D.

    2016-01-01

    The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264 PMID:27189737

  5. The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3- salvage transporter human Na+-HCO3- cotransport isoform 3.

    PubMed

    Park, Meeyoung; Ko, Shigeru B H; Choi, Joo Young; Muallem, Gaia; Thomas, Philip J; Pushkin, Alexander; Lee, Myeong-Sok; Kim, Joo Young; Lee, Min Goo; Muallem, Shmuel; Kurtz, Ira

    2002-12-27

    Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO(3)(-) secretion and HCO(3)(-) salvage in secretory epithelia. At least two luminal transporters mediate HCO(3)(-) salvage, the Na(+)/H(+) exchanger (NHE3) and the Na(+)-HCO(3)(-) cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na(+)-dependent, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO(3)(-) transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO(3)(-)-transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO(3)(-) transport by regulating the activity of all luminal HCO(3)(-) secretion and salvage mechanisms of secretory epithelial cells.

  6. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  7. Bundling dynamics regulates the active mechanics and transport in carbon nanotube networks and their nanocomposites

    NASA Astrophysics Data System (ADS)

    Hahm, Myung Gwan; Wang, Hailong; Jung, Hyun Young; Hong, Sanghyun; Lee, Sung-Goo; Kim, Sung-Ryong; Upmanyu, Moneesh; Jung, Yung Joon

    2012-05-01

    High-density carbon nanotube networks (CNNs) continue to attract interest as active elements in nanoelectronic devices, nanoelectromechanical systems (NEMS) and multifunctional nanocomposites. The interplay between the network nanostructure and its properties is crucial, yet current understanding remains limited to the passive response. Here, we employ a novel superstructure consisting of millimeter-long vertically aligned single walled carbon nanotubes (SWCNTs) sandwiched between polydimethylsiloxane (PDMS) layers to quantify the effect of two classes of mechanical stimuli, film densification and stretching, on the electronic and thermal transport across the network. The network deforms easily with an increase in the electrical and thermal conductivities, suggestive of a floppy yet highly reconfigurable network. Insight from atomistically informed coarse-grained simulations uncover an interplay between the extent of lateral assembly of the bundles, modulated by surface zipping/unzipping, and the elastic energy associated with the bent conformations of the nanotubes/bundles. During densification, the network becomes highly interconnected yet we observe a modest increase in bundling primarily due to the reduced spacing between the SWCNTs. The stretching, on the other hand, is characterized by an initial debundling regime as the strain accommodation occurs via unzipping of the branched interconnects, followed by rapid rebundling as the strain transfers to the increasingly aligned bundles. In both cases, the increase in the electrical and thermal conductivity is primarily due to the increase in bundle size; the changes in network connectivity have a minor effect on the transport. Our results have broad implications for filamentous networks of inorganic nanoassemblies composed of interacting tubes, wires and ribbons/belts.High-density carbon nanotube networks (CNNs) continue to attract interest as active elements in nanoelectronic devices, nanoelectromechanical systems

  8. Ctr9, a Protein in the Transcription Complex Paf1, Regulates Dopamine Transporter Activity at the Plasma Membrane*

    PubMed Central

    De Gois, Stéphanie; Slama, Patrick; Pietrancosta, Nicolas; Erdozain, Amaia M.; Louis, Franck; Bouvrais-Veret, Caroline; Daviet, Laurent; Giros, Bruno

    2015-01-01

    Dopamine (DA) is a major regulator of sensorimotor and cognitive functions. The DA transporter (DAT) is the key protein that regulates the spatial and temporal activity of DA release into the synaptic cleft via the rapid reuptake of DA into presynaptic termini. Several lines of evidence have suggested that transporter-interacting proteins may play a role in DAT function and regulation. Here, we identified the tetratricopeptide repeat domain-containing protein Ctr9 as a novel DAT binding partner using a yeast two-hybrid system. We showed that Ctr9 is expressed in dopaminergic neurons and forms a stable complex with DAT in vivo via GST pulldown and co-immunoprecipitation assays. In mammalian cells co-expressing both proteins, Ctr9 partially colocalizes with DAT at the plasma membrane. This interaction between DAT and Ctr9 results in a dramatic enhancement of DAT-mediated DA uptake due to an increased number of DAT transporters at the plasma membrane. We determined that the binding of Ctr9 to DAT requires residues YKF in the first half of the DAT C terminus. In addition, we characterized Ctr9, providing new insight into this protein. Using three-dimensional modeling, we identified three novel tetratricopeptide repeat domains in the Ctr9 sequence, and based on deletion mutation experiments, we demonstrated the role of the SH2 domain of Ctr9 in nuclear localization. Our results demonstrate that Ctr9 localization is not restricted to the nucleus, as previously described for the transcription complex Paf1. Taken together, our data provide evidence that Ctr9 modulates DAT function by regulating its trafficking. PMID:26048990

  9. Cell volume regulation by trout erythrocytes: characteristics of the transport systems activated by hypotonic swelling.

    PubMed

    Garcia-Romeu, F; Cossins, A R; Motais, R

    1991-01-01

    1. An osmolality reduction of the suspending medium leads to osmotic swelling of trout erythrocytes, which is followed by a volume readjustment towards the original level. The regulatory volume decrease (RVD) was not complete after 1 h. 2. During RVD the cells lost K+ and Cl- but gained Na+. This entry of Na+, which is about half the K+ loss, explains the incomplete volume recovery (it was complete when Na+ was replaced by impermeant N-methyl-D-glucamine). The cells also lose large quantities of taurine, which accounts for about 53% of the volume recovery. In addition RVD is accompanied by the activation of a pathway allowing some large organic cations which are normally impermeant, such as choline or tetramethyl-ammonium, to rapidly penetrate the cells. 3. The swelling-activated K+ loss is not significantly affected by replacement of Cl- by NO3-, indicating that K+ moves through a Cl(-)-independent K+ pathway. Furosemide, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and niflumic acid inhibit the K+ loss. From experiments performed in high-K(+)-containing media, it appears that these compounds block the K+ flux, not by inhibiting Cl- movements but by interfering with the K+ pathway. 4. All the volume-activated pathways (K+, Na+, taurine, choline) are fully inhibited by furosemide and by inhibitors of the anion exchanger such as DIDS and niflumic acid. The concentration required for 50% inhibition (IC50) of both inorganic cations and taurine appears to be similar. It is proposed that DIDS interacts with a unique target which controls all the volume-sensitive transport systems.

  10. Schisandra chinensis regulates drug metabolizing enzymes and drug transporters via activation of Nrf2-mediated signaling pathway

    PubMed Central

    He, Jin-Lian; Zhou, Zhi-Wei; Yin, Juan-Juan; He, Chang-Qiang; Zhou, Shu-Feng; Yu, Yang

    2015-01-01

    Drug metabolizing enzymes (DMEs) and drug transporters are regulated via epigenetic, transcriptional, posttranscriptional, and translational and posttranslational modifications. Phase I and II DMEs and drug transporters play an important role in the disposition and detoxification of a large number of endogenous and exogenous compounds. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a critical regulator of a variety of important cytoprotective genes that are involved in disposition and detoxification of xenobiotics. Schisandra chinensis (SC) is a commonly used traditional Chinese herbal medicine that has been primarily used to protect the liver because of its potent antioxidative and anti-inflammatory activities. SC can modulate some DMEs and drug transporters, but the underlying mechanisms are unclear. In this study, we aimed to explore the role of Nrf2 in the regulatory effect of SC extract (SCE) on selected DMEs and drug transporters in human hepatocellular liver carcinoma cell line (HepG2) cells. The results showed that SCE, schisandrin A, and schisandrin B significantly increased the expression of NAD(P)H: Nicotinamide Adenine Dinucleotide Phosphate-oxidase or:quinone oxidoreductase 1, heme oxygenase-1, glutamate–cysteine ligase, and glutathione S-transferase A4 at both transcriptional and posttranscriptional levels. Incubation of HepG2 cells with SCE resulted in a significant increase in the intracellular level of glutathione and total glutathione S-transferase content. SCE significantly elevated the messenger ribonucleic acid and protein levels of P-glycoprotein and multidrug resistance-associated protein 2 and 4, whereas the expression of organic anion transporting peptide 1A2 and 1B1 was significantly downregulated by SCE. Knockdown of Nrf2 by small interfering ribonucleic acid attenuated the regulatory effect of SCE on these DMEs and drug transporters. SCE significantly upregulated Nrf2 and promoted the translocation of Nrf2 from cytoplasm to

  11. The KDEL receptor couples to Gαq/11 to activate Src kinases and regulate transport through the Golgi

    PubMed Central

    Giannotta, Monica; Ruggiero, Carmen; Grossi, Mauro; Cancino, Jorge; Capitani, Mirco; Pulvirenti, Teodoro; Consoli, Grazia Maria Letizia; Geraci, Corrada; Fanelli, Francesca; Luini, Alberto; Sallese, Michele

    2012-01-01

    Membrane trafficking involves large fluxes of cargo and membrane across separate compartments. These fluxes must be regulated by control systems to maintain homoeostasis. While control systems for other key functions such as protein folding or the cell cycle are well known, the mechanisms that control secretory transport are poorly understood. We have previously described a signalling circuit operating at the Golgi complex that regulates intra-Golgi trafficking and is initiated by the KDEL receptor (KDEL-R), a protein previously known to mediate protein recycling from the Golgi to the endoplasmic reticulum (ER). Here, we investigated the KDEL-R signalling mechanism. We show that the KDEL-R is predicted to fold like a G-protein-coupled receptor (GPCR), and that it binds and activates the heterotrimeric signalling G-protein Gαq/11 which, in turn, regulates transport through the Golgi complex. These findings reveal an unexpected GPCR-like mode of action of the KDEL-R and shed light on a core molecular control mechanism of intra-Golgi traffic. PMID:22580821

  12. Down-regulation of Na+-coupled glutamate transporter EAAT3 and EAAT4 by AMP-activated protein kinase.

    PubMed

    Sopjani, Mentor; Alesutan, Ioana; Dërmaku-Sopjani, Miribane; Fraser, Scott; Kemp, Bruce E; Föller, Michael; Lang, Florian

    2010-06-01

    The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.

  13. Adaptive regulation of intestinal nutrient transporters.

    PubMed Central

    Diamond, J M; Karasov, W H

    1987-01-01

    Because most eukaryotic somatic cells are bathed in a constant internal milieu, most of their proteins are constitutive, unlike the adaptive enzymes of bacteria. However, intestinal mucosal cells, like bacteria, face a varying milieu. Hence, we tested for adaptive regulation of intestinal nutrient transporters, sought its functional significance, and compared it with regulation of bacterial proteins. All 12 transporters studied proved to be regulated by dietary substrate levels. Regulation in the intestine is slower than in bacteria and shows lower peak-to-basal activity levels. Regulatory patterns vary greatly among transporters: two sugars and two nonessential amino acids monotonically up-regulate their transporters, two vitamins and three minerals monotonically down-regulate their transporters, and two transporters of essential amino acids respond nonmonotonically to levels of their substrates. These varied patterns arise from trade-offs among four factors: transporter costs, calories yielded by metabolizable substrates, fixed daily requirements of essential nutrients, and toxicity of certain nutrients in large amounts. Based on these trade-offs, we predict the form of regulatory pattern for intestinal transporters not yet studied. PMID:3470788

  14. Abundance of amino acid transporters involved in mTORC1 activation in skeletal muscle of neonatal pigs is developmentally regulated

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously we demonstrated that the insulinand amino acid-induced activation of the mammalian target of rapamycin complex 1 (mTORC1) is developmentally regulated in neonatal pigs. Recent studies have indicated that members of the System A transporter (SNAT2), the System N transporter (SNAT3), the Sy...

  15. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    PubMed

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  16. The Structural Basis for the Metal Selective Activation of the Manganese Transport Regulator of Bacillus subtilis†,§

    PubMed Central

    Kliegman, Joseph I.; Griner, Sarah L.; Helmann, John D.; Brennan, Richard G.; Glasfeld, Arthur

    2008-01-01

    The manganese transport regulator (MntR) of Bacillus subtilis is activated by Mn2+ to repress transcription of genes encoding transporters involved in the uptake of manganese. MntR is also strongly activated by cadmium, both in vivo and in vitro, but it is poorly activated by other metal cations, including calcium and zinc. The previously published MntR•Mn2+ structure revealed a binuclear complex of manganese ions with a metal-metal separation of 3.3 Å (herein designated the AB conformer). Analysis of four additional crystal forms of MntR•Mn2+ reveals that the AB conformer is only observed in monoclinic crystals at 100 K, suggesting that this conformation may be stabilized by crystal packing forces. In contrast, monoclinic crystals analyzed at room temperature (at either pH 6.5 or 8.5), and a second hexagonal crystal form (analyzed at 100 K), all reveal the shift of one manganese ion by 2.5 Å thereby leading to a newly identified conformation (the AC conformer) with an internuclear distance of 4.4 Å. Significantly, the cadmium and calcium complexes of MntR also contain binuclear complexes with a 4.4 Å internuclear separation. In contrast, the zinc complex of MntR contains only one metal ion per subunit, in the A site. Isothermal titration calorimetry confirms the stoichiometry of Mn2+, Cd2+ and Zn2+ binding to MntR. We propose that the specificity of MntR activation is tied to productive binding of metal ions at two sites; the A site appears to act as a selectivity filter, determining whether the B or C site will be occupied and thereby fully activate MntR. PMID:16533030

  17. Regulation of auxin transport during gravitropism

    NASA Astrophysics Data System (ADS)

    Rashotte, A.; Brady, S.; Kirpalani, N.; Buer, C.; Muday, G.

    Plants respond to changes in the gravity vector by differential growth across the gravity-stimulated organ. The plant hormone auxin, which is normally basipetally transported, changes in direction and auxin redistribution has been suggested to drive this differential growth or gravitropism. The mechanisms by which auxin transport directionality changes in response to a change in gravity vector are largely unknown. Using the model plant, Arabidopsis thaliana, we have been exploring several regulatory mechanisms that may control auxin transport. Mutations that alter protein phosphorylation suggest that auxin transport in arabidopsis roots may be controlled via phosphorylation and this signal may facilitate gravitropic bending. The protein kinase mutant pinoid (pid9) has reduced auxin transport; whereas the protein phosphatase mutant, rcn1, has elevated transport, suggesting reciprocal regulation of auxin transport by reversible protein phosphorylation. In both of these mutants, the auxin transport defects are accompanied by gravitropic defects, linking phosphorylation signaling to gravity-induced changes in auxin transport. Additionally, auxin transport may be regulated during gravity response by changes in an endogenous auxin efflux inhibitor. Flavonoids, such as quercetin and kaempferol, have been implicated in regulation of auxin transport in vivo and in vitro. Mutants that make no flavonoids have reduced root gravitropic bending. Furthermore, changes in auxin-induced gene expression and flavonoid accumulation patterns have been observed during gravity stimulation. Current studies are examining whether there are spatial and temporal changes in flavonoid accumulation that precede gravitropic bending and whether the absence of these changes are the cause of the altered gravity response in plants with mutations that block flavonoid synthesis. These results support the idea that auxin transport may be regulated during gravity response by several mechanisms including

  18. Impacts of human activities on nutrient transport in the Yellow River: The role of the Water-Sediment Regulation Scheme.

    PubMed

    Li, Xinyu; Chen, Hontao; Jiang, Xueyan; Yu, Zhigang; Yao, Qingzhen

    2017-03-15

    Anthropogenic activities alter the natural states of large rivers and their surrounding environment. The Yellow River is a well-studied case of a large river with heavy human control. An artificial managed water and sediment release system, known as the Water-Sediment Regulation Scheme (WSRS), has been carried out annually in the Yellow River since 2002. Nutrient concentrations and composition display significant time and space variations during the WSRS period. To figure out the anthropogenic impact of nutrient changes and transport in the Yellow River, biogeochemical observations were carried out in both middle reaches and lower reaches of the Yellow River during 2014 WSRS period. WSRS has a direct impact on water oxidation-reduction environment in the middle reaches; concentrations of nitrite (NO2(-)) and ammonium (NH4(+)) increased, while nitrate (NO3(-)) concentration decreased by enhanced denitrification. WSRS changed transport of water and sediment; dissolved silicate (DSi) in the middle reaches was directly controlled by sediments release during the WSRS while in the lower reaches, DSi changed with both sediments and water released from middle reaches. During the WSRS, the differences of nutrient fluxes and concentrations between lower reaches and middle reaches were significant; dissolved inorganic phosphorous (DIP) and dissolved inorganic nitrogen (DIN) were higher in low reaches because of anthropogenic inputs. Human intervention, especially WSRS, can apparently change the natural states of both the mainstream and estuarine environments of the Yellow River within a short time.

  19. Transport to Rhebpress activity.

    PubMed

    Garrido, Amanda; Brandt, Marta; Djouder, Nabil

    2016-01-01

    The small GTPases from the rat sarcoma (Ras) superfamily are a heterogeneous group of proteins of about 21 kDa that act as molecular switches, modulating cell signaling pathways and controlling diverse cellular processes. They are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). Ras homolog enriched in brain (Rheb) is a member of the Ras GTPase superfamily and a key activator of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). We recently determined that microspherule protein 1 (MCRS1) maintains Rheb at lysosomal surfaces in an amino acid-dependent manner. MCRS1 depletion promotes the formation of the GDP-bound form of Rheb, which is then delocalized from the lysosomal platform and transported to endocytic recycling vesicles, leading to mTORC1 inactivation. During this delocalization process, Rheb-GDP remains farnesylated and associated with cellular endomembranes. These findings provide new insights into the regulation of small GTPases, whose activity depends on both their GTP/GDP switch state and their capacity to move between different cellular membrane-bound compartments. Dynamic spatial transport between compartments makes it possible to alter the proximity of small GTPases to their activatory sites depending on the prevailing physiological and cellular conditions.

  20. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

    PubMed

    Robert, Amélie; Herrmann, Harald; Davidson, Michael W; Gelfand, Vladimir I

    2014-07-01

    Intermediate filaments (IFs) form a dense and dynamic network that is functionally associated with microtubules and actin filaments. We used the GFP-tagged vimentin mutant Y117L to study vimentin-cytoskeletal interactions and transport of vimentin filament precursors. This mutant preserves vimentin interaction with other components of the cytoskeleton, but its assembly is blocked at the unit-length filament (ULF) stage. ULFs are easy to track, and they allow a reliable and quantifiable analysis of movement. Our results show that in cultured human vimentin-negative SW13 cells, 2% of vimentin-ULFs move along microtubules bidirectionally, while the majority are stationary and tightly associated with actin filaments. Rapid motor-dependent transport of ULFs along microtubules is enhanced ≥ 5-fold by depolymerization of actin cytoskeleton with latrunculin B. The microtubule-dependent transport of vimentin ULFs is further regulated by Rho-kinase (ROCK) and p21-activated kinase (PAK): ROCK inhibits ULF transport, while PAK stimulates it. Both kinases act on microtubule transport independently of their effects on actin cytoskeleton. Our study demonstrates the importance of the actin cytoskeleton to restrict IF transport and reveals a new role for PAK and ROCK in the regulation of IF precursor transport.-Robert, A., Herrmann, H., Davidson, M. W., and Gelfand, V. I. Microtubule-dependent transport of vimentin filament precursors is regulated by actin and by the concerted action of Rho- and p21-activated kinases.

  1. Regulation of Nucleocytoplasmic Transport in Skeletal Muscle

    PubMed Central

    Hall, Monica N.; Corbett, Anita H.; Pavlath, Grace K.

    2015-01-01

    Proper skeletal muscle function is dependent on spatial and temporal control of gene expression in multinucleated myofibers. In addition, satellite cells, which are tissue-specific stem cells that contribute critically to repair and maintenance of skeletal muscle, are also required for normal muscle physiology. Gene expression in both myofibers and satellite cells is dependent upon nuclear proteins that require facilitated nuclear transport. A unique challenge for myofibers is controlling the transcriptional activity of hundreds of nuclei in a common cytoplasm yet achieving nuclear selectivity in transcription at specific locations such as neuromuscular synapses and myotendinous junctions. Nucleocytoplasmic transport of macromolecular cargoes is regulated by a complex interplay among various components of the nuclear transport machinery, namely nuclear pore complexes, nuclear envelope proteins, and various soluble transport receptors. The focus of this review is to highlight what is known about the nuclear transport machinery and its regulation in skeletal muscle and to consider the unique challenges that multinucleated muscle cells as well as satellite cells encounter in regulating nucleocytoplasmic transport during cell differentiation and tissue adaptation. Understanding how regulated nucleocytoplasmic transport controls gene expression in skeletal muscle may lead to further insights into the mechanisms contributing to muscle growth and maintenance throughout the lifespan of an individual. PMID:21621074

  2. Cytoskeletal Network Morphology Regulates Intracellular Transport Dynamics.

    PubMed

    Ando, David; Korabel, Nickolay; Huang, Kerwyn Casey; Gopinathan, Ajay

    2015-10-20

    Intracellular transport is essential for maintaining proper cellular function in most eukaryotic cells, with perturbations in active transport resulting in several types of disease. Efficient delivery of critical cargos to specific locations is accomplished through a combination of passive diffusion and active transport by molecular motors that ballistically move along a network of cytoskeletal filaments. Although motor-based transport is known to be necessary to overcome cytoplasmic crowding and the limited range of diffusion within reasonable timescales, the topological features of the cytoskeletal network that regulate transport efficiency and robustness have not been established. Using a continuum diffusion model, we observed that the time required for cellular transport was minimized when the network was localized near the nucleus. In simulations that explicitly incorporated network spatial architectures, total filament mass was the primary driver of network transit times. However, filament traps that redirect cargo back to the nucleus caused large variations in network transport. Filament polarity was more important than filament orientation in reducing average transit times, and transport properties were optimized in networks with intermediate motor on and off rates. Our results provide important insights into the functional constraints on intracellular transport under which cells have evolved cytoskeletal structures, and have potential applications for enhancing reactions in biomimetic systems through rational transport network design.

  3. Redox regulation of intercellular transport.

    PubMed

    Benitez-Alfonso, Yoselin; Jackson, David; Maule, Andy

    2011-01-01

    Plant cells communicate with each other via plasmodesmata (PDs) in order to orchestrate specific responses to environmental and developmental cues. At the same time, environmental signals regulate this communication by promoting changes in PD structure that modify symplastic permeability and, in extreme cases, isolate damaged cells. Reactive oxygen species (ROS) are key messengers in plant responses to a range of biotic and abiotic stresses. They are also generated during normal metabolism, and mediate signaling pathways that modulate plant growth and developmental transitions. Recent research has suggested the participation of ROS in the regulation of PD transport. The study of several developmental and stress-induced processes revealed a co-regulation of ROS and callose (a cell wall polymer that regulates molecular flux through PDs). The identification of Arabidopsis mutants simultaneously affected in cell redox homeostasis and PD transport, and the histological detection of hydrogen peroxide and peroxidases in the PDs of the tomato vascular cambium provide new information in support of this novel regulatory mechanism. Here, we describe the evidence that supports a role for ROS in the regulation of callose deposition and/or in the formation of secondary PD, and discuss the potential importance of this mechanism during plant growth or defense against environmental stresses.

  4. Theaflavins, dimeric catechins, inhibit peptide transport across Caco-2 cell monolayers via down-regulation of AMP-activated protein kinase-mediated peptide transporter PEPT1.

    PubMed

    Takeda, Junko; Park, Ha-Young; Kunitake, Yuri; Yoshiura, Keiko; Matsui, Toshiro

    2013-06-15

    In the small intestine, peptide transporter 1 (PEPT1) plays a role in the transport of di- and tripeptides. In this study, we investigated whether theaflavins (TFs) affect the absorption of small peptides in human intestinal Caco-2 cells, since TFs do not penetrate through the cells and might be involved in intestinal transport systems. In transport experiments, the transport of glycyl-sarcosine (Gly-Sar, a model molecule for PEPT1 transport) and other dipeptides (Val-Tyr and Ile-Phe) were significantly reduced (P<0.05) in TFs-pretreated cells. In TF 3'-O-gallate-pretreated cells, Western blot analysis revealed attenuated expression of PEPT1 transporter and Gly-Sar transport was completely ameliorated by 10 μM Compound C, an AMP-activated protein kinase (AMPK) inhibitor. In conclusion, the present study demonstrated that TFs inhibit peptide transport across Caco-2 cell monolayers, probably through suppression of AMPK-mediated PEPT1 expression, which should be considered a new bioactivity of TFs in black tea.

  5. Susceptibility of juvenile and adult blood–brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity

    PubMed Central

    2012-01-01

    Background P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood–brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses. Methods Rats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (Kp,uu,brain) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-β (IL-1β), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray. Results We found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a

  6. Regulation of glucose transport in skeletal muscle.

    PubMed

    Barnard, R J; Youngren, J F

    1992-11-01

    The entry of glucose into muscle cells is achieved primarily via a carrier-mediated system consisting of protein transport molecules. GLUT-1 transporter isoform is normally found in the sarcolemmal (SL) membrane and is thought to be involved in glucose transport under basal conditions. With insulin stimulation, glucose transport is accelerated by translocating GLUT-4 transporters from an intracellular pool out to the T-tubule and SL membranes. Activation of transporters to increase the turnover number may also be involved, but the evidence is far from conclusive. When insulin binds to its receptor, it autophosphorylates tyrosine and serine residues on the beta-subunit of the receptor. The tyrosine residues are thought to activate tyrosine kinases, which in turn phosphorylate/activate as yet unknown second messengers. Insulin receptor antibodies, however, have been reported to increase glucose transport without increasing kinase activity. Insulin resistance in skeletal muscle is a major characteristic of obesity and diabetes mellitus, especially NIDDM. A decrease in the number of insulin receptors and the ability of insulin to activate receptor tyrosine kinase has been documented in muscle from NIDDM patients. Most studies report no change in the intracellular pool of GLUT-4 transporters available for translocation to the SL. Both the quality and quantity of food consumed can regulate insulin sensitivity. A high-fat, refined sugar diet, similar to the typical U.S. diet, causes insulin resistance when compared with a low-fat, complex-carbohydrate diet. On the other hand, exercise increases insulin sensitivity. After an acute bout of exercise, glucose transport in muscle increases to the same level as with maximum insulin stimulation. Although the number of GLUT-4 transporters in the sarcolemma increases with exercise, neither insulin or its receptor is involved. After an initial acute phase, which may involve calcium as the activator, a secondary phase of increased

  7. A phosphotyrosine switch regulates organic cation transporters.

    PubMed

    Sprowl, Jason A; Ong, Su Sien; Gibson, Alice A; Hu, Shuiying; Du, Guoqing; Lin, Wenwei; Li, Lie; Bharill, Shashank; Ness, Rachel A; Stecula, Adrian; Offer, Steven M; Diasio, Robert B; Nies, Anne T; Schwab, Matthias; Cavaletti, Guido; Schlatter, Eberhard; Ciarimboli, Giuliano; Schellens, Jan H M; Isacoff, Ehud Y; Sali, Andrej; Chen, Taosheng; Baker, Sharyn D; Sparreboom, Alex; Pabla, Navjotsingh

    2016-03-16

    Membrane transporters are key determinants of therapeutic outcomes. They regulate systemic and cellular drug levels influencing efficacy as well as toxicities. Here we report a unique phosphorylation-dependent interaction between drug transporters and tyrosine kinase inhibitors (TKIs), which has uncovered widespread phosphotyrosine-mediated regulation of drug transporters. We initially found that organic cation transporters (OCTs), uptake carriers of metformin and oxaliplatin, were inhibited by several clinically used TKIs. Mechanistic studies showed that these TKIs inhibit the Src family kinase Yes1, which was found to be essential for OCT2 tyrosine phosphorylation and function. Yes1 inhibition in vivo diminished OCT2 activity, significantly mitigating oxaliplatin-induced acute sensory neuropathy. Along with OCT2, other SLC-family drug transporters are potentially part of an extensive 'transporter-phosphoproteome' with unique susceptibility to TKIs. On the basis of these findings we propose that TKIs, an important and rapidly expanding class of therapeutics, can functionally modulate pharmacologically important proteins by inhibiting protein kinases essential for their post-translational regulation.

  8. Phosphorylation mechanisms in dopamine transporter regulation.

    PubMed

    Foster, James D; Vaughan, Roxanne A

    2016-11-09

    The dopamine transporter (DAT) is a plasma membrane phosphoprotein that actively translocates extracellular dopamine (DA) into presynaptic neurons. The transporter is the primary mechanism for control of DA levels and subsequent neurotransmission, and is the target for abused and therapeutic drugs that exert their effects by suppressing reuptake. The transport capacity of DAT is acutely regulated by signaling systems and drug exposure, providing neurons the ability to fine-tune DA clearance in response to specific conditions. Kinase pathways play major roles in these mechanisms, and this review summarizes the current status of DAT phosphorylation characteristics and the evidence linking transporter phosphorylation to control of reuptake and other functions. Greater understanding of these processes may aid in elucidation of their possible contributions to DA disease states and suggest specific phosphorylation sites as targets for therapeutic manipulation of reuptake.

  9. Ciliary transport regulates PDGF-AA/αα signaling via elevated mammalian target of rapamycin signaling and diminished PP2A activity.

    PubMed

    Umberger, Nicole L; Caspary, Tamara

    2015-01-15

    Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.

  10. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

    PubMed

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

    2017-01-06

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr(28), leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr(28) is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr(28) in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr(28) in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  11. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    SciTech Connect

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

    2016-10-07

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  12. BDNF regulates spontaneous correlated activity at early developmental stages by increasing synaptogenesis and expression of the K+/Cl- co-transporter KCC2.

    PubMed

    Aguado, Fernando; Carmona, Maria A; Pozas, Esther; Aguiló, Agustín; Martínez-Guijarro, Francisco J; Alcantara, Soledad; Borrell, Victor; Yuste, Rafael; Ibañez, Carlos F; Soriano, Eduardo

    2003-04-01

    Spontaneous neural activity is a basic property of the developing brain, which regulates key developmental processes, including migration, neural differentiation and formation and refinement of connections. The mechanisms regulating spontaneous activity are not known. By using transgenic embryos that overexpress BDNF under the control of the nestin promoter, we show here that BDNF controls the emergence and robustness of spontaneous activity in embryonic hippocampal slices. Further, BDNF dramatically increases spontaneous co-active network activity, which is believed to synchronize gene expression and synaptogenesis in vast numbers of neurons. In fact, BDNF raises the spontaneous activity of E18 hippocampal neurons to levels that are typical of postnatal slices. We also show that BDNF overexpression increases the number of synapses at much earlier stages (E18) than those reported previously. Most of these synapses were GABAergic, and GABAergic interneurons showed hypertrophy and a 3-fold increase in GAD expression. Interestingly, whereas BDNF does not alter the expression of GABA and glutamate ionotropic receptors, it does raise the expression of the recently cloned K(+)/Cl(-) KCC2 co-transporter, which is responsible for the conversion of GABA responses from depolarizing to inhibitory, through the control of the Cl(-) potential. Together, results indicate that both the presynaptic and postsynaptic machineries of GABAergic circuits may be essential targets of BDNF actions to control spontaneous activity. The data indicate that BDNF is a potent regulator of spontaneous activity and co-active networks, which is a new level of regulation of neurotrophins. Given that BDNF itself is regulated by neuronal activity, we suggest that BDNF acts as a homeostatic factor controlling the emergence, complexity and networking properties of spontaneous networks.

  13. Molecular mechanisms and regulation of iron transport.

    PubMed

    Chung, Jayong; Wessling-Resnick, Marianne

    2003-04-01

    Iron homeostasis is primarily maintained through regulation of its transport. This review summarizes recent discoveries in the field of iron transport that have shed light on the molecular mechanisms of dietary iron uptake, pathways for iron efflux to and between peripheral tissues, proteins implicated in organellar transport of iron (particularly the mitochondrion), and novel regulators that have been proposed to control iron assimilation. The transport of both transferrin-bound and nontransferrin-bound iron to peripheral tissues is discussed. Finally, the regulation of iron transport is also considered at the molecular level, with posttranscriptional, transcriptional, and posttranslational control mechanisms being reviewed.

  14. Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells.

    PubMed

    Nabokina, Svetlana M; Ramos, Mel Brendan; Valle, Judith E; Said, Hamid M

    2015-05-01

    Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5'-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5'- and 3'-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides -178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

  15. Nuclear Transport Factors: Global Regulation of Mitosis

    PubMed Central

    Forbes, Douglass J.; Travesa, Anna; Nord, Matthew; Bernis, Cyril

    2015-01-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear transport receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator the γ-TuRC complex and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores towards the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic. PMID:25982429

  16. Regulation of hepatic transporters by xenobiotic receptors.

    PubMed

    Klaassen, C D; Slitt, A L

    2005-08-01

    Chemicals that increase expression of phase-I and -II biotransformation enzymes in liver, as well as enhance hepatic uptake and biliary excretion are often referred to as microsomal enzyme inducers (MEIs). Early studies suggested that drug metabolism might be coordinately regulated along with drug efflux from hepatocytes as a means for the liver to rid itself of foreign chemicals. Since then, the identification and characterization of nuclear receptors (NRs) has aided in understanding of how various MEIs enhance xeniobiotic uptake, biotransformation, and excretion. In addition, the NRs by which several classes of MEIs induce phase-I and -II drug metabolizing enzymes have been elucidated (i.e. AHR, CAR, PXR, PPARalpha, Nrf2). Several transporter families which mediate uptake of chemicals into liver and excretion of chemicals from liver into blood and/or bile have been cloned and identified. In general, the organic anion transporting polypeptide family (Oatps) along with Organic cation transporter 1 (Oct1) and Organic anion transporter 2 mediate uptake of a large number of xenobiotics from blood into liver. Conversely, Multidrug resistance proteins (Mdrs), Multidrug resistance-associated proteins (Mrps), and Breast cancer resistance protein (Bcrp) mediate efflux of xenobiotics from liver into bile or blood. Recent studies have demonstrated that MEIs increase expression of various Oatps, Mrps, and Mdrs in liver, and some occur via activation of nuclear receptors.

  17. Cholesterol-lowering activity of sesamin is associated with down-regulation on genes of sterol transporters involved in cholesterol absorption.

    PubMed

    Liang, Yin Tong; Chen, Jingnan; Jiao, Rui; Peng, Cheng; Zuo, Yuanyuan; Lei, Lin; Liu, Yuwei; Wang, Xiaobo; Ma, Ka Ying; Huang, Yu; Chen, Zhen-Yu

    2015-03-25

    Sesame seed is rich in sesamin. The present study was to (i) investigate the plasma cholesterol-lowering activity of dietary sesamin and (ii) examine the interaction of dietary sesamin with the gene expression of sterol transporters, enzymes, receptors, and proteins involved in cholesterol metabolism. Thirty hamsters were divided into three groups fed the control diet (CON) or one of two experimental diets containing 0.2% (SL) and 0.5% (SH) sesamin, respectively, for 6 weeks. Plasma total cholesterol (TC) levels in hamsters given the CON, SL, and SH diets were 6.62 ± 0.40, 5.32 ± 0.40, and 5.00 ± 0.44 mmol/L, respectively, indicating dietary sesamin could reduce plasma TC in a dose-dependent manner. Similarly, the excretion of total fecal neutral sterols was dose-dependently increased with the amounts of sesamin in diets (CON, 2.65 ± 0.57; SL, 4.30 ± 0.65; and SH, 5.84 ± 1.27 μmol/day). Addition of sesamin into diets was associated with down-regulation of mRNA of intestinal Niemann-Pick C1 like 1 protein (NPC1L1), acyl-CoA:cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP-binding cassette transporters subfamily G members 5 and 8 (ABCG5 and ABCG8). Results also showed that dietary sesamin could up-regulate hepatic cholesterol-7α-hydroxylase (CYP7A1), whereas it down-regulated hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and liver X receptor alpha (LXRα). It was concluded that the cholesterol-lowering activity of sesamin was mediated by promoting the fecal excretion of sterols and modulating the genes involved in cholesterol absorption and metabolism.

  18. Nuclear transport factors: global regulation of mitosis.

    PubMed

    Forbes, Douglass J; Travesa, Anna; Nord, Matthew S; Bernis, Cyril

    2015-08-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

  19. A Drosophila ABC Transporter Regulates Lifespan

    PubMed Central

    Huang, He; Lu-Bo, Ying; Haddad, Gabriel G.

    2014-01-01

    MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling. PMID:25474322

  20. Regulation of ABC Transporters at the Blood-Brain Barrier

    PubMed Central

    Miller, David S.

    2015-01-01

    ATP Binding Cassette (ABC) transporters at the blood-brain barrier function as ATP-driven xenobiotic efflux pumps and limit delivery of small molecule drugs to the brain. Here I review recent progress in understanding the regulation of the expression and transport activity of these transporters and comment on how this new information might aid in improving drug delivery to the brain. PMID:25670036

  1. Regulation of Intestinal Glucose Absorption by Ion Channels and Transporters

    PubMed Central

    Chen, Lihong; Tuo, Biguang; Dong, Hui

    2016-01-01

    The absorption of glucose is electrogenic in the small intestinal epithelium. The major route for the transport of dietary glucose from intestinal lumen into enterocytes is the Na+/glucose cotransporter (SGLT1), although glucose transporter type 2 (GLUT2) may also play a role. The membrane potential of small intestinal epithelial cells (IEC) is important to regulate the activity of SGLT1. The maintenance of membrane potential mainly depends on the activities of cation channels and transporters. While the importance of SGLT1 in glucose absorption has been systemically studied in detail, little is currently known about the regulation of SGLT1 activity by cation channels and transporters. A growing line of evidence suggests that cytosolic calcium ([Ca2+]cyt) can regulate the absorption of glucose by adjusting GLUT2 and SGLT1. Moreover, the absorption of glucose and homeostasis of Ca2+ in IEC are regulated by cation channels and transporters, such as Ca2+ channels, K+ channels, Na+/Ca2+ exchangers, and Na+/H+ exchangers. In this review, we consider the involvement of these cation channels and transporters in the regulation of glucose uptake in the small intestine. Modulation of them may be a potential strategy for the management of obesity and diabetes. PMID:26784222

  2. Regulation of Intestinal Glucose Absorption by Ion Channels and Transporters.

    PubMed

    Chen, Lihong; Tuo, Biguang; Dong, Hui

    2016-01-14

    The absorption of glucose is electrogenic in the small intestinal epithelium. The major route for the transport of dietary glucose from intestinal lumen into enterocytes is the Na⁺/glucose cotransporter (SGLT1), although glucose transporter type 2 (GLUT2) may also play a role. The membrane potential of small intestinal epithelial cells (IEC) is important to regulate the activity of SGLT1. The maintenance of membrane potential mainly depends on the activities of cation channels and transporters. While the importance of SGLT1 in glucose absorption has been systemically studied in detail, little is currently known about the regulation of SGLT1 activity by cation channels and transporters. A growing line of evidence suggests that cytosolic calcium ([Ca(2+)]cyt) can regulate the absorption of glucose by adjusting GLUT2 and SGLT1. Moreover, the absorption of glucose and homeostasis of Ca(2+) in IEC are regulated by cation channels and transporters, such as Ca(2+) channels, K⁺ channels, Na⁺/Ca(2+) exchangers, and Na⁺/H⁺ exchangers. In this review, we consider the involvement of these cation channels and transporters in the regulation of glucose uptake in the small intestine. Modulation of them may be a potential strategy for the management of obesity and diabetes.

  3. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    PubMed

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-09

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

  4. Regulation of cilium length and intraflagellar transport.

    PubMed

    Broekhuis, Joost R; Leong, Weng Y; Jansen, Gert

    2013-01-01

    Primary cilia are highly conserved sensory organelles that extend from the surface of almost all vertebrate cells. The importance of cilia is evident from their involvement in many diseases, called ciliopathies. Primary cilia contain a microtubular axoneme that is used as a railway for transport of both structural components and signaling proteins. This transport machinery is called intraflagellar transport (IFT). Cilia are dynamic organelles whose presence on the cell surface, morphology, length and function are highly regulated. It is clear that the IFT machinery plays an important role in this regulation. However, it is not clear how, for example environmental cues or cell fate decisions are relayed to modulate IFT and cilium morphology or function. This chapter presents an overview of molecules that have been shown to regulate cilium length and IFT. Several examples where signaling modulates IFT and cilium function are used to discuss the importance of these systems for the cell and for understanding of the etiology of ciliopathies.

  5. AMPK and substrate availability regulate creatine transport in cultured cardiomyocytes.

    PubMed

    Darrabie, Marcus D; Arciniegas, Antonio Jose Luis; Mishra, Rajashree; Bowles, Dawn E; Jacobs, Danny O; Santacruz, Lucia

    2011-05-01

    Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in V(max) that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.

  6. 76 FR 70220 - New Jersey Regulations on Transportation of Regulated Medical Waste

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-10

    ... Pipeline and Hazardous Materials Safety Administration New Jersey Regulations on Transportation of... Federal hazardous material transportation law preempts regulations of the New Jersey Department of...'' requirements in the Federal hazardous material transportation law or the Hazardous Materials Regulations...

  7. Laboratory Exercise on Active Transport.

    ERIC Educational Resources Information Center

    Stalheim-Smith, Ann; Fitch, Greg K.

    1985-01-01

    Describes a laboratory exercise which demonstrates qualitatively the specificity of the transport mechanism, including a consideration of the competitive inhibition, and the role of adenosine triphosphate (ATP) in active transport. The exercise, which can be completed in two to three hours by groups of four students, consistently produces reliable…

  8. Activated transport in AMTEC electrodes

    NASA Technical Reports Server (NTRS)

    Williams, R. M.; Jeffries-Nakamura, B.; Ryan, M. A.; Underwood, M. L.; O'Connor, D.; Kikkert, S.

    1992-01-01

    Transport of alkali, metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. The temperature dependence of the diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and preexponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process.

  9. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

    Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

    2016-01-01

    Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

  10. Bile acid-regulated peroxisome proliferator-activated receptor-α (PPARα) activity underlies circadian expression of intestinal peptide absorption transporter PepT1/Slc15a1.

    PubMed

    Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

    2014-09-05

    Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter.

  11. 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

  12. 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.

  13. Activated transport in AMTEC electrodes

    SciTech Connect

    Williams, R.M.; Jeffries-Nakamura, B.; Ryan, M.A.; Underwood, M.L.; O`Connor, D.; Kikkert, S.

    1992-07-01

    Transport of alkali metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant, reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. Little can be learned about the detailed mass transport process from the ac impedance or current voltage curves of an electrode at one set of operating parameters, because the transport process includes a number of important physical parameters that are not all uniquely determined by one experiment. The temperature dependence of diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and pre-exponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process. 17 refs.

  14. Activated transport in AMTEC electrodes

    SciTech Connect

    Williams, R.M.; Jeffries-Nakamura, B.; Ryan, M.A.; Underwood, M.L.; O'Connor, D.; Kikkert, S.

    1992-01-01

    Transport of alkali metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant, reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. Little can be learned about the detailed mass transport process from the ac impedance or current voltage curves of an electrode at one set of operating parameters, because the transport process includes a number of important physical parameters that are not all uniquely determined by one experiment. The temperature dependence of diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and pre-exponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process. 17 refs.

  15. Transcriptional regulation of plant phosphate transporters

    PubMed Central

    Muchhal, Umesh S.; Raghothama, K. G.

    1999-01-01

    Phosphorus is acquired by plant roots primarily via the high-affinity inorganic phosphate (Pi) transporters. The transcripts for Pi transporters are highly inducible upon Pi starvation, which also results in enhanced Pi uptake when Pi is resupplied. Using antibodies specific to one of the tomato Pi transporters (encoded by LePT1), we show that an increase in the LePT1 transcript under Pi starvation leads to a concurrent increase in the transporter protein, suggesting a transcriptional regulation for Pi acquisition. LePT1 protein accumulates rapidly in tomato roots in response to Pi starvation. The level of transporter protein accumulation depends on the Pi concentration in the medium, and it is reversible upon resupply of Pi. LePT1 protein accumulates all along the roots under Pi starvation and is localized primarily in the plasma membranes. These results clearly demonstrate that plants increase their capacity for Pi uptake during Pi starvation by synthesis of additional transporter molecules. PMID:10318976

  16. Energetics of active transport processes.

    PubMed

    Essig, A; Caplan, S R

    1968-12-01

    Discussions of active transport usually assume stoichiometry between the rate of transport J(+) and the metabolic rate J(r). However, the observation of a linear relationship between J(+) and J(r) does not imply a stoichiometric relationship, i.e., complete coupling. Since coupling may possibly be incomplete, we examine systems of an arbitrary degree of coupling q, regarding stoichiometry as a limiting case. We consider a sodium pump, with J(+) and J(r) linear functions of the electrochemical potential difference, -X(+), and the chemical affinity of the metabolic driving reaction, A. The affinity is well defined even for various complex reaction pathways. Incorporation of a series barrier and a parallel leak does not affect the linearity of the composite observable system. The affinity of some region of the metabolic chain may be maintained constant, either by large pools of reactants or by regulation. If so, this affinity can be evaluated by two independent methods. Sodium transport is conveniently characterized by the open-circuit potential (Deltapsi)(I=0) and the natural limits, level flow (J(+))(X+=0), and static head X(0) (+) = (X(+))(J+=0). With high degrees of coupling -X(0) (+)/F approaches the electromotive force E(Na) (Ussing); -X(0) (+)/F cannot be identified with ((RT/F) ln f)(X+=0), where f is the flux ratio. The efficiency eta = -J(+)X(+)/J(r)A is of significance only when appreciable energy is being converted from one form to another. When either J(+) or -X(+) is small eta is low; the significant parameters are then the efficacies epsilon(J+) = J(+)/J(r)A and epsilon(X+) = -X(+)/J(r)A, respectively maximal at level flow and static head. Leak increases both J(+) and epsilon(J+) for isotonic saline reabsorption, but diminishes -X(0) (+) and epsilon(Xfemale symbol). Electrical resistance reflects both passive parameters and metabolism. Various fundamental relations are preserved despite coupling of passive ion and water flows.

  17. Regulation & Development of Membrane Transport Processes.

    DTIC Science & Technology

    1985-05-15

    Laboratory, Oak Ridge, Tennessee DAVID W. PLMPLIN Department of Anatomy, University of Maryland School of Medicine, Baltimore, Maryland MARILYN D. RESH...Muscle 265 Douglas M. Fambrough, Barry A. Wolitzky, and David W. Pumplin Index 283 REGULATION AND DEVELOPMENT OF MEMBRANE TRANSPORT PROCESSES 77, II PART 1...243 (Cell Physiol. 12). C 124-C132. 16. Huang. C. C.. Tsai. C. M.. and Canellakis, E. S. (1973) Bochiom. Biophys. Acta. 332, 59-68. 17. Hume . S. and

  18. Increased Mitochondrial Electron Transport Chain Activity at Complex I Is Regulated by N-Acetylcysteine in Lymphocytes of Patients with Systemic Lupus Erythematosus

    PubMed Central

    Doherty, Edward; Oaks, Zachary

    2014-01-01

    Abstract Aims: Systemic lupus erythematosus (SLE) patients' peripheral blood lymphocytes (PBL) show mitochondrial dysfunction and oxidative stress. To determine the electrochemical bases of mitochondrial dysfunction, we measured electron transport chain (ETC) activity and its regulation by N-acetylcysteine (NAC) that reversed glutathione depletion and improved disease activity in SLE. ETC activity was assessed in PBL of 69 SLE patients and 37 healthy donors. Negatively isolated T cells were examined in 7 SLE patients, 11 healthy donors, and 10 nonlupus inflammatory arthritis (IA) donors. Results: O2 consumption (in nmol/ml/min) by lupus PBL was increased at baseline (SLE: 2.492±0.196, control: 2.137±0.153; p=0.027) and with complex IV substrates (SLE: 7.722±0.419, control: 7.006±0.505; p=0.028). SLE PBL consumed more O2 upon in-chamber T-cell activation (p=0.012). After overnight T-cell stimulation, ETC activity of SLE PBL was 2.27-fold increased through complex I (SLE: 1.606±0.273, control: 0.709±0.169; p=0.001) and, to a lesser extent, through complex IV. Likewise, complex I activity was elevated in negatively isolated “untouched” T cells of SLE patients (1.816±0.180) relative to healthy controls (0.917±0.094; p=0.0003) and IA disease controls studied in parallel (1.057±0.199; p=0.0308). NAC diminished O2 consumption through complex I and H2O2 levels both in SLE and in control PBL. Innovation: O2 consumption was found to be increased in SLE patients' PBL relative to control subjects evaluated in parallel. ETC complex I is identified as the main source of oxidative stress in SLE. Conclusions: Lupus PBL exhibit increased O2 consumption through mitochondrial ETC complex I that is inhibited by NAC, which may have therapeutic efficacy through reducing oxidative stress in SLE. Antioxid. Redox Signal. 21, 56–65. PMID:24673154

  19. Sediment transport in two mediterranean regulated rivers.

    PubMed

    Lobera, G; Batalla, R J; Vericat, D; López-Tarazón, J A; Tena, A

    2016-01-01

    Mediterranean climate is characterized by highly irregular rainfall patterns with marked differences between wet and dry seasons which lead to highly variable hydrological fluvial regimes. As a result, and in order to ensure water availability and reduce its temporal variability, a high number of large dams were built during the 20th century (more than 3500 located in Mediterranean rivers). Dams modify the flow regime but also interrupt the continuity of sediment transfer along the river network, thereby changing its functioning as an ecosystem. Within this context, the present paper aims to assess the suspended sediment loads and dynamics of two climatically contrasting Mediterranean regulated rivers (i.e. the Ésera and Siurana) during a 2-yr period. Key findings indicate that floods were responsible for 92% of the total suspended sediment load in the River Siurana, while this percentage falls to 70% for the Ésera, indicating the importance of baseflows on sediment transport in this river. This fact is related to the high sediment availability, with the Ésera acting as a non-supply-limited catchment due to the high productivity of the sources (i.e. badlands). In contrast, the Siurana can be considered a supply-limited system due to its low geomorphic activity and reduced sediment availability, with suspended sediment concentration remaining low even for high magnitude flood events. Reservoirs in both rivers reduce sediment load up to 90%, although total runoff is only reduced in the case of the River Ésera. A remarkable fact is the change of the hydrological character of the River Ésera downstream for the dam, shifting from a humid mountainous river regime to a quasi-invariable pattern, whereas the Siurana experiences the opposite effect, changing from a flashy Mediterranean river to a more constant flow regime below the dam.

  20. Regulation of amino acid metabolic enzymes and transporters in plants.

    PubMed

    Pratelli, Réjane; Pilot, Guillaume

    2014-10-01

    Amino acids play several critical roles in plants, from providing the building blocks of proteins to being essential metabolites interacting with many branches of metabolism. They are also important molecules that shuttle organic nitrogen through the plant. Because of this central role in nitrogen metabolism, amino acid biosynthesis, degradation, and transport are tightly regulated to meet demand in response to nitrogen and carbon availability. While much is known about the feedback regulation of the branched biosynthesis pathways by the amino acids themselves, the regulation mechanisms at the transcriptional, post-transcriptional, and protein levels remain to be identified. This review focuses mainly on the current state of our understanding of the regulation of the enzymes and transporters at the transcript level. Current results describing the effect of transcription factors and protein modifications lead to a fragmental picture that hints at multiple, complex levels of regulation that control and coordinate transport and enzyme activities. It also appears that amino acid metabolism, amino acid transport, and stress signal integration can influence each other in a so-far unpredictable fashion.

  1. Factors regulating microglia activation

    PubMed Central

    Kierdorf, Katrin; Prinz, Marco

    2013-01-01

    Microglia are resident macrophages of the central nervous system (CNS) that display high functional similarities to other tissue macrophages. However, it is especially important to create and maintain an intact tissue homeostasis to support the neuronal cells, which are very sensitive even to minor changes in their environment. The transition from the “resting” but surveying microglial phenotype to an activated stage is tightly regulated by several intrinsic (e.g., Runx-1, Irf8, and Pu.1) and extrinsic factors (e.g., CD200, CX3CR1, and TREM2). Under physiological conditions, minor changes of those factors are sufficient to cause fatal dysregulation of microglial cell homeostasis and result in severe CNS pathologies. In this review, we discuss recent achievements that gave new insights into mechanisms that ensure microglia quiescence. PMID:23630462

  2. Rab proteins: The key regulators of intracellular vesicle transport

    SciTech Connect

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

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

  3. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  4. Rab proteins: the key regulators of intracellular vesicle transport.

    PubMed

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes.

  5. Regulation of Photosynthetic Electron Transport and Photoinhibition

    PubMed Central

    Roach, Thomas; Krieger-Liszkay, Anja Krieger

    2014-01-01

    Photosynthetic organisms and isolated photosystems are of interest for technical applications. In nature, photosynthetic electron transport has to work efficiently in contrasting environments such as shade and full sunlight at noon. Photosynthetic electron transport is regulated on many levels, starting with the energy transfer processes in antenna and ending with how reducing power is ultimately partitioned. This review starts by explaining how light energy can be dissipated or distributed by the various mechanisms of non-photochemical quenching, including thermal dissipation and state transitions, and how these processes influence photoinhibition of photosystem II (PSII). Furthermore, we will highlight the importance of the various alternative electron transport pathways, including the use of oxygen as the terminal electron acceptor and cyclic flow around photosystem I (PSI), the latter which seem particularly relevant to preventing photoinhibition of photosystem I. The control of excitation pressure in combination with the partitioning of reducing power influences the light-dependent formation of reactive oxygen species in PSII and in PSI, which may be a very important consideration to any artificial photosynthetic system or technical device using photosynthetic organisms. PMID:24678670

  6. Bursts of Active Transport in Living Cells

    NASA Astrophysics Data System (ADS)

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-01

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  7. Bursts of active transport in living cells.

    PubMed

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-15

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  8. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

    2012-01-01

    Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

  9. Regulation and roles of bicarbonate transporters in cancer.

    PubMed

    Gorbatenko, Andrej; Olesen, Christina W; Boedtkjer, Ebbe; Pedersen, Stine F

    2014-01-01

    A unifying feature of solid tumors is a markedly altered pH profile compared to normal tissues. This reflects that solid tumors, despite completely different origins, often share several phenotypic properties with implications for intra- and extracellular pH. These include: a metabolic shift in most cancer cells toward more acid-producing pathways, reflecting both oncogenic signaling and the development of hypoxia in poorly perfused regions of the tumors; the poorly perfused and often highly dense tumor microenvironment, reducing the diffusive flux of acid equivalents compared to that in normal tissues; and the markedly altered regulation of the expression and activity of pH-regulatory transport proteins in cancer cells. While some of these properties of tumors have been well described in recent years, the great majority of the research in this clinically important area has focused on proton transport, in particular via the Na(+)/H(+) exchanger 1 (SLC9A1, NHE1) and various H(+) ATPases. We have, however, recently demonstrated that at least under some conditions, including in vitro models of HER2 positive breast cancer, and measurements obtained directly in freshly dissected human mammary carcinomas, bicarbonate transporters such as the electroneutral Na(+), HCO(-) 3 cotransporter (SLC4A7, NBCn1), are upregulated and play central roles in pH regulation. In this review, we summarize and discuss the current knowledge regarding the regulation and roles of bicarbonate transporters in cancer. Furthermore, we present new analyses of publicly available expression data demonstrating widely altered expression levels of SLC4- and SLC26 family transporters in breast-, lung-, and colon cancer patients, and we hypothesize that bicarbonate transporter dysregulation may have both diagnostic and therapeutic potential in cancer treatment.

  10. Regulation and roles of bicarbonate transporters in cancer

    PubMed Central

    Gorbatenko, Andrej; Olesen, Christina W.; Boedtkjer, Ebbe; Pedersen, Stine F.

    2014-01-01

    A unifying feature of solid tumors is a markedly altered pH profile compared to normal tissues. This reflects that solid tumors, despite completely different origins, often share several phenotypic properties with implications for intra- and extracellular pH. These include: a metabolic shift in most cancer cells toward more acid-producing pathways, reflecting both oncogenic signaling and the development of hypoxia in poorly perfused regions of the tumors; the poorly perfused and often highly dense tumor microenvironment, reducing the diffusive flux of acid equivalents compared to that in normal tissues; and the markedly altered regulation of the expression and activity of pH-regulatory transport proteins in cancer cells. While some of these properties of tumors have been well described in recent years, the great majority of the research in this clinically important area has focused on proton transport, in particular via the Na+/H+ exchanger 1 (SLC9A1, NHE1) and various H+ ATPases. We have, however, recently demonstrated that at least under some conditions, including in vitro models of HER2 positive breast cancer, and measurements obtained directly in freshly dissected human mammary carcinomas, bicarbonate transporters such as the electroneutral Na+, HCO−3 cotransporter (SLC4A7, NBCn1), are upregulated and play central roles in pH regulation. In this review, we summarize and discuss the current knowledge regarding the regulation and roles of bicarbonate transporters in cancer. Furthermore, we present new analyses of publicly available expression data demonstrating widely altered expression levels of SLC4- and SLC26 family transporters in breast-, lung-, and colon cancer patients, and we hypothesize that bicarbonate transporter dysregulation may have both diagnostic and therapeutic potential in cancer treatment. PMID:24795638

  11. Harmonization - Two Years' of Transportation Regulation Lessons Learned

    SciTech Connect

    Colborn, K.

    2007-07-01

    The U.S. Department of Transportation issued modifications to the Hazardous Materials Regulations in October, 2004 as part of an ongoing effort to 'harmonize' U.S. regulations with those of the International Atomic Energy Agency. The harmonization effort had several predictable effects on low level radioactive materials shipment that were anticipated even prior to their implementation. However, after two years' experience with the new regulations, transporters have identified several effects on transportation which were not entirely apparent when the regulations were first implemented. This paper presents several case studies in the transportation of low level radioactive materials since the harmonization rules took effect. In each case, an analysis of the challenge posed by the regulatory revision is provided. In some cases, more than one strategy for compliance was considered, and the advantages and disadvantages of each are discussed. In several cases, regulatory interpretations were sought and obtained, and these are presented to clarify the legitimacy of the compliance approach. The presentation of interpretations will be accompanied by reports of clarifying discussions with the U.S. DOT about the interpretation and scope of the regulatory change. Specific transportation issues raised by the revised hazardous materials regulations are reviewed, including: The new definition of radioactive material in accordance with isotope-specific concentration and total activity limits. The new hazardous materials regulations (HMR) created a new definition for radioactive material. A case study is presented for soils contaminated with low levels of Th-230. These soils had been being shipped for years as exempt material under the old 2,000 pCi/g concentration limit. Under the new HMR, these same soils were radioactive material. Further, in rail-car quantities their activity exceeded an A2 value, so shipment of the material in gondolas appeared to require an IP-2 package

  12. Substrate-dependent regulation of ascorbate transport in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-02-26

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na{sup +}-dependent L-ascorbate transporter in the plasma membrane. The present study examined the effects of ascorbate deprivation and supplementation on the activity of the transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-({sup 14}C)ascorbate for 1 minute at 37C. They observed that the maximal uptake rate, V{sub max}, rapidly (<3 hours) increased when cultured cells were deprived of L-ascorbate. There was no change in the apparent affinity (K{sub m}) of the transport system for ascorbate. V{sub max} returned to normal following addition of L-ascorbate, but not D-isoascorbate, to the medium. The authors conclude that astrocytes adapt ascorbate transport rates to changes in substrate availability. Furthermore, the data suggest that the transport system located in the astroglial plasma membrane regulates intracellular ascorbate concentration, because changes in transport rate may compensate for regional differences and temporal fluctuations in extracellular ascorbate levels.

  13. Cholesterol transport and regulation in the mammary gland.

    PubMed

    Ontsouka, Edgar C; Albrecht, Christiane

    2014-03-01

    The milk-producing alveolar epithelial cells secrete milk that remains after birth the principal source of nutrients for neonates. Milk secretion and composition are highly regulated processes via integrated actions of hormones and local factors which involve specific receptors and downstream signal transduction pathways. Overall milk composition is similar among mammalian species, although the content of individual constituents such as lipids may significantly differ from one species to another. The milk lipid fraction is essentially composed of triglycerides, which represent more than 95 % of the total lipids in human and commercialized bovine milk. Though sterols, including cholesterol, which is the major milk sterol, represent less than 0.5 % of the total milk lipid fraction, they are of key importance for several biological processes. Cholesterol is required for the formation of biological membranes especially in rapidly growing organisms, and for the synthesis of sterol-based compounds. Cholesterol found in milk originates predominantly from blood uptake and, to a certain extent, from local synthesis in the mammary tissue. The present review summarizes current knowledge on cellular mechanisms and regulatory processes determining intra- and transcellular cholesterol transport in the mammary gland. Cholesterol exchanges between the blood, the mammary alveolar cells and the milk, and the likely role of active cholesterol transporters in these processes are discussed. In this context, the hormonal regulation and signal transduction pathways promoting active cholesterol transport as well as potential regulatory crosstalks are highlighted.

  14. A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity.

    PubMed

    Webb, Bradley A; White, Katharine A; Grillo-Hill, Bree K; Schönichen, André; Choi, Changhoon; Barber, Diane L

    2016-11-11

    The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHi in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHi However, the molecular mechanism for pH-dependent NHE1 activity remains incompletely resolved. We report that an evolutionarily conserved cluster of histidine residues located in the C-terminal cytoplasmic domain between two phosphatidylinositol 4,5-bisphosphate binding sites (PI(4,5)P2) of NHE1 confers pH-dependent PI(4,5)P2 binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P2 binding at pH 7.0 compared with pH 7.5. However, pH-sensitive binding is abolished by substitutions of the His-rich cluster to arginine (RXXR3) or alanine (AXXA3), mimicking protonated and neutral histidine residues, respectively, and the RXXR3 mutant had significantly greater PI(4,5)P2 binding than AXXA3. When expressed in cells, NHE1 activity and pHi were significantly increased with NHE1-RXXR3 and decreased with NHE1-AXXA3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-RXXR3 had significantly more contractile actin filaments and focal adhesions compared with fibroblasts expressing wild type NHE1, consistent with increased pHi enabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P2 binding regulating NHE1 activity and suggest that the evolutionarily conserved cluster of four histidines in the proximal cytoplasmic domain of NHE1 may constitute a proton modifier site. Moreover, a constitutively activated NHE1-RXXR3 mutant is a new tool that will be useful for studying how increased pHi contributes to cell behaviors, most notably the biology of cancer cells.

  15. Common folds and transport mechanisms of secondary active transporters.

    PubMed

    Shi, Yigong

    2013-01-01

    Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. Transporters of different functional families with little sequence similarity have repeatedly been found to exhibit similar folds, exemplified by the MFS, LeuT, and NhaA folds. Observations of multiple conformational states of the same transporter, represented by the LeuT superfamily members Mhp1, AdiC, vSGLT, and LeuT, led to proposals that structural changes are associated with substrate binding and transport. Despite recent biochemical and structural advances, our understanding of substrate recognition and energy coupling is rather preliminary. This review focuses on the common folds and shared transport mechanisms of secondary active transporters. Available structural information generally supports the alternating access model for substrate transport, with variations and extensions made by emerging structural, biochemical, and computational evidence.

  16. Single Molecule Analysis of Serotonin Transporter Regulation Using Quantum Dots

    NASA Astrophysics Data System (ADS)

    Chang, Jerry; Tomlinson, Ian; Warnement, Michael; Ustione, Alessandro; Carneiro, Ana; Piston, David; Blakely, Randy; Rosenthal, Sandra

    2011-03-01

    For the first time, we implement a novel, single molecule approach to define the localization and mobility of the brain's major target of widely prescribed antidepressant medications, the serotonin transporter (SERT). SERT labeled with single quantum dot (Qdot) revealed unsuspected features of transporter mobility with cholesterol-enriched membrane microdomains (often referred to as ``lipid rafts'') and cytoskeleton network linked to transporter activation. We document two pools of surface SERT proteins defined by their lateral mobility, one that exhibits relatively free diffusion in the plasma membrane and a second that displays significantly restricted mobility and localizes to cholesterol-enriched microdomains. Diffusion model prediction and instantaneous velocity analysis indicated that stimuli that act through p38 MAPK-dependent signaling pathways to activate SERT trigger rapid SERT movements within membrane microdomains. Cytoskeleton disruption showed that SERT lateral mobility behaves a membrane raft-constrained, cytoskeleton-associated manner. Our results identify an unsuspected aspect of neurotransmitter transporter regulation that we propose reflects the dissociation of inhibitory, SERT-associated cytoskeletal anchors.

  17. Political activity for physical activity: health advocacy for active transport

    PubMed Central

    2011-01-01

    Effective health advocacy is a priority for efforts to increase population participation in physical activity. Local councils are an important audience for this advocacy. The aim of the current study was to describe features of advocacy for active transport via submissions to city council annual plans in New Zealand, and the impact of an information sheet to encourage the health sector to be involved in this process. Written submissions to city council's annual consultation process were requested for 16 city councils over the period of three years (2007/08, 2008/09, and 2009/10). Submissions were reviewed and categories of responses were created. An advocacy information sheet encouraging health sector participation and summarising some of the evidence-base related to physical activity, active transport and health was released just prior to the 2009/10 submission time. Over the period of the study, city councils received 47,392 submissions, 17% of which were related to active transport. Most submissions came from city residents, with a small proportion (2%) from the health sector. The largest category of submissions was in support of pedestrian and cycling infrastructure, design and maintenance of facilities and additional features to support use of these transport modes. Health arguments featured prominently in justifications for active transport initiatives, including concerns about injury risk, obesity, physical inactivity, personal safety and facilities for people with disabilities. There was evidence that the information sheet was utilised by some health sector submitters (12.5%), providing tentative support for initiatives of this nature. In conclusion, the study provides novel information about the current nature of health advocacy for active transport and informs future advocacy efforts about areas for emphasis, such as health benefits of active transport, and potential alliances with other sectors such as environmental sustainability, transport and urban

  18. Regulation of inflammasome activation.

    PubMed

    Man, Si Ming; Kanneganti, Thirumala-Devi

    2015-05-01

    Inflammasome biology is one of the most exciting and rapidly growing areas in immunology. Over the past 10 years, inflammasomes have been recognized for their roles in the host defense against invading pathogens and in the development of cancer, auto-inflammatory, metabolic, and neurodegenerative diseases. Assembly of an inflammasome complex requires cytosolic sensing of pathogen-associated molecular patterns or danger-associated molecular patterns by a nucleotide-binding domain and leucine-rich repeat receptor (NLR) or absent in melanoma 2 (AIM2)-like receptors (ALR). NLRs and ALRs engage caspase-1, in most cases requiring the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC), to catalyze proteolytic cleavage of pro-interleukin-1β (pro-IL-1β) and pro-IL-18 and drive pyroptosis. Recent studies indicate that caspase-8, caspase-11, IL-1R-associated kinases (IRAK), and receptor-interacting protein (RIP) kinases contribute to inflammasome functions. In addition, post-translational modifications, including ubiquitination, deubiquitination, phosphorylation, and degradation control almost every aspect of inflammasome activities. Genetic studies indicate that mutations in NLRP1, NLRP3, NLRC4, and AIM2 are linked with the development of auto-inflammatory diseases, enterocolitis, and cancer. Overall, these findings transform our understanding of the basic biology and clinical relevance of inflammasomes. In this review, we provide an overview of the latest development of inflammasome research and discuss how inflammasome activities govern health and disease.

  19. 78 FR 75672 - New Jersey Regulations on Transportation of Regulated Medical Waste

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-12

    ... Pipeline and Hazardous Materials Safety Administration New Jersey Regulations on Transportation of... material transportation law, 49 U.S.C. 5101 et seq., and the Hazardous Materials Regulations (HMR), 49 CFR parts 171-180. Modes Affected: All transportation modes SUMMARY: Federal hazardous...

  20. Intrinsic and extrinsic negative regulators of nuclear protein transport processes

    PubMed Central

    Sekimoto, Toshihiro; Yoneda, Yoshihiro

    2012-01-01

    The nuclear–cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclear transport processes have been observed in stressed cells, which would change gene expressions. Some viruses interfere with nuclear transport in host cells to evade immune defense. Moreover, certain transport factors negatively regulate nuclear protein transport in cells. Understanding the regulatory mechanisms of nuclear–cytoplasmic trafficking not only provides important information about cellular processes, but also is of use for developing specific inhibitors for transport pathways. PMID:22672474

  1. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  2. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-03-30

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons.

  3. Organic anion transporting polypeptides (OATPs): regulation of expression and function.

    PubMed

    Svoboda, Martin; Riha, Juliane; Wlcek, Katrin; Jaeger, Walter; Thalhammer, Theresia

    2011-02-01

    Eleven members of the human organic anion transporter (OATP) family (grouped into six families) facilitate the Na(+)- independent transmembrane transport of various endo- and xenobiotics (bile acids, bilirubin, steroid hormone conjugates, thyroid hormones, prostaglandins, clinically used drugs, and toxins). OATPs are 12-transmembrane glycoproteins (643-722 amino acids) and contain many conserved structural features, for example, eleven cysteines in the large extracellular loop 5. They are important for proper transport, for which translocation of substrates through a central, positively-charged pore in a rocker-switch-type mechanism has been proposed. Although OATPs are expressed in various cells and tissues, some members show a more restricted pattern (well-studied OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis). In cancer, the distribution pattern is no longer maintained, and OATPs, like OATP1B3, become upregulated in malignant tissues (colon, breast, prostate). Studies in cell lines and animal models further revealed that the expression of OATPs is regulated in a cell- and tissue-specific way by cytokines and activation of nuclear receptors (LXR, FXR, PXR, CAR, HNF4). Also epigenetic mechanisms and postranslational modifications influence their expression and function. Therefore, changes in the expression of OATPs under pathological conditions will influence transport processes causing an altered accumulation of OATP substrates in cells of excretory organs (intestine, liver, kidney) and on various blood/organ barriers (such as brain, testis, placenta). For drugs, this may result in increased toxicity and adverse drug reactions. Therefore, it is important to improve the knowledge on the regulation and function of individual OATPs, and to apply it for therapeutic considerations.

  4. Regulation of divalent metal transporter-1 by serine phosphorylation

    PubMed Central

    Seo, Young Ah; Kumara, Ruvin; Wetli, Herbert; Wessling-Resnick, Marianne

    2016-01-01

    Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake. PMID:27681840

  5. Calcium transport in strongly calcifying laying birds: mechanisms and regulation.

    PubMed

    Bar, Arie

    2009-04-01

    Birds that lay long clutches (series of eggs laid sequentially before a "pause day"), among them the high-producing, strongly-calcifying Gallus gallus domesticus (domestic hen) and Coturnix coturnix japonica (Japanese quail), transfer about 10% of their total body calcium daily. They appear, therefore, to be the most efficient calcium-transporters among vertebrates. Such intensive transport imposes severe demands on ionic calcium (Ca2+) homeostasis, and activates at least two extremely effective mechanisms for Ca2+ transfer from food and bone to the eggshell. This review focuses on the development, action and regulation of the mechanisms associated with paracellular and transcellular Ca2+ transport in the intestine and the eggshell gland (ESG); it also considers some of the proteins (calbindin, Ca2+ATPase, Na+/Ca2+ exchange, epithelial calcium channels (TRPVs), osteopontin and carbonic anhydrase (CA) associated with this phenomenon. Calbindins are discussed in some detail, as they appear to be a major component of the transcellular transport system, and as only they have been studied extensively in birds. The review aims to gather old and new knowledge, which could form a conceptual basis, albeit not a completely accepted one, for our understanding of the mechanisms associated with this phenomenon. In the intestine, the transcellular pathway appears to compensate for low Ca2+ intake, but in birds fed adequate calcium the major drive for calcium absorption remains the electrochemical potential difference (ECPD) that facilitates paracellular transport. However, the mechanisms involved in Ca2+ transport into the ESG lumen are not yet established. In the ESG, the presence of Ca2+-ATPase and calbindin--two components of the transcellular transport pathway--and the apparently uphill transport of Ca2+ support the idea that Ca2+ is transported via the transcellular pathway. However, the positive (plasma with respect to mucosa) electrical potential difference (EPD) in the

  6. Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we examined the role of protein phosphorylation & dephosphorylation in the transport properties of the wheat root malate efflux transporter underlying Al resistance, TaALMT1. Preincubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurospo...

  7. Multiple-motor based transport and its regulation by Tau

    PubMed Central

    Vershinin, Michael; Carter, Brian C.; Razafsky, David S.; King, Stephen J.; Gross, Steven P.

    2007-01-01

    Motor-based intracellular transport and its regulation are crucial to the functioning of a cell. Disruption of transport is linked to Alzheimer's and other neurodegenerative diseases. However, many fundamental aspects of transport are poorly understood. An important issue is how cells achieve and regulate efficient long-distance transport. Mounting evidence suggests that many in vivo cargoes are transported along microtubules by more than one motor, but we do not know how multiple motors work together or can be regulated. Here we first show that multiple kinesin motors, working in conjunction, can achieve very long distance transport and apply significantly larger forces without the need of additional factors. We then demonstrate in vitro that the important microtubule-associated protein, tau, regulates the number of engaged kinesin motors per cargo via its local concentration on microtubules. This function of tau provides a previously unappreciated mechanism to regulate transport. By reducing motor reattachment rates, tau affects cargo travel distance, motive force, and cargo dispersal. We also show that different isoforms of tau, at concentrations similar to those in cells, have dramatically different potency. These results provide a well defined mechanism for how altered tau isoform levels could impair transport and thereby lead to neurodegeneration without the need of any other pathway. PMID:17190808

  8. SNAT2 and LAT1 transporter abundance is developmentally regulated in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we demonstrated that the insulin and amino acid–induced activation of the mammalian target of rapamycin complex 1 (mTORC1), is developmentally regulated in neonatal pigs. Recent studies have indicated an important role of the System A transporters (SNAT2 and SLC1A5) and the L transporter...

  9. P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters*

    PubMed Central

    Jiménez, Esperanza; Zafra, Francisco; Pérez-Sen, Raquel; Delicado, Esmerilda G.; Miras-Portugal, Maria Teresa; Aragón, Carmen; López-Corcuera, Beatriz

    2011-01-01

    The sodium- and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5′-diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y1 and P2Y13 because the effects are partially reversed by the specific antagonists N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate and pyridoxal-5′-phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y12 receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNA-mediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca2+ mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y1 receptor. Sensitivity to 2-methylthioadenosine 5′-diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization. PMID:21245148

  10. Reprint of "Nuclear transport factors: global regulation of mitosis".

    PubMed

    Forbes, Douglass J; Travesa, Anna; Nord, Matthew S; Bernis, Cyril

    2015-06-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator – the γ-TuRC complex – and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

  11. Adrenocortical Activity and Emotion Regulation.

    ERIC Educational Resources Information Center

    Stansbury, Kathy; Gunnar, Megan R.

    1994-01-01

    This essay argues that the activity of the hypothalamic-pituitary-adrenocortical (HPA) system does not appear to be related to emotion regulation processes in children, although individual differences in emotion processes related to negative emotion temperaments appear to be associated with individual differences in HPA reactivity among normally…

  12. Regulation of Glutamine Transport in Escherichia coli.

    PubMed Central

    Willis, R C; Iwata, K K; Furlong, C E

    1975-01-01

    The formation of the high-affinity (Km equal to 0.2 muM) L-glutamine transport system of Escherichia coli strain 7 (Lin) appears to be subject to the same major control as the glutamine synthetase (EC 6.3.1.2) of this gram-negative organism. Culture of cells under nitrogen-limited conditions provides maximum derepression of both the glutamine synthetase and the glutamine transport system. Nutritional conditions providing a rich supply of ammonium salts or available sources of nitrogen, i.e., conditions which repress the formation of glutamine synthetase, provide three- and 20-fold repression, respectively, of the glutamine transport system. Culture of cells with glutamine supplements of 2 mM does not increase the repression of high-affinity glutamine transport system beyond the level observed in the absence of glutamine. A second kinetically distinct low-affinity component of glutamine. A second kinetically distinct low-affinity component of glutamine uptake is observed in cells cultured with a glutamine-depleted nutrient broth. This second component is associated with the appearance of glutaminase A (EC 3.5.1.2) and asparaginase I (EC 3.5.1.1), a periplasmic enzyme. Parallel changes were observed in the levels of the high-affinity glutamine transport system and the glutamine synthetase when cells were cultured with the carbon sources: glucose, glycerol, or succinate. PMID:238938

  13. Regulation of expression of the arabinose and glucose transporter genes in the thermophilic archaeon Sulfolobus solfataricus.

    PubMed

    Lubelska, Joanna M; Jonuscheit, Melanie; Schleper, Christa; Albers, Sonja-Verena; Driessen, Arnold J M

    2006-10-01

    Sugar uptake in Sulfolobus solfataricus, a thermoacidophilic archaeon, occurs through high-affinity binding of protein-dependent ABC transporters. We have investigated the expression patterns of two sugar transport operons, that is, the glucose and arabinose transporters. Analysis of the araS promoter activity, and the mRNA and protein levels in S. solfataricus cells grown on different carbon sources showed that expression of the arabinose transporter gene cluster is highly regulated and dependent on the presence of arabinose in the medium. Glucose in the growth medium repressed the expression of the arabinose transport genes. By means of primer extension, the transcriptional start site for the arabinose operon was mapped. Interestingly, expression of the arabinose transporter is down-regulated by addition of a selective set of amino acids to the medium. Expression of the glucose transporter genes appeared constitutive. These data confirm the earlier observation of a catabolite repression-like system in S. solfataricus.

  14. Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation*

    PubMed Central

    Yang, Liling; Wang, Shuxing; Sung, Backil; Lim, Grewo; Mao, Jianren

    2008-01-01

    Glutamate transporters play a crucial role in physiological glutamate homeostasis, neurotoxicity, and glutamatergic regulation of opioid tolerance. However, how the glutamate transporter turnover is regulated remains poorly understood. Here we show that chronic morphine exposure induced posttranscriptional down-regulation of the glutamate transporter EAAC1 in C6 glioma cells with a concurrent decrease in glutamate uptake and increase in proteasome activity, which were blocked by the selective proteasome inhibitor MG-132 or lactacystin but not the lysosomal inhibitor chloroquin. At the cellular level, chronic morphine induced the PTEN (phosphatase and tensin homolog deleted on chromosome Ten)-mediated up-regulation of the ubiquitin E3 ligase Nedd4 via cAMP/protein kinase A signaling, leading to EAAC1 ubiquitination and proteasomal degradation. Either Nedd4 or PTEN knockdown with small interfering RNA prevented the morphine-induced EAAC1 degradation and decreased glutamate uptake. These data indicate that cAMP/protein kinase A signaling serves as an intracellular regulator upstream to the activation of the PTEN/Nedd4-mediated ubiquitin-proteasome system activity that is critical for glutamate transporter turnover. Under an in vivo condition, chronic morphine exposure also induced posttranscriptional down-regulation of the glutamate transporter EAAC1, which was prevented by MG-132, and transcriptional up-regulation of PTEN and Nedd4 within the spinal cord dorsal horn. Thus, inhibition of the ubiquitin-proteasome-mediated glutamate transporter degradation may be an important mechanism for preventing glutamate overexcitation and may offer a new strategy for treating certain neurological disorders and improving opioid therapy in chronic pain management. PMID:18539596

  15. AXONAL TRANSPORT: CARGO-SPECIFIC MECHANISMS OF MOTILITY AND REGULATION

    PubMed Central

    Maday, Sandra; Twelvetrees, Alison E.; Moughamian, Armen J.; Holzbaur, Erika L. F.

    2014-01-01

    Axonal transport is essential for neuronal function, and many neurodevelopmental and neurodegenerative diseases result from mutations in the axonal transport machinery. Anterograde transport supplies distal axons with newly synthesized proteins and lipids, including synaptic components required to maintain presynaptic activity. Retrograde transport is required to maintain homeostasis by removing aging proteins and organelles from the distal axon for degradation and recycling of components. Retrograde axonal transport also plays a major role in neurotrophic and injury response signaling. This review provides an overview of the axonal transport pathway and discusses its role in neuronal function. PMID:25374356

  16. 76 FR 18737 - Defense Transportation Regulation, Part IV

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-05

    ... Rules for the Defense Personal Property Program (DP3) in the Defense Transportation Regulation (DTR...). Implementation timelines will be based on completion of Defense Personal Property System (DPS) Phase III... Internet at http://www.regulations.gov as they are received without change, including any...

  17. Caulis Sinomenii extracts activate DA/NE transporter and inhibit 5HT transporter.

    PubMed

    Zhao, Gang; Bi, Cheng; Qin, Guo-Wei; Guo, Li-He

    2009-08-01

    Caulis Sinomenii (QFT) has analgesic, sedative, and anxiolytic-like actions, and is proven effective for improving drug dependence that is known to be associated with abnormal monoaminergic transmission. We assessed whether QFT would be biologically active in functionally regulating monoamine transporters using CHO cells expressing dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. D8, N1, or S6 cells, respectively). Here, we showed that its primary extracts, such as QA, QC, QE, QD, and QB (QFT ethanol, chloroform, ethyl acetate, alkaloid-free chloroform, and alkaloid-containing chloroform extract, respectively), and secondary extracts, such as QE-2, - 3, - 5, - 7, QD-1, - 2, - 3, - 4, - 5, and QB-1, - 2, - 3, - 4, - 5 (fractioned from QE, QD, and QB, respectively), in differing degrees, either increased DA/ NE uptake by corresponding D8/N1 cells or decreased 5HT uptake by S6 cells; wherein, QE-2, QD-3, and QE-7 were potent DA/NE uptake activators while both QE-7 and QB-5 were potent 5HT uptake inhibitors. Furthermore, the enhancement of DA/NE uptake was dependent of DAT/NET activity, and the inhibition of 5HT uptake was typical of competition. Thus, QFT extracts, especially QE-2 and QE-7 (both with stronger potencies), are novel monoamine transporter modulators functioning as DAT/ NET activators and/or SERT inhibitors, and would likely improve neuropsychological disorders through regulating monoamine transporters.

  18. Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis

    NASA Technical Reports Server (NTRS)

    Brown, D. E.; Rashotte, A. M.; Murphy, A. S.; Normanly, J.; Tague, B. W.; Peer, W. A.; Taiz, L.; Muday, G. K.

    2001-01-01

    Polar transport of the plant hormone auxin controls many aspects of plant growth and development. A number of synthetic compounds have been shown to block the process of auxin transport by inhibition of the auxin efflux carrier complex. These synthetic auxin transport inhibitors may act by mimicking endogenous molecules. Flavonoids, a class of secondary plant metabolic compounds, have been suggested to be auxin transport inhibitors based on their in vitro activity. The hypothesis that flavonoids regulate auxin transport in vivo was tested in Arabidopsis by comparing wild-type (WT) and transparent testa (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis, chalcone synthase. In a comparison between tt4 and WT plants, phenotypic differences were observed, including three times as many secondary inflorescence stems, reduced plant height, decreased stem diameter, and increased secondary root development. Growth of WT Arabidopsis plants on naringenin, a biosynthetic precursor to those flavonoids with auxin transport inhibitor activity in vitro, leads to a reduction in root growth and gravitropism, similar to the effects of synthetic auxin transport inhibitors. Analyses of auxin transport in the inflorescence and hypocotyl of independent tt4 alleles indicate that auxin transport is elevated in plants with a tt4 mutation. In hypocotyls of tt4, this elevated transport is reversed when flavonoids are synthesized by growth of plants on the flavonoid precursor, naringenin. These results are consistent with a role for flavonoids as endogenous regulators of auxin transport.

  19. The transport properties of activated carbon fibers

    SciTech Connect

    di Vittorio, S.L. . Dept. of Materials Science and Engineering); Dresselhaus, M.S. . Dept. of Electrical Engineering and Computer Science Massachusetts Inst. of Tech., Cambridge, MA . Dept. of Physics); Endo, M. . Dept. of Electrical Engineering); Issi, J-P.; Piraux, L.

    1990-07-01

    The transport properties of activated isotropic pitch-based carbon fibers with surface area 1000 m{sup 2}/g have been investigated. We report preliminary results on the electrical conductivity, the magnetoresistance, the thermal conductivity and the thermopower of these fibers as a function of temperature. Comparisons are made to transport properties of other disordered carbons. 19 refs., 4 figs.

  20. The Transport Properties of Activated Carbon Fibers

    DOE R&D Accomplishments Database

    di Vittorio, S. L.; Dresselhaus, M. S.; Endo, M.; Issi, J-P.; Piraux, L.

    1990-07-01

    The transport properties of activated isotropic pitch-based carbon fibers with surface area 1000 m{sup 2}/g have been investigated. We report preliminary results on the electrical conductivity, the magnetoresistance, the thermal conductivity and the thermopower of these fibers as a function of temperature. Comparisons are made to transport properties of other disordered carbons.

  1. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. 10 figs., 2 tabs.

  2. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

    The ability to change cytoplasmic Ca{sup 2+} levels ((Ca{sup 2+})) by cells has made this cation a key regulator of many biological processes. Cytoplasmic (Ca{sup 2+}) is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic (Ca{sup 2+}) and active Ca{sup 2+} transport systems that lower cytosolic (Ca{sup 2+}). The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  3. Prediction of cis-regulatory elements for drug-activated transcription factors in the regulation of drug-metabolising enzymes and drug transporters.

    PubMed

    Podvinec, Michael; Meyer, Urs A

    2006-06-01

    The expression of drug-metabolising enzymes is affected by many endogenous and exogenous factors, including sex, age, diet and exposure to xenobiotics and drugs. To understand fully how the organism metabolises a drug, these alterations in gene expression must be taken into account. The central process, the definition of likely regulatory elements in the genes coding for enzymes and transporters involved in drug disposition, can be vastly accelerated using existing and emerging bioinformatics methods to unravel the regulatory networks causing drug-mediated induction of genes. Here, various approaches to predict transcription factor interactions with regulatory DNA elements are reviewed.

  4. Novel aspects of cholinergic regulation of colonic ion transport

    PubMed Central

    Bader, Sandra; Diener, Martin

    2015-01-01

    Nicotinic receptors are not only expressed by excitable tissues, but have been identified in various epithelia. One aim of this study was to investigate the expression of nicotinic receptors and their involvement in the regulation of ion transport across colonic epithelium. Ussing chamber experiments with putative nicotinic agonists and antagonists were performed at rat colon combined with reverse transcription polymerase chain reaction (RT-PCR) detection of nicotinic receptor subunits within the epithelium. Dimethylphenylpiperazinium (DMPP) and nicotine induced a tetrodotoxin-resistant anion secretion leading to an increase in short-circuit current (Isc) across colonic mucosa. The response was suppressed by the nicotinic receptor antagonist hexamethonium. RT-PCR experiments revealed the expression of α2, α4, α5, α6, α7, α10, and β4 nicotinic receptor subunits in colonic epithelium. Choline, the product of acetylcholine hydrolysis, is known for its affinity to several nicotinic receptor subtypes. As a strong acetylcholinesterase activity was found in colonic epithelium, the effect of choline on Isc was examined. Choline induced a concentration-dependent, tetrodotoxin-resistant chloride secretion which was, however, resistant against hexamethonium, but was inhibited by atropine. Experiments with inhibitors of muscarinic M1 and M3 receptors revealed that choline-evoked secretion was mainly due to a stimulation of epithelial M3 receptors. Although choline proved to be only a partial agonist, it concentration-dependently desensitized the response to acetylcholine, suggesting that it might act as a modulator of cholinergically induced anion secretion. Thus the cholinergic regulation of colonic ion transport – up to now solely explained by cholinergic submucosal neurons stimulating epithelial muscarinic receptors – is more complex than previously assumed. PMID:26236483

  5. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption.

    PubMed

    Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P

    2015-09-01

    Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.

  6. Ion transport in a pH-regulated nanopore.

    PubMed

    Yeh, Li-Hsien; Zhang, Mingkan; Qian, Shizhi

    2013-08-06

    Fundamental understanding of ion transport phenomena in nanopores is crucial for designing the next-generation nanofluidic devices. Due to surface reactions of dissociable functional groups on the nanopore wall, the surface charge density highly depends upon the proton concentration on the nanopore wall, which in turn affects the electrokinetic transport of ions, fluid, and particles within the nanopore. Electrokinetic ion transport in a pH-regulated nanopore, taking into account both multiple ionic species and charge regulation on the nanopore wall, is theoretically investigated for the first time. The model is verified by the experimental data of nanopore conductance available in the literature. The results demonstrate that the spatial distribution of the surface charge density at the nanopore wall and the resulting ion transport phenomena, such as ion concentration polarization (ICP), ion selectivity, and conductance, are significantly affected by the background solution properties, such as the pH and salt concentration.

  7. Quercetin up-regulates expressions of peroxisome proliferator-activated receptor γ, liver X receptor α, and ATP binding cassette transporter A1 genes and increases cholesterol efflux in human macrophage cell line.

    PubMed

    Lee, Seung-Min; Moon, Jiyoung; Cho, Yoonsu; Chung, Ji Hyung; Shin, Min-Jeong

    2013-02-01

    Cholesterol-laden macrophages trigger accumulation of foam cells and increase the risk of developing atherosclerosis. We hypothesized that quercetin could lower the content of cholesterol in macrophages by regulating the expression of the ATP binding cassette transporter A1 (ABCA1) gene in differentiated human acute monocyte leukemia cell line (THP-1) cells and thereby reducing the chance of forming foam cells. Quercetin, in concentrations up to 30 μM, was not cytotoxic to differentiated THP-1 cells. Quercetin up-regulated both ABCA1 messenger RNA and protein expression in differentiated THP-1 cells, and its maximum effects were demonstrated at 0.3 μM for 4 to 8 hours in incubation. In addition, quercetin increased protein levels of peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptor α (LXRα) within 2 hours of treatment. Because PPARγ and LXRα are important transcriptional factors for ABCA1, quercetin-induced up-regulation of ABCA1 may be mediated by increased expression levels of the PPARγ and LXRα genes. Furthermore, quercetin-enhanced cholesterol efflux from differentiated THP-1 cells to both high-density lipoprotein (HDL) and apolipoprotein A1. Quercetin at the dose of 0.15 μM elevated the cholesterol efflux only for HDL. At the dose of 0.3 μM, quercetin demonstrated effects both on HDL and apolipoprotein A1. Our data demonstrated that quercetin increased the expressions of PPARγ, LXRα, and ABCA1 genes and cholesterol efflux from THP-1 macrophages. Quercetin-induced expression of PPARγ and LXRα might subsequently affect up-regulation of their target gene ABCA1. Taken together, ingestion of quercetin or quercetin-rich foods could be an effective way to improve cholesterol efflux from macrophages, which would contribute to lowering the risk of atherosclerosis.

  8. Defense Transportation Regulation Part 2, Cargo Movement.

    DTIC Science & Technology

    1998-08-01

    regarding any aspect of CPP should be forwarded to the attention of the appropriate area command or HQ MTMC. Addresses and phone numbers are as...A pop-up menu displays. (1) Type the interface name, i.e., MTMC-TFG, and press [ENTER]. (2) Type the phone number that was assigned to you with your...who will be responsible for maintaining the activity TFG record. (3) Phone . Enter DSN or commercial telephone number of the POC indicated above. (4

  9. Role of nuclear receptors in the regulation of drug transporters in the brain.

    PubMed

    Chan, Gary N Y; Hoque, Md Tozammel; Bendayan, Reina

    2013-07-01

    ATP-binding cassette membrane-associated drug efflux transporters and solute carrier influx transporters, expressed at the blood-brain barrier, blood-cerebrospinal fluid barrier, and in brain parenchyma, are important determinants of drug disposition in the central nervous system. Targeting the regulatory pathways that govern the expression of these transporters could provide novel approaches to selectively alter drug permeability into the brain. Nuclear receptors are ligand-activated transcription factors which regulate the gene expression of several metabolic enzymes and drug efflux/influx transporters. Although efforts have primarily been focused on investigating these regulatory pathways in peripheral organs (i.e., liver and intestine), recent findings demonstrate their significance in the brain. This review addresses the role of nuclear receptors in the regulation of drug transporter functional expression in the brain. An in-depth understanding of these pathways could guide the development of novel pharmacotherapy with either enhanced efficacy in the central nervous system or minimal associated neurotoxicity.

  10. ATM and GLUT1-S490 Phosphorylation Regulate GLUT1 Mediated Transport in Skeletal Muscle

    PubMed Central

    Andrisse, Stanley; Patel, Gaytri D.; Chen, Joseph E.; Webber, Andrea M.; Spears, Larry D.; Koehler, Rikki M.; Robinson-Hill, Rona M.; Ching, James K.; Jeong, Imju; Fisher, Jonathan S.

    2013-01-01

    Objective The glucose and dehydroascorbic acid (DHA) transporter GLUT1 contains a phosphorylation site, S490, for ataxia telangiectasia mutated (ATM). The objective of this study was to determine whether ATM and GLUT1-S490 regulate GLUT1. Research Design and Methods L6 myoblasts and mouse skeletal muscles were used to study the effects of ATM inhibition, ATM activation, and S490 mutation on GLUT1 localization, trafficking, and transport activity. Results In myoblasts, inhibition of ATM significantly diminished cell surface GLUT1, glucose and DHA transport, GLUT1 externalization, and association of GLUT1 with Gα-interacting protein-interacting protein, C-terminus (GIPC1), which has been implicated in recycling of endosomal proteins. In contrast, ATM activation by doxorubicin (DXR) increased DHA transport, cell surface GLUT1, and the GLUT1/GIPC1 association. S490A mutation decreased glucose and DHA transport, cell surface GLUT1, and interaction of GLUT1 with GIPC1, while S490D mutation increased transport, cell surface GLUT1, and the GLUT1/GIPC1 interaction. ATM dysfunction or ATM inhibition reduced DHA transport in extensor digitorum longus (EDL) muscles and decreased glucose transport in EDL and soleus. In contrast, DXR increased DHA transport in EDL. Conclusions These results provide evidence that ATM and GLUT1-S490 promote cell surface GLUT1 and GLUT1-mediated transport in skeletal muscle associated with upregulation of the GLUT1/GIPC1 interaction. PMID:23776597

  11. Ratchet transport powered by chiral active particles

    PubMed Central

    Ai, Bao-quan

    2016-01-01

    We numerically investigate the ratchet transport of mixtures of active and passive particles in a transversal asymmetric channel. A big passive particle is immersed in a ‘sea’ of active particles. Due to the chirality of active particles, the longitudinal directed transport is induced by the transversal asymmetry. For the active particles, the chirality completely determines the direction of the ratchet transport, the counterclockwise and clockwise particles move to the opposite directions and can be separated. However, for the passive particle, the transport behavior becomes complicated, the direction is determined by competitions among the chirality, the self-propulsion speed, and the packing fraction. Interestingly, within certain parameters, the passive particle moves to the left, while active particles move to the right. In addition, there exist optimal parameters (the chirality, the height of the barrier, the self-propulsion speed and the packing fraction) at which the rectified efficiency takes its maximal value. Our findings could be used for the experimental pursuit of the ratchet transport powered by chiral active particles. PMID:26795952

  12. The connexion between active cation transport and metabolism in erythrocytes

    PubMed Central

    Whittam, R.; Ager, Margaret E.

    1965-01-01

    1. A study has been made of the dependence on the concentrations of internal Na+ and external K+ of lactate and phosphate production in human erythrocytes. 2. Lactate production was stimulated by Na+ and K+ but only when they were internal and external respectively. The stimulation was counteracted by ouabain. The production of phosphate was affected in the same way. 3. There is a quantitative correlation between these effects and those previously found for cation movements and the membrane adenosine triphosphatase. 4. It is concluded that the rate of energy production in glycolysis is partly controlled by the magnitude of active transport; the extent of this regulation is shown to vary from 25 to 75% of a basal rate that is independent of active transport. 5. The activity of the membrane adenosine triphosphatase was also compared with rates of Na+ and K+ transport. The latter were varied by altering the concentrations of internal Na+ and external K+, and by inhibiting with ouabain. 6. A threefold variation of active transport rate was accompanied by a parallel change in the membrane adenosine-triphosphatase activity. The results show a constant stoicheiometry for the number of ions moved/mol. of ATP hydrolysed, independent of the electrochemical gradient against which the ions were moved. 7. Calculations show that the amount of ATP hydrolysed would provide enough energy for the osmotic work. The results are discussed in relation to possible mechanisms for active transport. PMID:16749106

  13. Regulation of ABC Efflux Transporters at Blood-Brain Barrier in Health and Neurological Disorders

    PubMed Central

    Qosa, Hisham; Miller, David S.; Pasinelli, Piera; Trotti, Davide

    2015-01-01

    The strength of the blood-brain barrier (BBB) in providing protection to the central nervous system from exposure to circulating chemicals is maintained by tight junctions between endothelial cells and by a broad range of transporter proteins that regulate exchange between CNS and blood. The most important transporters that restrict the permeability of large number of toxins as well as therapeutic agents are the ABC transporters. Among them, P-gp, BCRP, MRP1 and MRP2 are the utmost studied. These efflux transporters are neuroprotective, limiting the brain entry of neurotoxins; however, they could also restrict the entry of many therapeutics and contribute to CNS pharmacoresistance. Characterization of several regulatory pathways that govern expression and activity of ABC efflux transporters in the endothelium of brain capillaries have led to an emerging consensus that these processes are complex and contain several cellular and molecular elements. Alterations in ABC efflux transporters expression and/or activity occur in several neurological diseases. Here, we review the signaling pathways that regulate expression and transport activity of P-gp, BCRP, MRP1 and MRP2 as well as how their expression/activity changes in neurological diseases. PMID:26187753

  14. pH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones.

    PubMed

    Howe, Ethan N W; Busschaert, Nathalie; Wu, Xin; Berry, Stuart N; Ho, Junming; Light, Mark E; Czech, Dawid D; Klein, Harry A; Kitchen, Jonathan A; Gale, Philip A

    2016-07-06

    Gated ion transport across biological membranes is an intrinsic process regulated by protein channels. Synthetic anion carriers (anionophores) have potential applications in biological research; however, previously reported examples are mostly nonspecific, capable of mediating both electrogenic and electroneutral (nonelectrogenic) transport processes. Here we show the transmembrane Cl(-) transport studies of synthetic phenylthiosemicarbazones mimicking the function of acid-sensing (proton-gated) ion channels. These anionophores have remarkable pH-switchable transport properties with up to 640-fold increase in transport efficacy on going from pH 7.2 to 4.0. This "gated" process is triggered by protonation of the imino nitrogen and concomitant conformational change of the anion-binding thiourea moiety from anti to syn. By using a combination of two cationophore-coupled transport assays, with either monensin or valinomycin, we have elucidated the fundamental transport mechanism of phenylthiosemicarbazones which is shown to be nonelectrogenic, inseparable H(+)/Cl(-) cotransport. This study demonstrates the first examples of pH-switchable nonelectrogenic anion transporters.

  15. Immune regulation of human colonic electrolyte transport in vitro.

    PubMed Central

    Stack, W A; Keely, S J; O'Donoghue, D P; Baird, A W

    1995-01-01

    The role of lamina propria cells in regulating human colonic ion transport was investigated in vitro. Normal human colonic mucosae were mounted in Ussing chambers, and short circuit current changes (delta SCC) were monitored in response to immune cell activation. Anti-human immunoglobulin E (anti-IgE) and formyl-Methionyl-Leucyl-Phenylalanine (fMLP) were used to stimulate mast cells and phagocytes respectively. Anti-IgE (100 micrograms/ml) and fMLP (100 microM) evoked rapid onset, inward delta SCC (mean (SEM) max delta SCC 19.3 (2.8) and 29.4 (4.7) microA/0.63 cm2 respectively). A pharmacological approach was used to identify the charge carrying ion species and to characterise mediators involved in the SCC response. Responses to each secretagogue were significantly attenuated by bumetanide, indicating that the delta SCC was at least partly due to electrogenic chloride secretion. Piroxicam reduced the delta SCC to mast cell and phagocyte activation by 91.1 (3.4)% and 48.2 (25.2)% respectively, implicating eicosanoids as mediators of the responses. Mepyramine (100 microM) reduced the SCC responses to anti-IgE by 79.6 (12.0)% but did not significantly alter delta SCC responses to fMLP. Desensitisation to repeated anti-IgE or fMLP stimulation, and cross desensitisation between each of the stimuli, were features of immune cell activation. In summary, we have shown that activation of immune cells can stimulate electrogenic chloride secretion. Such events in vivo will result in gradient driven secretory diarrhoea, which may occur as a protective response to enteric-dwelling parasites, or as a feature of local bowel inflammation. PMID:7698700

  16. Stochastic steps in secondary active sugar transport.

    PubMed

    Adelman, Joshua L; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D F; Choe, Seungho; Abramson, Jeff; Rosenberg, John M; Wright, Ernest M; Grabe, Michael

    2016-07-05

    Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

  17. Stochastic steps in secondary active sugar transport

    PubMed Central

    Adelman, Joshua L.; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D. F.; Choe, Seungho; Abramson, Jeff; Rosenberg, John M.; Wright, Ernest M.; Grabe, Michael

    2016-01-01

    Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state. PMID:27325773

  18. Palmitate-induced interleukin 6 production is mediated by protein kinase C and nuclear-factor kappaB activation and leads to glucose transporter 4 down-regulation in skeletal muscle cells.

    PubMed

    Jové, Mireia; Planavila, Anna; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2005-07-01

    The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. Here, we report that exposure of C2C12 skeletal muscle cells to 0.5 mm palmitate results in increased mRNA levels (3.5-fold induction; P < 0.05) and secretion (control 375 +/- 57 vs. palmitate 1129 +/- 177 pg/ml; P < 0.001) of the proinflammatory cytokine IL-6. Palmitate increased nuclear factor-kappaB activation and coincubation of the cells with palmitate and the nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate prevented both IL-6 expression and secretion. Furthermore, incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C, and phorbol myristate acetate, that down-regulates protein kinase C in long-term incubations, abolished induction of IL-6 production. Finally, exposure of skeletal muscle cells to palmitate caused a fall in the mRNA levels of glucose transporter 4 and insulin-stimulated glucose uptake, whereas in the presence of anti-IL-6 antibody, which neutralizes the biological activity of mouse IL-6 in cell culture, these reductions were prevented. These findings suggest that IL-6 may mediate several of the prodiabetic effects of palmitate.

  19. Hormone- and light-regulated nucleocytoplasmic transport in plants: current status.

    PubMed

    Lee, Yew; Lee, Hak-Soo; Lee, June-Seung; Kim, Seong-Ki; Kim, Soo-Hwan

    2008-01-01

    The gene regulation mechanisms underlying hormone- and light-induced signal transduction in plants rely not only on post-translational modification and protein degradation, but also on selective inclusion and exclusion of proteins from the nucleus. For example, plant cells treated with light or hormones actively transport many signalling regulatory proteins, transcription factors, and even photoreceptors and hormone receptors into the nucleus, while actively excluding other proteins. The nuclear envelope (NE) is the physical and functional barrier that mediates this selective partitioning, and nuclear transport regulators transduce hormone- or light-initiated signalling pathways across the membrane to mediate nuclear activities. Recent reports revealed that mutating the proteins regulating nuclear transport through the pores, such as nucleoporins, alters the plant's response to a stimulus. In this review, recent works are introduced that have revealed the importance of regulated nucleocytoplasmic partitioning. These important findings deepen our understanding about how co-ordinated plant hormone and light signal transduction pathways facilitate communication between the cytoplasm and the nucleus. The roles of nucleoporin components within the nuclear pore complex (NPC) are also emphasized, as well as nuclear transport cargo, such as Ran/TC4 and its binding proteins (RanBPs), in this process. Recent findings concerning these proteins may provide a possible direction by which to characterize the regulatory potential of hormone- or light-triggered nuclear transport.

  20. Mechanisms of dopamine transporter regulation in normal and disease states.

    PubMed

    Vaughan, Roxanne A; Foster, James D

    2013-09-01

    The dopamine (DA) transporter (DAT) controls the spatial and temporal dynamics of DA neurotransmission by driving reuptake of extracellular transmitter into presynaptic neurons. Many diseases such as depression, bipolar disorder, Parkinson's disease (PD), and attention deficit hyperactivity disorder (ADHD) are associated with abnormal DA levels, implicating DAT as a factor in their etiology. Medications used to treat these disorders and many addictive drugs target DAT and enhance dopaminergic signaling by suppressing transmitter reuptake. We now understand that the transport and binding properties of DAT are regulated by complex and overlapping mechanisms that provide neurons with the ability to modulate DA clearance in response to physiological demands. These processes are controlled by endogenous signaling pathways and affected by exogenous transporter ligands, demonstrating their importance for normal neurotransmission, drug abuse, and disease treatments. Increasing evidence supports the disruption of these mechanisms in DA disorders, implicating dysregulation of transport in disease etiologies and suggesting these processes as potential points for therapeutic manipulation of DA availability.

  1. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

    PubMed Central

    Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

    2015-01-01

    Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

  2. Regulation of amniotic fluid volume: mathematical model based on intramembranous transport mechanisms.

    PubMed

    Brace, Robert A; Anderson, Debra F; Cheung, Cecilia Y

    2014-11-15

    Experimentation in late-gestation fetal sheep has suggested that regulation of amniotic fluid (AF) volume occurs primarily by modulating the rate of intramembranous transport of water and solutes across the amnion into underlying fetal blood vessels. In order to gain insight into intramembranous transport mechanisms, we developed a computer model that allows simulation of experimentally measured changes in AF volume and composition over time. The model included fetal urine excretion and lung liquid secretion as inflows into the amniotic compartment plus fetal swallowing and intramembranous absorption as outflows. By using experimental flows and solute concentrations for urine, lung liquid, and swallowed fluid in combination with the passive and active transport mechanisms of the intramembranous pathway, we simulated AF responses to basal conditions, intra-amniotic fluid infusions, fetal intravascular infusions, urine replacement, and tracheoesophageal occlusion. The experimental data are consistent with four intramembranous transport mechanisms acting in concert: 1) an active unidirectional bulk transport of AF with all dissolved solutes out of AF into fetal blood presumably by vesicles; 2) passive bidirectional diffusion of solutes, such as sodium and chloride, between fetal blood and AF; 3) passive bidirectional water movement between AF and fetal blood; and 4) unidirectional transport of lactate into the AF. Further, only unidirectional bulk transport is dynamically regulated. The simulations also identified areas for future study: 1) identifying intramembranous stimulators and inhibitors, 2) determining the semipermeability characteristics of the intramembranous pathway, and 3) characterizing the vesicles that are the primary mediators of intramembranous transport.

  3. Down-regulation of the rat serotonin transporter upon exposure to a selective serotonin reuptake inhibitor.

    PubMed

    Horschitz, S; Hummerich, R; Schloss, P

    2001-07-20

    The serotonin transporter (SERT) terminates serotonergic neurotransmission by rapid reuptake of 5-hydroxytryptamine (5-HT) into the nerve terminal or axonal varicosities. SERT represents the target of various antidepressants which inhibit 5-HT transport and are widely used for the pharmacotherapy of depression. Here, we have analyzed the function of SERT stably expressed in HEK 293 cells upon exposure to citalopram, a selective serotonin reuptake inhibitor (SSRI), with respect to 5-HT transport activity and protein expression as estimated by ligand binding experiments. Our results show that long-term exposure to an SSRI causes a down-regulation of transport activity as revealed by a reduction of the maximal transport rate, without affecting substrate affinity, accompanied by a decrease in ligand binding sites.

  4. Regulation of ABC transporters blood-brain barrier: the good, the bad, and the ugly.

    PubMed

    Miller, David S

    2015-01-01

    The brain capillary endothelial cells that constitute the blood-brain barrier express multiple ABC transport proteins on the luminal, blood-facing, plasma membrane. These transporters function as ATP-driven efflux pumps for xenobiotics and endogenous metabolites. High expression of these ABC transporters at the barrier is a major obstacle to the delivery of therapeutics, including chemotherapeutics, to the CNS. Here, I review the signals that alter ABC transporter expression and transport function with an emphasis on P-glycoprotein, Mrp2, and breast cancer resistance protein (BCRP), the efflux transporters for which we have the most detailed picture of regulation. Recent work shows that transporter protein expression can be upregulated in response to inflammatory and oxidative stress, therapeutic drugs, diet, and persistent environmental pollutants; as a consequence, drug delivery to the brain is reduced (potentially bad and ugly). In contrast, basal transport activity of P-glycoprotein and BCRP can be reduced through complex signaling pathways that involve events in and on the brain capillary endothelial cells. Targeting these signaling events provides opportunities to rapidly and reversibly increase brain accumulation of drugs that are substrates for the transporters (potentially good). The clinical usefulness of targeting signaling to reduce efflux transporter activity and improve drug delivery to the CNS remains to be established.

  5. Compendium of federal and state radioactive materials transportation laws and regulations: Transportation Legislative Database (TLDB)

    SciTech Connect

    Not Available

    1989-10-01

    The Transportation Legislative Database (TLDB) is an on-line information service containing detailed information on legislation and regulations regarding the transportation of radioactive materials in the United States. The system is dedicated to serving the legislative and regulatory information needs of the US Department of Energy and other federal agencies; state, tribal, and local governments; the hazardous materials transportation industry; and interested members of the general public. In addition to the on-line information service, quarterly and annual Legal Developments Reports are produced using information from the TLDB. These reports summarize important changes in federal and state legislation, regulations, administrative agency rulings, and judicial decisions over the reporting period. Information on significant legal developments at the tribal and local levels is also included on an as-available basis. Battelle's Office of Transportation Systems and Planning (OTSP) will also perform customized searches of the TLDB and produce formatted printouts in response to specific information requests.

  6. L-leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the involvement of the mammalian target of rapamycin (mTOR) pathway in the L-leucine-induced up-regulation of system A amino acid transport.

    PubMed Central

    Peyrollier, K; Hajduch, E; Blair, A S; Hyde, R; Hundal, H S

    2000-01-01

    Amino acid availability is known to regulate diverse cell processes including the activation of p70 S6 kinase, initiation factors involved in mRNA translation, gene expression and cellular amino acid uptake. Essential amino acids, in particular the branched-chain amino acids (e.g. leucine), have been shown to be the dominant players in mediating these effects, although the precise nature by which they regulate these processes remain poorly understood. In this study we have investigated the mechanisms involved in the leucine-induced modulation of p70 S6 kinase and addressed whether this kinase participates in the up-regulation of the System A amino acid transporter in L6 muscle cells. Incubation of muscle cells that had been amino acid-deprived for 1 h with L-leucine (2 mM) led to a rapid (>2-fold) activation of p70 S6 kinase, which was suppressed by both wortmannin and rapamycin. Consistent with this finding, addition of leucine caused a rapid ( approximately 5-fold) but transient stimulation of phosphatidylinositol 3-kinase (PI3K). PI3K activation was inhibited by wortmannin and was not dependent upon insulin receptor substrate-1 activation. Unlike stimulation by insulin, activation of neither protein kinase B nor p42/p44 mitogen-activated protein kinase accompanied the leucine-induced stimulation of PI3K. However, the leucine-induced activation of PI3K and p70 S6 kinase did result in the concomitant inactivation of glycogen synthase kinase-3 (GSK-3). Leucine enhanced System A transport by approximately 50%. We have shown previously that this stimulation is protein-synthesis-dependent and in the current study we show that it was blocked by both wortmannin and rapamycin. Our findings indicate that PI3K and the mammalian target of rapamycin are components of a nutrient signalling pathway that regulates the activation of p70 S6 kinase and induction of System A in L6 cells. The activation of this pathway by leucine is also responsible for the inactivation of GSK-3

  7. Development of novel active transport membrande devices

    SciTech Connect

    Laciak, D.V.

    1994-11-01

    Air Products has undertaken a research program to fabricate and evaluate gas separation membranes based upon promising ``active-transport`` (AT) materials recently developed in our laboratories. Active Transport materials are ionic polymers and molten salts which undergo reversible interaction or reaction with ammonia and carbon dioxide. The materials are useful for separating these gases from mixtures with hydrogen. Moreover, AT membranes have the unique property of possessing high permeability towards ammnonia and carbon dioxide but low permeability towards hydrogen and can thus be used to permeate these components from a gas stream while retaining hydrogen at high pressure.

  8. Transport and transcriptional regulation of oil production in plants.

    PubMed

    Manan, Sehrish; Chen, Beibei; She, Guangbiao; Wan, Xiaochun; Zhao, Jian

    2016-08-23

    Triacylglycerol (TAG) serves as an energy reservoir and phospholipids as build blocks of biomembrane to support plant life. They also provide human with foods and nutrients. Multi-compartmentalized biosynthesis, trafficking or cross-membrane transport of lipid intermediates or precursors and their regulatory mechanisms are not fully understood. Recent progress has aided our understanding of how fatty acids (FAs) and phospholipids are transported between the chloroplast, the cytoplasm, and the endoplasmic reticulum (ER), and how the ins and outs of lipids take place in the peroxisome and other organelles for lipid metabolism and function. In addition, information regarding the transcriptional regulation network associated with FA and TAG biosynthesis has been further enriched. Recent breakthroughs made in lipid transport and transcriptional regulation has provided significant insights into our comprehensive understanding of plant lipid biology. This review attempts to highlight the recent progress made on lipid synthesis, transport, degradation, and their regulatory mechanisms. Metabolic engineering, based on these knowledge-powered technologies for production of edible oils or biofuels, is reviewed. The biotechnological application of metabolic enzymes, transcription factors and transporters, for oil production and composition improvement, are discussed in a broad context in order to provide a fresh scenario for researchers and to guide future research and applications.

  9. Health Impacts of Active Transportation in Europe

    PubMed Central

    Rojas-Rueda, David; de Nazelle, Audrey; Andersen, Zorana J.; Braun-Fahrländer, Charlotte; Bruha, Jan; Bruhova-Foltynova, Hana; Desqueyroux, Hélène; Praznoczy, Corinne; Ragettli, Martina S.; Tainio, Marko; Nieuwenhuijsen, Mark J.

    2016-01-01

    Policies that stimulate active transportation (walking and bicycling) have been related to heath benefits. This study aims to assess the potential health risks and benefits of promoting active transportation for commuting populations (age groups 16–64) in six European cities. We conducted a health impact assessment using two scenarios: increased cycling and increased walking. The primary outcome measure was all-cause mortality related to changes in physical activity level, exposure to fine particulate matter air pollution with a diameter <2.5 μm, as well as traffic fatalities in the cities of Barcelona, Basel, Copenhagen, Paris, Prague, and Warsaw. All scenarios produced health benefits in the six cities. An increase in bicycle trips to 35% of all trips (as in Copenhagen) produced the highest benefits among the different scenarios analysed in Warsaw 113 (76–163) annual deaths avoided, Prague 61 (29–104), Barcelona 37 (24–56), Paris 37 (18–64) and Basel 5 (3–9). An increase in walking trips to 50% of all trips (as in Paris) resulted in 19 (3–42) deaths avoided annually in Warsaw, 11(3–21) in Prague, 6 (4–9) in Basel, 3 (2–6) in Copenhagen and 3 (2–4) in Barcelona. The scenarios would also reduce carbon dioxide emissions in the six cities by 1,139 to 26,423 (metric tonnes per year). Policies to promote active transportation may produce health benefits, but these depend of the existing characteristics of the cities. Increased collaboration between health practitioners, transport specialists and urban planners will help to introduce the health perspective in transport policies and promote active transportation. PMID:26930213

  10. Fluid transport by active elastic membranes

    NASA Astrophysics Data System (ADS)

    Evans, Arthur A.; Lauga, Eric

    2011-09-01

    A flexible membrane deforming its shape in time can self-propel in a viscous fluid. Alternatively, if the membrane is anchored, its deformation will lead to fluid transport. Past work in this area focused on situations where the deformation kinematics of the membrane were prescribed. Here we consider models where the deformation of the membrane is not prescribed, but instead the membrane is internally forced. Both the time-varying membrane shape and the resulting fluid motion result then from a balance between prescribed internal active stresses, internal passive resistance, and external viscous stresses. We introduce two specific models for such active internal forcing: one where a distribution of active bending moments is prescribed, and one where active inclusions exert normal stresses on the membrane by pumping fluid through it. In each case, we asymptotically calculate the membrane shape and the fluid transport velocities for small forcing amplitudes, and recover our results using scaling analysis.

  11. Regulation of transport across cell membranes by the serum- and glucocorticoid-inducible kinase SGK1.

    PubMed

    Lang, Florian; Stournaras, Christos; Alesutan, Ioana

    2014-02-01

    The serum- and glucocorticoid-inducible kinase 1 (SGK1) is genomically upregulated by cell stress including energy depletion and hyperosmotic shock as well as a variety of hormones including glucocorticoids, mineralocorticoids and TGFβ. SGK1 is activated by insulin, growth factors and oxidative stress via phosphatidylinositide-3-kinase, 3-phosphoinositide-dependent kinase PDK1 and mTOR. SGK1 is a powerful stimulator of Na(+)/K(+)-ATPase, carriers (e.g., NCC, NKCC, NHE1, NHE3, SGLT1, several amino acid transporters) and ion channels (e.g., ENaC, SCN5A, TRPV4-6, ORAI1/STIM1, ROMK, KCNE1/KCNQ1, GluR6, CFTR). Mechanisms employed by SGK1 in transport regulation include direct phosphorylation of target transport proteins, phosphorylation and thus activation of other transport regulating kinases, stabilization of membrane proteins by phosphorylation and thus inactivation of the ubiquitin ligase NEDD4-2, as well as stimulation of transport protein expression by upregulation transcription factors (e.g., nuclear factor kappa-B [NFκB]) and by fostering of protein translation. SGK1 sensitivity of pump, carrier and channel activities participate in the regulation of epithelial transport, cardiac and neuronal excitability, degranulation, platelet function, migration, cell proliferation and apoptosis. SGK1-sensitive functions do not require the presence of SGK1 but are markedly upregulated by SGK1. Accordingly, the phenotype of SGK1 knockout mice is mild. The mice are, however, less sensitive to excessive activation of transport by glucocorticoids, mineralocorticoids, insulin and inflammation. Moreover, excessive SGK1 activity contributes to the pathophysiology of hypertension, obesity, diabetes, thrombosis, stroke, inflammation, autoimmune disease, fibrosis and tumor growth.

  12. Transcriptional Regulation and Transport of Terpenoid Indole Alkaloid in Catharanthus roseus: Exploration of New Research Directions

    PubMed Central

    Liu, Jiaqi; Cai, Junjun; Wang, Rui; Yang, Shihai

    2016-01-01

    As one of the model medicinal plants for exploration of biochemical pathways and molecular biological questions on complex metabolic pathways, Catharanthus roseus synthesizes more than 100 terpenoid indole alkaloids (TIAs) used for clinical treatment of various diseases and for new drug discovery. Given that extensive studies have revealed the major metabolic pathways and the spatial-temporal biosynthesis of TIA in C. roseus plant, little is known about subcellular and inter-cellular trafficking or long-distance transport of TIA end products or intermediates, as well as their regulation. While these transport processes are indispensable for multi-organelle, -tissue and -cell biosynthesis, storage and their functions, great efforts have been made to explore these dynamic cellular processes. Progress has been made in past decades on transcriptional regulation of TIA biosynthesis by transcription factors as either activators or repressors; recent studies also revealed several transporters involved in subcellular and inter-cellular TIA trafficking. However, many details and the regulatory network for controlling the tissue-or cell-specific biosynthesis, transport and storage of serpentine and ajmalicine in root, catharanthine in leaf and root, vindoline specifically in leaf and vinblastine and vincristine only in green leaf and their biosynthetic intermediates remain to be determined. This review is to summarize the progress made in biosynthesis, transcriptional regulation and transport of TIAs. Based on analysis of organelle, tissue and cell-type specific biosynthesis and progresses in transport and trafficking of similar natural products, the transporters that might be involved in transport of TIAs and their synthetic intermediates are discussed; according to transcriptome analysis and bioinformatic approaches, the transcription factors that might be involved in TIA biosynthesis are analyzed. Further discussion is made on a broad context of transcriptional and

  13. Neuroinflammation activates efflux transport by NFκB

    PubMed Central

    Yu, Chuanhui; Argyropoulos, George; Zhang, Yan; Kastin, Abba J.; Hsuchou, Hung; Pan, Weihong

    2009-01-01

    Background/aims Although it is known that drug delivery across the blood-brain barrier (BBB) may be hampered by efflux transport activity of the multidrug resistance (mdr) gene product P-glycoprotein, it is not clear how inflammation regulates efflux transporters. In rat brain endothelial (RBE4) cells of BBB origin, the proinflammatory cytokine TNF mainly induces transcriptional upregulation of mdr1b, and to a lesser extent mdr1a, resulting in greater efflux of the substrates (Yu C et al., Cell Physiol Biochem, 2007). This study further determined the mechanisms by which TNF activates mdr1b promoter activity. Methods/Results Luciferase reporter assays and DNA binding studies show that (a) maximal basal promoter activity was conferred by a 476 bp sequence upstream to the mdr1b transcriptional initiation site; (2) TNF induced upregulation of promoter activity by NFkB nuclear translocation; and (3) the NFκB binding site of the mdr1b promoter was solely responsible for basal and TNF-activated gene transcription, whereas the p53 binding site was not involved. Binding of the p65 subunit of NFκB to nuclear DNA from RBE4 cells was shown by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. Conclusion Thus, NFκB mediated TNF-induced upregulation of mdr1b promoter activity, illustrating how inflammation activates BBB efflux transport. PMID:19088456

  14. Increased synthesis of folate transporters regulates folate transport in conditions of ethanol exposure and folate deficiency.

    PubMed

    Thakur, Shilpa; More, Deepti; Rahat, Beenish; Khanduja, Krishan Lal; Kaur, Jyotdeep

    2016-01-01

    Excessive alcohol consumption and dietary folate inadequacy are the main contributors leading to folate deficiency (FD). The present study was planned to study regulation of folate transport in conditions of FD and ethanol exposure in human embryonic kidney cell line. Also, the reversible nature of effects mediated by ethanol exposure and FD was determined by folate repletion and ethanol removal. For ethanol treatment, HEK293 cells were grown in medium containing 100 mM ethanol, and after treatment, one group of cells was shifted on medium that was free from ethanol. For FD treatment, cells were grown in folate-deficient medium followed by shifting of one group of cells on folate containing medium. FD as well as ethanol exposure resulted in an increase in folate uptake which was due to an increase in expression of folate transporters, i.e., reduced folate carrier, proton-coupled folate transporter, and folate receptor, both at the mRNA and protein level. The effects mediated by ethanol exposure and FD were reversible on removal of treatment. Promoter region methylation of folate transporters remained unaffected after FD and ethanol exposure. As far as transcription rate of folate transporters is concerned, an increase in rate of synthesis was observed in both ethanol exposure and FD conditions. Additionally, mRNA life of folate transporters was observed to be reduced by FD. An increased expression of folate transporters under ethanol exposure and FD conditions can be attributed to enhanced rate of synthesis of folate transporters.

  15. A new look at water transport regulation in plants.

    PubMed

    Martínez-Vilalta, Jordi; Poyatos, Rafael; Aguadé, David; Retana, Javier; Mencuccini, Maurizio

    2014-10-01

    Plant function requires effective mechanisms to regulate water transport at a variety of scales. Here, we develop a new theoretical framework describing plant responses to drying soil, based on the relationship between midday and predawn leaf water potentials. The intercept of the relationship (Λ) characterizes the maximum transpiration rate per unit of hydraulic transport capacity, whereas the slope (σ) measures the relative sensitivity of the transpiration rate and plant hydraulic conductance to declining water availability. This framework was applied to a newly compiled global database of leaf water potentials to estimate the values of Λ and σ for 102 plant species. Our results show that our characterization of drought responses is largely consistent within species, and that the parameters Λ and σ show meaningful associations with climate across species. Parameter σ was ≤1 in most species, indicating a tight coordination between the gas and liquid phases of water transport, in which canopy transpiration tended to decline faster than hydraulic conductance during drought, thus reducing the pressure drop through the plant. The quantitative framework presented here offers a new way of characterizing water transport regulation in plants that can be used to assess their vulnerability to drought under current and future climatic conditions.

  16. Transport and biological activities of bile acids.

    PubMed

    Zwicker, Brittnee L; Agellon, Luis B

    2013-07-01

    Bile acids have emerged as important biological molecules that support the solubilization of various lipids and lipid-soluble compounds in the gut, and the regulation of gene expression and cellular function. Bile acids are synthesized from cholesterol in the liver and eventually released into the small intestine. The majority of bile acids are recovered in the distal end of the small intestine and then returned to the liver for reuse. The components of the mechanism responsible for the recycling of bile acids within the enterohepatic circulation have been identified whereas the mechanism for intracellular transport is less understood. Recently, the ileal lipid binding protein (ILBP; human gene symbol FABP6) was shown to be needed for the efficient transport of bile acids from the apical side to the basolateral side of enterocytes in the distal intestine. This review presents an overview of the transport of bile acids between the liver and the gut as well as within hepatocytes and enterocytes. A variety of pathologies is associated with the malfunction of the bile acid transport system.

  17. Modeling regulation of zinc uptake via ZIP transporters in yeast and plant roots.

    PubMed

    Claus, Juliane; Chavarría-Krauser, Andrés

    2012-01-01

    In yeast (Saccharomyces cerevisiae) and plant roots (Arabidopsis thaliana) zinc enters the cells via influx transporters of the ZIP family. Since zinc is both essential for cell function and toxic at high concentrations, tight regulation is essential for cell viability. We provide new insight into the underlying mechanisms, starting from a general model based on ordinary differential equations and adapting it to the specific cases of yeast and plant root cells. In yeast, zinc is transported by the transporters ZRT1 and ZRT2, which are both regulated by the zinc-responsive transcription factor ZAP1. Using biological data, parameters were estimated and analyzed, confirming the different affinities of ZRT1 and ZRT2 reported in the literature. Furthermore, our model suggests that the positive feedback in ZAP1 production has a stabilizing function at high influx rates. In plant roots, various ZIP transporters play a role in zinc uptake. Their regulation is largely unknown, but bZIP transcription factors are thought to be involved. We set up three putative models based on: an activator only, an activator with dimerization and an activator-inhibitor pair. These were fitted to measurements and analyzed. Simulations show that the activator-inhibitor model outperforms the other two in providing robust and stable homeostasis at reasonable parameter ranges.

  18. Integrated regulation of motor-driven organelle transport by scaffolding proteins.

    PubMed

    Fu, Meng-meng; Holzbaur, Erika L F

    2014-10-01

    Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment.

  19. Glucocorticoid regulation of amino acid transport in anucleate rat hepatoma (HTC) cells

    PubMed Central

    1981-01-01

    The transport of alpha-aminoisobutyric acid (AIB) by rat hepatoma tissue culture (HTC) cells is rapidly and reversibly inhibited by dexamethasone and other glucocorticoids. To investigate the role of the nucleus in the regulation of transport and to determine whether steroid hormones or steroid-receptor complexes may have direct effects on cytoplasmic or membrane functions, we have examined the regulation of transport by dexamethasone in anucleate HTC cells. Cytoplasts prepared from suspension cultures of HTC cells fully retain active transport of AIB with the same kinetic properties as intact cells. However, the uptake of AIB is not inhibited by dexamethasone or other corticosteroids. Neither is the inhibited rate of transport, manifested by cytoplasts prepared from dexamethasone-treated cells, restored to normal upon removal of the hormone. Anucleate cells exhibit specific, saturable binding of [3H]dexamethasone; however, the binding is reduced compared with that of intact cells. The nucleus is thus required for the glucocorticoid regulation of amino acid transport in HTC cells. PMID:7217203

  20. Ergostatrien-3β-ol from Antrodia camphorata inhibits diabetes and hyperlipidemia in high-fat-diet treated mice via regulation of hepatic related genes, glucose transporter 4, and AMP-activated protein kinase phosphorylation.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Cheng-Hsiu; Shih, Chun-Ching

    2015-03-11

    This study was designed to explore the effects and mechanism of ergostatrien-3β-ol (EK100) from the submerged whole broth of Antrodia camphorata on diabetes and dyslipidemia in high fat diet (HFD)-fed mice for 12 weeks. The C57BL/6J mouse fed with a high fat diet (HFD) could induce insulin resistance and hyperlipidemia. After 8 week of induction, mice were receiving EK100 (at three dosages) or fenofibrate (Feno) or rosiglitazone (Rosi) or vehicle by oral gavage 4 weeks afterward. HFD-fed mice display increased blood glucose, glycated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), insulin, and leptin levels. These blood markers were significantly lower in EK100-treated mice, and finally ameliorated insulin resistance. EK100 treatment exhibited reduced hepatic ballooning degeneration and size of visceral adipocytes. Glucose transporter 4 (GLUT4) proteins and phosphorylation of Akt in skeletal muscle were significantly increased in EK100- and Rosi-treated mice. EK100, Feno, and Rosi treatment led to significant increases in phosphorylation of AMP-activated protein kinase (phospho-AMPK) protein in both skeletal muscle and liver. Moreover, EK100 caused a decrease in hepatic expressions of phosphenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase), and decreased glucose production. EK100 lowered blood TG level by inhibition of hepatic fatty acid synthesis by dampening sterol response element binding protein-1c (SREBP-1c) but increasing expression of peroxisome proliferator activated receptor α (PPARα). Moreover, EK100-treated mice reduced blood TC levels by decreased hepatic expressions of SREBP2, which plays a major role in the regulation of cholesterol synthesis. EK100 increased high-density lipoprotein cholesterol (HDL-C) concentrations by increasing expressions of apolipoprotein A-I (apo A-I) in liver tissue. Our findings manifest that EK100 may have therapeutic potential in treating type 2 diabetes associated with hyperlipidemia

  1. Regulation of Cisplatin cytotoxicity by cu influx transporters.

    PubMed

    Abada, Paolo; Howell, Stephen B

    2010-01-01

    Platinum drugs are an important class of cancer chemotherapeutics. However, the use of these drugs is limited by the development of resistance during treatment with decreased accumulation being a common mechanism. Both Cu transporters CTR1 and CTR2 influence the uptake and cytotoxicity of cisplatin. Although it is structurally similar to CTR1, CTR2 functions in a manner opposite to that of CTR1 with respect to Pt drug uptake. Whereas knockout of CTR1 reduces Pt drug uptake, knockdown of CTR2 enhances cisplatin uptake and cytotoxicity. CTR2 is subject to transcriptional and posttranscriptional regulation by both Cu and cisplatin; this regulation is partly dependent on the Cu chaperone ATOX1. Insight into the mechanisms by which CTR1 and CTR2 regulate sensitivity to the Pt-containing drugs has served as the basis for novel pharmacologic strategies for improving their efficacy.

  2. Regulation of Cisplatin Cytotoxicity by Cu Influx Transporters

    PubMed Central

    Abada, Paolo; Howell, Stephen B.

    2010-01-01

    Platinum drugs are an important class of cancer chemotherapeutics. However, the use of these drugs is limited by the development of resistance during treatment with decreased accumulation being a common mechanism. Both Cu transporters CTR1 and CTR2 influence the uptake and cytotoxicity of cisplatin. Although it is structurally similar to CTR1, CTR2 functions in a manner opposite to that of CTR1 with respect to Pt drug uptake. Whereas knockout of CTR1 reduces Pt drug uptake, knockdown of CTR2 enhances cisplatin uptake and cytotoxicity. CTR2 is subject to transcriptional and posttranscriptional regulation by both Cu and cisplatin; this regulation is partly dependent on the Cu chaperone ATOX1. Insight into the mechanisms by which CTR1 and CTR2 regulate sensitivity to the Pt-containing drugs has served as the basis for novel pharmacologic strategies for improving their efficacy. PMID:21274436

  3. Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars1[W][OA

    PubMed Central

    Fan, Ren-Chun; Peng, Chang-Cao; Xu, Yan-Hong; Wang, Xiao-Fang; Li, Yan; Shang, Yi; Du, Shu-Yuan; Zhao, Rui; Zhang, Xiao-Yan; Zhang, Ling-Yun; Zhang, Da-Peng

    2009-01-01

    Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability. PMID:19502355

  4. Serum- and glucocorticoid-inducible kinase 1 in the regulation of renal and extrarenal potassium transport.

    PubMed

    Lang, Florian; Vallon, Volker

    2012-02-01

    Serum- and glucocorticoid inducible-kinase 1 (SGK1) is an early gene transcriptionally upregulated by cell stress such as cell shrinkage and hypoxia and several hormones including gluco- and mineralocorticoids. It is activated by insulin and growth factors. SGK1 is a powerful regulator of a wide variety of channels and transporters. The present review describes the role of SGK1 in the regulation of potassium (K(+)) channels, K(+) transporters and K(+) homeostasis. SGK1-regulated K(+) channels include renal outer medullary K+ channel, Kv1.3, Kv1.5, KCNE1/KCNQ1, KCNQ4 and, via regulation of calcium (Ca(2+)) entry, Ca(2+)-sensitive K(+) channels. SGK1-sensitive transporters include sodium-potassium-chloride cotransporter 2 and sodium/potassium-adenosine triphosphatase. SGK1-dependent regulation of K(+) channels and K(+) transport contributes to the stimulation of renal K(+) excretion following high K(+) intake, to insulin-induced cellular K(+) uptake and hypokalemia, to inhibition of insulin release by glucocorticoids, to stimulation of mast cell degranulation and gastric acid secretion, and to cardiac repolarization. Thus, SGK1 has a profound effect on K(+) homeostasis and on a multitude of K(+)-sensitive cellular functions.

  5. Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease.

    PubMed

    Mori, Nozomu; Miyashita, Takenori; Inamoto, Ryuhei; Matsubara, Ai; Mori, Terushige; Akiyama, Kosuke; Hoshikawa, Hiroshi

    2017-04-01

    Ion transport and its regulation in the endolymphatic sac (ES) are reviewed on the basis of recent lines of evidence. The morphological and physiological findings demonstrate that epithelial cells in the intermediate portion of the ES are more functional in ion transport than those in the other portions. Several ion channels, ion transporters, ion exchangers, and so on have been reported to be present in epithelial cells of ES intermediate portion. An imaging study has shown that mitochondria-rich cells in the ES intermediate portion have a higher activity of Na(+), K(+)-ATPase and a higher Na(+) permeability than other type of cells, implying that molecules related to Na(+) transport, such as epithelial sodium channel (ENaC), Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) and thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC), may be present in mitochondria-rich cells. Accumulated lines of evidence suggests that Na(+) transport is most important in the ES, and that mitochondria-rich cells play crucial roles in Na(+) transport in the ES. Several lines of evidence support the hypothesis that aldosterone may regulate Na(+) transport in ES, resulting in endolymph volume regulation. The presence of molecules related to acid/base transport, such as H(+)-ATPase, Na(+)-H(+) exchanger (NHE), pendrin (SLC26A4), Cl(-)-HCO3(-) exchanger (SLC4A2), and carbonic anhydrase in ES epithelial cells, suggests that acid/base transport is another important one in the ES. Recent basic and clinical studies suggest that aldosterone may be involved in the effect of salt-reduced diet treatment in Meniere's disease.

  6. Synaptic activation modifies microtubules underlying transport of postsynaptic cargo.

    PubMed

    Maas, Christoph; Belgardt, Dorthe; Lee, Han Kyu; Heisler, Frank F; Lappe-Siefke, Corinna; Magiera, Maria M; van Dijk, Juliette; Hausrat, Torben J; Janke, Carsten; Kneussel, Matthias

    2009-05-26

    Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.

  7. Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport

    PubMed Central

    Quizon, Pamela M.; Sun, Wei-Lun; Yuan, Yaxia; Midde, Narasimha M.; Zhan, Chang-Guo; Zhu, Jun

    2016-01-01

    Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission. PMID:27966610

  8. A novel iron-regulated metal transporter from plants identified by functional expression in yeast.

    PubMed

    Eide, D; Broderius, M; Fett, J; Guerinot, M L

    1996-05-28

    Iron is an essential nutrient for virtually all organisms. The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake. Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd. IRT1 is predicted to be an integral membrane protein with a metal-binding domain. Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis. Related sequences were also found in the genomes of rice, yeast, nematodes, and humans. In Arabidopsis, IRT1 is expressed in roots, is induced by iron deficiency, and has altered regulation in plant lines bearing mutations that affect the iron uptake system. These results provide the first molecular insight into iron transport by plants.

  9. Regional differences in rat conjunctival ion transport activities.

    PubMed

    Yu, Dongfang; Thelin, William R; Rogers, Troy D; Stutts, M Jackson; Randell, Scott H; Grubb, Barbara R; Boucher, Richard C

    2012-10-01

    Active ion transport and coupled osmotic water flow are essential to maintain ocular surface health. We investigated regional differences in the ion transport activities of the rat conjunctivas and compared these activities with those of cornea and lacrimal gland. The epithelial sodium channel (ENaC), sodium/glucose cotransporter 1 (Slc5a1), transmembrane protein 16 (Tmem16a, b, f, and g), cystic fibrosis transmembrane conductance regulator (Cftr), and mucin (Muc4, 5ac, and 5b) mRNA expression was characterized by RT-PCR. ENaC proteins were measured by Western blot. Prespecified regions (palpebral, fornical, and bulbar) of freshly isolated conjunctival tissues and cell cultures were studied electrophysiologically with Ussing chambers. The transepithelial electrical potential difference (PD) of the ocular surface was also measured in vivo. The effect of amiloride and UTP on the tear volume was evaluated in lacrimal gland excised rats. All selected genes were detected but with different expression patterns. We detected αENaC protein in all tissues, βENaC in palpebral and fornical conjunctiva, and γENaC in all tissues except lacrimal glands. Electrophysiological studies of conjunctival tissues and cell cultures identified functional ENaC, SLC5A1, CFTR, and TMEM16. Fornical conjunctiva exhibited the most active ion transport under basal conditions amongst conjunctival regions. PD measurements confirmed functional ENaC-mediated Na(+) transport on the ocular surface. Amiloride and UTP increased tear volume in lacrimal gland excised rats. This study demonstrated that the different regions of the conjunctiva exhibited a spectrum of ion transport activities. Understanding the specific functions of distinct regions of the conjunctiva may foster a better understanding of the physiology maintaining hydration of the ocular surface.

  10. Regional differences in rat conjunctival ion transport activities

    PubMed Central

    Yu, Dongfang; Thelin, William R.; Rogers, Troy D.; Stutts, M. Jackson; Randell, Scott H.; Grubb, Barbara R.

    2012-01-01

    Active ion transport and coupled osmotic water flow are essential to maintain ocular surface health. We investigated regional differences in the ion transport activities of the rat conjunctivas and compared these activities with those of cornea and lacrimal gland. The epithelial sodium channel (ENaC), sodium/glucose cotransporter 1 (Slc5a1), transmembrane protein 16 (Tmem16a, b, f, and g), cystic fibrosis transmembrane conductance regulator (Cftr), and mucin (Muc4, 5ac, and 5b) mRNA expression was characterized by RT-PCR. ENaC proteins were measured by Western blot. Prespecified regions (palpebral, fornical, and bulbar) of freshly isolated conjunctival tissues and cell cultures were studied electrophysiologically with Ussing chambers. The transepithelial electrical potential difference (PD) of the ocular surface was also measured in vivo. The effect of amiloride and UTP on the tear volume was evaluated in lacrimal gland excised rats. All selected genes were detected but with different expression patterns. We detected αENaC protein in all tissues, βENaC in palpebral and fornical conjunctiva, and γENaC in all tissues except lacrimal glands. Electrophysiological studies of conjunctival tissues and cell cultures identified functional ENaC, SLC5A1, CFTR, and TMEM16. Fornical conjunctiva exhibited the most active ion transport under basal conditions amongst conjunctival regions. PD measurements confirmed functional ENaC-mediated Na+ transport on the ocular surface. Amiloride and UTP increased tear volume in lacrimal gland excised rats. This study demonstrated that the different regions of the conjunctiva exhibited a spectrum of ion transport activities. Understanding the specific functions of distinct regions of the conjunctiva may foster a better understanding of the physiology maintaining hydration of the ocular surface. PMID:22814399

  11. 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

  12. SLC6 transporters: structure, function, regulation, disease association and therapeutics.

    PubMed

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

    2013-01-01

    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.

  13. Regulation of nitrate transport in citrus rootstocks depending on nitrogen availability.

    PubMed

    Cerezo, Miguel; Camañes, Gemma; Flors, Víctor; Primo-Millo, Eduardo; García-Agustín, Pilar

    2007-09-01

    Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO(3) (-). The V(max) for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks.Additionally, we studied the regulation of root NO(3) (-) uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO(3) (-) and down-regulated by the N status and by NO(3) (-) itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status.The use of various metabolic uncouplers or inhibitors indicated that NO(3) (-) net uptake mediated by iHATS and LATS was an active transport system in both rootstocks.

  14. Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

    PubMed Central

    Cerezo, Miguel; Camañes, Gemma; Flors, Víctor; Primo-Millo, Eduardo

    2007-01-01

    Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO3−. The Vmax for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks. Additionally, we studied the regulation of root NO3− uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO3− and down-regulated by the N status and by NO3− itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status. The use of various metabolic uncouplers or inhibitors indicated that NO3− net uptake mediated by iHATS and LATS was an active transport system in both rootstocks. PMID:19516998

  15. GSK-3 regulates transport of kinesin-1 driven cargos in vivo

    NASA Astrophysics Data System (ADS)

    Leidel, Christina; Weaver, Carole; Szpankowski, Lukasz; Goldstein, Lawrence S. B.; Shubeita, George T.; CenterNonlinear Dynamics, Department of Physics, University of Texas At Austin Collaboration; Hhmi, Department of Cellular; Molecular Medicine, Univ. Of California Collaboration

    2011-03-01

    The Glycogen Synthase Kinase 3 (GSK-3) has been linked to many aspects of the development of Alzheimer's disease and was proposed to play a role in the transport of the Amyloid Precursor Protein (APP) by kinesin-1 motors. Using Drosophila embryos and larvae with altered GSK-3 expression, we characterize motor transport of cargos including APP and lipid droplets using DIC microscopy, high-resolution video tracking, fluorescence, and in vivo stall force measurements with optical tweezers. By comparing cargo velocities and run lengths we find that GSK-3 is a required negative regulator of in vivo transport. Stall force measurements on lipid droplets reveal that enhanced transport under conditions of reduced GSK-3 is a result of a larger number of active motors hauling the cargo. Our findings have implications on the use of GSK-3 inhibitors in treatment of Alzheimer's disease.

  16. Iodide transport and its regulation in the thyroid gland

    SciTech Connect

    Price, D.J.

    1987-01-01

    This study was undertaken to examine the autoregulatory mechanism of iodide induced suppression of subsequently determined iodide transport activity in the thyroid gland. Two model systems were developed to identify the putative, transport-related, iodine-containing, inhibitory factor responsible for autoregulation. The first system was a maternal and fetal rabbit thyroid tissue slice preparation in which iodide pretreatment inhibited the maternal /sup 125/I-T/M ratio by 30% and had no significant effect on fetal iodide transport. In the second system, the role of protein synthesis in the autoregulatory phenomenon was studied. Cat thyroid slices pretreated with0.1 mM cycloheximide for 60 min prior to preexposure to excess iodide demonstrated a significant reduction in the degree of iodide included autoregulation. In both of these systems iodide induced suppression of cAMP accumulation remained intact. These findings suggest (1) fetal rabbit thyroid lacks the autoregulatory mechanism of iodide transport and (2) protein synthesis is involved in the mechanism of thyroid autoregulation of iodide transport.

  17. Monocarboxylate transporters: new players in body weight regulation.

    PubMed

    Carneiro, L; Pellerin, L

    2015-02-01

    Over the last two decades, several genes have been identified that appear to play a role in the regulation of energy homeostasis and body weight. For a small subset of them, a reduction or an absence of expression confers a resistance to the development of obesity. Recently, a knockin mouse for a member of the monocarboxylate transporter (MCT) family, MCT1, was demonstrated to exhibit a typical phenotype of resistance to diet-induced obesity and a protection from its associated metabolic perturbations. Such findings point out at MCTs as putatively new therapeutic targets in the context of obesity. Here, we will review what is known about MCTs and their possible metabolic roles in different organs and tissues. Based on the description of the phenotype of the MCT1 knockin mouse, we will also provide some insights about their putative roles in weight gain regulation.

  18. Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins.

    PubMed

    Geisler, Markus; Bailly, Aurélien; Ivanchenko, Maria

    2016-04-01

    Plant development and architecture are greatly influenced by the polar distribution of the essential hormone auxin. The directional influx and efflux of auxin from plant cells depends primarily on AUX1/LAX, PIN, and ABCB/PGP/MDR families of auxin transport proteins. The functional analysis of these proteins has progressed rapidly within the last decade thanks to the establishment of heterologous auxin transport systems. Heterologous co-expression allowed also for the testing of protein-protein interactions involved in the regulation of transporters and identified relationships with members of the FK506-Binding Protein (FKBP) and cyclophilin protein families, which are best known in non-plant systems as cellular receptors for the immunosuppressant drugs, FK506 and cyclosporin A, respectively. Current evidence that such interactions affect membrane trafficking, and potentially the activity of auxin transporters is reviewed. We also propose that FKBPs andcyclophilins might integrate the action of auxin transport inhibitors, such as NPA, on members of the ABCB and PIN family, respectively. Finally, we outline open questions that might be useful for further elucidation of the role of immunophilins as regulators (servants) of auxin transporters (masters).

  19. Active learning in transportation engineering education

    NASA Astrophysics Data System (ADS)

    Weir, Jennifer Anne

    The objectives of this research were (1) to develop experimental active-based-learning curricula for undergraduate courses in transportation engineering and (2) to assess the effectiveness of an active-learning-based traffic engineering curriculum through an educational experiment. The researcher developed a new highway design course as a pilot study to test selected active-learning techniques before employing them in the traffic engineering curriculum. Active-learning techniques, including multiple-choice questions, short problems completed by individual students or small groups, and group discussions, were used as active interludes within lectures. The researcher also collected and analyzed student performance and attitude data from control and experimental classes to evaluate the relative effectiveness of the traditional lecture (control) approach and the active-learning (experimental) approach. The results indicate that the active-learning approach adopted for the experimental class did have a positive impact on student performance as measured by exam scores. The students in the experimental class also indicated slightly more positive attitudes at the end of the course than the control class, although the difference was not significant. The author recommends that active interludes similar to those in the experimental curricula be used in other courses in civil engineering.

  20. Human dopamine transporter gene: differential regulation of 18-kb haplotypes

    PubMed Central

    Zhao, Ying; Xiong, Nian; Liu, Yang; Zhou, Yanhong; Li, Nuomin; Qing, Hong; Lin, Zhicheng

    2013-01-01

    Aim Since previous functional studies of short haplotypes and polymorphic sites of SLC6A3 have shown variant-dependent and drug-sensitive promoter activity, this study aimed to understand whether a large SLC6A3 regulatory region, containing these small haplotypes and polymorphic sites, can display haplotype-dependent promoter activity in a drug-sensitive and pathway-related manner. Materials & methods By creating and using a single copy number luciferase-reporter vector, we examined regulation of two different SLC6A3 haplotypes (A and B) of the 5′ 18-kb promoter and two known downstream regulatory variable number tandem repeats by 17 drugs in four different cellular models. Results The two regulatory haplotypes displayed up to 3.2-fold difference in promoter activity. The regulations were drug selective (37.5% of the drugs showed effects), and both haplotype and cell type dependent. Pathway analysis revealed at least 13 main signaling hubs targeting SLC6A3, including histone deacetylation, AKT, PKC and CK2 α-chains. Conclusion SLC6A3 may be regulated via either its promoter or the variable number tandem repeats independently by specific signaling pathways and in a haplotype-dependent manner. Furthermore, we have developed the first pathway map for SLC6A3 regulation. These findings provide a framework for understanding complex and variant-dependent regulations of SLC6A3. PMID:24024899

  1. The plasminogen activator system: biology and regulation.

    PubMed

    Irigoyen, J P; Muñoz-Cánoves, P; Montero, L; Koziczak, M; Nagamine, Y

    1999-10-01

    The regulation of plasminogen activation involves genes for two plasminogen activators (tissue type and urokinase type), two specific inhibitors (type 1 and type 2), and a membrane-anchored urokinase-type plasminogen-activator-specific receptor. This system plays an important role in various biological processes involving extracellular proteolysis. Recent studies have revealed that the system, through interplay with integrins and the extracellular matrix protein vitronectin, is also involved in the regulation of cell migration and proliferation in a manner independent of proteolytic activity. The genes are expressed in many different cell types and their expression is under the control of diverse extracellular signals. Gene expression reflects the levels of the corresponding mRNA, which should be the net result of synthesis and degradation. Thus, modulation of mRNA stability is an important factor in overall regulation. This review summarizes current understanding of the biology and regulation of genes involved in plasminogen activation at different levels.

  2. Engineering intracellular active transport systems as in vivo biomolecular tools.

    SciTech Connect

    Bachand, George David; Carroll-Portillo, Amanda

    2006-11-01

    Active transport systems provide essential functions in terms of cell physiology and metastasis. These systems, however, are also co-opted by invading viruses, enabling directed transport of the virus to and from the cell's nucleus (i.e., the site of virus replication). Based on this concept, fundamentally new approaches for interrogating and manipulating the inner workings of living cells may be achievable by co-opting Nature's active transport systems as an in vivo biomolecular tool. The overall goal of this project was to investigate the ability to engineer kinesin-based transport systems for in vivo applications, specifically the collection of effector proteins (e.g., transcriptional regulators) within single cells. In the first part of this project, a chimeric fusion protein consisting of kinesin and a single chain variable fragment (scFv) of an antibody was successfully produced through a recombinant expression system. The kinesin-scFv retained both catalytic and antigenic functionality, enabling selective capture and transport of target antigens. The incorporation of a rabbit IgG-specific scFv into the kinesin established a generalized system for functionalizing kinesin with a wide range of target-selective antibodies raised in rabbits. The second objective was to develop methods of isolating the intact microtubule network from live cells as a platform for evaluating kinesin-based transport within the cytoskeletal architecture of a cell. Successful isolation of intact microtubule networks from two distinct cell types was demonstrated using glutaraldehyde and methanol fixation methods. This work provides a platform for inferring the ability of kinesin-scFv to function in vivo, and may also serve as a three-dimensional scaffold for evaluating and exploiting kinesin-based transport for nanotechnological applications. Overall, the technology developed in this project represents a first-step in engineering active transport system for in vivo applications. Further

  3. Glucose Transporters at the Blood-Brain Barrier: Function, Regulation and Gateways for Drug Delivery.

    PubMed

    Patching, Simon G

    2017-03-01

    Glucose transporters (GLUTs) at the blood-brain barrier maintain the continuous high glucose and energy demands of the brain. They also act as therapeutic targets and provide routes of entry for drug delivery to the brain and central nervous system for treatment of neurological and neurovascular conditions and brain tumours. This article first describes the distribution, function and regulation of glucose transporters at the blood-brain barrier, the major ones being the sodium-independent facilitative transporters GLUT1 and GLUT3. Other GLUTs and sodium-dependent transporters (SGLTs) have also been identified at lower levels and under various physiological conditions. It then considers the effects on glucose transporter expression and distribution of hypoglycemia and hyperglycemia associated with diabetes and oxygen/glucose deprivation associated with cerebral ischemia. A reduction in glucose transporters at the blood-brain barrier that occurs before the onset of the main pathophysiological changes and symptoms of Alzheimer's disease is a potential causative effect in the vascular hypothesis of the disease. Mutations in glucose transporters, notably those identified in GLUT1 deficiency syndrome, and some recreational drug compounds also alter the expression and/or activity of glucose transporters at the blood-brain barrier. Approaches for drug delivery across the blood-brain barrier include the pro-drug strategy whereby drug molecules are conjugated to glucose transporter substrates or encapsulated in nano-enabled delivery systems (e.g. liposomes, micelles, nanoparticles) that are functionalised to target glucose transporters. Finally, the continuous development of blood-brain barrier in vitro models is important for studying glucose transporter function, effects of disease conditions and interactions with drugs and xenobiotics.

  4. OXYGEN TRANSPORT IN THE MICROCIRCULATION AND ITS REGULATION

    PubMed Central

    Pittman, Roland N.

    2012-01-01

    Cells require energy to carry out their functions and they typically use oxidative phosphorylation to generate the needed ATP. Thus, cells have a continuous need for oxygen which they receive by diffusion from the blood through the interstitial fluid. The circulatory system pumps oxygen-rich blood through a network of increasingly minute vessels, the microcirculation. The structure of the microcirculation is such that all cells have at least one nearby capillary for diffusive exchange of oxygen and red blood cells release the oxygen bound to hemoglobin as they traverse capillaries. This review focuses first on the historical development of techniques to measure oxygen at various sites in the microcirculation, including the blood, interstitium and cells. Next, approaches are described as to how these techniques have been employed to make discoveries about different aspects of oxygen transport. Finally, ways in which oxygen might participate in the regulation of blood flow toward matching oxygen supply to oxygen demand is discussed. Overall, the transport of oxygen to the cells of the body is one of the most critical functions of the cardiovascular system and it is in the microcirculation where the final local determinants of oxygen supply, oxygen demand and their regulation are decided. PMID:23025284

  5. Transportation in the Interstitial Space of the Brain Can Be Regulated by Neuronal Excitation

    NASA Astrophysics Data System (ADS)

    Shi, Chunyan; Lei, Yiming; Han, Hongbin; Zuo, Long; Yan, Junhao; He, Qingyuan; Yuan, Lan; Liu, Huipo; Xu, Ge; Xu, Weiguo

    2015-12-01

    The transportation of substances in the interstitial space (ISS) is crucial for the maintenance of brain homeostasis, however its link to neuronal activity remains unclear. Here, we report a marked reduction in substance transportation in the ISS after neuronal excitation. Using a tracer-based method, water molecules in the interstitial fluid (ISF) could be specifically visualized in magnetic resonance (MR) imaging. We first observed the flow of ISF in the thalamus and caudate nucleus of a rat. The ISF flow was then modulated using a painful stimulation model. We demonstrated that the flow of ISF slowed significantly following neuronal activity in the thalamus. This reduction in ISF flow continued for hours and was not accompanied by slow diffusion into the ISS. This observation suggests that the transportation of substances into the ISS can be regulated with a selective external stimulation.

  6. Transportation in the Interstitial Space of the Brain Can Be Regulated by Neuronal Excitation.

    PubMed

    Shi, Chunyan; Lei, Yiming; Han, Hongbin; Zuo, Long; Yan, Junhao; He, Qingyuan; Yuan, Lan; Liu, Huipo; Xu, Ge; Xu, Weiguo

    2015-12-03

    The transportation of substances in the interstitial space (ISS) is crucial for the maintenance of brain homeostasis, however its link to neuronal activity remains unclear. Here, we report a marked reduction in substance transportation in the ISS after neuronal excitation. Using a tracer-based method, water molecules in the interstitial fluid (ISF) could be specifically visualized in magnetic resonance (MR) imaging. We first observed the flow of ISF in the thalamus and caudate nucleus of a rat. The ISF flow was then modulated using a painful stimulation model. We demonstrated that the flow of ISF slowed significantly following neuronal activity in the thalamus. This reduction in ISF flow continued for hours and was not accompanied by slow diffusion into the ISS. This observation suggests that the transportation of substances into the ISS can be regulated with a selective external stimulation.

  7. Transportation in the Interstitial Space of the Brain Can Be Regulated by Neuronal Excitation

    PubMed Central

    Shi, Chunyan; Lei, Yiming; Han, Hongbin; Zuo, Long; Yan, Junhao; He, Qingyuan; Yuan, Lan; Liu, Huipo; Xu, Ge; Xu, Weiguo

    2015-01-01

    The transportation of substances in the interstitial space (ISS) is crucial for the maintenance of brain homeostasis, however its link to neuronal activity remains unclear. Here, we report a marked reduction in substance transportation in the ISS after neuronal excitation. Using a tracer-based method, water molecules in the interstitial fluid (ISF) could be specifically visualized in magnetic resonance (MR) imaging. We first observed the flow of ISF in the thalamus and caudate nucleus of a rat. The ISF flow was then modulated using a painful stimulation model. We demonstrated that the flow of ISF slowed significantly following neuronal activity in the thalamus. This reduction in ISF flow continued for hours and was not accompanied by slow diffusion into the ISS. This observation suggests that the transportation of substances into the ISS can be regulated with a selective external stimulation. PMID:26631412

  8. Regulation of renal peripheral benzodiazepine receptors by anion transport inhibitors

    SciTech Connect

    Basile, A.S.; Lueddens, W.M.; Skolnick, P.

    1988-01-01

    The in vitro and in vivo regulation of (/sup 3/H)Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of (/sup 3/H)Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approx. = 30 - 130 ..mu..M). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki=100-1000 ..mu..M). Administration of furosemide to rats for five days resulted in a profound diuresis accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of (/sup 3/H)Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors. 36 references, 4 tables.

  9. Regulation of lysosomal ion homeostasis by channels and transporters.

    PubMed

    Xiong, Jian; Zhu, Michael X

    2016-08-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H(+), Ca(2+), Na(+), K(+), and Cl(-) across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease.

  10. 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

  11. Regulation of lysosomal ion homeostasis by channels and transporters

    PubMed Central

    Xiong, Jian; Zhu, Michael X.

    2016-01-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H+, Ca2+, Na+, K+, and Cl− across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease. PMID:27430889

  12. Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport

    SciTech Connect

    Gracz, L.M.; Wang, W.; Parsons, S.M.

    1988-07-12

    Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP/sub 1/ synaptic vesicles banding at 1.055 g/mL actively transported (/sup 3/H)acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more (/sup 3/H)AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP/sub 1/ vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP/sub 2/ synaptic vesicle originally described in intact Torpedo marmorata electric organ. The properties of the receptor for vesamicol were studied by measuring binding of (/sup 3/H)vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP/sub 1/ vesicles the VP/sub 2/ vesicles had a ratio of (/sup 3/H)AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. The Hill coefficients ..cap alpha../sub H/ and equilibrium dissociation constants K for vesamicol binding to VP/sub 1/ and VP/sub 2/ vesicles were essentially the same. The positive Hill coefficient suggests that the vesamicol receptor exists as a homotropic oligomeric complex. The results demonstrate that VP/sub 1/ and VP/sub 2/ synaptic vesicles exhibit functional differences in the AcCh transport system, presumably as a result of regulatory phenomena.

  13. Dopamine transporters participate in the physiological regulation of prolactin.

    PubMed

    Demaria, J E; Nagy, G M; Lerant, A A; Fekete, M I; Levenson, C W; Freeman, M E

    2000-01-01

    Three populations of hypothalamic neuroendocrine dopaminergic (NEDA) neurons, arising from the arcuate and periventricular nuclei of the hypothalamus release dopamine (DA) that acts at the pituitary gland to regulate the secretion of PRL. It is generally accepted that NEDA neurons lack functional DA transporters (DATs), which are responsible for uptake of DA from the synaptic cleft into the presynaptic axon terminal. This study localized DATs to the hypothalamo-pituitary axis and evaluated the effect of DAT blockade on the hypothalamo-pituitary regulation of PRL. After 7 days of treatment with cocaine (a nonspecific amine transporter blocker) or mazindol (a specific DAT blocker), the relative abundance of PRL messenger RNA (mRNA) in the anterior lobe (AL) of OVX rats was significantly decreased, whereas the relative abundance of tyrosine hydroxylase mRNA in the hypothalamus was significantly increased. The effect of cocaine or mazindol administration on DA turnover and serum PRL concentration was examined in estradiol (E2)-treated OVX rats. E2 administration (i.v.) resulted in a significant increase in serum PRL within 4 h; however, cocaine or mazindol administration abolished the E2-induced increase of PRL. Cocaine or mazindol significantly increased the concentration of DA at the site of the axon terminals within the median eminence (ME), intermediate lobe (IL) and neural lobe (NL), indicating blockade of uptake. Because formation of DOPAC requires uptake of DA, concentrations of DOPAC in the ME, IL and NL decreased following treatment with either cocaine or mazindol. These data, together with the presence of immunopositive DAT in the ME, pituitary stalk, IL, and NL, suggest that a functional DAT system is present within all three populations of NEDA neurons. Moreover, similarity between the effects of cocaine and mazindol treatment indicate that blockade of the DAT, but not other amine transporters, is responsible for suppression of PRL gene expression and

  14. Single-molecule detection with active transport

    NASA Astrophysics Data System (ADS)

    Ball, David Allan

    A glass capillary is used near the focal region of a custom-built confocal microscope to investigate the use of active transport for single-molecule detection in solution, with both one and two-photon laser excitation. The capillary tip has a diameter of several microns and is carefully aligned nearby to the sub-micron laser beam waist, collinear to the optical axis, so that a negative pressure-difference causes molecules to be drawn into the capillary, along the laser beam axis. The flow of solution, which is characterized by fluorescence correlation spectroscopy (FCS), can increase the single-molecule detection rate for slowly diffusing proteins by over a factor of 100, while the mean rate of photons during each burst is similar to that for random diffusional transport. Also, the flow is along the longest axis of the ellipsoidally-shaped confocal volume, which results in more collected photons per molecule than that for transverse flow at the same speed. When transport is dominated by flow, FCS can no longer distinguish molecules with differing translational diffusion, and hence a fluorescence fluctuation spectroscopy method based on differences in fluorescence brightness is investigated as a means for assaying different solution components, for applications in pharmaceutical drug discovery. Multi-channel fluctuation spectroscopy techniques can also be used for assays with the flow system and hence this dissertation also reports the characterization of a prototype 4-channel single-photon detector with a two-wavelength polarization-resolved optical set-up.

  15. Glycosylation regulates prestin cellular activity.

    PubMed

    Rajagopalan, Lavanya; Organ-Darling, Louise E; Liu, Haiying; Davidson, Amy L; Raphael, Robert M; Brownell, William E; Pereira, Fred A

    2010-03-01

    Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by

  16. 50 CFR 404.7 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Wildlife and Fisheries JOINT REGULATIONS (UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR AND NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT... MARINE NATIONAL MONUMENT § 404.7 Regulated activities. Except as provided in §§ 404.8, 404.9 and...

  17. Transport systems of Ventricaria ventricosa: asymmetry of the hyper- and hypotonic regulation mechanisms.

    PubMed

    Bisson, M A; Beilby, M J

    2008-01-01

    Hyper- and hypotonic stresses elicit apparently symmetrical responses in the alga Ventricaria. With hypertonic stress, membrane potential difference (PD) between the vacuole and the external medium becomes more positive, conductance at positive PDs (Gmpos) increases and KCl is actively taken up to increase turgor. With hypotonic stress, the membrane PD becomes more negative, conductance at negative PDs (Gmneg) increases and KCl is lost to decrease turgor. We used inhibitors that affect active transport to determine whether agents that inhibit the K(+) pump and hypertonic regulation also inhibit hypotonic regulatory responses. Cells whose turgor pressure was held low by the pressure probe (turgor-clamped) exhibited the same response as cells challenged by hyperosmotic medium, although the response was maintained longer than in osmotically challenged cells, which regulate turgor. The role of active K(+) transport was confirmed by the effects of decreased light, dichlorophenyldimethyl urea and diethylstilbestrol, which induced a uniformly low conductance (quiet state). Cells clamped to high turgor exhibited the same response as cells challenged by hypo-osmotic medium, but the response was similarly transient, making effects of inhibitors hard to determine. Unlike clamped cells, cells challenged by hypo-osmotic medium responded to inhibitors with rapid, transient, negative-going PDs, with decreased Gmneg and increased Gmpos (linearized I-V), achieving the quiet state as PD recovered. These changes are different from those exerted on the pump state, indicating that different transport systems are responsible for turgor regulation in the two cases.

  18. Regulation of Sodium Channel Activity by Capping of Actin Filaments

    PubMed Central

    Shumilina, Ekaterina V.; Negulyaev, Yuri A.; Morachevskaya, Elena A.; Hinssen, Horst; Khaitlina, Sofia Yu

    2003-01-01

    Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells. PMID:12686620

  19. Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity

    PubMed Central

    Ormerod, Kiel G.

    2017-01-01

    Abstract Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. Here, we describe an analysis of how Ca2+ signals within Drosophila astrocyte-like glia regulate excitability in the nervous system. We find that Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, we find that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. We observe that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. Our data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability. PMID:28303263

  20. Copper transporter 2 regulates intracellular copper and sensitivity to cisplatin.

    PubMed

    Huang, Carlos P; Fofana, Mariama; Chan, Jefferson; Chang, Christopher J; Howell, Stephen B

    2014-03-01

    Mammalian cells express two copper (Cu) influx transporters, CTR1 and CTR2. CTR1 serves as an influx transporter for both Cu and cisplatin (cDDP). In mouse embryo fibroblasts, reduction of CTR1 expression renders cells resistant to cDDP whereas reduction of CTR2 makes them hypersensitive both in vitro and in vivo. To investigate the role of CTR2 on intracellular Cu and cDDP sensitivity its expression was molecularly altered in the human epithelial 2008 cancer cell model. Intracellular exchangeable Cu(+) was measured with the fluorescent probe Coppersensor-3 (CS3). The ability of CS3 to report on changes in intracellular Cu(+) was validated by showing that Cu chelators reduced its signal, and that changes in signal accompanied alterations in expression of the major Cu influx transporter CTR1 and the two Cu efflux transporters, ATP7A and ATP7B. Constitutive knock down of CTR2 mRNA by ∼50% reduced steady-state exchangeable Cu by 22-23% and increased the sensitivity of 2008 cells by a factor of 2.6-2.9 in two separate clones. Over-expression of CTR2 increased exchangeable Cu(+) by 150% and rendered the 2008 cells 2.5-fold resistant to cDDP. The results provide evidence that CS3 can quantitatively assess changes in exchangeable Cu(+), and that CTR2 regulates both the level of exchangeable Cu(+) and sensitivity to cDDP in a model of human epithelial cancer. This study introduces CS3 and related sensors as novel tools for probing and assaying Cu-dependent sensitivity to anticancer therapeutics.

  1. Dopamine Transporter Activity Is Modulated by α-Synuclein.

    PubMed

    Butler, Brittany; Saha, Kaustuv; Rana, Tanu; Becker, Jonas P; Sambo, Danielle; Davari, Paran; Goodwin, J Shawn; Khoshbouei, Habibeh

    2015-12-04

    The duration and strength of the dopaminergic signal are regulated by the dopamine transporter (DAT). Drug addiction and neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine-induced DAT-mediated dopamine efflux, and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT·α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells and that the amphetamine-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases dopamine efflux and enhances DAT localization in cholesterol-rich membrane microdomains.

  2. Serotonin transporter activity in platelets and canine aggression.

    PubMed

    Rosado, Belén; García-Belenguer, Sylvia; Palacio, Jorge; Chacón, Gema; Villegas, Ainara; Alcalde, Ana I

    2010-10-01

    Several studies have suggested an inhibitory action of the serotonergic system in the regulation of canine aggression, but the role of the serotonin (5-HT) transporter (5-HTT) has not been investigated. Platelet 5-HT uptake has been proposed as a peripheral marker of brain 5-HTT. The aim of the study was to investigate the relationship between platelet 5-HTT activity and canine aggression by measuring the rate of 5-HT uptake mediated by 5-HTT in platelets and serum concentrations of 5-HT in both aggressive (n=14) and non-aggressive dogs (n=17). Aggressive dogs showed significantly higher 5-HT uptake by 5-HTT in platelets and lower serum concentrations of 5-HT, compared with the control group. These results suggested an association between an alteration in the serotonergic system and canine aggression, possibly mediated by an increased 5-HT transport.

  3. Regulation of the divalent metal ion transporter via membrane budding

    PubMed Central

    Mackenzie, KimberlyD; Foot, Natalie J; Anand, Sushma; Dalton, Hazel E; Chaudhary, Natasha; Collins, Brett M; Mathivanan, Suresh; Kumar, Sharad

    2016-01-01

    The release of extracellular vesicles (EVs) is important for both normal physiology and disease. However, a basic understanding of the targeting of EV cargoes, composition and mechanism of release is lacking. Here we present evidence that the divalent metal ion transporter (DMT1) is unexpectedly regulated through release in EVs. This process involves the Nedd4-2 ubiquitin ligase, and the adaptor proteins Arrdc1 and Arrdc4 via different budding mechanisms. We show that mouse gut explants release endogenous DMT1 in EVs. Although we observed no change in the relative amount of DMT1 released in EVs from gut explants in Arrdc1 or Arrdc4 deficient mice, the extent of EVs released was significantly reduced indicating an adaptor role in biogenesis. Furthermore, using Arrdc1 or Arrdc4 knockout mouse embryonic fibroblasts, we show that both Arrdc1 and Arrdc4 are non-redundant positive regulators of EV release. Our results suggest that DMT1 release from the plasma membrane into EVs may represent a novel mechanism for the maintenance of iron homeostasis, which may also be important for the regulation of other membrane proteins. PMID:27462458

  4. p53 regulates thymic Notch1 activation.

    PubMed

    Laws, Amy M; Osborne, Barbara A

    2004-03-01

    Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP)/CD8+ single positive (SP) transition, but regulation of Notchactivation is not well understood. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. We hypothesize that p53 regulates Notch activity during T cell development. We found that Notch1 expression and activation were negatively regulated by p53in several thymoma lines. Additionally, NIC was elevated in Trp53(-/-) thymocytes as compared to Trp53(+/+) thymocytes. To determine if elevated Notch1 activation in Trp53(-/-) thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP/CD8+ SP T cell ratio was decreased in Trp53(-/-) splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8+SP T cells in Trp53(-/-) mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, we suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation.

  5. Regulation of TRPM8 channel activity

    PubMed Central

    Yudin, Yevgen; Rohacs, Tibor

    2011-01-01

    Transient Receptor Potential Melastatin 8 (TRPM8) is a Ca2+ permeable non-selective cation channel directly activated by cold temperatures and chemical agonists such as menthol. It is a well established sensor of environmental cold temperatures, found in peripheral sensory neurons, where its activation evokes depolarization and action potentials. The activity of TRPM8 is regulated by a number of cellular signaling pathways, most notably by phosphoinositides and the activation of phospholipase C. This review will summarize current knowledge on the physiological and pathophysiological roles of TRPM8 and its regulation by various intracellular messenger molecules and signaling pathways. PMID:22061619

  6. Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells

    PubMed Central

    Jouan, Elodie; Le Vée, Marc; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2016-01-01

    Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP) activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP), organic anion-transporting polypeptides (OATPs) and organic cation transporter 1 (OCT1), and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP). Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2), OCT1 and bile salt export pump) or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3) in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR)- and nuclear factor erythroid 2-related factor 2 (Nrf2)-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation. PMID:28036031

  7. The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation

    PubMed Central

    Sasser, Terry L.; Padolina, Mark; Fratti, Rutilio A.

    2013-01-01

    Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function. PMID:22970809

  8. Transport in active systems crowded by obstacles

    NASA Astrophysics Data System (ADS)

    Huang, Mu-Jie; Schofield, Jeremy; Kapral, Raymond

    2017-02-01

    The reactive and diffusive dynamics of a single chemically powered Janus motor in a crowded medium of moving but passive obstacles is investigated using molecular simulation. It is found that the reaction rate of the catalytic motor reaction decreases in a crowded medium as the volume fraction of obstacles increases as a result of a reduction in the Smoluchowski diffusion-controlled reaction rate coefficient that contributes to the overall reaction rate. A continuum model is constructed and analyzed to interpret the dependence of the steady-state reaction rate observed in simulations on the volume fraction of obstacles in the system. The steady-state concentration fields of reactant and product are shown to be sensitive to the local structure of obstacles around the Janus motor. It is demonstrated that the active motor exhibits enhanced diffusive motion at long times with a diffusion constant that decreases as the volume fraction of crowding species increases. In addition, the dynamical properties of a passive tracer particle in a system containing many active Janus motors is studied to investigate how an active environment influences the transport of non-active species. The diffusivity of a passive tracer particle in an active medium is found to be enhanced in systems with forward-moving Janus motors due to the cooperative dynamics of these motors.

  9. Fingolimod effects in neuroinflammation: Regulation of astroglial glutamate transporters?

    PubMed

    Lee, De-Hyung; Seubert, Silvia; Huhn, Konstantin; Brecht, Lukas; Rötger, Caroline; Waschbisch, Anne; Schlachetzki, Johannes; Klausmeyer, Alice; Melms, Arthur; Wiese, Stefan; Winkler, Jürgen; Linker, Ralf A

    2017-01-01

    Fingolimod is an oral sphingosine-1-phosphate-receptor modulator which reduces the recirculation of immune cells and may also directly target glial cells. Here we investigate effects of fingolimod on expression of astroglial glutamate transporters under pro-inflammatory conditions. In astrocyte cell culture, the addition of pro-inflammatory cytokines led to a significant downregulation of glutamate transporters glutamate transporter-1 (slc1a2/SLC1A2) and glutamate aspartate transporter (slc1a3/SLC1A3) expression on the mRNA or protein level. In this setting, the direct application of fingolimod-1 phosphate (F1P) on astrocytes did not change expression levels of slc1a2 and slc1a3 mRNA. The analysis of both transporters on the protein level by Western Blot and immunocytochemistry did also not reveal any effect of F1P. On a functional level, the addition of conditioned supernatants from F1P treated astrocytes to neuronal cell culture did not result in increased neurite growth. In experimental autoimmune encephalomyelitis as a model of multiple sclerosis, fingolimod treatment reduced T cell and macrophages/microglia mediated inflammation and also diminished astrocyte activation. At the same time, fingolimod restored the reduced expression of slc1a2 and slc1a3 in the inflamed spinal cord on the mRNA level and of SLC1A2 and SLC1A3 on the protein level, presumably via indirect, anti-inflammatory mechanisms. These findings provide further evidence for a predominantly peripheral effect of the compound in neuroinflammation.

  10. Fingolimod effects in neuroinflammation: Regulation of astroglial glutamate transporters?

    PubMed Central

    Lee, De-Hyung; Seubert, Silvia; Huhn, Konstantin; Brecht, Lukas; Rötger, Caroline; Waschbisch, Anne; Schlachetzki, Johannes; Klausmeyer, Alice; Melms, Arthur; Wiese, Stefan; Winkler, Jürgen; Linker, Ralf A.

    2017-01-01

    Fingolimod is an oral sphingosine-1-phosphate-receptor modulator which reduces the recirculation of immune cells and may also directly target glial cells. Here we investigate effects of fingolimod on expression of astroglial glutamate transporters under pro-inflammatory conditions. In astrocyte cell culture, the addition of pro-inflammatory cytokines led to a significant downregulation of glutamate transporters glutamate transporter-1 (slc1a2/SLC1A2) and glutamate aspartate transporter (slc1a3/SLC1A3) expression on the mRNA or protein level. In this setting, the direct application of fingolimod-1 phosphate (F1P) on astrocytes did not change expression levels of slc1a2 and slc1a3 mRNA. The analysis of both transporters on the protein level by Western Blot and immunocytochemistry did also not reveal any effect of F1P. On a functional level, the addition of conditioned supernatants from F1P treated astrocytes to neuronal cell culture did not result in increased neurite growth. In experimental autoimmune encephalomyelitis as a model of multiple sclerosis, fingolimod treatment reduced T cell and macrophages/microglia mediated inflammation and also diminished astrocyte activation. At the same time, fingolimod restored the reduced expression of slc1a2 and slc1a3 in the inflamed spinal cord on the mRNA level and of SLC1A2 and SLC1A3 on the protein level, presumably via indirect, anti-inflammatory mechanisms. These findings provide further evidence for a predominantly peripheral effect of the compound in neuroinflammation. PMID:28273090

  11. Posttranslational Regulation of Organic Anion Transporters by Ubiquitination: Known and Novel.

    PubMed

    Xu, Da; Wang, Haoxun; You, Guofeng

    2016-09-01

    Organic anion transporters (OATs) encoded by solute carrier 22 family are localized in the epithelia of multiple organs, where they mediate the absorption, distribution, and excretion of a diverse array of negatively charged environmental toxins and clinically important drugs. Alterations in the expression and function of OATs play important roles in intra- and interindividual variability of the therapeutic efficacy and the toxicity of many drugs. As a result, the activity of OATs must be under tight regulation so as to carry out their normal functions. The regulation of OAT transport activity in response to various stimuli can occur at several levels such as transcription, translation, and posttranslational modification. Posttranslational regulation is of particular interest, because it usually happens within a very short period of time (minutes to hours) when the body has to deal with rapidly changing amounts of substances as a consequence of variable intake of drugs, fluids, or meals as well as metabolic activity. This review article highlights the recent advances from our laboratory in uncovering several posttranslational mechanisms underlying OAT regulation. These advances offer the promise of identifying targets for novel strategies that will maximize therapeutic efficacy in drug development.

  12. The riboswitch regulates a thiamine pyrophosphate ABC transporter of the oral spirochete Treponema denticola.

    PubMed

    Bian, Jiang; Shen, Hongwu; Tu, Youbin; Yu, Aiming; Li, Chunhao

    2011-08-01

    Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B₁), is an essential cofactor in all living systems. Microorganisms either synthesize TPP via de novo biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. The oral spirochete Treponema denticola is an important pathogen that is associated with human periodontal diseases. It lacks a de novo TPP biosynthesis pathway and needs exogenous TPP for growth, suggesting that it may obtain exogenous TPP via a thiamine transporter. In this study, we identified a gene cluster that encodes a TPP ABC transporter which consists of a TPP-binding protein (TDE0143), a transmembrane permease (TDE0144), and a cytosolic ATPase (TDE0145). Transcriptional and translational analyses showed that the genes encoding these three proteins are cotranscribed and form an operon (tbpABC(Td)) that is initiated by a σ⁷⁰-like promoter. The expression level of this operon is negatively regulated by exogenous TPP and is mediated by a TPP-sensing riboswitch (Td(thi-)(box)). Genetic and biochemical studies revealed that the TDE0143 deletion mutant (T. denticola ΔtbpA) had a decreased ability to transport exogenous TPP, and the mutant failed to grow when exogenous TPP was insufficient. These results taken together indicate that the tbpABC(Td) operon encodes an ABC transporter that is required for the uptake of exogenous TPP and that the expression of this operon is regulated by a TPP-binding riboswitch via a feedback inhibition mechanism.

  13. Regulation of Auxin Transport by Phosphorylation and Flavonoids during Gravitropism in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Muday, Gloria K.

    2005-01-01

    The focus of this research includes: 1) Regulation of Axin transport by flavonoids during gravitropism; 2) Phosphorylation control of auxin transport during gravity response; 3) Ethylene regulation of gravitropic curvature; 4) IBA transport and gravitropic response; and 5) Other collaborative projects.

  14. New mechanisms that regulate Saccharomyces cerevisiae short peptide transporter achieve balanced intracellular amino acid concentrations.

    PubMed

    Melnykov, Artem V

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is able to take up large quantities of amino acids in the form of di- and tripeptides via a short peptide transporter, Ptr2p. It is known that PTR2 can be induced by certain peptides and amino acids, and the mechanisms governing this upregulation are understood at the molecular level. We describe two new opposing mechanisms of regulation that emphasize potential toxicity of amino acids: the first is upregulation of PTR2 in a population of cells, caused by amino acid secretion that accompanies peptide uptake; the second is loss of Ptr2p activity, due to transporter internalization following peptide uptake. Our findings emphasize the importance of proper amino acid balance in the cell and extend understanding of peptide import regulation in yeast.

  15. Calcium-Mediated Regulation of Proton-Coupled Sodium Transport - Final Report

    SciTech Connect

    Schumaker, Karen S

    2013-10-24

    The long-term goal of our experiments was to understand mechanisms that regulate energy coupling by ion currents in plants. Activities of living organisms require chemical, mechanical, osmotic or electrical work, the energy for which is supplied by metabolism. Adenosine triphosphate (ATP) has long been recognized as the universal energy currency, with metabolism supporting the synthesis of ATP and the hydrolysis of ATP being used for the subsequent work. However, ATP is not the only energy currency in living organisms. A second and very different energy currency links metabolism to work by the movement of ions passing from one side of a membrane to the other. These ion currents play a major role in energy capture and they support a range of physiological processes from the active transport of nutrients to the spatial control of growth and development. In Arabidopsis thaliana (Arabidopsis), the activity of a plasma membrane Na+/H+ exchanger, SALT OVERLY SENSITIVE1 (SOS1), is essential for regulation of sodium ion homeostasis during plant growth in saline conditions. Mutations in SOS1 result in severely reduced seedling growth in the presence of salt compared to the growth of wild type. SOS1 is a secondary active transporter coupling movement of sodium ions out of the cell using energy stored in the transplasma membrane proton gradient, thereby preventing the build-up of toxic levels of sodium in the cytosol. SOS1 is regulated by complexes containing the SOS2 and CALCINEURIN B-LIKE10 (CBL10) or SOS3 proteins. CBL10 and SOS3 (also identified as CBL4) encode EF-hand calcium sensors that interact physically with and activate SOS2, a serine/threonine protein kinase. The CBL10/SOS2 or SOS3/SOS2 complexes then activate SOS1 Na+/H+ exchange activity. We completed our studies to understand how SOS1 activity is regulated. Specifically, we asked: (1) how does CBL10 regulate SOS1 activity? (2) What role do two putative CBL10-interacting proteins play in SOS1 regulation? (3) Are

  16. Impact of river regulation on potential sediment mobilization and transport in an Alpine catchment

    NASA Astrophysics Data System (ADS)

    Costa, Anna; Molnar, Peter; Lane, Stuart N.; Bakker, Maarten

    2015-04-01

    The upper Rhône basin (upstream of Lake Geneva) has been heavily affected by human activities during the last century. The most evident impacts are related to river regulation, specifically flow impoundement, flow abstraction and channelization. In the last century and mainly since 1960, several large dams have been built along the main tributaries of the Rhône River, resulting in the water storage of a volume equal to 20% of the total annual river flow. The dams are part of hydropower systems which abstract water from streams and transfer it through complex networks (intakes, tunnels and pumping stations) to the reservoirs. Hydropower production leads to regulated flow in the Rhône: mostly an increase of winter flows, a reduction of summer flows, and a decrease of flood peaks. The sediment supply into Lake Geneva has decreased following dam construction (Loizeau & Dominik, 2000) due to the storage of sediment in upstream reservoirs, in rivers with reduced sediment transport capacity due to flow abstraction, and due to the development of sediment mining. Our hypothesis is that streamflow regulation itself has dramatically impacted the sediment transport dynamics of the system. We investigate the impacts of flow regulation on the sediment transport regime, by analysing the effects on potential sediment transport capacity (bedload). By the use of different bedload transport formulae (Meyer-Peter Müller, Wilcock and Crowe), the potential sediment transport capacity is computed at different cross sections within the basin. Potential sediment mobility occurs when the applied bed shear stress exceeds a critical value, τ>τc. The applied bed shear stress is computed as τ=ρghS, with water depth (h) measured from rating curves. We obtain an estimate of the energy slope (S) from the analysis of the river cross section, assuming uniform flow. The critical value of bed shear stress τc is computed using empirical formulae as a function of the grain diameter (ds). To

  17. Iron-induced turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter requires lysine residues.

    PubMed

    Kerkeb, Loubna; Mukherjee, Indrani; Chatterjee, Iera; Lahner, Brett; Salt, David E; Connolly, Erin L

    2008-04-01

    Iron is an essential micronutrient but is toxic if accumulated at high levels. Thus, iron uptake and distribution in plants are controlled by precise regulatory mechanisms. IRON-REGULATED TRANSPORTER1 (IRT1) is the major high affinity iron transporter responsible for iron uptake from the soil in Arabidopsis (Arabidopsis thaliana). Previously, we showed that IRT1 is subject to posttranscriptional regulation; when expressed from the constitutive cauliflower mosaic virus 35S promoter, IRT1 protein accumulates only in iron-deficient roots. IRT1 contains an intracellular loop that may be critical for posttranslational regulation by metals. Of particular interest are a histidine (His) motif (HGHGHGH) that might bind metals and two lysine residues that could serve as attachment sites for ubiquitin. We constructed a set of mutant IRT1 alleles: IRT1H154Q, IRT1H156Q, IRT1H158Q, IRT1H160Q, IRT14HQ (quadruple His mutant), IRT1K146R, IRT1K171R, and a double mutant (IRT1K146R,K171R). Mutation of the His or lysine residues did not eliminate the ability of IRT1 to transport iron or zinc. Expression of each of the IRT1 variants and an IRT1intact construct in plants from the 35S promoter revealed that either K146 or K171 is required for iron-induced protein turnover, and 35S-IRT1K146R,K171R plants contain higher levels of iron as compared to 35S-IRT1 and wild type. Furthermore, accumulation of metals in 35S-IRT1K146R,K171R plants was not associated with an increase in ferric chelate reductase activity; this result indicates that, at least under conditions when iron is abundant, reduction of ferric iron may not be the rate-limiting step in iron uptake by strategy I plants such as Arabidopsis.

  18. Differential regulation of placental amino acid transport by saturated and unsaturated fatty acids.

    PubMed

    Lager, Susanne; Jansson, Thomas; Powell, Theresa L

    2014-10-15

    Fatty acids are critical for normal fetal development but may also influence placental function. We have previously reported that oleic acid (OA) stimulates amino acid transport in primary human trophoblasts (PHTs). In other tissues, saturated and unsaturated fatty acids have distinct effects on cellular signaling, for instance, palmitic acid (PA) but not OA reduces IκBα expression. We hypothesized that saturated and unsaturated fatty acids differentially affect trophoblast amino acid transport and cellular signaling. To test this hypothesis, PHTs were cultured in docosahexaenoic acid (DHA; 50 μM), OA (100 μM), or PA (100 μM). DHA and OA were also combined to test whether DHA could counteract the OA stimulatory effect on amino acid transport. The effects of fatty acids were compared against a vehicle control. Amino acid transport was measured by isotope-labeled tracers. Activation of inflammatory-related signaling pathways and the mechanistic target of rapamycin (mTOR) pathway were determined by Western blot analysis. Exposure of PHTs to DHA for 24 h reduced amino acid transport and phosphorylation of p38 MAPK, STAT3, mTOR, eukaryotic initiation factor 4E-binding protein 1, and ribosomal protein (rp)S6. In contrast, OA increased amino acid transport and phosphorylation of ERK, mTOR, S6 kinase 1, and rpS6. The combination of DHA with OA increased amino acid transport and rpS6 phosphorylation. PA did not affect amino acid transport but reduced IκBα expression. In conclusion, these fatty acids differentially regulated placental amino acid transport and cellular signaling. Taken together, these findings suggest that dietary fatty acids could alter the intrauterine environment by modifying placental function, thereby having long-lasting effects on the developing fetus.

  19. A general method for determining secondary active transporter substrate stoichiometry.

    PubMed

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-25

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na(+)-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

  20. Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes

    PubMed Central

    1992-01-01

    Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP- binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed. PMID:1447289

  1. 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

  2. 77 FR 51706 - Transportation of Household Goods in Interstate Commerce; Consumer Protection Regulations...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-08-27

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF TRANSPORTATION Federal Motor Carrier Safety Administration 49 CFR Part 375 RIN 2126-AB41 Transportation of Household Goods in Interstate Commerce; Consumer Protection Regulations: Household Goods Motor Carrier...

  3. Regulation of dopamine transporter function by protein-protein interactions: new discoveries and methodological challenges.

    PubMed

    Eriksen, Jacob; Jørgensen, Trine Nygaard; Gether, Ulrik

    2010-04-01

    The dopamine transporter (DAT) plays a key role in regulating dopaminergic signalling in the brain by mediating rapid clearance of dopamine from the synaptic clefts. The psychostimulatory actions of cocaine and amphetamine are primarily the result of a direct interaction of these compounds with DAT leading to attenuated dopamine clearance and for amphetamine even increased dopamine release. In the last decade, intensive efforts have been directed towards understanding the molecular and cellular mechanisms governing the activity and availability of DAT in the plasma membrane of the pre-synaptic neurons. This has led to the identification of a plethora of different kinases, receptors and scaffolding proteins that interact with DAT and hereby either modulate the catalytic activity of the transporter or regulate its trafficking and degradation. Several new tools for studying DAT regulation in live cells have also recently become available such as fluorescently tagged cocaine analogues and fluorescent substrates. Here we review the current knowledge about the role of protein-protein interactions in DAT regulation as well as we describe the most recent methodological developments that have been established to overcome the challenges associated with the study of DAT in endogenous systems.

  4. Identification of glucose-6-phosphate transporter as a key regulator functioning at the autophagy initiation step.

    PubMed

    Ahn, Hye-Hyun; Oh, Yumin; Lee, Huikyong; Lee, WonJae; Chang, Jae-Woong; Pyo, Ha-Kyung; Nah, Do hyung; Jung, Yong-Keun

    2015-07-22

    Autophagy is a catabolic process involving autophagosome formation via lysosome. However, the initiation step of autophagy is largely unknown. We found an interaction between ULK1 and ATG9 in mammalian cells and utilized the interaction to identify novel regulators of autophagy upstream of ULK1. We established a cell-based screening assay employing bimolecular fluorescence complementation. By performing gain-of-function screening, we identified G6PT as an autophagy activator. G6PT enhanced the interaction between N-terminal Venus-tagged ULK1 and C-terminal Venus-tagged ATG9, and increased autophagic flux independent of its transport activity. G6PT negatively regulated mTORC1 activity, demonstrating that G6PT functions upstream of mTORC1 in stimulating autophagy.

  5. Regulation of glomerulotubular balance: flow-activated proximal tubule function.

    PubMed

    Wang, Tong; Weinbaum, Sheldon; Weinstein, Alan M

    2017-03-07

    The purpose of this review is to summarize our knowledge and understanding of the physiological importance and the mechanisms underlying flow-activated proximal tubule transport. Since the earliest micropuncture studies of mammalian proximal tubule, it has been recognized that tubular flow is an important regulator of sodium, potassium, and acid-base transport in the kidney. Increased fluid flow stimulates Na(+) and HCO3(-) absorption in the proximal tubule via stimulation of Na/H-exchanger isoform 3 (NHE3) and H(+)-ATPase. In the proximal tubule, brush border microvilli are the major flow sensors, which experience changes in hydrodynamic drag and bending moment as luminal flow velocity changes and which transmit the force of altered flow to cytoskeletal structures within the cell. The signal to NHE3 depends upon the integrity of the actin cytoskeleton; the signal to the H(+)-ATPase depends upon microtubules. We have demonstrated that alterations in fluid drag impact tubule function by modulating ion transporter availability within the brush border membrane of the proximal tubule. Beyond that, there is evidence that transporter activity within the peritubular membrane is also modulated by luminal flow. Secondary messengers that regulate the flow-mediated tubule function have also been delineated. Dopamine blunts the responsiveness of proximal tubule transporters to changes in luminal flow velocity, while a DA1 antagonist increases flow sensitivity of solute reabsorption. IP3 receptor-mediated intracellular Ca(2+) signaling is critical to transduction of microvillus drag. In this review, we summarize our findings of the regulatory mechanism of flow-mediated Na(+) and HCO3(-) transport in the proximal tubule and review available information about flow sensing and regulatory mechanism of glomerulotubular balance.

  6. Berberine acutely activates the glucose transport activity of GLUT1.

    PubMed

    Cok, Alexandra; Plaisier, Christina; Salie, Matthew J; Oram, Daniel S; Chenge, Jude; Louters, Larry L

    2011-07-01

    Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine- activated glucose uptake reaching maximum stimulation of five-fold at >40 μM. Significant activation (P < 0.05) was measured within 5 min reaching a maximum by 30 min. The berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the K(m) of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the V(max) of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.

  7. Insulin regulation of sugar transport in giant muscle fibres of the barnacle

    PubMed Central

    Baker, P. F.; Carruthers, A.

    1983-01-01

    1. Sugar transport in the giant muscle cells of Balanus nubilus is accelerated during contractile activity and exposure to porcine insulin. The characteristics of hexose-transfer regulation in the giant muscle cells have been examined by studying the transport of 3-O-methylglucose (a non-metabolized sugar) in both intact giant fibres and fibres subjected to internal solute control by internal dialysis. 2. Sugar transport in barnacle muscle is mediated by a saturable process which is inhibited by both phloretin and cytochalasin B. Insulin increases the capacity of the transport system with little effect on its apparent affinity for sugar. Under the same conditions insulin increases 3-O-methylglucose-displaceable cytochalasin B binding. The effects of insulin on transport are half-maximal at 5 μM-insulin and are abolished by both insulin antibody and phloretin. The intact barnacle releases an insulin-like material in response to a rise in blood glucose levels. 3. Insulin increases the cyclic GMP (cGMP) content and reduces the cyclic AMP (cAMP) content of barnacle muscle. Experiments with fibres injected with aequorin show that insulin also lowers cytosolic ionized Ca levels. The changes in cyclic nucleotide levels induced by insulin precede the effects on sugar transport and cytosolic ionized Ca. During repetitive contractile activity, cAMP, cGMP and ionized Ca levels are raised. 4. Agents which raise the cAMP content of barnacle muscle normally inhibit sugar transport. Dibutyryl cAMP also inhibits transport. Alterations in cytosolic ionized Ca levels in intact fibres are without effect on sugar transport. Nevertheless, stimulation of transport by insulin is blunted when cytosolic ionized Ca is lowered by intracellular injection of the Ca-chelating agent, EGTA. 5. Sugar uptake in the internally dialysed fibre is inhibited by intracellular application of cAMP. Internal application of Ca and cGMP stimulate sugar uptake in the dialysed fibre. Cyclic AMP reduces the

  8. Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment.

    PubMed

    Kleist, Thomas J; Luan, Sheng

    2016-03-01

    Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL-CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses.

  9. Methamphetamine Regulation of Firing Activity of Dopamine Neurons.

    PubMed

    Lin, Min; Sambo, Danielle; Khoshbouei, Habibeh

    2016-10-05

    Methamphetamine (METH) is a substrate for the dopamine transporter that increases extracellular dopamine levels by competing with dopamine uptake and increasing reverse transport of dopamine via the transporter. METH has also been shown to alter the excitability of dopamine neurons. The mechanism of METH regulation of the intrinsic firing behaviors of dopamine neurons is less understood. Here we identified an unexpected and unique property of METH on the regulation of firing activity of mouse dopamine neurons. METH produced a transient augmentation of spontaneous spike activity of midbrain dopamine neurons that was followed by a progressive reduction of spontaneous spike activity. Inspection of action potential morphology revealed that METH increased the half-width and produced larger coefficients of variation of the interspike interval, suggesting that METH exposure affected the activity of voltage-dependent potassium channels in these neurons. Since METH has been shown to affect Ca(2+) homeostasis, the unexpected findings that METH broadened the action potential and decreased the amplitude of afterhyperpolarization led us to ask whether METH alters the activity of Ca(2+)-activated potassium (BK) channels. First, we identified BK channels in dopamine neurons by their voltage dependence and their response to a BK channel blocker or opener. While METH suppressed the amplitude of BK channel-mediated unitary currents, the BK channel opener NS1619 attenuated the effects of METH on action potential broadening, afterhyperpolarization repression, and spontaneous spike activity reduction. Live-cell total internal reflection fluorescence microscopy, electrophysiology, and biochemical analysis suggest METH exposure decreased the activity of BK channels by decreasing BK-α subunit levels at the plasma membrane.

  10. Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

    PubMed

    Noor, Sina Ibne; Pouyssegur, Jacques; Deitmer, Joachim W; Becker, Holger M

    2017-01-01

    Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.

  11. RirA is the iron response regulator of the rhizobactin 1021 biosynthesis and transport genes in Sinorhizobium meliloti 2011.

    PubMed

    Viguier, Caroline; O Cuív, Páraic; Clarke, Paul; O'Connell, Michael

    2005-05-15

    The genes encoding the biosynthesis and transport of rhizobactin 1021, a siderophore produced by Sinorhizobium meliloti, are negatively regulated by iron. Mutagenesis of rirA, the rhizobial iron regulator, resulted in abolition of the iron responsive regulation of the biosynthesis and transport genes. Bioassay analysis revealed that the siderophore is produced in the presence of iron in a rirA mutant. RNA analysis and GFP fusions supported the conclusion that RirA is the mediator of iron-responsive transcriptional repression of the two transcripts encoding the biosynthesis and transport genes. RirA in S. meliloti appears to fulfil the role often observed for Fur in other bacterial species. The regulator was found to mediate the iron-responsive expression of two additional genes, smc02726 and dppA1, repressing the former while activating the latter. The rirA mutant nodulated the host plant Medicago sativa (alfalfa) and fixed nitrogen as effectively as the wild type.

  12. Cortisol and IGF-1 synergistically up-regulate taurine transport by the rat skeletal muscle cell line, L6.

    PubMed

    Park, Sung-Hee; Lee, Haemi; Park, Taesun

    2004-01-01

    This study was undertaken to evaluate effects of exercise-induced hormones, cortisol, IGF-1, and beta-endorphin, on the regulation of taurine transport activity in rat skeletal myoblasts, L6 cells. Challenge of L6 cells with cortisol (100 nM) for 24 hrs resulted in a 165% increase in taurine transport activity, 220% increase in Vmax of the taurine transporter, and 55% increase in taurine transporter/ beta-actin mRNA level compared with untreated control cells. Neither IGF-1 (1 approximately 100 nM) nor beta-endorphin (1 approximately 20 nM), added in the incubation medium separately for 24 hrs, affected taurine uptake by L6 cells. However, when cells were co-treated with IGF-1 (10 nM) plus cortisol (100 nM), taurine transport activity (37% increase, p < 0.05), Vmax of the transporter (54%, p < 0.05), and taurine transporter/ beta-actin mRNA level were further increased compared to the value for cells treated with cortisol alone. These results suggest that taurine transport by skeletal muscle cells appear to be synergistically up-regulated during a prolonged exercise via elevated levels of cortisol and IGF-1 in muscle.

  13. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

  14. Post-translational modification by acetylation regulates the mitochondrial carnitine/acylcarnitine transport protein.

    PubMed

    Giangregorio, Nicola; Tonazzi, Annamaria; Console, Lara; Indiveri, Cesare

    2017-02-01

    The carnitine/acylcarnitine transporter (CACT; SLC25A20) mediates an antiport reaction allowing entry of acyl moieties in the form of acylcarnitines into the mitochondrial matrix and exit of free carnitine. The transport function of CACT is crucial for the β-oxidation pathway. In this work, it has been found that CACT is partially acetylated in rat liver mitochondria as demonstrated by anti-acetyl-lys antibody immunostaining. Acetylation was reversed by the deacetylase Sirtuin 3 in the presence of NAD(+). After treatment of the mitochondrial extract with the deacetylase, the CACT activity, assayed in proteoliposomes, increased. The half-saturation constant of the CACT was not influenced, while the V max was increased by deacetylation. Sirtuin 3 was not able to deacetylate the CACT when incubation was performed in intact mitoplasts, indicating that the acetylation sites are located in the mitochondrial matrix. Prediction on the localization of acetylated residues by bioinformatics correlates well with the experimental data. Recombinant CACT treated with acetyl-CoA was partially acetylated by non-enzymatic mechanism with a corresponding decrease of transport activity. The experimental data indicate that acetylation of CACT inhibits its transport activity, and thus may contribute to the regulation of the mitochondrial β-oxidation pathway.

  15. Characteristics and pharmacological regulation of epithelial Na+ channel (ENaC) and epithelial Na+ transport.

    PubMed

    Marunaka, Yoshinori

    2014-01-01

    Epithelial Na(+) transport participates in control of various body functions and conditions: e.g., homeostasis of body fluid content influencing blood pressure, control of amounts of fluids covering the apical surface of alveolar epithelial cells at appropriate levels for normal gas exchange, and prevention of bacterial/viral infection. Epithelial Na(+) transport via the transcellular pathway is mediated by the entry step of Na(+) across the apical membrane via Epithelial Na(+) Channel (ENaC) located at the apical membrane, and the extrusion step of Na(+) across the basolateral membrane via the Na(+),K(+)-ATPase located at the basolateral membrane. The rate-limiting step of the epithelial Na(+) transport via the transcellular pathway is generally recognized to be the entry step of Na(+) across the apical membrane via ENaC. Thus, up-/down-regulation of ENaC essentially participates in regulatory systems of blood pressure and normal gas exchange. Amount of ENaC-mediated Na(+) transport is determined by the number of ENaCs located at the apical membrane, activity (open probability) of individual ENaC located at the apical membrane, single channel conductance of ENaC located at the apical membrane, and driving force for the Na(+) entry via ENaCs across the apical membrane. In the present review article, I discuss the characteristics of ENaC and how these factors are regulated.

  16. Regulation of L-cystine transport in Salmonella typhimurium.

    PubMed Central

    Baptist, E W; Kredich, N M

    1977-01-01

    A kinetic analysis of L-cystine uptake in wild-type Salmonella typhimurium indicates the presence of at least two, and possibly three, separate transport systems. CTS-1 accounts for the majority of uptake at 20 muM L-cystine, with a Vmax of 9.5 nmol/min per mg and a Km of 2.0 muM; CTS-2 is a low-capacity, higher-affinity system with a Vmax of 0.22 nmol/min per mg and a Km of 0.05 muM; a third, nonsaturable process has been designated CTS-3. We find that wild-type CTS-1 levels are at least 11 times higher in sulfur-limited cells than in L-cystine-grown cells. Pleiotropic cysteine auxotrophs of the types cysE (lacking serine transacetylase) and cysB- (lacking a regulatory element of positive control) have very low levels of CTS-1 even when grown under conditions of sulfur limitation, which response is analogous to that previously observed for cysteine biosynthetic enzymes (N . M. Kredich, J. Biol. Chem. 246:3474-3484, 1971). CTS-1 is induced in cysE mutants by growth in the presence of O-acetyl-L-serine (the product of serine transacetylase), again paralleling the behavior of the cysteine biosynthetic pathway. Strain DW25, a prototrophic cysBc mutant, which is constitutive for cysteine biosynthesis, is also derepressed for CTS-1 when grown on L-cystine. Since CTS-1 is regulated by sulfur limitation, O-acetyl-L-serine, and the cysB gene product, the same three conditions controlling cysteine biosynthesis, we propose that this transport system is a part of the cysteine regulon. PMID:326753

  17. Pharmacogenetics, pharmacogenomics and epigenetics of Nrf2-regulated xenobiotic-metabolizing enzymes and transporters by dietary phytochemical and cancer chemoprevention.

    PubMed

    Wu, Tien-Yuan; Khor, Tin Oo; Lee, Jong Hun; Cheung, Ka Lung; Shu, Limin; Chen, Chi; Kong, Ah-Ng

    2013-07-01

    Cancer chemopreventive activities of various phytochemicals have been attributed to the modulation of xenobiotic disposition, which includes absorption, distribution, metabolism, and excretion. The interaction between xenobiotics and xenobiotic-metabolizing enzymes (XMEs) is bidirectional. XMEs are responsible for the biotransformation of xenobiotics such as bioactivation and detoxification. Conversely, xenobiotics affect XMEs through transcriptional regulation (induction or suppression) and post-translational interactions (inhibition or activation). Similar relationships also exist between xenobiotics and their transporters. Studies conducted over the past decade have demonstrated that the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), plays a critical role in the regulation of detoxifying enzymes and transporters through a signaling system that senses and responds to redox imbalance. The role of Nrf2 in the interaction between chemopreventive phytochemicals and detoxifying enzymes/transporters has become an important topic in cancer chemoprevention. In this review, the genetic and epigenetic factors that contribute to Nrf2-mediated regulation of detoxifying XMEs and transporters are discussed in the context of cancer chemoprevention. Phytochemicals may modulate the genome as well as epigenome, altering the regulation of XMEs and transporters, which may be critical for both cancer chemoprevention and the prevention of other oxidative stress- and inflammatory-related diseases, including cardiovascular, metabolic and neurological pathologies. The pharmacogenomic expression of XMEs and transporters, with an emphasis on both genomics and epigenetics, will also be discussed.

  18. Rab 11 regulates constitutive dopamine transporter trafficking and function in N2A neuroblastoma cells.

    PubMed

    Furman, Cheryse A; Lo, Charles B; Stokes, Stephanie; Esteban, Jose A; Gnegy, Margaret E

    2009-09-29

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission which undergoes constitutive and substrate-mediated trafficking to and from the membrane. Although, considerable research has been done to elucidate the regulation of substrate-stimulated DAT trafficking, less is known about which trafficking proteins are involved in constitutive DAT trafficking. Rab proteins are GTPases known to regulate the trafficking of proteins to and from specific endocytic compartments. Rabs 8 and 11, in particular, are involved in trafficking proteins from intracellular compartments to the plasma membrane. In this study, we sought to determine whether Rabs 8 and 11 would modulate DAT activity and trafficking in N2A neuroblastoma cells. We used Rab mutations known to confer constitutively active or dominant negative activity of these proteins to investigate the role of Rab activity in constitutive DAT trafficking and function. We found that constitutively active Rab 11 upregulates DAT function and surface expression while neither the constitutively active nor the dominant negative mutant of Rab 8 had any effect on DA uptake. Furthermore, immunofluorescence experiments revealed that dominant negative Rab 11 overexpression results in decreased surface DAT indicating a necessary function of Rab 11 in DAT trafficking to the plasma membrane. These data show for the first time a functional role of Rab proteins in the constitutive recycling of DAT to the plasma membrane.

  19. Translational and post-translational regulation of mouse cation transport regulator homolog 1

    PubMed Central

    Nomura, Yuki; Hirata, Yoko; Kiuchi, Kazutoshi; Oh-hashi, Kentaro

    2016-01-01

    Cation transport regulator homolog 1 (Chac1) is an endoplasmic reticulum (ER) stress inducible gene that has a function as a γ-glutamyl cyclotransferase involved in the degradation of glutathione. To characterize the translation and stability of Chac1, we found that the Kozak-like sequence present in the 5′ untranslated region (5′UTR) of the Chac1 mRNA was responsible for Chac1 translation. In addition, the short form (ΔChac1), which translated from the second ATG codon, was generated in the absence of the 5′UTR. The proteasome pathway predominantly participated in the stability of the Chac1 protein; however, its expression was remarkably up-regulated by co-transfection with ubiquitin genes. Using an immunoprecipitation assay, we revealed that ubiquitin molecule was directly conjugated to Chac1, and that mutated Chac1 with all lysine residues replaced by arginine was also ubiquitinated. Finally, we showed that WT Chac1 but not ΔChac1 reduced the intracellular level of glutathione. Taken together, our results suggest that the Chac1 protein expression is regulated in translational and post-translational fashion due to the Kozak-like sequence in the 5′UTR and the ubiquitin-mediated pathways. The bidirectional roles of ubiquitination in regulating Chac1 stabilization might give us a new insight into understanding the homeostasis of glutathione under pathophysiological conditions. PMID:27302742

  20. Function and Regulation of the C4-Dicarboxylate Transporters in Campylobacter jejuni

    PubMed Central

    Wösten, Marc M. S. M.; van de Lest, Chris H. A.; van Dijk, Linda; van Putten, Jos P. M.

    2017-01-01

    C4-dicarboxylates are important molecules for the human pathogen C.jejuni, as they are used as carbon and electron acceptor molecules, as sugars cannot be utilized by this microaerophilic organism. Based on the genome analysis, C. jejuni may possess five different C4–dicarboxylate transporters: DctA, DcuA, DcuB, and two homologs of DcuC. Here, we investigated the regulation and function of various C4–dicarboxylate transporters in C. jejuni. Transcription of the dctA and dcuC homologs is constitutive, while dcuA and dcuB are both directly regulated by the two-component RacR/RacS system in response to limited oxygen availability and the presence of nitrate. The DctA transporter is the only C4-dicarboxylate transporter to allow C. jejuni to grow on C4-carbon sources such as aspartate, fumarate, and succinate at high oxygen levels (10% O2) and is indispensable for the uptake of succinate from the medium under these conditions. Both DcuA and DcuB can sequester aspartate from the medium under low-oxygen conditions (0.3% O2). However, under these conditions, DcuB is the only transporter to secrete succinate to the environment. Under low-oxygen conditions, nitrate prevents the secretion of succinate to the environment and was able to overrule the phenotype of the C4-transporter mutants, indicating that the activity of the aspartate–fumarate–succinate pathway in C. jejuni is strongly reduced by the addition of nitrate in the medium. PMID:28223978

  1. A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

    Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)

  2. 50 CFR 665.904 - Regulated activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Regulated activities. 665.904 Section 665.904 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Marianas Trench...

  3. 50 CFR 665.904 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Regulated activities. 665.904 Section 665.904 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Marianas Trench...

  4. 50 CFR 665.964 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Regulated activities. 665.964 Section 665.964 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Rose Atoll Marine...

  5. 50 CFR 665.964 - Regulated activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Regulated activities. 665.964 Section 665.964 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Rose Atoll Marine...

  6. Ion channels/transporters as epigenetic regulators? -a microRNA perspective.

    PubMed

    Jiang, XiaoHua; Zhang, Jie Ting; Chan, Hsiao Chang

    2012-09-01

    MicroRNA (miRNA) alterations in response to changes in an extracellular microenvironment have been observed and considered as one of the major mechanisms for epigenetic modifications of the cell. While enormous efforts have been made in the understanding of the role of miRNAs in regulating cellular responses to the microenvironment, the mechanistic insight into how extracellular signals can be transduced into miRNA alterations in cells is still lacking. Interestingly, recent studies have shown that ion channels/transporters, which are known to conduct or transport ions across the cell membrane, also exhibit changes in levels of expression and activities in response to changes of extracellular microenvironment. More importantly, alterations in expression and function of ion channels/transporters have been shown to result in changes in miRNAs that are known to change in response to alteration of the microenvironment. In this review, we aim to summarize the recent data demonstrating the ability of ion channels/transporters to transduce extracellular signals into miRNA changes and propose a potential link between cells and their microenvironment through ion channels/transporters. At the same time, we hope to provide new insights into epigenetic regulatory mechanisms underlying a number of physiological and pathological processes, including embryo development and cancer metastasis.

  7. Macrophage Independent Regulation of Reverse Cholesterol Transport by Liver X Receptors

    PubMed Central

    Breevoort, Sarah R.; Angdisen, Jerry; Schulman, Ira G.

    2014-01-01

    Objective The ability of high density lipoprotein (HDL) particles to accept cholesterol from peripheral cells such as lipid-laden macrophages and to transport cholesterol to the liver for catabolism and excretion in a process termed reverse cholesterol transport (RCT) is believed to underlie the beneficial cardiovascular effects of elevated HDL. The liver X receptors (LXRα and LXRβ) regulate RCT by controlling the efflux of cholesterol from macrophages to HDL and the excretion, catabolism and absorption of cholesterol in the liver and intestine. Importantly, treatment with LXR agonists increases RCT and decreases atherosclerosis in animal models. Nevertheless, LXRs are expressed in multiple tissues involved in RCT and their tissue specific contributions to RCT are still not well defined. Approach and Results Utilizing tissue-specific LXR deletions together with in vitro and in vivo assays of cholesterol efflux and fecal cholesterol excretion we demonstrate that macrophage LXR activity is neither necessary nor sufficient for LXR agonist-stimulated RCT. In contrast, the ability of LXR agonists primarily acting in the intestine to increase HDL mass and HDL function appears to underlie the ability of LXR agonists to stimulate RCT in vivo. Conclusions We demonstrate that activation of LXR in macrophages makes little or no contribution to LXR agonist-stimulated RCT. Unexpectedly our studies suggest that the ability of macrophages to efflux cholesterol to HDL in vivo is not regulated by macrophage activity but is primarily determined by the quantity and functional activity of HDL. PMID:24947527

  8. Regulation of the norepinephrine transporter by endothelins: a potential therapeutic target.

    PubMed

    Vatta, Marcelo S; Bianciotti, Liliana G; Guil, María J; Hope, Sandra I

    2015-01-01

    Neuronal norepinephrine (NE) uptake is a crucial step in noradrenergic neurotransmission that regulates NE concentration in the synaptic cleft. It is a key mechanism mediated by the NE transporter (NET) which takes the neurotransmitter into the presynaptic neuron terminal or the adrenal medulla chromaffin cell. The activity of NET is short and long terms modulated by phosphorylation mediated by protein kinases A, C, and G and calcium-calmodulin-dependent protein kinase, whereas the transporter availability at the cell surface is regulated by glycosylation. Several neuropeptides like angiotensins II, III, and 1-7, bradykinin, natriuretic peptides, as well as endothelins (ETs) regulate a wide variety of biological effects, including noradrenergic transmission and in particular neuronal NE uptake. Diverse reports, including studies from our laboratory, show that ETs differentially modulate the activity and expression of NET not only in normal conditions but also in diverse cardiovascular diseases such as congestive heart failure and hypertension. Current literature supports a key role for the interaction between ETs and NE in maintaining neurotransmission homeostasis and further suggests that this interaction may represent a potential therapeutic target for various diseases, particularly hypertension.

  9. Transportation as a "Related Service": Issues that Involve Transition Activities.

    ERIC Educational Resources Information Center

    Missouri Univ., Columbia. Missouri LINC.

    The paper discusses transportation as a related service for students with disabilities expecially as related to school-to-work transition activities. First, the legislative and legal basis for providing transportation services is discussed in the form of answers to frequently asked questions: why provide transportation? what is the basis for…

  10. Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity.

    PubMed

    Ozcan, Nuran; Krämer, Reinhard; Morbach, Susanne

    2005-07-01

    The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.

  11. Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria.

    PubMed Central

    Silver, S; Walderhaug, M

    1992-01-01

    Regulation of chromosomally determined nutrient cation and anion uptake systems shows important similarities to regulation of plasmid-determined toxic ion resistance systems that mediate the outward transport of deleterious ions. Chromosomally determined transport systems result in accumulation of K+, Mg2+, Fe3+, Mn2+, PO4(3-), SO4(2-), and additional trace nutrients, while bacterial plasmids harbor highly specific resistance systems for AsO2-, AsO4(3-), CrO4(2-), Cd2+, Co2+, Cu2+, Hg2+, Ni2+, SbO2-, TeO3(2-), Zn2+, and other toxic ions. To study the regulation of these systems, we need to define both the trans-acting regulatory proteins and the cis-acting target operator DNA regions for the proteins. The regulation of gene expression for K+ and PO4(3-) transport systems involves two-component sensor-effector pairs of proteins. The first protein responds to an extracellular ionic (or related) signal and then transmits the signal to an intracellular DNA-binding protein. Regulation of Fe3+ transport utilizes the single iron-binding and DNA-binding protein Fur. The MerR regulatory protein for mercury resistance both represses and activates transcription. The ArsR regulatory protein functions as a repressor for the arsenic and antimony(III) efflux system. Although the predicted cadR regulatory gene has not been identified, cadmium, lead, bismuth, zinc, and cobalt induce this system in a carefully regulated manner from a single mRNA start site. The cadA Cd2+ resistance determinant encodes an E1(1)-1E2-class efflux ATPase (consisting of two polypeptides, rather than the one earlier identified). Cadmium resistance is also conferred by the czc system (which confers resistances to zinc and cobalt in Alcaligenes species) via a complex efflux pump consisting of four polypeptides. These two cadmium efflux systems are not otherwise related. For chromate resistance, reduced cellular accumulation is again the resistance mechanism, but the regulatory components are not identified

  12. Air pollution exposure: An activity pattern approach for active transportation

    NASA Astrophysics Data System (ADS)

    Adams, Matthew D.; Yiannakoulias, Nikolaos; Kanaroglou, Pavlos S.

    2016-09-01

    In this paper, we demonstrate the calculation of personal air pollution exposure during trips made by active transportation using activity patterns without personal monitors. We calculate exposure as the inhaled dose of particulate matter 2.5 μg or smaller. Two modes of active transportation are compared, and they include cycling and walking. Ambient conditions are calculated by combining mobile and stationary monitoring data in an artificial neural network space-time model. The model uses a land use regression framework and has a prediction accuracy of R2 = 0.78. Exposure is calculated at 10 m or shorter intervals during the trips using inhalation rates associated with both modes. The trips are children's routes between home and school. The average dose during morning cycling trips was 2.17 μg, during morning walking trips was 3.19 μg, during afternoon cycling trips was 2.19 μg and during afternoon walking trips was 3.23 μg. The cycling trip dose was significantly lower than the walking trip dose. The air pollution exposure during walking or cycling trips could not be strongly predicted by either the school or household ambient conditions, either individually or in combination. Multiple linear regression models regressing both the household and school ambient conditions against the dose were only able to account for, at most, six percent of the variance in the exposure. This paper demonstrates that incorporating activity patterns when calculating exposure can improve the estimate of exposure compared to its calculation from ambient conditions.

  13. Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell-to-cell transport in plants occurs through cytoplasmic channels called “plasmodesmata” and is regulated by developmental and environmental factors. Callose deposition modulates plasmodesmal transport in vivo, but little is known about the mechanisms that regulate this process. Here we report a ...

  14. 14 CFR 223.21 - Free and reduced-rate transportation authorized by statute or regulation.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 4 2012-01-01 2012-01-01 false Free and reduced-rate transportation authorized by statute or regulation. 223.21 Section 223.21 Aeronautics and Space OFFICE OF THE SECRETARY... International Travel § 223.21 Free and reduced-rate transportation authorized by statute or regulation. (a)...

  15. 14 CFR 223.21 - Free and reduced-rate transportation authorized by statute or regulation.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Free and reduced-rate transportation authorized by statute or regulation. 223.21 Section 223.21 Aeronautics and Space OFFICE OF THE SECRETARY... International Travel § 223.21 Free and reduced-rate transportation authorized by statute or regulation. (a)...

  16. 14 CFR 223.21 - Free and reduced-rate transportation authorized by statute or regulation.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Free and reduced-rate transportation authorized by statute or regulation. 223.21 Section 223.21 Aeronautics and Space OFFICE OF THE SECRETARY... International Travel § 223.21 Free and reduced-rate transportation authorized by statute or regulation. (a)...

  17. 14 CFR 223.21 - Free and reduced-rate transportation authorized by statute or regulation.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Free and reduced-rate transportation authorized by statute or regulation. 223.21 Section 223.21 Aeronautics and Space OFFICE OF THE SECRETARY... International Travel § 223.21 Free and reduced-rate transportation authorized by statute or regulation. (a)...

  18. 14 CFR 223.21 - Free and reduced-rate transportation authorized by statute or regulation.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Free and reduced-rate transportation authorized by statute or regulation. 223.21 Section 223.21 Aeronautics and Space OFFICE OF THE SECRETARY... International Travel § 223.21 Free and reduced-rate transportation authorized by statute or regulation. (a)...

  19. ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Li, C; Ramjeesingh, M; Wang, W; Garami, E; Hewryk, M; Lee, D; Rommens, J M; Galley, K; Bear, C E

    1996-11-08

    The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

  20. 75 FR 2126 - Regulations Governing the Conduct of Open Seasons for Alaska Natural Gas Transportation Projects...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-14

    ... Gas Transportation Projects; Notice of Alaska Natural Gas Transportation Projects Open Season Pre... season for an Alaska Natural Gas Transportation Project. The Workshop is being hosted by the Alaska... docket, FERC enacted regulations under the Alaska Natural Gas Pipeline Act which established...

  1. Identification and Regulation of Genes for Cobalamin Transport in the Cyanobacterium Synechococcus sp. Strain PCC 7002

    PubMed Central

    Pérez, Adam A.; Rodionov, Dmitry A.

    2016-01-01

    ABSTRACT The cyanobacterium Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis of l-methionine by methionine synthase (MetH). Synechococcus sp. strain PCC 7002 is unable to synthesize cobalamin de novo, and because of the large size of this tetrapyrrole, an active-transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. With more sophisticated in silico prediction tools, a btuB-cpdA-btuC-btuF operon encoding components putatively required for a B12 uptake (btu) system was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. Global transcriptional profiling by high-throughput RNA sequencing of a cobalamin-independent form of Synechococcus sp. strain PCC 7002 grown in the absence or presence of cobalamin confirmed regulation of the btu operon by cobalamin. Pérez et al. (A. A. Pérez, Z. Liu, D. A. Rodionov, Z. Li, and D. A. Bryant, J Bacteriol 198:2743–2752, 2016, http://dx.doi.org/10.1128/JB.00475-16) developed a cobalamin-dependent yellow fluorescent protein reporter system in a Synechococcus sp. strain PCC 7002 variant that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of the btu uptake system by assessing the ability of targeted mutants to transport cobalamin. The btuB promoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to a yfp reporter. The combined data indicate that the btuB-cpdA-btuF-btuC operon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch. IMPORTANCE With a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin

  2. Different transporter systems regulate extracellular GABA from vesicular and non-vesicular sources

    PubMed Central

    Song, Inseon; Volynski, Kirill; Brenner, Tanja; Ushkaryov, Yuri; Walker, Matthew; Semyanov, Alexey

    2013-01-01

    Tonic GABA type A (GABAA) conductance is a key factor regulating neuronal excitability and computation in neuronal networks. The magnitude of the tonic GABAA conductance depends on the concentration of ambient GABA originating from vesicular and non-vesicular sources and is tightly regulated by GABA uptake. Here we show that the transport system regulating ambient GABA responsible for tonic GABAA conductances in hippocampal CA1 interneurons depends on its source. In mice, GABA from vesicular sources is regulated by mouse GABA transporter 1 (mGAT1), while that from non-vesicular sources by mouse GABA transporters 3/4 (mGAT3/4). This finding suggests that the two transporter systems do not just provide backup for each other, but regulate distinct signaling pathways. This allows individual tuning of the two signaling systems and indicates that drugs designed to act at specific transporters will have distinct therapeutic actions. PMID:23494150

  3. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model.

    PubMed

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  4. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

    PubMed Central

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay ® (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  5. Src regulates the activity of SIRT2

    SciTech Connect

    Choi, You Hee; Kim, Hangun; Lee, Sung Ho; Jin, Yun-Hye; Lee, Kwang Youl

    2014-07-25

    Highlights: • Src decreases the protein levels of Sirt2. • Src inhibitor and knockdown of Src increase the protein levels of Sirt2. • Src interacts with and phosphorylates Sirt2. • Src regulate the activity of Sirt2. - Abstract: SIRT2 is a mammalian member of the Sirtuin family of NAD{sup +}-dependent protein deacetylases. The tyrosine kinase Src is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. The function of SIRT2 is modulated by post-translational modifications; however, the precise molecular signaling mechanism of SIRT2 through interactions with c-Src has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by c-Src. We found that the protein levels of SIRT2 were decreased by c-Src, and subsequently rescued by the addition of a Src specific inhibitor, SU6656, or by siRNA-mediated knockdown of c-Src. The c-Src interacts with and phosphorylates SIRT2 at Tyr104. c-Src also showed the ability to regulate the deacetylation activity of SIRT2. Investigation on the phosphorylation of SIRT2 suggested that this was the method of c-Src-mediated SIRT2 regulation.

  6. Proteolytic Processing Regulates Placental Growth Factor Activities*

    PubMed Central

    Hoffmann, Daniel C.; Willenborg, Sebastian; Koch, Manuel; Zwolanek, Daniela; Müller, Stefan; Becker, Ann-Kathrin A.; Metzger, Stephanie; Ehrbar, Martin; Kurschat, Peter; Hellmich, Martin; Hubbell, Jeffrey A.; Eming, Sabine A.

    2013-01-01

    Placental growth factor (PlGF) is a critical mediator of blood vessel formation, yet mechanisms of its action and regulation are incompletely understood. Here we demonstrate that proteolytic processing regulates the biological activity of PlGF. Specifically, we show that plasmin processing of PlGF-2 yields a protease-resistant core fragment comprising the vascular endothelial growth factor receptor-1 binding site but lacking the carboxyl-terminal domain encoding the heparin-binding domain and an 8-amino acid peptide encoded by exon 7. We have identified plasmin cleavage sites, generated a truncated PlGF118 isoform mimicking plasmin-processed PlGF, and explored its biological function in comparison with that of PlGF-1 and -2. The angiogenic responses induced by the diverse PlGF forms were distinct. Whereas PlGF-2 increased endothelial cell chemotaxis, vascular sprouting, and granulation tissue formation upon skin injury, these activities were abrogated following plasmin digestion. Investigation of PlGF/Neuropilin-1 binding and function suggests a critical role for heparin-binding domain/Neuropilin-1 interaction and its regulation by plasmin processing. Collectively, here we provide new mechanistic insights into the regulation of PlGF-2/Neuropilin-1-mediated tissue vascularization and growth. PMID:23645683

  7. Transporting Radioactive Waste: An Engineering Activity. Grades 5-12.

    ERIC Educational Resources Information Center

    HAZWRAP, The Hazardous Waste Remedial Actions Program.

    This brochure contains an engineering activity for upper elementary, middle school, and high school students that examines the transportation of radioactive waste. The activity is designed to inform students about the existence of radioactive waste and its transportation to disposal sites. Students experiment with methods to contain the waste and…

  8. Genetic and chemical reductions in protein phosphatase activity alter auxin transport, gravity response, and lateral root growth

    NASA Technical Reports Server (NTRS)

    Rashotte, A. M.; DeLong, A.; Muday, G. K.; Brown, C. S. (Principal Investigator)

    2001-01-01

    Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.

  9. Components in seminal plasma regulating sperm transport and elimination.

    PubMed

    Troedsson, M H T; Desvousges, A; Alghamdi, A S; Dahms, B; Dow, C A; Hayna, J; Valesco, R; Collahan, P T; Macpherson, M L; Pozor, M; Buhi, W C

    2005-10-01

    binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.

  10. Epigenetic regulation of ADME-related genes: focus on drug metabolism and transport.

    PubMed

    Zhong, Xiao-bo; Leeder, J Steven

    2013-10-01

    Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence. Examples of such modifications include DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture. Epigenetic modifications are crucial for packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors. Recently, epigenetics has become one of the fastest-growing areas of science and has now become a central issue in biologic studies of development and disease pathogenesis. The interest in epigenetics is also true for studies of drug metabolism and transport. In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic. The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide interindividual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient's epigenetic profiles.

  11. Identification of a CysB-regulated gene involved in glutathione transport in Escherichia coli.

    PubMed

    Parry, Jesse; Clark, David P

    2002-03-19

    Growth of Escherichia coli using the tripeptide glutathione as a sulfur source is well documented, but transport of glutathione into E. coli is uncharacterized. We have found that the ybiK gene, at 18.7 min, appears to be involved in the transport of glutathione and have therefore renamed ybiK as spt for sulfur peptide transport. The ybiK/spt gene is the first of what appear to be five cotranscribed genes, three of which show high homology to the peptide transport operon dpp. When the lacZ gene encoding beta-galactosidase was fused to the promoter of ybiK/spt, expression of the ybiK-lacZ fusion was repressed in rich media. This was shown to be due to the presence of exogenous cysteine. The ybiK-lacZ fusion was found to be regulated by cysB, the transcriptional activator for the cysteine regulon. Mutations in the cysB or ybiK genes led to severe growth inhibition when cells were given glutathione as the sole sulfur source. In particular, strains of E. coli containing mutations in both the ybiK and cysA genes were unable to grow when the sole sulfur source provided was glutathione whereas single cysA mutants grew well with glutathione. In contrast, no such defects were seen when L-djenkolic acid or cysteine were used as the sole sulfur source.

  12. Molecular regulation of sucrose catabolism and sugar transport for development, defence and phloem function.

    PubMed

    Li, Jun; Wu, Limin; Foster, Ryan; Ruan, Yong-Ling

    2017-03-17

    Sucrose (Suc) is the major end product of photosynthesis in mesophyll cells of most vascular plants. It is loaded into phloem of mature leaves for long-distance translocation to non-photosynthetic organs where it is unloaded for diverse uses. Clearly, Suc transport and metabolism is central to plant growth and development and the functionality of the entire vascular system. Despite of vast information in the literature about the physiological roles of individual sugar metabolic enzymes and transporters, there is a lack of systematic evaluation about their molecular regulation from transcriptional to post-translational levels. Knowledge on this topic is essential for understanding and improving plant development, optimising resource distribution and increasing crop productivity. We therefore focused our analyses on molecular control of key players in Suc metabolism and transport, including (i) the identification of promoter elements responsive to sugars and hormones or targeted by transcription factors and microRNAs degrading transcripts of target genes and (ii) modulation of enzyme and transporter activities through protein-protein interactions and other post-translational modifications. We highlighted major remaining questions and discussed opportunities to exploit current understanding to gain new insights into molecular control of carbon partitioning for improving plant performance.

  13. The role of membrane fatty-acid transporters in regulating skeletal muscle substrate use during exercise.

    PubMed

    Pelsers, Maurice M A L; Stellingwerff, Trent; van Loon, Luc J C

    2008-01-01

    While endogenous carbohydrates form the main substrate source during high-intensity exercise, long-chain fatty acids (LCFA) represent the main substrate source during more prolonged low- to moderate-intensity exercise. Adipose tissue lipolysis is responsible for the supply of LCFA to the contracting muscle. Once taken up by skeletal muscle tissue, LCFA can either serve as a substrate for oxidative phosphorylation or can be directed towards esterification into triacylglycerol. Myocellular uptake of LCFA comprises a complex and incompletely understood process. Although LCFA can enter the cell via passive diffusion, more recent reports indicate that LCFA uptake is tightly regulated by plasma membrane-located transport proteins (fatty acid translocase [FAT/CD36], plasmalemmal-located fatty acid binding protein [FABPpm] and fatty acid transport protein [FATP]). Depending on cardiac and skeletal muscle energy demands, some of these LCFA transporters can translocate rapidly from intracellular pools to the plasma membrane to allow greater LCFA uptake. This translocation process can be induced by insulin and/or muscle contraction. However, the precise signalling pathways responsible for activating the translocation machinery remain to be elucidated. This article will provide an overview on the effects of diet, acute exercise and exercise training on the expression and/or translocation of the various LCFA transporters in skeletal muscle tissue (FAT/CD36, FABPpm, FATP).

  14. Tumor marker nucleoporin 88 kDa regulates nucleocytoplasmic transport of NF-{kappa}B

    SciTech Connect

    Takahashi, Nozomi Kilsdonk, Jeroen W.J. van; Ostendorf, Benedikt; Smeets, Ruben; Bruggeman, Sophia W.M.; Alonso, Angel; Loo, Fons van de; Schneider, Matthias; Berg, Wim B. van den; Swart, Guido W.M.

    2008-09-26

    Nucleoporin 88 kDa (Nup88) is a tumor marker, overexpressed in various types of cancer. In Drosophila Nup88 (mbo) was reported to selectively mediate the nucleocytoplasmic transport of NF-{kappa}B, an ubiquitous transcription factor involved in immune responses, apoptosis, and cancer. We addressed the function of Nup88 in mammalian cells. Selective depletion of Nup88 by small interfering RNA (siRNA) inhibited NF-{kappa}B-dependent reporter gene activation and the nuclear translocation of NF-{kappa}B without affecting the upstream activation pathway in NIH3T3 cells. In contrast, nuclear translocation of glucocorticoid receptor was not reduced by the depletion of Nup88. In metastatic melanoma cells overexpressing Nup88, constitutive activation of NF-{kappa}B was found both in nucleus and cytoplasm. Nup88 depletion in these cells reduced TNF-induced nuclear accumulation of NF-{kappa}B subunits. We conclude that Nup88 regulates the activity of NF-{kappa}B at the level of nucleocytoplasmic transport. Overexpression of Nup88 in tumor cells may, thus be involved in the constitutive NF-{kappa}B activation.

  15. The Role of Flexible Loops in Folding, Trafficking and Activity of Equilibrative Nucleoside Transporters.

    PubMed

    Aseervatham, Jaya; Tran, Lucky; Machaca, Khaled; Boudker, Olga

    2015-01-01

    Equilibrative nucleoside transporters (ENTs) are integral membrane proteins, which reside in plasma membranes of all eukaryotic cells and mediate thermodynamically downhill transport of nucleosides. This process is essential for nucleoside recycling, and also plays a key role in terminating adenosine-mediated cellular signaling. Furthermore, ENTs mediate the uptake of many drugs, including anticancer and antiviral nucleoside analogues. The structure and mechanism, by which ENTs catalyze trans-membrane transport of their substrates, remain unknown. To identify the core of the transporter needed for stability, activity, and for its correct trafficking to the plasma membrane, we have expressed human ENT deletion mutants in Xenopus laevis oocytes and determined their localization, transport properties and susceptibility to inhibition. We found that the carboxyl terminal trans-membrane segments are essential for correct protein folding and trafficking. In contrast, the soluble extracellular and intracellular loops appear to be dispensable, and must be involved in the fine-tuning of transport regulation.

  16. (−)-Epicatechin-3-O-β-d-allopyranoside from Davallia formosana, Prevents Diabetes and Hyperlipidemia by Regulation of Glucose Transporter 4 and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

    Shih, Chun-Ching; Wu, Jin-Bin; Jian, Jia-Ying; Lin, Cheng-Hsiu; Ho, Hui-Ya

    2015-01-01

    The purpose of this experiment was to determine the antidiabetic and lipid-lowering effects of (−)-epicatechin-3-O-β-d-allopyranoside (BB) from the roots and stems of Davallia formosana in mice. Animal treatment was induced by high-fat diet (HFD) or low-fat diet (control diet, CD). After eight weeks of HFD or CD exposure, the HFD mice were treating with BB or rosiglitazone (Rosi) or fenofibrate (Feno) or water through gavage for another four weeks. However, at 12 weeks, the HFD-fed group had enhanced blood levels of glucose, triglyceride (TG), and insulin. BB treatment significantly decreased blood glucose, TG, and insulin levels. Moreover, visceral fat weights were enhanced in HFD-fed mice, accompanied by increased blood leptin concentrations and decreased adiponectin levels, which were reversed by treatment with BB. Muscular membrane protein levels of glucose transporter 4 (GLUT4) were reduced in HFD-fed mice and significantly enhanced upon administration of BB, Rosi, and Feno. Moreover, BB treatment markedly increased hepatic and skeletal muscular expression levels of phosphorylation of AMP-activated (adenosine monophosphate) protein kinase (phospho-AMPK). BB also decreased hepatic mRNA levels of phosphenolpyruvate carboxykinase (PEPCK), which are associated with a decrease in hepatic glucose production. BB-exerted hypotriglyceridemic activity may be partly associated with increased mRNA levels of peroxisome proliferator activated receptor α (PPARα), and with reduced hepatic glycerol-3-phosphate acyltransferase (GPAT) mRNA levels in the liver, which decreased triacylglycerol synthesis. Nevertheless, we demonstrated BB was a useful approach for the management of type 2 diabetes and dyslipidemia in this animal model. PMID:26492243

  17. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

    PubMed

    Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Cheng, Lailiang; Hao, Yu-Jin

    2016-03-01

    Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants.

  18. Copper transporter 2 regulates endocytosis and controls tumor growth and sensitivity to cisplatin in vivo.

    PubMed

    Blair, Brian G; Larson, Christopher A; Adams, Preston L; Abada, Paolo B; Pesce, Catherine E; Safaei, Roohangiz; Howell, Stephen B

    2011-01-01

    Copper transporter 2 (CTR2) is one of the four copper transporters in mammalian cells that influence the cellular pharmacology of cisplatin and carboplatin. CTR2 was knocked down using a short hairpin RNA interference. Robust expression of CTR2 was observed in parental tumors grown in vivo, whereas no staining was found in the tumors formed from cells in which CTR2 had been knocked down. Knockdown of CTR2 reduced growth rate by 5.8-fold, increased the frequency of apoptotic cells, and decreased the vascular density, but it did not change copper content. Knockdown of CTR2 increased the tumor accumulation of cis-diamminedichloroplatinum(II) [cisplatin (cDDP)] by 9.1-fold and greatly increased its therapeutic efficacy. Because altered endocytosis has been implicated in cDDP resistance, uptake of dextran was used to quantify the rate of macropinocytosis. Knockdown of CTR2 increased dextran uptake 2.5-fold without reducing exocytosis. Inhibition of macropinocytosis with either amiloride or wortmannin blocked the increase in macropinocytosis mediated by CTR2 knockdown. Stimulation of macropinocytosis by platelet-derived growth factor coordinately increased dextran and cDDP uptake. Knockdown of CTR2 was associated with activation of the Rac1 and cdc42 GTPases that control macropinocytosis but not activation of the phosphoinositide-3 kinase pathway. We conclude that CTR2 is required for optimal tumor growth and that it is an unusually strong regulator of cisplatin accumulation and cytotoxicity. CTR2 regulates the transport of cDDP in part through control of the rate of macropinocytosis via activation of Rac1 and cdc42. Selective knockdown of CTR2 in tumors offers a strategy for enhancing the efficacy of cDDP.

  19. Copper Transporter 2 Regulates Endocytosis and Controls Tumor Growth and Sensitivity to Cisplatin In Vivo

    PubMed Central

    Blair, Brian G.; Larson, Christopher A.; Adams, Preston L.; Abada, Paolo B.; Pesce, Catherine E.; Safaei, Roohangiz

    2011-01-01

    Copper transporter 2 (CTR2) is one of the four copper transporters in mammalian cells that influence the cellular pharmacology of cisplatin and carboplatin. CTR2 was knocked down using a short hairpin RNA interference. Robust expression of CTR2 was observed in parental tumors grown in vivo, whereas no staining was found in the tumors formed from cells in which CTR2 had been knocked down. Knockdown of CTR2 reduced growth rate by 5.8-fold, increased the frequency of apoptotic cells, and decreased the vascular density, but it did not change copper content. Knockdown of CTR2 increased the tumor accumulation of cis-diamminedichloroplatinum(II) [cisplatin (cDDP)] by 9.1-fold and greatly increased its therapeutic efficacy. Because altered endocytosis has been implicated in cDDP resistance, uptake of dextran was used to quantify the rate of macropinocytosis. Knockdown of CTR2 increased dextran uptake 2.5-fold without reducing exocytosis. Inhibition of macropinocytosis with either amiloride or wortmannin blocked the increase in macropinocytosis mediated by CTR2 knockdown. Stimulation of macropinocytosis by platelet-derived growth factor coordinately increased dextran and cDDP uptake. Knockdown of CTR2 was associated with activation of the Rac1 and cdc42 GTPases that control macropinocytosis but not activation of the phosphoinositide-3 kinase pathway. We conclude that CTR2 is required for optimal tumor growth and that it is an unusually strong regulator of cisplatin accumulation and cytotoxicity. CTR2 regulates the transport of cDDP in part through control of the rate of macropinocytosis via activation of Rac1 and cdc42. Selective knockdown of CTR2 in tumors offers a strategy for enhancing the efficacy of cDDP. PMID:20930109

  20. Regulation of vesicle transport and cell motility by Golgi-localized Dbs

    PubMed Central

    Fitzpatrick, Ethan R; Hu, Tinghui; Ciccarelli, Bryan T; Whitehead, Ian P

    2014-01-01

    DBS/MCF2L has been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration. PMID:25483302

  1. Salvinorin A Regulates Dopamine Transporter Function Via A Kappa Opioid Receptor and ERK1/2-Dependent Mechanism

    PubMed Central

    Kivell, Bronwyn; Uzelac, Zeljko; Sundaramurthy, Santhanalakshmi; Rajamanickam, Jeyaganesh; Ewald, Amy; Chefer, Vladimir; Jaligam, Vanaja; Bolan, Elizabeth; Simonson, Bridget; Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Prisinzano, Thomas; Gomes, Ivone; Devi, Lakshmi A.; Jayanthi, Lankupalle D.; Sitte, Harald H.; Ramamoorthy, Sammanda; Shippenberg, Toni S.

    2014-01-01

    Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP+ accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP+). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signaling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists. PMID:25107591

  2. Salvinorin A regulates dopamine transporter function via a kappa opioid receptor and ERK1/2-dependent mechanism.

    PubMed

    Kivell, Bronwyn; Uzelac, Zeljko; Sundaramurthy, Santhanalakshmi; Rajamanickam, Jeyaganesh; Ewald, Amy; Chefer, Vladimir; Jaligam, Vanaja; Bolan, Elizabeth; Simonson, Bridget; Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Prisinzano, Thomas E; Gomes, Ivone; Devi, Lakshmi A; Jayanthi, Lankupalle D; Sitte, Harald H; Ramamoorthy, Sammanda; Shippenberg, Toni S

    2014-11-01

    Salvinorin A (SalA), a selective κ-opioid receptor (KOR) agonist, produces dysphoria and pro-depressant like effects. These actions have been attributed to inhibition of striatal dopamine release. The dopamine transporter (DAT) regulates dopamine transmission via uptake of released neurotransmitter. KORs are apposed to DAT in dopamine nerve terminals suggesting an additional target by which SalA modulates dopamine transmission. SalA produced a concentration-dependent, nor-binaltorphimine (BNI)- and pertussis toxin-sensitive increase of ASP(+) accumulation in EM4 cells coexpressing myc-KOR and YFP-DAT, using live cell imaging and the fluorescent monoamine transporter substrate, trans 4-(4-(dimethylamino)-styryl)-N-methylpyridinium) (ASP(+)). Other KOR agonists also increased DAT activity that was abolished by BNI pretreatment. While SalA increased DAT activity, SalA treatment decreased serotonin transporter (SERT) activity and had no effect on norepinephrine transporter (NET) activity. In striatum, SalA increased the Vmax for DAT mediated DA transport and DAT surface expression. SalA up-regulation of DAT function is mediated by KOR activation and the KOR-linked extracellular signal regulated kinase-½ (ERK1/2) pathway. Co-immunoprecipitation and BRET studies revealed that DAT and KOR exist in a complex. In live cells, DAT and KOR exhibited robust FRET signals under basal conditions. SalA exposure caused a rapid and significant increase of the FRET signal. This suggests that the formation of KOR and DAT complexes is promoted in response to KOR activation. Together, these data suggest that enhanced DA transport and decreased DA release resulting in decreased dopamine signalling may contribute to the dysphoric and pro-depressant like effects of SalA and other KOR agonists.

  3. Pregnane X receptor regulates drug metabolism and transport in the vasculature and protects from oxidative stress

    PubMed Central

    Swales, Karen E.; Moore, Rick; Truss, Nicola J.; Tucker, Arthur; Warner, Timothy D.; Negishi, Masahiko; Bishop-Bailey, David

    2012-01-01

    Aims Circulating endogenous, dietary, and foreign chemicals can contribute to vascular dysfunction. The mechanism by which the vasculature protects itself from these chemicals is unknown. This study investigates whether the pregnane X receptor (PXR), the major transcriptional regulator of hepatic drug metabolism and transport that responds to such xenobiotics, mediates vascular protection by co-ordinating a defence gene programme in the vasculature. Methods and results PXR was detected in primary human and rat aortic endothelial and smooth muscle cells (SMC) and blood vessels including the human and rat aorta. Metabolic PXR target genes cytochrome P450 3A, 2B, 2C, and glutathione S-transferase mRNA and activity were induced by PXR ligands in rodent and human vascular cells and absent in the aortas from PXR-null mice stimulated in vivo or in rat aortic SMC expressing dominant-negative PXR. Activation of aortic PXR by classical agonists had several protective effects: increased xenobiotic metabolism demonstrated by bioactivation of the pro-drug clopidogrel, which reduced adenosine diphosphate-induced platelet aggregation; increased expression of multidrug resistance protein 1, mediating chemical efflux from the vasculature; and protection from reactive oxygen species-mediated cell death. Conclusion PXR co-ordinately up-regulates drug metabolism, transport, and antioxidant genes to protect the vasculature from endogenous and exogenous insults, thus representing a novel gatekeeper for vascular defence. PMID:22166712

  4. Insights into the post-transcriptional regulation of the mitochondrial electron transport chain

    PubMed Central

    Sirey, Tamara M.; Ponting, Chris P.

    2016-01-01

    The regulation of the mitochondrial electron transport chain is central to the control of cellular homeostasis. There are significant gaps in our understanding of how the expression of the mitochondrial and nuclear genome-encoded components of the electron transport chain are co-ordinated, and how the assembly of the protein complexes that constitute the electron transport chain are regulated. Furthermore, the role post-transcriptional gene regulation may play in modulating these processes needs to be clarified. This review summarizes the current knowledge regarding the post-transcriptional gene regulation of the electron transport chain and highlights how noncoding RNAs may contribute significantly both to complex electron transport chain regulatory networks and to mitochondrial dysfunction. PMID:27911731

  5. Heat transport in active harmonic chains

    SciTech Connect

    Zheng, Mei C.; Ellis, Fred M.; Kottos, Tsampikos; Fleischmann, Ragnar; Geisel, Theo; Prosen, Tomaz

    2011-08-15

    We show that a harmonic lattice model with amplifying and attenuating elements, when coupled to two thermal baths, exhibits unique heat transport properties. Some of these novel features include anomalous nonequilibrium steady-state heat currents, negative differential thermal conductance, as well as nonreciprocal heat transport. We find that when these elements are arranged in a PT-symmetric manner, the domain of existence of the nonequilibrium steady state is maximized. We propose an electronic experimental setup based on resistive-inductive-capacitive (RLC) transmission lines, where our predictions can be tested.

  6. Expression and regulated nuclear transport of transducers of regulated CREB 1 in retinal ganglion cells.

    PubMed

    Deng, J; Zhang, X-L; Wang, J-W; Teng, L-L; Ge, J; Takemori, H; Xiong, Z-Q; Zhou, Y

    2009-03-31

    Calcium- and cAMP-dependent activation of CREB and transcription of cAMP-responsive element (CRE)-target genes play critical roles in various physiological and pathological conditions. TORCs (transducers of regulated CREB) represent a new family of conserved CREB coactivators that function as intracellular calcium- and cAMP-sensitive coincidence detectors, controlling the kinetics of CRE-mediated responses and long-term potentiation of synaptic transmission. Here we examined the expression and activity-dependent translocation of TORCs in adult retinal ganglion cells (RGCs), the primary target of acute retinal ischemic injury as well as chronic retinal degenerative diseases. We found that both mRNAs of TORC1 and TORC2, but not TORC3, were enriched in adult rat retina. Comparing with TORC2, TORC1 protein was highly and selectively expressed in RGCs. At resting condition, TORC1 protein was localized in the cytoplasm but not nucleus of RGCs. Activation of N-methyl-D-aspartate (NMDA) receptors by intravitreous injection of NMDA or increase of cAMP signaling by administration of forskolin triggered nuclear accumulation of TORC1. Furthermore, transient retinal ischemic injury resulted in peri-nuclear and nuclear accumulation of TORC1 as well as transcription of BDNF in RGCs. Our results demonstrate that TORC1 is enriched in RGCs and its subcellular location could be regulated by Ca(2+) and cAMP, suggesting that manipulation of TORC1 activity may promote survival of RGCs in some optic disease conditions.

  7. Team Regulation, Regulation of Social Activities or Co-Regulation: Different Labels for Effective Regulation of Learning in CSCL

    ERIC Educational Resources Information Center

    Saab, Nadira

    2012-01-01

    Computer-supported collaborative learning (CSCL) is an approach to learning in which learners can actively and collaboratively construct knowledge by means of interaction and joint problem solving. Regulation of learning is especially important in the domain of CSCL. Next to the regulation of task performance, the interaction between learners who…

  8. Greater-than-Class C low-level radioactive waste transportation regulations and requirements study. National Low-Level Waste Management Program

    SciTech Connect

    Tyacke, M.; Schmitt, R.

    1993-07-01

    The purpose of this report is to identify the regulations and requirements for transporting greater-than-Class C (GTCC) low-level radioactive waste (LLW) and to identify planning activities that need to be accomplished in preparation for transporting GTCC LLW. The regulations and requirements for transporting hazardous materials, of which GTCC LLW is included, are complex and include several Federal agencies, state and local governments, and Indian tribes. This report is divided into five sections and three appendices. Section 1 introduces the report. Section 2 identifies and discusses the transportation regulations and requirements. The regulations and requirements are divided into Federal, state, local government, and Indian tribes subsections. This report does not identify the regulations or requirements of specific state, local government, and Indian tribes, since the storage, treatment, and disposal facility locations and transportation routes have not been specifically identified. Section 3 identifies the planning needed to ensure that all transportation activities are in compliance with the regulations and requirements. It is divided into (a) transportation packaging; (b) transportation operations; (c) system safety and risk analysis, (d) route selection; (e) emergency preparedness and response; and (f) safeguards and security. This section does not provide actual planning since the details of the Department of Energy (DOE) GTCC LLW Program have not been finalized, e.g., waste characterization and quantity, storage, treatment and disposal facility locations, and acceptance criteria. Sections 4 and 5 provide conclusions and referenced documents, respectively.

  9. Regulation of adenosine transport by acute and chronic ethanol exposure

    SciTech Connect

    Nagy, L.E.; Casso, D.; Diamond, I.; Gordon, A.S. )

    1989-02-09

    Chronic exposure to ethanol results in a desensitization of adenosine receptor-stimulated cAMP production. Since adenosine is released by cells and is known to desensitize its own as well as other receptors, it may be involved in ethanol-induced desensitization of adenosine receptor function. Therefore, we have examine the acute and chronic effects of ethanol on the transport of adenosine via the nucleoside transport. Acute exposure to ethanol caused an inhibition of adenosine uptake in S49 lymphoma cells. This decrease in uptake resulted in accumulation of extracellular adenosine after ethanol exposure. The effect of ethanol was specific to nucleoside transport. Uptake of uridine, also transported by the nucleoside transporter, was inhibited by ethanol to the same degree as adenosine uptake, while neither isoleucine nor deoxyglucose uptake was altered by ethanol treatment. Inhibition of adenosine uptake by ethanol was non-competitive and dependent on the concentration of ethanol. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol. There was no longer an acute inhibition of adenosine uptake, nor was these accumulation of extracellular adenosine. Chronic ethanol exposure also resulted in a decrease in the absolute rate of adenosine uptake. Binding studies using a high affinity lignad for the nucleoside transporter, nitrobenzylthioinosine (NBMPR), indicate that this decreased uptake was due to a decrease in the maximal number of binding sites. These ethanol-induced changes in adenosine transport may be important for the acute and chronic effects of ethanol.

  10. Activities and regulation of peptidoglycan synthases.

    PubMed

    Egan, Alexander J F; Biboy, Jacob; van't Veer, Inge; Breukink, Eefjan; Vollmer, Waldemar

    2015-10-05

    Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein-protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein-protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

  11. Molecular mechanisms regulating NLRP3 inflammasome activation

    PubMed Central

    Jo, Eun-Kyeong; Kim, Jin Kyung; Shin, Dong-Min; Sasakawa, Chihiro

    2016-01-01

    Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inflammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome. PMID:26549800

  12. 75 FR 51392 - Federal Management Regulation; Transportation Management

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-20

    ... From the Federal Register Online via the Government Publishing Office GENERAL SERVICES... Governmentwide Policy, General Services Administration (GSA). ACTION: Final rule. SUMMARY: The General Services... transportation management. This final rule updates definitions and corrects mailing and Web site addresses....

  13. 78 FR 76098 - Rail Transportation of Grain, Rate Regulation Review

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-16

    ...) Opening 4-5, Rate Regulation Reforms, EP 715; Alliance for Rail Competition, Montana Wheat & Barley Committee, Colorado Wheat Administrative Committee, Idaho Barley Commission, Idaho Wheat Commission,...

  14. N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

    PubMed Central

    Mudgil, Yashwanti; Ghawana, Sanjay; Jones, Alan M.

    2013-01-01

    Background N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development. Methodology/Principal Findings Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem. Conclusion/Significance NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients. PMID:24223735

  15. Regulation of monocarboxylate transporter 1 in skeletal muscle cells by intracellular signaling pathways.

    PubMed

    Narumi, Katsuya; Furugen, Ayako; Kobayashi, Masaki; Otake, Sho; Itagaki, Shirou; Iseki, Ken

    2010-01-01

    Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibers also use lactic acid as a respiratory fuel. Monocarboxylate transporter (MCT) 1 has been suggested to play a major role in influx of L-lactic acid for oxidation. The regulation mechanism of MCT1 was characterized utilizing rhabdomyosarcoma cells as an in vitro skeletal muscle model. The uptake of L-lactic acid via MCT1 was studied in the presence of various intracellular regulatory pathways, including pathways mediated by protein kinases A, C and G (PKA, PKC and PKG), protein tyrosine kinase (PTK), and Ca2+/calmodulin modulators. The results showed that PKG-, PTK-, and Ca2+/calmodulin-mediated regulatory pathways play no role in the regulation of L-lactic acid uptake, but a role for PKC- and PKA-mediated pathways was apparent. Uptake of L-lactic acid appeared to be stimulated by phorbol 12-myristate 13-acetate (PMA, a PKC activator) via an increase in Vmax of transport processes with no alteration in Km. In parallel, PMA treatment also resulted in an increase in the level of MCT1 expression. On the other hand, exposure to 8-Br-cAMP, a cAMP analog, and to forskolin, an adenylyl cyclase activator, resulted in a significant decrease in L-lactic acid uptake. Additionally, 8-Br-cAMP reduced Vmax but not Km values. Parallel to the decrease in Vmax of L-lactic acid uptake, the level of MCT1 expression was decreased in response to incubation with 8-Br-cAMP. These results indicate the possible involvement of a PKC- and PKA-mediated pathway associated with expression of MCT1 and lactate transport.

  16. Epigenetic Regulation of Dopamine Transporter mRNA Expression in Human Neuroblastoma Cells

    PubMed Central

    Green, Ashley L.; Hossain, Muhammad M.; Tee, Siew C.; Zarbl, Helmut; Guo, Grace L.; Richardson, Jason R.

    2016-01-01

    The dopamine transporter (DAT) is a key regulator of dopaminergic neurotransmission. As such, proper regulation of DAT expression is important to maintain homeostasis, and disruption of DAT expression can lead to neurobehavioral dysfunction. Based on genomic features within the promoter of the DAT gene, there is potential for DAT expression to be regulated through epigenetic mechanisms, including DNA methylation and histone acetylation. However, the relative contribution of these mechanisms to DAT expression has not been empirically determined. Using pharmacologic and genetic approaches, we demonstrate that inhibition of DNA methyltransferase (DNMT) activity increased DAT mRNA approximately 1.5–2 fold. This effect was confirmed by siRNA knockdown of DNMT1. Likewise, the histone deacetylase (HDAC) inhibitors valproate and butyrate also increased DAT mRNA expression, but the response was much more robust with expression increasing over tenfold. Genetic knockdown of HDAC1 by siRNA also increased DAT expression, but not to the extent seen with pharmacological inhibition, suggesting additional isoforms of HDAC or other targets may contribute to the observed effect. Together, these data identify the relative contribution of DNMTs and HDACs in regulating expression. These finding may aid in understanding the mechanistic basis for changes in DAT expression in normal and pathophysiological states. PMID:25963949

  17. Study of active cooling for supersonic transports

    NASA Technical Reports Server (NTRS)

    Brewer, G. D.; Morris, R. E.

    1975-01-01

    The potential benefits of using the fuel heat sink of hydrogen fueled supersonic transports for cooling large portions of the aircraft wing and fuselage are examined. The heat transfer would be accomplished by using an intermediate fluid such as an ethylene glycol-water solution. Some of the advantages of the system are: (1) reduced costs by using aluminum in place of titanium, (2) reduced cabin heat loads, and (3) more favorable environmental conditions for the aircraft systems. A liquid hydrogen fueled, Mach 2.7 supersonic transport aircraft design was used for the reference uncooled vehicle. The cooled aircraft designs were analyzed to determine their heat sink capability, the extent and location of feasible cooled surfaces, and the coolant passage size and spacing.

  18. Modeling of active transmembrane transport in a mixture theory framework.

    PubMed

    Ateshian, Gerard A; Morrison, Barclay; Hung, Clark T

    2010-05-01

    This study formulates governing equations for active transport across semi-permeable membranes within the framework of the theory of mixtures. In mixture theory, which models the interactions of any number of fluid and solid constituents, a supply term appears in the conservation of linear momentum to describe momentum exchanges among the constituents. In past applications, this momentum supply was used to model frictional interactions only, thereby describing passive transport processes. In this study, it is shown that active transport processes, which impart momentum to solutes or solvent, may also be incorporated in this term. By projecting the equation of conservation of linear momentum along the normal to the membrane, a jump condition is formulated for the mechano-electrochemical potential of fluid constituents which is generally applicable to nonequilibrium processes involving active transport. The resulting relations are simple and easy to use, and address an important need in the membrane transport literature.

  19. [Model of active peristaltic transport in biosystems].

    PubMed

    Klochkov, B N; Romanov, A S

    2013-01-01

    A nonlinear distributed mathematical model of soft vessel with the nonmonotonous static characteristic is proposed and considered. The model describes space-time dynamics of vessel clearance change. Wave phenomena in vessels of different nature and the possibility of peristaltic fluid pumping are discussed and analyzed. The model is rather common in character and represents a description of the whole class of transport phenomena. Lymphatic vessels are particularly considered.

  20. Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism

    PubMed Central

    Giudetti, Anna M.; Stanca, Eleonora; Siculella, Luisa; Gnoni, Gabriele V.; Damiano, Fabrizio

    2016-01-01

    The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs). The citrate carrier (CiC) and the carnitine/acylcarnitine transporter (CACT) are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism. PMID:27231907

  1. RhoA regulates resistance to irinotecan by regulating membrane transporter and apoptosis signaling in colorectal cancer

    PubMed Central

    Ruihua, Huang; Mengyi, Zhang; Chong, Zhao; Meng, Qiu; Xin, Ma; Qiulin, Tang; Feng, Bi; Ming, Liu

    2016-01-01

    Colorectal cancer (CRC) is a major cause of mortality and morbidity worldwide. While surgery remains the mainstay of treatment in early stage CRC, chemotherapy is usually given to prolong the overall survival and improve the quality of life for metastatic colorectal cancer (mCRC). But drug resistance is one of the major hurdles of mCRC treatment, and the underlying mechanisms are still largely unknown. In this study, we show that, compared with parental cells, RhoA is up-regulated in irinotecan (CPT-11)-resistant CRC cells. Furthermore, inhibition of RhoA in drug resistant cells, at least partially, rescues the resistance against irinotecan and increases the sensitivity to other chemotherapeutic drug by inhibiting expression of MDR1, MRP1and GSTP1, promotes apoptosis by suppressing the expression of BCL-XL and Bcl-2 and increasing Bax expression, and significantly decreases side population cells. Our results suggest that, in addition to survival, proliferation, migration, adhesion, cell cycle and gene transcription, RhoA is also involved in chemoresistance by regulating the expression of membrane transporter and apoptosis protein in colorectal cancer. They raise an interesting possibility that the expression of RhoA may indicate a poor prognosis due to the high probability to therapy resistance and, on the other hand, inhibition of RhoA activity and function may overcome chemoresistance and improve the effectiveness of clinical treatment of CRC. PMID:27888624

  2. In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers

    PubMed Central

    Findlay, Heather E.; Harris, Nicola J.; Booth, Paula J.

    2016-01-01

    Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks. PMID:27996025

  3. PPAR-α, a lipid-sensing transcription factor, regulates blood-brain barrier efflux transporter expression.

    PubMed

    More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary Ny; Miller, David S; Cannon, Ronald E

    2017-04-01

    Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR- α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood-brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR- α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR- α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood-brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

  4. Active and passive calcium transport systems in plant cells. Progress report, May 1986--January 1991

    SciTech Connect

    Sze, H.

    1991-12-31

    The ability to change cytoplasmic Ca{sup 2+} levels ([Ca{sup 2+}]) by cells has made this cation a key regulator of many biological processes. Cytoplasmic [Ca{sup 2+}] is determined by the coordination of passive Ca{sup 2+} fluxes which increase cytosolic [Ca{sup 2+}] and active Ca{sup 2+} transport systems that lower cytosolic [Ca{sup 2+}]. The mechanisms by which plant cells achieve this is poorly understood. We have initially used isolated vesicles from the plasma membrane or organellar membranes to study Ca{sup 2+} transport systems in oat roots (a monocot) and carrot suspension cells (a dicot). The objectives of the proposal were to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells.

  5. Regulation of the transportation of hazardous materials: a critique and a proposal

    SciTech Connect

    Marten, B.M.

    1981-01-01

    This article is a description and analysis of the system of regulation of hazardous-materials transportation and a proposal for reform. It has four parts. Part I surveys the present scheme of federal, state, and local safety regulation of hazardous-materials transportation. Part II describes the failures of the present regulatory system and presents a framework for analyzing alternative approaches to preventing hazardous-materials-transportation accidents. Part III compares the present system with one such alternative, a system of strict liability. Part IV is a proposal for reforming hazardous-materials-transportation regulation by deregulating materials that are not extremely hazardous, and imposing strict liability on all transporters of hazardous materials.

  6. Cell volume regulatory ion transport in the regulation of cell migration.

    PubMed

    Jakab, M; Ritter, M

    2006-01-01

    Cell migration is typically accomplished by the generation of protrusive mechanical forces and is achieved by repeated spatially and temporally coordinated cycles including the formation of a leading edge, the formation of new and disruption of older adhesions to the substratum, actomyosin based contractions and retraction of the trailing edge. Beside the well-described roles of the cytoskeleton and cell adhesions during these processes, a growing body of evidence indicates that the precise regulation of the cell volume is an indispensable prerequisite for coordinated cell migration. On the one hand during cell migration cell volume is continuously tormented by mechanical and morphological alterations, which pose changes to the intracellular hydrostatic pressure, metabolic changes and the formation or degradation of macromolecules like actin, which distort the osmotic equilibrium and the action of chemoattractants, hormones and transmitters, which frequently alter the electrical properties of a cell and thus cause cell swelling or shrinkage, respectively. On the other hand, a migrating cell actively has to govern cell volume regulatory ion transport mechanisms in order to create the appropriate micro- or even nanoenvironment in the intra- and/or extracellular space, which is necessary to guarantee the correct polarity and hence direction of movement of a migrating cell. This chapter will focus on the role of the cell volume regulatory ion transport mechanisms as they participate in the regulation of cell migration and special emphasis is given to their interplay with the cytoskeleton, their meaning for substrate adhesion and to the polarized fashion of their subcellular distribution.

  7. 77 FR 25371 - Transportation of Household Goods in Interstate Commerce; Consumer Protection Regulations...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-30

    ... Goods in Interstate Commerce; Consumer Protection Regulations: Released Rates of Motor Carriers of..., 1995) abolished the Interstate Commerce Commission (ICC), which previously had jurisdiction over the... goods motor carriers that provide for-hire transportation in interstate or foreign commerce....

  8. Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

    PubMed

    Serpinskaya, Anna S; Tuphile, Karine; Rabinow, Leonard; Gelfand, Vladimir I

    2014-01-01

    Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks.

  9. Calcium and cargoes as regulators of myosin 5a activity

    SciTech Connect

    Sellers, James R. Thirumurugan, Kavitha; Sakamoto, Takeshi; Hammer, John A.; Knight, Peter J.

    2008-04-25

    Myosin 5a is a two-headed actin-dependent motor that transports various cargoes in cells. Its enzymology and mechanochemistry have been extensively studied in vitro. It is a processive motor that takes multiple 36 nm steps on actin. The enzymatic activity of myosin 5 is regulated by an intramolecular folding mechanism whereby its lever arms fold back against the coiled-coil tail such that the motor domains directly bind the globular tail domains. We show that the structure seen in individual folded molecules is consistent with electron density map of two-dimensional crystals of the molecule. In this compact state, the actin-activated MgATPase activity of the molecule is markedly inhibited and the molecule cannot move processively on surface bound actin filaments. The actin-activated MgATPase activity of myosin 5a is activated by increasing the calcium concentration or by binding of a cargo-receptor molecule, melanophilin, in vitro. However, calcium binding to the calmodulin light chains results in dissociation of some of the calmodulin which disrupts the ability of myosin 5a to move on actin filaments in vitro. Thus we propose that the physiologically relevant activation pathway in vivo involves binding of cargo-receptor proteins.

  10. Regulation of polymorphonuclear cell activation by thrombopoietin.

    PubMed Central

    Brizzi, M F; Battaglia, E; Rosso, A; Strippoli, P; Montrucchio, G; Camussi, G; Pegoraro, L

    1997-01-01

    Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation. PMID:9120001

  11. Approach to novel functional foods for stress control 4. Regulation of serotonin transporter by food factors.

    PubMed

    Ito, Mikiko; Haito, Sakiko; Furumoto, Mari; Kawai, Yoshichika; Terao, Junji; Miyamoto, Ken-ichi

    2005-11-01

    Serotonin transporters (SERTs) are pre-synaptic proteins specialized for the clearance of serotonin following vesicular release at central nervous system (CNS) and enteric nervous system synapses. SERTs are high affinity targets in vivo for antidepressants such as serotonin selective reuptake inhibitors (SSRIs). These include 'medical' psychopharmacological agents such as analgesics and antihistamines, a plant extract called St John's Wort (Hypericum). Osteoclasts are the primary cells responsible for bone resorption. They arise by the differentiation of osteoclast precursors of the monocyte/macrophage lineage. The expression of SERTs was increased in RANKL-induced osteoclast-like cells. Using RANKL stimulation of RAW264.7 cells as a model system for osteoclast differentiation, we studied the direct effects of food factor on serotonin uptake. The SSRIs (fluoxetine and fluvoxamine) inhibited markedly (approximately 95%) in serotonin transport in differentiated osteoclast cells. The major components of St. John's Wort, hyperforin and hypericine were significantly decreased in serotonin transport activity. Thus, a new in vitro model using RANKL-induced osteoclast-like cells may be useful to analyze the regulation of SERT by food factors and SSRIs.

  12. AGAP2 regulates retrograde transport between early endosomes and the TGN

    PubMed Central

    Shiba, Yoko; Römer, Winfried; Mardones, Gonzalo A.; Burgos, Patricia V.; Lamaze, Christophe; Johannes, Ludger

    2010-01-01

    The retrograde transport route links early endosomes and the TGN. Several endogenous and exogenous cargo proteins use this pathway, one of which is the well-explored bacterial Shiga toxin. ADP-ribosylation factors (Arfs) are ~20 kDa GTP-binding proteins that are required for protein traffic at the level of the Golgi complex and early endosomes. In this study, we expressed mutants and protein fragments that bind to Arf-GTP to show that Arf1, but not Arf6 is required for transport of Shiga toxin from early endosomes to the TGN. We depleted six Arf1-specific ARF-GTPase-activating proteins and identified AGAP2 as a crucial regulator of retrograde transport for Shiga toxin, cholera toxin and the endogenous proteins TGN46 and mannose 6-phosphate receptor. In AGAP2-depleted cells, Shiga toxin accumulates in transferrin-receptor-positive early endosomes, suggesting that AGAP2 functions in the very early steps of retrograde sorting. A number of other intracellular trafficking pathways are not affected under these conditions. These results establish that Arf1 and AGAP2 have key trafficking functions at the interface between early endosomes and the TGN. PMID:20551179

  13. Ca2+-transport in sea urchin unfertilized eggs: regulation by endogenous sulfated polysaccharides and K+.

    PubMed

    Landeira-Fernandez, Ana M; Aquino, Rafael S; Mourão, Paulo A S; de Meis, Leopoldo

    2006-10-01

    Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca(2+) by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K(+) leading to an enhancement of the Ca(2+) transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca(2+) at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K(+) but not by Li(+) and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K(+). These data suggest that the regulation of the SERCA pump by K(+) and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.

  14. An Abiotic Glass-Bead Collector Exhibiting Active Transport

    NASA Astrophysics Data System (ADS)

    Goto, Youhei; Kanda, Masato; Yamamoto, Daigo; Shioi, Akihisa

    2015-09-01

    Animals relocate objects as needed by active motion. Active transport is ubiquitous in living organisms but has been difficult to realize in abiotic systems. Here we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. This is a biomimetic active transport with loadings and unloadings, because the transport was performed by a carrier and the motion of the carrier was maintained by the energy of the chemical reaction. The oil droplet produced fluctuation of the local number density of the beads on the floor, followed by its autocatalytic growth. This mechanism may inspire the technologies based on active transport wherein chemical and physical substances migrate as in living organisms.

  15. Pathophysiological regulation of renal SLC22A organic ion transporters in acute kidney injury: pharmacological and toxicological implications.

    PubMed

    Saito, Hideyuki

    2010-01-01

    The kidneys play a primary role in maintaining homeostasis and detoxification of diverse hydrophilic xenobiotics as well as endogenous by-products. Solute carrier (SLC)22A organic ion transporter family members mediate renal excretion of both endogenous and exogenous substances. Thus, the functional and molecular variations of renal SLC22A transporters under acute kidney injury (AKI) have an impact on systemic clearance of their substrate drugs, resulting in altered pharmacokinetics or unexpected adverse events caused by the accumulation of drugs. Recently, there have been significant advances in our understanding of the regulatory mechanisms for transcription, membrane trafficking and/or kidney-specific expression of SLC22A6/OAT1, SLC22A8/OAT3 and SLC22A2/OCT2. Hepatocyte nuclear factor (HNF)-1alpha/beta and HNF-4 appear to play key roles in the transcriptional regulation of OAT1 and OAT3. Furthermore, OAT1 activity/function is modulated via phosphorylation mediated by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. AKI affects renal disposition of organic ions in association with the deteriorated glomerular filtration and tubular transport functions. Thus, dysfunctional regulation of SLC22A transporters during AKI induced by ischemia or toxicants, such as cisplatin, inorganic mercury or uranyl nitrate, cause uremic syndromes or adverse drug reactions. Indoxyl sulfate, a uremic toxin and substrate of OAT1 and OAT3, appears to mediate the progression of AKI evoked by renal ischemia and cisplatin treatment. Precise mechanisms for regulation of the SLC22A transporters in AKI require studies based on the transcription, trafficking, phosphorylation and endogenous factor-dependent modulation. Such analysis will provide a better understanding of the pathophysiological implications of SLC22A transporters.

  16. A general method for determining secondary active transporter substrate stoichiometry

    PubMed Central

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-01

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein’s ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter’s mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na+-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures. DOI: http://dx.doi.org/10.7554/eLife.21016.001 PMID:28121290

  17. 14 CFR 1300.3 - Supplementary regulations of the Air Transportation Stabilization Board.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Supplementary regulations of the Air Transportation Stabilization Board. 1300.3 Section 1300.3 Aeronautics and Space AIR TRANSPORTATION SYSTEM STABILIZATION OFFICE OF MANAGEMENT AND BUDGET AVIATION DISASTER RELIEF-AIR CARRIER GUARANTEE LOAN...

  18. 75 FR 6370 - Regulations Governing the Conduct of Open Seasons for Alaska Natural Gas Transportation Projects...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-09

    ... Energy Regulatory Commission Regulations Governing the Conduct of Open Seasons for Alaska Natural Gas Transportation Projects; Notice of Alaska Natural Gas Transportation Projects Open Season Pre-Filing Workshop... hold a workshop on the procedures and process for holding and commenting on an open season for...

  19. In vitro characterization and endocrine regulation of cholesterol and phospholipid transport in the mammary gland.

    PubMed

    Ontsouka, Corneille Edgar; Huang, Xiao; Aliyev, Eldar; Albrecht, Christiane

    2017-01-05

    Cell-based studies previously showed that the ATP-binding cassette transporter A1 (ABCA1) transfers cholesterol across mammary epithelial cells (MEC). Data for phospholipid transport are lacking, and it is unclear from which cellular source the transported cholesterol stems, whether this transport activates signaling pathways, and how lactogenic hormones regulate it. To clarify these aspects, lipid transport and expressional analyses were performed in bovine primary (bMEC) and/or immortalized (MAC-T) MEC cultures. Lipid efflux and ABCA1, ABCG1 and liver X receptorα mRNA levels were higher in MAC-T than bMEC. In MAC-T, the transported cholesterol originated mainly from the plasma membrane. ABCA1 dependent cholesterol efflux was higher than phosphatidylcholine efflux, was suppressed by probucol (ABCA1 inhibitor), AG490 (janus kinase-2 inhibitor), PD98059 (mitogen activated protein kinase kinase inhibitor) and pretreatment with β-cyclodextrin (lowering membrane cholesterol). Insulin was the only hormone significantly increasing cholesterol efflux. In conclusion, this study gives novel mechanistic and regulatory insights into the transport of cholesterol and phospholipids in MEC.

  20. Oregon Pupil Transportation Manual. 1987 Revised Regulations and Responsibilities.

    ERIC Educational Resources Information Center

    Oregon State Dept. of Education, Salem.

    This manual provides school bus drivers and school officials in Oregon with material relating to safe and efficient school transportation. The guide is not intended to take the place of motor vehicle laws or school laws, but is a reference for questions that arise in connection with the school bus driver's job. Chapter 1 enumerates laws governing…

  1. Active transportation safety features around schools in Canada.

    PubMed

    Pinkerton, Bryn; Rosu, Andrei; Janssen, Ian; Pickett, William

    2013-10-31

    The purpose of this study was to describe the presence and quality of active transportation safety features in Canadian school environments that relate to pedestrian and bicycle safety. Variations in these features and associated traffic concerns as perceived by school administrators were examined by geographic status and school type. The study was based on schools that participated in 2009/2010 Health Behaviour in School-aged Children (HBSC) survey. ArcGIS software version 10 and Google Earth were used to assess the presence and quality of ten different active transportation safety features. Findings suggest that there are crosswalks and good sidewalk coverage in the environments surrounding most Canadian schools, but a dearth of bicycle lanes and other traffic calming measures (e.g., speed bumps, traffic chokers). Significant urban/rural inequities exist with a greater prevalence of sidewalk coverage, crosswalks, traffic medians, and speed bumps in urban areas. With the exception of bicycle lanes, the active transportation safety features that were present were generally rated as high quality. Traffic was more of a concern to administrators in urban areas. This study provides novel information about active transportation safety features in Canadian school environments. This information could help guide public health efforts aimed at increasing active transportation levels while simultaneously decreasing active transportation injuries.

  2. Active Transportation Safety Features around Schools in Canada

    PubMed Central

    Pinkerton, Bryn; Rosu, Andrei; Janssen, Ian; Pickett, William

    2013-01-01

    The purpose of this study was to describe the presence and quality of active transportation safety features in Canadian school environments that relate to pedestrian and bicycle safety. Variations in these features and associated traffic concerns as perceived by school administrators were examined by geographic status and school type. The study was based on schools that participated in 2009/2010 Health Behaviour in School-aged Children (HBSC) survey. ArcGIS software version 10 and Google Earth were used to assess the presence and quality of ten different active transportation safety features. Findings suggest that there are crosswalks and good sidewalk coverage in the environments surrounding most Canadian schools, but a dearth of bicycle lanes and other traffic calming measures (e.g., speed bumps, traffic chokers). Significant urban/rural inequities exist with a greater prevalence of sidewalk coverage, crosswalks, traffic medians, and speed bumps in urban areas. With the exception of bicycle lanes, the active transportation safety features that were present were generally rated as high quality. Traffic was more of a concern to administrators in urban areas. This study provides novel information about active transportation safety features in Canadian school environments. This information could help guide public health efforts aimed at increasing active transportation levels while simultaneously decreasing active transportation injuries. PMID:24185844

  3. Entropic Ratchet transport of interacting active Brownian particles

    SciTech Connect

    Ai, Bao-Quan; He, Ya-Feng; Zhong, Wei-Rong

    2014-11-21

    Directed transport of interacting active (self-propelled) Brownian particles is numerically investigated in confined geometries (entropic barriers). The self-propelled velocity can break thermodynamical equilibrium and induce the directed transport. It is found that the interaction between active particles can greatly affect the ratchet transport. For attractive particles, on increasing the interaction strength, the average velocity first decreases to its minima, then increases, and finally decreases to zero. For repulsive particles, when the interaction is very weak, there exists a critical interaction at which the average velocity is minimal, nearly tends to zero, however, for the strong interaction, the average velocity is independent of the interaction.

  4. Active urea transport in lower vertebrates and mammals.

    PubMed

    Bankir, Lise

    2014-01-01

    Some unicellular organisms can take up urea from the surrounding fluids by an uphill pumping mechanism. Several active (energy-dependent) urea transporters (AUTs) have been cloned in these organisms. Functional studies show that active urea transport also occurs in elasmobranchs, amphibians, and mammals. In the two former groups, active urea transport may serve to conserve urea in body fluids in order to balance external high ambient osmolarity or prevent desiccation. In mammals, active urea transport may be associated with the need to either store and/or reuse nitrogen in the case of low nitrogen supply, or to excrete nitrogen efficiently in the case of excess nitrogen intake. There are probably two different families of AUTs, one with a high capacity able to establish only a relatively modest transepithelial concentration difference (renal tubule of some frogs, pars recta of the mammalian kidney, early inner medullary collecting duct in some mammals eating protein-poor diets) and others with a low capacity but able to maintain a high transepithelial concentration difference that has been created by another mechanism or in another organ (elasmobranch gills, ventral skin of some toads, and maybe mammalian urinary bladder). Functional characterization of these transporters shows that some are coupled to sodium (symports or antiports) while others are sodium-independent. In humans, only one genetic anomaly, with a mild phenotype (familial azotemia), is suspected to concern one of these transporters. In spite of abundant functional evidence for such transporters in higher organisms, none have been molecularly identified yet.

  5. Sarcoplasmic reticulum Ca2+ ATPase pump is a major regulator of glucose transport in the healthy and diabetic heart.

    PubMed

    Waller, Amanda P; Kalyanasundaram, Anuradha; Hayes, Summer; Periasamy, Muthu; Lacombe, Véronique A

    2015-05-01

    Despite intensive research, the pathways that mediate calcium (Ca(2+))-stimulated glucose transport in striated muscle remain elusive. Since the sarcoplasmic reticulum calcium ATPase (SERCA) pump tightly regulates cytosolic [Ca(2+)], we investigated whether the SERCA pump is a major regulator of cardiac glucose transport. We used healthy and insulin-deficient diabetic transgenic (TG) mice expressing SERCA1a in the heart. Active cell surface glucose transporter (GLUT)-4 was measured by a biotinylated photolabeled assay in the intact perfused myocardium and isolated myocytes. In healthy TG mice, cardiac-specific SERCA1a expression increased active cell-surface GLUT4 and glucose uptake in the myocardium, as well as whole body glucose tolerance. Diabetes reduced active cell-surface GLUT4 content and glucose uptake in the heart of wild type mice, all of which were preserved in diabetic TG mice. Decreased basal AS160 and increased proportion of calmodulin-bound AS160 paralleled the increase in cell surface GLUT4 content in the heart of TG mice, suggesting that AS160 regulates GLUT trafficking by a Ca(2+)/calmodulin dependent pathway. In addition, cardiac-specific SERCA1a expression partially rescues hyperglycemia during diabetes. Collectively, these data suggested that the SERCA pump is a major regulator of cardiac glucose transport by an AS160 dependent mechanism during healthy and insulin-deficient state. Our data further indicated that cardiac-specific SERCA overexpression rescues diabetes induced-alterations in cardiac glucose transport and improves whole body glucose homeostasis. Therefore, findings from this study provide novel mechanistic insights linking upregulation of the SERCA pump in the heart as a potential therapeutic target to improve glucose metabolism during diabetes.

  6. Regulation of transport in the connecting tubule and cortical collecting duct

    PubMed Central

    Staruschenko, Alexander

    2012-01-01

    The central goal of this overview article is to summarize recent findings in renal epithelial transport, focusing chiefly on the connecting tubule (CNT) and the cortical collecting duct (CCD). Mammalian CCD and CNT are involved in fine tuning of electrolyte and fluid balance through reabsorption and secretion. Specific transporters and channels mediate vectorial movements of water and solutes in these segments. Although only a small percent of the glomerular filtrate reaches the CNT and CCD, these segments are critical for water and electrolyte homeostasis since several hormones, e.g. aldosterone and arginine vasopressin, exert their main effects in these nephron sites. Importantly, hormones regulate the function of the entire nephron and kidney by affecting channels and transporters in the CNT and CCD. Knowledge about the physiological and pathophysiological regulation of transport in the CNT and CCD and particular roles of specific channels/transporters has increased tremendously over the last two decades. Recent studies shed new light on several key questions concerning the regulation of renal transport. Precise distribution patterns of transport proteins in the CCD and CNT will be reviewed, and their physiological roles and mechanisms mediating ion transport in these segments will be also covered. Special emphasis will be given to pathophysiological conditions appearing as a result of abnormalities in renal transport in the CNT and CCD. PMID:23227301

  7. Glutamine synthetase activity and the expression of three glul paralogues in zebrafish during transport.

    PubMed

    Dhanasiri, Anusha K S; Fernandes, Jorge M O; Kiron, Viswanath

    2012-01-01

    The enzyme glutamine synthetase (GS; glutamate-ammonia ligase, EC 6.3.1.2) plays an important role in the nitrogen metabolism of fish. In this study the GS activity and the corresponding genes were examined to understand how they are regulated in zebrafish in response to hyperammonemic stress during a 72 h simulated transport. Whole body ammonia levels, the activity of the enzyme GS and the mRNA expression of the splice variants of three paralogues of glul, glutamine synthetase gene (glula, glulb and glulc) were examined in brain, liver and kidney of zebrafish. Whole body ammonia reached significantly higher levels by 48 h, while brain showed higher levels as early as 24 h, compared to the values at the start of the transport. The GS activities in brain, liver and kidney were significantly higher at the end of 72 h transport than those at the start. However, only the expression of mRNA of glulb-002 and glulb-003 were significantly upregulated during the simulated transport. In silico analysis of the putative promoter regions of glul paralogues revealed glucocorticoid receptor binding sites. However, glucocorticoid response elements of glulb were not different. The up-regulation of GS enzyme activity and hitherto unreported mRNA expression of glul paralogues during zebrafish transport indicate a physiological response of fish to ammonia.

  8. Mammalian Glucose Transporter Activity Is Dependent upon Anionic and Conical Phospholipids*

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Quigley, Andrew; Carpenter, Elisabeth P.; Hruz, Paul W.

    2016-01-01

    The regulated movement of glucose across mammalian cell membranes is mediated by facilitative glucose transporters (GLUTs) embedded in lipid bilayers. Despite the known importance of phospholipids in regulating protein structure and activity, the lipid-induced effects on the GLUTs remain poorly understood. We systematically examined the effects of physiologically relevant phospholipids on glucose transport in liposomes containing purified GLUT4 and GLUT3. The anionic phospholipids, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol, were found to be essential for transporter function by activating it and stabilizing its structure. Conical lipids, phosphatidylethanolamine and diacylglycerol, enhanced transporter activity up to 3-fold in the presence of anionic phospholipids but did not stabilize protein structure. Kinetic analyses revealed that both lipids increase the kcat of transport without changing the Km values. These results allowed us to elucidate the activation of GLUT by plasma membrane phospholipids and to extend the field of membrane protein-lipid interactions to the family of structurally and functionally related human solute carriers. PMID:27302065

  9. Renalase regulates peripheral and central dopaminergic activities.

    PubMed

    Quelhas-Santos, Janete; Serrão, Maria Paula; Soares-Silva, Isabel; Fernandes-Cerqueira, Cátia; Simões-Silva, Liliana; Pinho, Maria João; Remião, Fernando; Sampaio-Maia, Benedita; Desir, Gary V; Pestana, Manuel

    2015-01-15

    Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency.

  10. Renalase regulates peripheral and central dopaminergic activities

    PubMed Central

    Serrão, Maria Paula; Soares-Silva, Isabel; Fernandes-Cerqueira, Cátia; Simões-Silva, Liliana; Pinho, Maria João; Remião, Fernando; Sampaio-Maia, Benedita; Desir, Gary V.; Pestana, Manuel

    2014-01-01

    Renalase is a recently identified FAD/NADH-dependent amine oxidase mainly expressed in kidney that is secreted into blood and urine where it was suggested to metabolize catecholamines. The present study evaluated central and peripheral dopaminergic activities in the renalase knockout (KO) mouse model and examined the changes induced by recombinant renalase (RR) administration on plasma and urine catecholamine levels. Compared with wild-type (WT) mice, KO mice presented increased plasma levels of epinephrine (Epi), norepinephrine (NE), and dopamine (DA) that were accompanied by increases in the urinary excretion of Epi, NE, DA. In addition, the KO mice presented an increase in urinary DA-to-l-3,4-dihydroxyphenylalanine (l-DOPA) ratios without changes in renal tubular aromatic-l-amino acid decarboxylase (AADC) activity. By contrast, the in vivo administration of RR (1.5 mg/kg sc) to KO mice was accompanied by significant decreases in plasma levels of Epi, DA, and l-DOPA as well as in urinary excretion of Epi, DA, and DA-to-l-DOPA ratios notwithstanding the accompanied increase in renal AADC activity. In addition, the increase in renal DA output observed in renalase KO mice was accompanied by an increase in the expression of the L-type amino acid transporter like (LAT) 1 that is reversed by the administration of RR in these animals. These results suggest that the overexpression of LAT1 in the renal cortex of the renalase KO mice might contribute to the enhanced l-DOPA availability/uptake and consequently to the activation of the renal dopaminergic system in the presence of renalase deficiency. PMID:25411385

  11. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    SciTech Connect

    Matt Vaughn Greg Harrington Daniel R Bush

    2002-08-06

    This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose

  12. Inhibition of Human Drug Transporter Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin

    PubMed Central

    Chedik, Lisa; Bruyere, Arnaud; Le Vee, Marc; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Potin, Sophie; Fardel, Olivier

    2017-01-01

    act as regulators of the activity of various ABC and SLC drug transporters, but only when used at high and non-relevant concentrations, making unlikely any contribution of these transporter activity alterations to pyrethroid toxicity in environmentally exposed humans. PMID:28099443

  13. How Phosphorylation and ATPase Activity Regulate Anion Flux though the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

    PubMed

    Zwick, Matthias; Esposito, Cinzia; Hellstern, Manuel; Seelig, Anna

    2016-07-08

    The cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), mutations of which cause cystic fibrosis, belongs to the ATP-binding cassette (ABC) transporter family and works as a channel for small anions, such as chloride and bicarbonate. Anion channel activity is known to depend on phosphorylation by cAMP-dependent protein kinase A (PKA) and CFTR-ATPase activity. Whereas anion channel activity has been extensively investigated, phosphorylation and CFTR-ATPase activity are still poorly understood. Here, we show that the two processes can be measured in a label-free and non-invasive manner in real time in live cells, stably transfected with CFTR. This study reveals three key findings. (i) The major contribution (≥90%) to the total CFTR-related ATP hydrolysis rate is due to phosphorylation by PKA and the minor contribution (≤10%) to CFTR-ATPase activity. (ii) The mutant CFTR-E1371S that is still conductive, but defective in ATP hydrolysis, is not phosphorylated, suggesting that phosphorylation requires a functional nucleotide binding domain and occurs in the post-hydrolysis transition state. (iii) CFTR-ATPase activity is inversely related to CFTR anion flux. The present data are consistent with a model in which CFTR is in a closed conformation with two ATPs bound. The open conformation is induced by ATP hydrolysis and corresponds to the post-hydrolysis transition state that is stabilized by phosphorylation and binding of chloride channel potentiators.

  14. Hormonal regulation of ion and water transport in anuran amphibians.

    PubMed

    Uchiyama, Minoru; Konno, Norifumi

    2006-05-15

    Amphibians occupy a wide variety of ecological habitats, and their adaptation is made possible through the specialization of the epithelia of their osmoregulatory organs, such as the skin, kidney, and urinary bladder, which control the hydromineral and acid-base balance of their internal medium. Amphibians can change drastically plasma Na+, Cl-, and urea levels and excretion rates in response to environmental stimuli such as acute desiccation and changes in external salinity. Several hormones and the autonomic nervous system act to control osmoregulation. Several ion channels including an epithelial sodium channel (ENaC), a urea transporter (UT), and water channels (AQPs) are found in epithelial tissues of their osmoregulatory organs. This mini review examines the currents status of our knowledge about hormone receptors for arginine vasotocin, angiotensin II and aldosterone, and membrane ion channels and transporters, such as ENaC, UT, and AQPs in amphibians.

  15. Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma

    PubMed Central

    2012-01-01

    Background The Breast Cancer Resistance Protein (BCRP/ABCG2) is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC) in both in vivo and in vitro models with different degree of malignancy. Methods In cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot. Results ABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 μM doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p<0.01). The gene expression was also investigated in 51 adult liver tissues with HCC and related cirrhosis; normal liver tissue was used as control. ABCG2 gene expression was higher in HCC than both cirrhotic paired tissue and normal tissue. This up-regulation was greater (p<0.05) in pathological poorly differentiated grade G3/G4 than in well-differentiated G1/G2 HCC. Conclusions Our results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors. PMID:23153066

  16. Dioscin Protects ANIT-Induced Intrahepatic Cholestasis Through Regulating Transporters, Apoptosis and Oxidative Stress

    PubMed Central

    Yao, Hong; Xu, Youwei; Yin, Lianhong; Tao, Xufeng; Xu, Lina; Qi, Yan; Han, Xu; Sun, Pengyuan; Liu, Kexin; Peng, Jinyong

    2017-01-01

    Intrahepatic cholestasis, a clinical syndrome, is caused by excessive accumulation of bile acids in body and liver. Proper regulation of bile acids in liver cells is critical for liver injury. We previously reported the effects of dioscin against α-naphthylisothio- cyanate (ANIT)-induced cholestasis in rats. However, the pharmacological and mechanism data are limited. In our work, the animals of rats and mice, and Sandwich-cultured hepatocytes (SCHs) were caused by ANIT, and dioscin was used for the treatment. The results showed that dioscin markedly altered relative liver weights, restored ALT, AST, ALP, TBIL, GSH, GSH-Px, MDA, SOD levels, and rehabilitated ROS level and cell apoptosis. In mechanism study, dioscin not only significantly regulated the protein levels of Ntcp, OAT1, OCT1, Bsep and Mrp2 to accelerate bile acids excretion, but also regulated the expression levels of Bak, Bcl-xl, Bcl-2, Bax, Caspase 3 and Caspase 9 in vivo and in vitro to improve apoptosis. In addition, dioscin markedly inhibited PI3K/Akt pathway and up-regulated the levels of Nrf2, GCLc, GCLm, NQO1 and HO-1 against oxidative stress (OS) caused by bile acids. These results were further validated by inhibition of PI3K and Akt using the inhibitors of wortmannin and perifosine in SCHs. Our data showed that dioscin had good action against ANIT-caused intrahepatic cholestasis through regulating transporters, apoptosis and OS. This natural product can be considered as one active compound to treat intrahepatic cholestasis in the future. PMID:28337145

  17. Aberrant regulation of choline metabolism by mitochondrial electron transport system inhibition in neuroblastoma cells

    PubMed Central

    Baykal, Ahmet T.; Jain, Mohit R.

    2009-01-01

    Anomalous choline metabolic patterns have been consistently observed in vivo using Magnetic Resonance Spectroscopy (MRS) analysis of patients with neurodegenerative diseases and tissues from cancer patient. It remains unclear; however, what signaling events may have triggered these choline metabolic aberrancies. This study investigates how changes in choline and phospholipid metabolism are regulated by distinct changes in the mitochondrial electron transport system (ETS). We used specific inhibitors to down regulate the function of individual protein complexes in the ETS of SH-SY5Y neuroblastoma cells. Interestingly, we found that dramatic elevation in the levels of phosphatidylcholine metabolites could be induced by the inhibition of individual ETS complexes, similar to in vivo observations. Such interferences produced divergent metabolic patterns, which were distinguishable via principal component analysis of the cellular metabolomes. Functional impairments in ETS components have been reported in several central nervous system (CNS) diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD); however, it remains largely unknown how the suppression of individual ETS complex function could lead to specific dysfunction in different cell types, resulting in distinct disease phenotypes. Our results suggest that the inhibition of each of the five ETS complexes might differentially regulate phospholipase activities within choline metabolic pathways in neuronal cells, which could contribute to the overall understanding of mitochondrial diseases. PMID:19774105

  18. The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport

    PubMed Central

    Johnson, Adam J.; Wu, Ming J.

    2016-01-01

    The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2’s regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport. PMID:28009816

  19. Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport

    PubMed Central

    Held, Aaron; Sargeant, John; Thorsen, Kevin; Hay, Jesse C.

    2016-01-01

    Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport—peflin is a negative regulator of transport. PMID:27276012

  20. Hedgehog signaling regulates drug sensitivity by targeting ABC transporters ABCB1 and ABCG2 in epithelial ovarian cancer.

    PubMed

    Chen, Yi; Bieber, Marcia M; Teng, Nelson N H

    2014-08-01

    A major challenge of successful chemotherapy in ovarian cancer is overcoming intrinsic or acquired multi-drug resistance caused by active drug efflux mediated by ATP-binding cassette (ABC) transporters. Regulation of these transporters in ovarian cancer is poorly understood. We have found that abnormal expression of the hedgehog (Hh) signaling pathway transcription factor Gli1 is involved in the regulation of ABC transporters ABCB1 and ABCG2 in ovarian cancer. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work has demonstrated that Gli1 is abnormally activated in a portion of ovarian cancers. Inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels and enhances the response of ovarian cancer cells to certain chemotherapeutic drugs. The underlying mechanism is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABCB1 and ABCG2 genes. This study provides new understanding of ABC gene regulation by Hh signaling pathway, which may lead to the identification of new markers to detect and to anticipate ovarian cancer chemotherapy drug sensitivity.

  1. 75 FR 38168 - Hazardous Materials: International Regulations for the Safe Transport of Radioactive Material (TS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-01

    ... International Atomic Energy Agency's (IAEA) ``Regulations for the Safe Transport of Radioactive Material'' (TS-R... (NRC) will jointly be submitting comments on the draft document to the IAEA. We are requesting input... visit http://www.regulations.gov . SUPPLEMENTARY INFORMATION: I. Background The IAEA works with...

  2. 75 FR 41994 - Federal Management Regulation; Home-to-Work Transportation

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-20

    ... 3090-AJ05 Federal Management Regulation; Home-to-Work Transportation AGENCY: Office of Governmentwide... Administration is amending the Federal Management Regulation (FMR) to clarify existing Home-to-Work... Register on September 12, 2000 (65 FR 54966) to establish policy regarding home-to-work...

  3. Regulation of beta-galactoside transport and accumulation in heterofermentative lactic acid bacteria.

    PubMed Central

    Romano, A H; Brino, G; Peterkofsky, A; Reizer, J

    1987-01-01

    Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacillus brevis and Lactobacillus buchneri transported methyl-beta-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrast, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low cellular concentration of the galactoside, i.e., only two- to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibrium of TMG between the extra- and intracellular milieux. The means by which glucose

  4. [Sanitary epidemiologic safety and technical regulations on railway transport].

    PubMed

    Leksin, A G

    2009-01-01

    The author necessitated that requirements on sanitary and epidemiologic safety of life support systems for engine driver cabin (microclimate maintainance system, protection from vibration and noise, illumination, workplace ergonomics, etc) should be included into technical regulations for railway vehicles, both newly constructed and modernized.

  5. The cystic fibrosis transmembrane conductance regulator Cl⁻ channel: a versatile engine for transepithelial ion transport.

    PubMed

    Li, Hongyu; Cai, Zhiwei; Chen, Jeng-Haur; Ju, Min; Xu, Zhe; Sheppard, David N

    2007-08-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) transporter superfamily that forms a Cl(-) channel with complex regulation. CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory domain (RD). The MSDs assemble to form a low conductance (6-10 pS) anion-selective pore with deep intracellular and shallow extracellular vestibules separated by a selectivity filter. The NBDs form a head-to-tail dimer with two ATP-binding sites (termed sites 1 and 2) located at the dimer interface. Anion flow through CFTR is gated by the interaction of ATP with sites 1 and 2 powering cycles of NBD dimer association and dissociation and hence, conformational changes in the MSDs that open and close the channel pore. The RD is an unstructured domain with multiple consensus phosphorylation sites, phosphorylation of which stimulates CFTR function by enhancing the interaction of ATP with the NBDs. Tight spatial and temporal control of CFTR activity is achieved by macromolecular signalling complexes in which scaffolding proteins colocalise CFTR and plasma membrane receptors with protein kinases and phosphatases. Moreover, a macromolecular complex composed of CFTR and metabolic enzymes (a CFTR metabolon) permits CFTR activity to be coupled tightly to metabolic pathways within cells so that CFTR inhibition conserves vital energy stores. CFTR is expressed in epithelial tissues throughout the body, lining ducts and tubes. It functions to control the quantity and composition of epithelial secretions by driving either the absorption or secretion of salt and water. Of note, in the respiratory airways CFTR plays an additional important role in host defence. Malfunction of CFTR disrupts transepithelial ion transport leading to a wide spectrum of human disease.

  6. Classroom Activities in Transportation: Technology Education.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison.

    This curriculum supplement was designed to correlate directly with "A Guide to Curriculum Planning in Technology Education," published by the Wisconsin Department of Public Instruction. It is also a companion book to three other classroom activity compilations, one in each of the other three major systems of technology--manufacturing,…

  7. Promoting physical activity and reducing climate change: opportunities to replace short car trips with active transportation.

    PubMed

    Maibach, Edward; Steg, Linda; Anable, Jillian

    2009-10-01

    Automobile use is a significant contributor to climate change, local air pollution, pedestrian injuries and deaths, declines in physical activity and obesity. A significant proportion of car use is for short trips that can relatively easily be taken with active transportation options--walking or cycling--or with public transportation. In this commentary, we review a number of immediate, practical opportunities to implement policies and programs that reduce short car trips and increase active transportation.

  8. Active Transportation to School: Findings from a National Survey

    ERIC Educational Resources Information Center

    Fulton, Janet E.; Shisler, Jessica L.; Yore, Michelle M.; Caspersen, Carl J.

    2005-01-01

    In the past, active transportation to school offered an important source of daily physical activity for youth; more recently, however, factors related to distance, safety, or physical or social environments may have contributed to the proportion of children who travel to school by motorized vehicle. The authors examine the characteristics of…

  9. Rab27A Regulates Transport of Cell Surface Receptors Modulating Multinucleation and Lysosome-Related Organelles in Osteoclasts

    PubMed Central

    Shimada-Sugawara, Megumi; Sakai, Eiko; Okamoto, Kuniaki; Fukuda, Mitsunori; Izumi, Tetsuro; Yoshida, Noriaki; Tsukuba, Takayuki

    2015-01-01

    Rab27A regulates transport of lysosome-related organelles (LROs) and release of secretory granules in various types of cells. Here, we identified up-regulation of Rab27A during differentiation of osteoclasts (OCLs) from bone-marrow macrophages (BMMs), by DNA microarray analysis. Rab27A deficiency in OCLs, using small interfering RNA (siRNA) knockdown in RAW-D cell line or BMMs derived from ashen mice, which display genetic defects in Rab27A expression, induced multinucleated and giant cells. Upon stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), essential cytokines for OCL differentiation, phosphorylation levels of extracellular signal-regulated kinase (Erk), proto-oncogene tyrosine-protein kinase (Src), and p-38 were slightly enhanced in ashen BMMs than in wild-type BMMs. The cell surface level of c-fms, an M-CSF receptor, was slightly higher in ashen BMMs than in wild-type BMMs, and down-regulation of RANK, a RANKL receptor, was delayed. In addition to receptors, OCLs derived from ashen mice exhibited aberrant actin ring formation, abnormal subcellular localization of lysosome-associated membrane protein (LAMP2) and cathepsin K (CTSK), and marked reduction in resorbing activity. Thus, these findings suggest that Rab27A regulates normal transport of cell surface receptors modulating multinucleation and LROs in OCLs. PMID:25882854

  10. Norepinephrine transporter function and desipramine: residual drug effects versus short-term regulation.

    PubMed

    Ordway, Gregory A; Jia, Weihong; Li, Jing; Zhu, Meng-Yang; Mandela, Prashant; Pan, Jun

    2005-04-30

    Previous research has shown that exposure of norepinephrine transporter (NET)-expressing cells to desipramine (DMI) downregulates the norepinephrine transporter, although changes in the several transporter parameters do not demonstrate the same time course. Exposures to desipramine for <1 day reduces only radioligand binding and uptake capacity while transporter-immunoreactivity is unaffected. Recent demonstration of persistent drug retention in cells following desipramine exposures raises the possibility that previous reported changes in the norepinephrine transporter may be partly accountable by residual drug. In this study, potential effects of residual desipramine on norepinephrine transporter binding and uptake were re-evaluated following exposures of PC12 cells to desipramine using different methods to remove residual drug. Using a method that minimizes residual drug, exposure of intact PC12 cells to desipramine for 4h had no effect on uptake capacity or [(3)H]nisoxetine binding to the norepinephrine transporter, while exposures for > or =16 h reduced uptake capacity. Desipramine-induced reductions in binding to the transporter required >24 h or greater periods of desipramine exposure. This study confirms that uptake capacity of the norepinephrine transporter is reduced earlier than changes in radioligand binding, but with a different time course than originally shown. Special pre-incubation procedures are required to abolish effects of residual transporter inhibitor when studying inhibitor-induced transporter regulation.

  11. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium.

    PubMed Central

    Mason, S. J.; Paradiso, A. M.; Boucher, R. C.

    1991-01-01

    1 The role of extracellular nucleotides in regulation of ion transport activities (short circuit current, Isc) of human respiratory epithelia was studied. 2 Application of nucleotides to the apical or basolateral membrane of human nasal epithelium induced a concentration-dependent increase in Isc. 3 The rank order of potency of purine- or pyrimidine-induced changes in Isc of normal human nasal epithelium when applied to the apical membrane (UTP greater than or equal to ATP greater than ATP gamma S greater than 2MeSATP greater than ADP beta S much greater than beta gamma MeATP greater than or equal to alpha beta MeATP) or basolateral membrane (2MeSATP greater than UTP greater than ATP greater than ATP gamma S greater than alpha beta MeATP greater than beta gamma MeATP) is consistent with involvement of a P2 purinoceptor. A similar rank order of potencies was observed for nucleotide effects on intracellular calcium measured by Fura-2 fluorescence using microspectrofluorimetry. 4 Similar nucleotide potency in the regulation of ion transport and intracellular calcium in cystic fibrosis (CF) airway epithelium (UTP greater than or equal to ATP) was observed, suggesting purinoceptors might be used to stimulate ion transport processes that would promote hydration of airway secretions and facilitate their clearance from CF lungs. 5 These data provide evidence for the regulation of ion transport by P2 purinoceptors in normal and cystic fibrosis human airway epithelium. PMID:1718521

  12. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples1[OPEN

    PubMed Central

    Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Hao, Yu-Jin

    2016-01-01

    Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H+-pumping activities of vacuolar H+-ATPase (VHA) and/or vacuolar H+-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H+-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. PMID:26637549

  13. Epithelial pH and ion transport regulation by proton pumps and exchangers.

    PubMed

    Harvey, B J; Ehrenfeld, J

    1988-01-01

    This study reports on the interaction between transepithelial Na+ transport and H+ secretory and intracellular pH (pHi) regulating mechanisms in the model 'tight' epithelium of frog skin. We have used 22Na isotope fluxes and fixed end-point titration to measure undirectional Na+ fluxes, net Na absorption (J(net)Na) and proton secretion (J(net)H), and electrophysiological techniques (double-barrelled ion-sensitive microelectrodes and cell membrane current--voltage relations) to determine intracellular activities of Na+, Cl- and H+ and the conductance of apical membranes to Na+ (gNa) and of basolateral membranes to K+ (gK). In dilute mucosal solutions or in the absence of a permeant anion (Cl-) or counter-current (open-circuit conditions) to accompany Na+ uptake, the J(net)Na is electrically coupled to J(net)H via an electrogenic apical H+-ATPase (located in mitochondria-rich cells). Both fluxes proceed via mitochondria-rich cells and are inhibited by blockers of carbonic anhydrase and H+-ATPase and stimulated by aldosterone and acid load. In high NaCl-containing mucosal solutions or in short-circuit conditions, the J(net)Na becomes uncoupled from J(net)H and proceeds mainly via the principal cells in the epithelium, in which pHi is regulated by basolateral Na+/H+ and Cl-/HCO3- exchangers. Under these conditions, J(net)Na, gNa and gK vary directly and in parallel with pHi, when pHi is changed by permeable weak acids or bases. There is also co-variance between gNa and pHi accompanying spontaneous variations in J(net)Na and when Na+ transport is stimulated by aldosterone or inhibited with ouabain. We conclude that the level of intracellular H+, modulated by H+ pump and Na+/H+ and Cl-/HCO3- exchangers provides an intrinsic regulation of epithelial Na+ transport.

  14. Identification of a TRAP transporter for malonate transport and its expression regulated by GtrA from Sinorhizobium meliloti.

    PubMed

    Chen, Ai-Min; Wang, Yong-Bao; Jie, Sun; Yu, Ai-Yuan; Luo, Li; Yu, Guan-Qiao; Zhu, Jia-Bi; Wang, Yan-Zhang

    2010-09-01

    Sinorhizobium meliloti can live as a saprophyte in soil or as a nitrogen-fixing symbiont inside the root nodule cells of alfalfa and related legumes by utilizing different organic compounds as its carbon source. Here we have identified the matPQMAB operon in S. meliloti 1021. Within this operon, matP, matQ and the M region of the fused gene matMA encode an extracytoplasmic solute receptor, a small transmembrane protein and a large transmembrane protein, consisting of three components of the tripartite ATP-independent periplasmic (TRAP) transporter for malonate transport. The A region of the fused gene matMA and matB encode malonate-metabolizing enzymes, malonyl-CoA decarboxylase and malonyl-CoA synthetase. The null mutant of each matPQMAB gene is unable to grow on M9 minimal medium containing malonate as the sole carbon source. However, these mutants can induce the formation of efficient nitrogen-fixing root nodules on alfalfa. The matPQMAB operon is expressed in free-living bacterial cells and symbiotic bacterial cells from infection threads and root nodules. The GntR family transcriptional regulator, GtrA, specifically binds the promoter of the matPQMAB operon, positively regulating its expression. Moreover, the matPQMAB can be transcriptionally induced by malonate. These results suggested that a C(3)-dicarboxylic acid TRAP transporter is responsible for malonate transport in S. meliloti.

  15. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

    PubMed Central

    Sesma, Juliana I.; Kreda, Silvia M.; Okada, Seiko F.; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C.; O'Neal, Wanda K.; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori

    2013-01-01

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins. PMID:23467297

  16. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

    PubMed

    Sesma, Juliana I; Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C; O'Neal, Wanda K; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori; Lazarowski, Eduardo R

    2013-05-15

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

  17. Regulation of the Na(+)-coupled glutamate transporter EAAT3 by PIKfyve.

    PubMed

    Klaus, Fabian; Gehring, Eva-Maria; Zürn, Agathe; Laufer, Joerg; Lindner, Ricco; Strutz-Seebohm, Nathalie; Tavaré, Jeremy M; Rothstein, Jeffrey D; Boehmer, Christoph; Palmada, Monica; Gruner, Ivonne; Lang, Undine E; Seebohm, Guiscard; Lang, Florian

    2009-01-01

    The Na(+), glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end,EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I(glu)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I(glu). Coexpression of either, SGK1 orPIKfyve, significantly enhanced I(glu) in EAAT3 expressing oocytes. The increased I(glu) was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I(glu) and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressingEAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant (K127N)SGK1 did not significantly alter I(glu) in EAAT3 expressing oocytes and completely reversed the stimulating effect ofPIKfyve coexpression on I(glu). The stimulating effect of PIKfyve on I(glu) was abolished by replacement of the serine by alanine in the SGK consensus sequence ((S318A)PIKfyve). Moreover, additional coexpression of(S318A)PIKfyve significantly blunted I(glu) in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.

  18. Promoter Analysis of the Human Ascorbic Acid Transporters SVCT1 & 2: Mechanisms of Adaptive Regulation in Liver Epithelial Cells

    PubMed Central

    Reidling, Jack C.; Rubin, Stanley A.

    2010-01-01

    Ascorbic acid, the active form of vitamin C, is a vital antioxidant in the human liver, yet the molecular mechanisms involved in the regulation of ascorbic acid transporters (hSVCT1 and hSVCT2) in liver cells are poorly understood. Therefore, we characterized the minimal promoter regions of hSVCT1 & 2 in cultured human liver epithelial cells (HepG2) and examined the effects of ascorbic acid deprivation and supplementation on activity and regulation of the transport systems. Identified minimal promoters required for basal activity were found to include multiple cis-regulatory elements, whereas mutational analysis demonstrated that HNF-1 sites in the hSVCT1 promoter and KLF/Sp1 sites in the hSVCT2 promoter were essential for activities. When cultured in ascorbic acid deficient or supplemented media, HepG2 cells demonstrated significant (P < 0.01) and specific reciprocal changes in [14C]-Ascorbic acid uptake, and in hSVCT1 mRNA and protein levels as well as hSVCT1 promoter activity. However, no significant changes in hSVCT2 expression or promoter activity were observed during ascorbic acid deficient or supplemented conditions. We mapped the ascorbic acid responsive region in the hSVCT1 promoter and determined that HNF-1 sites are important for the adaptive regulation response. The results of these studies further characterize the hSVCT1 and 2 promoters, establish that ascorbic acid uptake by human liver epithelial cells is adaptively regulated, and show that transcriptional mechanisms via HNF-1 in the hSVCT1 promoter may, in part, be involved in this regulation. PMID:20471816

  19. 76 FR 12364 - Agency Information Collection Activities: Bonded Warehouse Regulations

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-07

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Bonded Warehouse... Bonded Warehouse Regulations. This request for comment is being made pursuant to the Paperwork Reduction... concerning the following information collection: Title: Bonded Warehouse Regulations. OMB Number:...

  20. Advocacy for active transport: advocate and city council perspectives

    PubMed Central

    2010-01-01

    Background Effective advocacy is an important part of efforts to increase population participation in physical activity. Research about effective health advocacy is scarce, however, the health sector can learn from the experiences and knowledge of community advocates and those who are on the receiving end of this advocacy. The aim of this study is to explore advocacy for active transport from the perspectives of community advocates and representatives from City councils. Methods Cycling and walking advocates were identified from the local contact list of Cycling Advocates Network and Living Streets Aotearoa. Semi-structured telephone interviews were conducted with cycle and walking advocates from throughout New Zealand. Advocates also nominated a suitable council officer at their local City council to be interviewed. Interviews were recorded and transcribed and categories of responses for each of the questions created. Results Several processes were used by advocates to engage with council staff, including formal council submissions, meetings, stakeholder forums and partnership in running community events promoting active transport. Several other agencies were identified as being influential for active transport, some as potential coalition partners and others as potential adversaries. Barriers to improving conditions for active transport included a lack of funding, a lack of will-power among either council staff or councillors, limited council staff capacity (time or training) and a culture of providing infrastructure for motor vehicles instead of people. Several suggestions were made about how the health sector could contribute to advocacy efforts, including encouraging political commitment, engaging the media, communicating the potential health benefits of active transport to the general public and being role models in terms of personal travel mode choice and having workplaces that support participation in active transport. Conclusions There is potential for the

  1. Cyfip1 Regulates Presynaptic Activity during Development

    PubMed Central

    Hsiao, Kuangfu; Harony-Nicolas, Hala; Buxbaum, Joseph D.

    2016-01-01

    Copy number variations encompassing the gene encoding Cyfip1 have been associated with a variety of human diseases, including autism and schizophrenia. Here we show that juvenile mice hemizygous for Cyfip1 have altered presynaptic function, enhanced protein translation, and increased levels of F-actin. In developing hippocampus, reduced Cyfip1 levels serve to decrease paired pulse facilitation and increase miniature EPSC frequency without a change in amplitude. Higher-resolution examination shows these changes to be caused primarily by an increase in presynaptic terminal size and enhanced vesicle release probability. Short hairpin-mediated knockdown of Cyfip1 coupled with expression of mutant Cyfip1 proteins indicates that the presynaptic alterations are caused by dysregulation of the WAVE regulatory complex. Such dysregulation occurs downstream of Rac1 as acute exposure to Rac1 inhibitors rescues presynaptic responses in culture and in hippocampal slices. The data serve to highlight an early and essential role for Cyfip1 in the generation of normally functioning synapses and suggest a means by which changes in Cyfip1 levels could impact the generation of neural networks and contribute to abnormal and maladaptive behaviors. SIGNIFICANCE STATEMENT Several developmental brain disorders have been associated with gene duplications and deletions that serve to increase or decrease levels of encoded proteins. Cyfip1 is one such protein, but the role it plays in brain development is poorly understood. We asked whether decreased Cyfip1 levels altered the function of developing synapses. The data show that synapses with reduced Cyfip1 are larger and release neurotransmitter more rapidly. These effects are due to Cyfip1's role in actin polymerization and are reversed by expression of a Cyfip1 mutant protein retaining actin regulatory function or by inhibiting Rac1. Thus, Cyfip1 has a more prominent early role regulating presynaptic activity during a stage of development when

  2. The Shoelace Antenna: Measurements of Driven Transport and Prospects for Active Edge Control

    NASA Astrophysics Data System (ADS)

    Golfinopoulos, Theodore; Labombard, B.; Brunner, D.; Terry, J. L.; Baek, S. G.; Ennever, P.; Edlund, E.; Han, W.; Burke, W. M.; Wolfe, S. M.; Irby, J. H.; Hughes, J. W.; Fitzgerald, E. W.; Granetz, R. S.; Greenwald, M. J.; Leccacorvi, R.; Marmar, E. S.; Pierson, S. Z.; Porkolab, M.; Vieira, R. F.; Wukitch, S. J.; Alcator C-Mod Team

    2016-10-01

    The Shoelace antenna was built to drive edge fluctuations in the Alcator C-Mod tokamak, matching the wavenumber (k = 1.5/cm) and frequency (50< f<200 kHz) of the Quasi-Coherent Mode (QCM). This fluctuation is responsible for regulating transport across the plasma boundary in the steady-state, ELM-free Enhanced D α(EDA) H-mode; the goal of the Shoelace antenna is to regulate edge transport actively via the same mechanism. Initial experiments demonstrated that the antenna drove a resonant response in the edge plasma in steady-state EDA and transient, non-ELMy H-modes, but transport measurements were unavailable. In 2016, the Shoelace antenna was relocated to enable direct measurements of driven transport by a reciprocating Mirror Langmuir Probe, while also making available gas puff imaging and reflectometer data to provide radial localization of the driven fluctuation. This talk will describe these measurements, and compare them to those of the intrinsic QCM in the context of assessing the feasibility of achieving active control of edge transport using direct coupling to edge modes. This work is supported by USDoE Award DE-FC02-99ER54512.

  3. Active and passive calcium transport systems in plant cells: Progress report, January 1986--June 1989

    SciTech Connect

    Sze, H.

    1989-01-01

    The objectives of this proposal are to identify and characterize active (energy-dependent) and passive calcium transport systems that work together to regulate calcium levels in the cytoplasm of plant cells. Several different energy-dependent Ca transport systems have been identified and characterized from oat root tissue (a monocot tissue) and carrot suspension cells (a dicot tissue). They are described in more detail below. I also have included in this progress report our continuing studies to understand the mode of action of the Helminthosporium maydis T toxin. This study was initially supported by a preceding DOE grant. The time needed to complete the study overlapped partly with the present grant period.

  4. Mitochondrial Ion Channels/Transporters as Sensors and Regulators of Cellular Redox Signaling

    PubMed Central

    Ryu, Shin-Young; Jhun, Bong Sook; Hurst, Stephen

    2014-01-01

    Abstract Significance: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. Recent Advances: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. Critical Issues: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. Future Directions: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters. Antioxid. Redox Signal. 21, 987–1006. PMID:24180309

  5. SNAT3-mediated glutamine transport in perisynaptic astrocytes in situ is regulated by intracellular sodium.

    PubMed

    Todd, Alison C; Marx, Mari-Carmen; Hulme, Sarah R; Bröer, Stefan; Billups, Brian

    2017-03-08

    The release of glutamine from astrocytes adjacent to synapses in the central nervous system is thought to play a vital role in the mechanism of glutamate recycling and is therefore important for maintaining excitatory neurotransmission. Here we investigate the nature of astrocytic membrane transport of glutamine in rat brainstem slices, using electrophysiological recording and fluorescent imaging of pHi and Nai+. Glutamine application to perisynaptic astrocytes induced a membrane current, caused by activation of system A (SA) family transporters. A significant electroneutral component was also observed, which was mediated by the system N (SN) family transporters. This response was stimulated by glutamine (KM of 1.57 mM), histidine, and asparagine, but not by leucine or serine, indicating activation of the SNAT3 isoform of SN. We hypothesized that increasing the [Na(+) ]i would alter the SNAT3 transporter equilibrium, thereby stimulating glutamine release. In support of this hypothesis, we show that SNAT3 transport can be driven by changing cation concentration and that manipulations to raise [Na(+) ]i (activation of excitatory amino acid transporters (EAATs), SA transporters or AMPA receptors) all directly influence SNAT3 transport rate. A kinetic model of glutamine fluxes is presented, which shows that EAAT activation causes the release of glutamine, driven mainly by the increased [Na(+) ]i . These data demonstrate that SNAT3 is functionally active in perisynaptic astrocytes in situ. As a result, astrocytic Nai+ signaling, as would be stimulated by neighboring synaptic activity, has the capacity to stimulate astrocytic glutamine release to support glutamate recycling.

  6. The Zinc Transport Systems and Their Regulation in Pathogenic Fungi.

    PubMed

    Jung, Won Hee

    2015-09-01

    Zinc is an essential micronutrient required for many enzymes that play essential roles in a cell. It was estimated that approximately 3% of the total cellular proteins are required for zinc for their functions. Zinc has long been considered as one of the key players in host-pathogen interactions. The host sequesters intracellular zinc by utilizing multiple cellular zinc importers and exporters as a means of nutritional immunity. To overcome extreme zinc limitation within the host environment, pathogenic microbes have successfully evolved a number of mechanisms to secure sufficient concentrations of zinc for their survival and pathogenesis. In this review, we briefly discuss the zinc uptake systems and their regulation in the model fungus Saccharomyces cerevisiae and in major human pathogenic fungi such as Aspergillus fumigatus, Candida albicans, and Cryptococcus gattii.

  7. Topology mapping of insulin-regulated glucose transporter GLUT4 using computational biology.

    PubMed

    Chakraborty, Chiranjib; Bandyopadhyay, Sanghamitra; Maulik, Ujjwal; Agoramoorthy, Govindasamy

    2013-01-01

    The type 2 diabetes is increasing rapidly around the globe. The primary cause for this is insulin resistance due to the disruption of the insulin signal transduction mechanism. Insulin signal transduction stimulates glucose transport through the glucose transporter GLUT4, by promoting the exocytosis process. Understanding the structural topology of GLUT4 mechanism will increase our understanding of the dynamic activities about glucose transport and its regulation in the membrane environment. However, little is known about the topology of GLUT4. In this article, we have determined the amino acid composition, disulfide topology, structure conformation pattern of GLUT4. The amino acid composition portrays that leucine composition is the highest contributing to 15.5% among all other amino acids. Three cysteine residues such as Cys223, Cys361, and Cys363 were observed and the last two were associated with one disulfide bond formation. We have generated surface cavities to know the clefts/pockets on the surface of this protein that showed few irregular cavities placed mostly in the transmembrane-helical part. Besides, topology mapping of 12 transmembrane-helixes was done to predict N- and O-glycosylation sites and to show the highly glycosylated GLUT4 that includes both N- and O-glycosylation sites. Furthermore, hydrophobic segment and molecular charge distribution were analyzed. This article shows that bioinformatics tools can provide a rapid methodology to predict the topology of GLUT4. It also provides insights into the structural details and structural functioning relationships in the human GLUT4. The results can be of great help to advance future drug development research using GLUT4 as a target protein.

  8. Prolactin increases hepatic Na+/taurocholate co-transport activity and messenger RNA post partum.

    PubMed Central

    Ganguly, T C; Liu, Y; Hyde, J F; Hagenbuch, B; Meier, P J; Vore, M

    1994-01-01

    We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity. Images Figure 2 PMID:7945260

  9. Adipocyte glucose transport regulation by eicosanoid precursors and inhibitors

    SciTech Connect

    Lee, H.C.C.

    1987-01-01

    Glucose uptake and free fatty acid release by adipocytes are increased by catecholamines. The mechanism of the stimulatory action of catecholamines on glucose uptake may be via eicosanoid production from release fatty acids. Rats were fed iso-nutrient diets with high or low safflower oil. After one month, 5 rats per diet group were fed diets with aspirin or without aspirin for 2 days. Isolated adipocytes from epididymal fat pads were incubated at 37/sup 0/C, gassed with 95% O/sub 2/-5% CO/sub 2/ in KRB buffer with 3% bovine serum albumin and with or without eicosanoid modifiers; a stimulator (10/sup -5/ M norepinephrine, N), or inhibitors (167 ..mu..l of antiserum to prostaglandin E (AntiE) per 1600 ..mu..l or 23mM Asp), or combinations of these. At 2-, 5-, and 10-min incubation, samples of incubation mixtures were taken to measure 2-deoxy glucose transport using /sup 3/H-2-deoxy glucose, /sup 14/C-inulin, and liquid scintillation counter.

  10. The spatial and temporal regulation of the hormonal signal. Role of mitochondria in the formation of a protein complex required for the activation of cholesterol transport and steroids synthesis.

    PubMed

    Poderoso, Cecilia; Duarte, Alejandra; Cooke, Mariana; Orlando, Ulises; Gottifredi, Vanesa; Solano, Angela R; Lemos, Jose R; Podestá, Ernesto J

    2013-05-22

    The mitochondria are critical for steroidogenesis since the ability of cholesterol to move into mitochondria to be available for cytochrome P450, CYP11A1, determines the efficacy of steroid production. Several proteins kinases, such as PKA, MEK and ERK which are essential to complete steroidogenesis, form a mitochondria-associated complex. The protein-protein interactions between kinases and key factors during the transport of cholesterol takes place in the contact sites between the two mitochondrial membranes; however, no mitochondrial targeting sequence has been described for these kinases. Here we discuss the possibility that mitochondrial reorganization may be mediating a compartmentalized cellular response. This reorganization could allow the physical interaction between the hormone-receptor complex and the enzymatic and lipidic machinery necessary for the complete steroid synthesis and release. The movement of organelles in specialized cells could impact on biological processes that include, but are not limited to, steroid synthesis.

  11. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability.

    PubMed

    Yeyati, Patricia L; Schiller, Rachel; Mali, Girish; Kasioulis, Ioannis; Kawamura, Akane; Adams, Ian R; Playfoot, Christopher; Gilbert, Nick; van Heyningen, Veronica; Wills, Jimi; von Kriegsheim, Alex; Finch, Andrew; Sakai, Juro; Schofield, Christopher J; Jackson, Ian J; Mill, Pleasantine

    2017-02-28

    Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive "actin gate" that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics.

  12. KDM3A coordinates actin dynamics with intraflagellar transport to regulate cilia stability

    PubMed Central

    Schiller, Rachel; Kawamura, Akane; Gilbert, Nick; Wills, Jimi; von Kriegsheim, Alex

    2017-01-01

    Cilia assembly and disassembly are coupled to actin dynamics, ensuring a coherent cellular response during environmental change. How these processes are integrated remains undefined. The histone lysine demethylase KDM3A plays important roles in organismal homeostasis. Loss-of-function mouse models of Kdm3a phenocopy features associated with human ciliopathies, whereas human somatic mutations correlate with poor cancer prognosis. We demonstrate that absence of KDM3A facilitates ciliogenesis, but these resulting cilia have an abnormally wide range of axonemal lengths, delaying disassembly and accumulating intraflagellar transport (IFT) proteins. KDM3A plays a dual role by regulating actin gene expression and binding to the actin cytoskeleton, creating a responsive “actin gate” that involves ARP2/3 activity and IFT. Promoting actin filament formation rescues KDM3A mutant ciliary defects. Conversely, the simultaneous depolymerization of actin networks and IFT overexpression mimics the abnormal ciliary traits of KDM3A mutants. KDM3A is thus a negative regulator of ciliogenesis required for the controlled recruitment of IFT proteins into cilia through the modulation of actin dynamics. PMID:28246120

  13. A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides in Geobacillus stearothermophilus▿

    PubMed Central

    Shulami, Smadar; Zaide, Galia; Zolotnitsky, Gennady; Langut, Yael; Feld, Geoff; Sonenshein, Abraham L.; Shoham, Yuval

    2007-01-01

    Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His6-XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the −53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 μM XynC. The His6-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (ΔCp) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system. PMID:17142383

  14. Regulation of transepithelial ion transport in the rat late distal colon by the sympathetic nervous system.

    PubMed

    Zhang, X; Li, Y; Zhang, X; Duan, Z; Zhu, J

    2015-01-01

    The colorectum (late distal colon) is innervated by the sympathetic nervous system, and many colorectal diseases are related to disorders of the sympathetic nervous system. The sympathetic regulation of colorectal ion transport is rarely reported. The present study aims to investigate the effect of norepinephrine (NE) in the normal and catecholamine-depleted condition to clarify the regulation of the sympathetic adrenergic system in ion transport in the rat colorectum. NE-induced ion transport in the rats colorectum was measured by short-circuit current (I(sc)) recording; the expression of beta-adrenoceptors and NE transporter (NET) were quantified by real-time PCR, and western blotting. When the endogenous catecholamine was depleted by reserpine, the baseline I(sc) in the colorectum was increased significantly comparing to controls. NE evoked downward deltaI(sc) in colorectum of treated rats was 1.8-fold of controls. The expression of beta(2)-adrenoceptor protein in the colorectal mucosa was greater than the control, though the mRNA level was reduced. However, NET expression was significantly lower in catecholamine-depleted rats compared to the controls. In conclusion, the sympathetic nervous system plays an important role in regulating basal ion transport in the colorectum. Disorders of sympathetic neurotransmitters result in abnormal ion transport, beta-adrenoceptor and NET are involved in the process.

  15. Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration

    PubMed Central

    Simpkins, Jessica A.; Rickel, Kirby E.; Madeo, Marianna; Ahlers, Bethany A.; Carlisle, Gabriel B.; Nelson, Heidi J.; Cardillo, Andrew L.; Weber, Emily A.; Vitiello, Peter F.; Pearce, David A.

    2016-01-01

    ABSTRACT Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling. PMID:27142334

  16. Dopamine transporter occupancy by RTI-55, inhibition of dopamine transport and stimulation of locomotor activity

    SciTech Connect

    Gatley, S.J.; Gifford, A.N.; Volkow, N.D.

    1997-05-01

    Cocaine analogs such as RTI-55 (or {beta}CIT) with a higher affinity for the DAT are potentially useful as therapeutic drugs in cocaine abuse as well as for radiopharmaceutical use. Previously we showed that in mice RTI-55 (2 mg/Kg, i/p) reduced H-3 cocaine striatum-to-cerebellum ratios (St/Cb, {lg_bullet}) from 1.6 to 1.2 at 3 h after administration, with recovery by 12 h. In the present study we demonstrate a very similar time-course for transport {triangle} measured in striatal homo within 2 min of sacrifice. The maximum inhibition of uptake at about 1 h corresponded to about 80% of the control uptake rate, similar to the percent reduction in St/Cb. The time-course of the effect of this dose of RTI-55 on locomotor activity ({sq_bullet}) was complex, with a drop in the activity measure at 7 h, after a further injection of RTI-55, but activity remained higher than in saline controls. In spite of this complexity, which may be associated with stereotypies and/or exhaustion, the duration of increased activity is consistent with the duration of transporter blockade. These experiments support the notion that PET/SPECT measures of transporter occupancy accurately reflect transporter inhibition.

  17. Transport of active ellipsoidal particles in ratchet potentials

    SciTech Connect

    Ai, Bao-Quan Wu, Jian-Chun

    2014-03-07

    Rectified transport of active ellipsoidal particles is numerically investigated in a two-dimensional asymmetric potential. The out-of-equilibrium condition for the active particle is an intrinsic property, which can break thermodynamical equilibrium and induce the directed transport. It is found that the perfect sphere particle can facilitate the rectification, while the needlelike particle destroys the directed transport. There exist optimized values of the parameters (the self-propelled velocity, the torque acting on the body) at which the average velocity takes its maximal value. For the ellipsoidal particle with not large asymmetric parameter, the average velocity decreases with increasing the rotational diffusion rate, while for the needlelike particle (very large asymmetric parameter), the average velocity is a peaked function of the rotational diffusion rate. By introducing a finite load, particles with different shapes (or different self-propelled velocities) will move to the opposite directions, which is able to separate particles of different shapes (or different self-propelled velocities)

  18. Regulation of blood oxygen transport in hibernating mammals.

    PubMed

    Revsbech, Inge G; Fago, Angela

    2017-03-21

    Along with the periodic reductions in O2 requirements of mammalian hibernators during winter, the O2 affinity of the blood of mammalian hibernators is seasonally regulated to help match O2 supply to consumption, contributing to limit tissue oxidative stress, particularly at arousals. Specifically, mammalian hibernators consistently show an overall increase in the blood-O2 affinity, which causes a decreased O2 unloading to tissues, while having similar or lower tissue O2 tensions during hibernation. This overview explores how the decreased body temperature and concentration of red blood cell 2,3-diphosphoglycerate (DPG) that occur in hibernation contribute separately or in combination to the concurrent increase in the O2 affinity of the hemoglobin, the O2 carrier protein of the blood. Most mammalian hemoglobins are responsive to changes in DPG concentrations, including that of the hibernating brown bear, although the smaller hibernators, such as golden-mantled ground squirrel, chipmunks, and dormice, have hemoglobins with low sensitivity to DPG. While the effect of DPG on oxygenation may vary, the decrease in body temperature invariably increases hemoglobin's O2 affinity in all hibernating species. However, the temperature sensitivity of hemoglobin oxygenation is low in hibernators compared to human, apparently due in part to endothermic allosteric quaternary transition in ground squirrels and dissociation of chloride ions in brown bears. A low heat of blood oxygenation in temporal heterotherms, like hibernators, may thus contribute to reduce heat loss, as found in regional heterotherms, like polar mammals, although the significance would be low in winter hibernation.

  19. Self-regulated oscillation of transport and topology of magnetic islands in toroidal plasmas

    PubMed Central

    Ida, K.; Kobayashi, T.; Evans, T. E.; Inagaki, S.; Austin, M. E.; Shafer, M. W.; Ohdachi, S.; Suzuki, Y.; Itoh, S.-I.; Itoh, K.

    2015-01-01

    The coupling between the transport and magnetic topology is an important issue because the structure of magnetic islands, embedded in a toroidal equilibrium field, depends on the nature of the transport at the edge of the islands. Measurements of modulated heat pulse propagation in the DIII-D tokamak have revealed the existence of self-regulated oscillations in the radial energy transport into magnetic islands that are indicative of bifurcations in the island structure and transport near the q = 2 surface. Large amplitude heat pulses are seen in one state followed by small amplitude pulses later in the discharge resulting in a repeating cycle of island states. These two states are interpreted as a bifurcation of magnetic island with high and low heat pulse accessibility. This report describes the discovery of a bifurcation in the coupled dynamics between the transport and topology of magnetic islands in tokamak plasmas. PMID:26530273

  20. Self-regulated oscillation of transport and topology of magnetic islands in toroidal plasmas.

    PubMed

    Ida, K; Kobayashi, T; Evans, T E; Inagaki, S; Austin, M E; Shafer, M W; Ohdachi, S; Suzuki, Y; Itoh, S-I; Itoh, K

    2015-11-04

    The coupling between the transport and magnetic topology is an important issue because the structure of magnetic islands, embedded in a toroidal equilibrium field, depends on the nature of the transport at the edge of the islands. Measurements of modulated heat pulse propagation in the DIII-D tokamak have revealed the existence of self-regulated oscillations in the radial energy transport into magnetic islands that are indicative of bifurcations in the island structure and transport near the q = 2 surface. Large amplitude heat pulses are seen in one state followed by small amplitude pulses later in the discharge resulting in a repeating cycle of island states. These two states are interpreted as a bifurcation of magnetic island with high and low heat pulse accessibility. This report describes the discovery of a bifurcation in the coupled dynamics between the transport and topology of magnetic islands in tokamak plasmas.

  1. Self-regulated oscillation of transport and topology of magnetic islands in toroidal plasmas

    SciTech Connect

    Ida, K.; Kobayashi, T.; Evans, T. E.; Inagaki, S.; Austin, M. E.; Shafer, M. W.; Ohdachi, S.; Suzuki, Y.; Itoh, S. -I.; Itoh, K.

    2015-11-04

    The coupling between the transport and magnetic topology is an important issue because the structure of magnetic islands, embedded in a toroidal equilibrium field, depends on the nature of the transport at the edge of the islands. Measurements of modulated heat pulse propagation in the DIII-D tokamak have revealed the existence of self-regulated oscillations in the radial energy transport into magnetic islands that are indicative of bifurcations in the island structure and transport near the q = 2 surface. Furthermore, large amplitude heat pulses are seen in one state followed by small amplitude pulses later in the discharge resulting in a repeating cycle of island states. We interpret these two states as a bifurcation of magnetic island with high and low heat pulse accessibility. This report describes the discovery of a bifurcation in the coupled dynamics between the transport and topology of magnetic islands in tokamak plasmas.

  2. Self-regulated oscillation of transport and topology of magnetic islands in toroidal plasmas

    DOE PAGES

    Ida, K.; Kobayashi, T.; Evans, T. E.; ...

    2015-11-04

    The coupling between the transport and magnetic topology is an important issue because the structure of magnetic islands, embedded in a toroidal equilibrium field, depends on the nature of the transport at the edge of the islands. Measurements of modulated heat pulse propagation in the DIII-D tokamak have revealed the existence of self-regulated oscillations in the radial energy transport into magnetic islands that are indicative of bifurcations in the island structure and transport near the q = 2 surface. Furthermore, large amplitude heat pulses are seen in one state followed by small amplitude pulses later in the discharge resulting inmore » a repeating cycle of island states. We interpret these two states as a bifurcation of magnetic island with high and low heat pulse accessibility. This report describes the discovery of a bifurcation in the coupled dynamics between the transport and topology of magnetic islands in tokamak plasmas.« less

  3. Discovery of an auto-regulation mechanism for the maltose ABC transporter MalFGK2.

    PubMed

    Bao, Huan; Duong, Franck

    2012-01-01

    The maltose transporter MalFGK(2), together with the substrate-binding protein MalE, is one of the best-characterized ABC transporters. In the conventional model, MalE captures maltose in the periplasm and delivers the sugar to the transporter. Here, using nanodiscs and proteoliposomes, we instead find that MalE is bound with high-affinity to MalFGK2 to facilitate the acquisition of the sugar. When the maltose concentration exceeds the transport capacity, MalE captures maltose and dissociates from the transporter. This mechanism explains why the transport rate is high when MalE has low affinity for maltose, and low when MalE has high affinity for maltose. Transporter-bound MalE facilitates the acquisition of the sugar at low concentrations, but also captures and dissociates from the transporter past a threshold maltose concentration. In vivo, this maltose-forced dissociation limits the rate of transport. Given the conservation of the substrate-binding proteins, this mode of allosteric regulation may be universal to ABC importers.

  4. 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.

  5. Hydrogen peroxide stimulates the active transport of serotonin into human platelets

    SciTech Connect

    Bosin, T.R. )

    1991-03-11

    The effect of hydrogen peroxide on the active transport of serotonin (5-HT) by human platelets was investigated. Platelets were exposed to either a single dose of H{sub 2}O{sub 2} or to H{sub 2}O{sub 2} generated by the glucose/glucose oxidase or xanthine/xanthine oxidase enzyme systems. H{sub 2}{sub 2} produced a rapid, dose-dependent and time-dependent increase in 5-HT transport which was maximal after a 2 min incubation and decreased with continued incubation. Catalase completely prevented H{sub 2}O{sub 2}-induced stimulation and fluoxetine totally blocked 5-HT uptake into stimulated platelets. The glucose/glucose oxidase and the xanthine/xanthine oxidase generating systems produced a similar response to that of H{sub 2}O{sub 2}. In the xanthine/xanthine oxidase system, superoxide dismutase failed to alter the stimulation, while catalase effectively prevented the response. The kinetics of 5-HT transport indicated that H{sub 2}O{sub 2} treatment did not alter the K{sub m} of 5-HT transport but significantly increased the maximal rate of 5-HT transport. These data demonstrated that exposure of human platelets to H{sub 2}O{sub 2} resulted in a stimulation of the active transport of 5-HT and suggested that H{sub 2}O{sub 2} may function to regulate this process.

  6. Zinc transporter Slc39a14 regulates inflammatory signaling associated with hypertrophic adiposity

    PubMed Central

    Troche, Catalina; Beker Aydemir, Tolunay

    2015-01-01

    Zinc is a signaling molecule in numerous metabolic pathways, the coordination of which occurs through activity of zinc transporters. The expression of zinc transporter Zip14 (Slc39a14), a zinc importer of the solute carrier 39 family, is stimulated under proinflammatory conditions. Adipose tissue upregulates Zip14 during lipopolysaccharide-induced endotoxemia. A null mutation of Zip14 (KO) revealed that phenotypic changes in adipose include increased cytokine production, increased plasma leptin, hypertrophied adipocytes, and dampened insulin signaling. Adipose tissue from KO mice had increased levels of preadipocyte markers, lower expression of the differentiation marker (PPARγ), and activation of NF-κB and STAT3 pathways. Our overall hypothesis was that ZIP14 would play a role in adipocyte differentiation and inflammatory obesity. Global Zip14 KO causes systemic endotoxemia. The observed metabolic changes in adipose metabolism were reversed when oral antibiotics were administrated, indicating that circulating levels of endotoxin were in part responsible for the adipose phenotype. To evaluate a mechanism, 3T3-L1 cells were differentiated into adipocytes and treated with siRNA to knock down Zip14. These cells had an impaired ability to mobilize zinc, which caused dysregulation of inflammatory pathways (JAK2/STAT3 and NF-κB). The Zip14 deletion may limit the availability of intracellular zinc, yielding the unique phenotype of inflammation coupled with hypertrophy. Taken together, these results suggest that aberrant zinc distribution observed with Zip14 ablation impacts adipose cytokine production and metabolism, ultimately increasing fat deposition when exposed to endotoxin. To our knowledge, this is the first investigation into the mechanistic role of ZIP14 in adipose tissue regulation and metabolism. PMID:26646099

  7. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  8. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  9. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  10. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  11. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  12. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  13. Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells.

    PubMed

    Komaba, Shigeru; Coluccio, Lynne M

    2015-01-01

    Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM.

  14. Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thévenieau, France; Nicaud, Jean-Marc

    2015-09-01

    Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast.

  15. Disrupted sleep-wake regulation in type 1 equilibrative nucleoside transporter knockout mice.

    PubMed

    Kim, T; Ramesh, V; Dworak, M; Choi, D-S; McCarley, R W; Kalinchuk, A V; Basheer, R

    2015-09-10

    The type 1 equilibrative nucleoside transporter (ENT1) is implicated in regulating levels of extracellular adenosine ([AD]ex). In the basal forebrain (BF) levels of [AD]ex increase during wakefulness and closely correspond to the increases in the electroencephalogram (EEG) delta (0.75-4.5Hz) activity (NRδ) during subsequent non-rapid eye movement sleep (NREMS). Thus in the BF, [AD]ex serves as a biochemical marker of sleep homeostasis. Waking EEG activity in theta range (5-9Hz, Wθ) is also described as a marker of sleep homeostasis. An hour-by-hour temporal relationship between the Wθ and NRδ is unclear. In this study we examined the relationship between these EEG markers of sleep homeostasis during spontaneous sleep-wakefulness and during sleep deprivation (SD) and recovery sleep in the ENT1 gene knockout (ENT1KO) mouse. We observed that baseline NREMS amount was decreased during the light period in ENT1KO mice, accompanied by a weak correlation between Wθ of each hour and NRδ of its subsequent hour when compared to their wild-type (WT) littermates. Perfusion of low dose of adenosine into BF not only strengthened the Wθ-NRδ relationship, but also increased NREMS to match with the WT littermates suggesting decreased [AD]ex in ENT1KO mice. However, the SD-induced [AD]ex increase in the BF and the linear correlation between the EEG markers of sleep homeostasis were unaffected in ENT1KO mice suggesting that during SD, sources other than ENT1 contribute to increase in [AD]ex. Our data provide evidence for a differential regulation of wakefulness-associated [AD]ex during spontaneous vs prolonged waking.

  16. Volume regulation of intestinal cells of echinoderms: Putative role of ion transporters (Na(+)/K(+)-ATPase and NKCC).

    PubMed

    Castellano, Giovanna C; Souza, Marta M; Freire, Carolina A

    2016-11-01

    Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na(+)/K(+)-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na(+)/K(+)-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells.

  17. The canonical Notch pathway effector RBP-J regulates neuronal plasticity and expression of GABA transporters in hippocampal networks.

    PubMed

    Liu, Shuxi; Wang, Yue; Worley, Paul F; Mattson, Mark P; Gaiano, Nicholas

    2015-05-01

    Activation of the Notch pathway in neurons is essential for learning and memory in various species from invertebrates to mammals. However, it remains unclear how Notch signaling regulates neuronal plasticity, and whether the transcriptional regulator and canonical pathway effector RBP-J plays a role. Here, we report that conditional disruption of RBP-J in the postnatal hippocampus leads to defects in long-term potentiation, long-term depression, and in learning and memory. Using gene expression profiling and chromatin immunoprecipitation, we identified two GABA transporters, GAT2 and BGT1, as putative Notch/RBP-J pathway targets, which may function downstream of RBP-J to limit the accumulation of GABA in the Schaffer collateral pathway. Our results reveal an essential role for canonical Notch/RBP-J signaling in hippocampal synaptic plasticity and suggest that role, at least in part, is mediated by the regulation of GABAergic signaling.

  18. Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

    PubMed Central

    Echtay, Karim S.; Winkler, Edith; Frischmuth, Karina; Klingenberg, Martin

    2001-01-01

    Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609–613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2′-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl− but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters. PMID:11171965

  19. Kinetics and regulation of lactose transport and metabolism in Kluyveromyces lactis JA6.

    PubMed

    Santos, A M; Silveira, W B; Fietto, L G; Brandão, R L; Castro, I M

    2014-07-01

    Kluyveromyces lactis strains are able to assimilate lactose. They have been used industrially to eliminate this sugar from cheese whey and in other industrial products. In this study, we investigated specific features and the kinetic parameters of the lactose transport system in K. lactis JA6. In lactose grown cells, lactose was transported by a system transport with a half-saturation constant (K s) of 1.49 ± 0.38 mM and a maximum velocity (V max) of 0.96 ± 0.12 mmol. (g dry weight)(-1) h(-1) for lactose. The transport system was constitutive and energy-dependent. Results obtained by different approaches showed that the lactose transport system was regulated by glucose at the transcriptional level and by glucose and other sugars at a post-translational level. In K. lactis JA6, galactose metabolization was under glucose control. These findings indicated that the regulation of lactose-galactose regulon in K. lactis was similar to the regulation of galactose regulon in Saccharomyces cerevisiae.

  20. Regulation of plants' phosphate uptake in common mycorrhizal networks: Role of intraradical fungal phosphate transporters

    PubMed Central

    Walder, Florian; Boller, Thomas; Wiemken, Andres; Courty, Pierre-Emmanuel

    2016-01-01

    ABSTRACT We have recently identified two genes coding for inorganic phosphate transporters (Pht) in sorghum (Sorghum bicolor) and flax (Linum usitatissimum) that were induced in roots colonized by arbuscular mycorrhizal (AM) fungi. Mycorrhizal acquisition of inorganic phosphorus (Pi) was strongly affected by the combination of plant and AM fungal species, but the expression level of these genes coding for AM-inducible Pi transporters did not explain differences in plant phosphorus acquisition where flax and sorghum are sharing a common mycorrhizal network. In the present study, we investigated the possible role of fungal Pi transporters in the regulation of mycorrhizal Pi acquisition by measuring their expression in roots of flax and sorghum. One Pi transporter of Rhizophagus irregularis (RiPT5) showed a positive correlation with mycorrhizal Pi acquisition of sorghum. This indicates that a possible involvement in the regulation of mycorrhizal Pi acquisition. In general, expression of AMF Pi transporters was more related to mycorrhizal Pi acquisition of sorghum than of flax, indicating plant species-specific differences in the regulation of mycorrhizal Pi acquisition. PMID:26751110

  1. Plasmodesmata localizing proteins regulate transport and signaling during systemic acquired immunity in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Systemic acquired resistance (SAR) in plants is mediated by the signaling molecules azelaic acid (AzA),glycerol-3-phosphate (G3P), and salicylic acid (SA).Here, we show that AzA and G3P transport occurs via the symplastic route, which is regulated by channels known as plasmodesmata (PD). In contrast...

  2. Impact of Clean Air Regulations on Nitrogen Fate and Transport in Neuse River Basin

    EPA Science Inventory

    We investigated impacts of Clean Air Act (CAA) nitrogen emissions regulations on the fate and transport of nitrogen for two watersheds in the Neuse River Basin. The Soil and Water Assessment Tool (SWAT) and the Community Multi-Scale Air Quality (CMAQ) models were used. Two scenar...

  3. Impact of Clean Air Act Regulations on Nitrogen Fate and Transport in Neuse River Basin

    EPA Science Inventory

    This study investigated impacts of Clean Air Act Amendment (CAAA) NOx emissions regulations on the fate and transport of nitrogen for two watersheds in the Neuse River Basin, North Carolina, USA from 1990 to 2020. The Soil and Water Assessment Tool (SWAT) and the Community Multi-...

  4. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

    PubMed

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  5. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

    SciTech Connect

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  6. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  7. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  8. Localization of the calcium-regulated citrate transport process in proximal tubule cells.

    PubMed

    Hering-Smith, Kathleen S; Mao, Weibo; Schiro, Faith R; Coleman-Barnett, Joycelynn; Pajor, Ana M; Hamm, L Lee

    2014-06-01

    Urinary citrate is an important inhibitor of calcium-stone formation. Most of the citrate reabsorption in the proximal tubule is thought to occur via a dicarboxylate transporter NaDC1 located in the apical membrane. OK cells, an established opossum kidney proximal tubule cell line, transport citrate but the characteristics change with extracellular calcium such that low calcium solutions stimulate total citrate transport as well as increase the apparent affinity for transport. The present studies address several fundamental properties of this novel process: the polarity of the transport process, the location of the calcium-sensitivity and whether NaDC1 is present in OK cells. OK cells grown on permeable supports exhibited apical >basolateral citrate transport. Apical transport of both citrate and succinate was sensitive to extracellular calcium whereas basolateral transport was not. Apical calcium, rather than basolateral, was the predominant determinant of changes in transport. Also 2,3-dimethylsuccinate, previously identified as an inhibitor of basolateral dicarboxylate transport, inhibited apical citrate uptake. Although the calcium-sensitive transport process in OK cells is functionally not typical NaDC1, NaDC1 is present in OK cells by Western blot and PCR. By immunolocalization studies, NaDC1 was predominantly located in discrete apical membrane or subapical areas. However, by biotinylation, apical NaDC1 decreases in the apical membrane with lowering calcium. In sum, OK cells express a calcium-sensitive/regulated dicarboxylate process at the apical membrane which responds to variations in apical calcium. Despite the functional differences of this process compared to NaDC1, NaDC1 is present in these cells, but predominantly in subapical vesicles.

  9. Estrone Sulfate Transport and Steroid Sulfatase Activity in Colorectal Cancer: Implications for Hormone Replacement Therapy

    PubMed Central

    Gilligan, Lorna C.; Gondal, Ali; Tang, Vivien; Hussain, Maryam T.; Arvaniti, Anastasia; Hewitt, Anne-Marie; Foster, Paul A.

    2017-01-01

    Hormone replacement therapy (HRT) affects the incidence and potential progression of colorectal cancer (CRC). As HRT primarily consists of estrone sulfate (E1S), understanding whether this conjugated estrogen is transported and metabolized in CRC will define its potential effect in this malignancy. Here, we show that a panel of CRC cell lines (Colo205, Caco2, HCT116, HT-29) have steroid sulfatase (STS) activity, and thus can hydrolyze E1S. STS activity is significantly higher in CRC cell lysate, suggesting the importance of E1S transport in intracellular STS substrate availability. As E1S transport is regulated by the expression pattern of certain solute carrier organic anion transporter polypeptides, we show that in CRC OATP4A1 is the most abundantly expressed transporter. All four CRC cell lines rapidly transported E1S into cells, with this effect significantly inhibited by the competitive OATP inhibitor BSP. Transient knockdown of OATP4A1 significantly disrupted E1S uptake. Examination of estrogen receptor status showed ERα was present in Colo205 and Caco2 cells. None of the cells expressed ERβ. Intriguingly, HCT116 and HT29 cells strongly expressed the G protein coupled estrogen receptor (GPER), and that stimulation of this receptor with estradiol (E2) and G1, a GPER agonist, significantly (p < 0.01) increased STS activity. Furthermore, tamoxifen and fulvestrant, known GPER agonist, also increased CRC STS activity, with this effect inhibited by the GPER antagonist G15. These results suggest that CRC can take up and hydrolyze E1S, and that subsequent GPER stimulation increases STS activity in a potentially novel positive feedback loop. As elevated STS expression is associated with poor prognosis in CRC, these results suggest HRT, tamoxifen and fulvestrant may negatively impact CRC patient outcomes. PMID:28326039

  10. Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

    PubMed Central

    Busby, Jason N.; Fritz, Georg; Moreland, Nicole J.; Cook, Gregory M.; Lott, J. Shaun; Baker, Edward N.

    2014-01-01

    Bacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains. PMID:25049090

  11. GPR30 regulates glutamate transporter GLT-1 expression in rat primary astrocytes.

    PubMed

    Lee, Eunsook; Sidoryk-Wêgrzynowicz, Marta; Wang, Ning; Webb, Anton; Son, Deok-Soo; Lee, Kyuwon; Aschner, Michael

    2012-08-03

    The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury.

  12. GPR30 Regulates Glutamate Transporter GLT-1 Expression in Rat Primary Astrocytes*

    PubMed Central

    Lee, Eunsook; Sidoryk-Wêgrzynowicz, Marta; Wang, Ning; Webb, Anton; Son, Deok-Soo; Lee, Kyuwon; Aschner, Michael

    2012-01-01

    The G protein-coupled estrogen receptor GPR30 contributes to the neuroprotective effects of 17β-estradiol (E2); however, the mechanisms associated with this protection have yet to be elucidated. Given that E2 increases astrocytic expression of glutamate transporter-1 (GLT-1), which would prevent excitotoxic-induced neuronal death, we proposed that GPR30 mediates E2 action on GLT-1 expression. To investigate this hypothesis, we examined the effects of G1, a selective agonist of GPR30, and GPR30 siRNA on astrocytic GLT-1 expression, as well as glutamate uptake in rat primary astrocytes, and explored potential signaling pathways linking GPR30 to GLT-1. G1 increased GLT-1 protein and mRNA levels, subject to regulation by both MAPK and PI3K signaling. Inhibition of TGF-α receptor suppressed the G1-induced increase in GLT-1 expression. Silencing GPR30 reduced the expression of both GLT-1 and TGF-α and abrogated the G1-induced increase in GLT-1 expression. Moreover, the G1-induced increase in GLT-1 protein expression was abolished by a protein kinase A inhibitor and an NF-κB inhibitor. G1 also enhanced cAMP response element-binding protein (CREB), as well as both NF-κB p50 and NF-κB p65 binding to the GLT-1 promoter. Finally, to model dysfunction of glutamate transporters, manganese was used, and G1 was found to attenuate manganese-induced impairment in GLT-1 protein expression and glutamate uptake. Taken together, the present data demonstrate that activation of GPR30 increases GLT-1 expression via multiple pathways, suggesting that GPR30 is worthwhile as a potential target to be explored for developing therapeutics of excitotoxic neuronal injury. PMID:22645130

  13. Regulation of GLUT transporters by flavonoids in androgen-sensitive and -insensitive prostate cancer cells.

    PubMed

    Gonzalez-Menendez, Pedro; Hevia, David; Rodriguez-Garcia, Aida; Mayo, Juan C; Sainz, Rosa M

    2014-09-01

    Cancer cells show different metabolic requirements from normal cells. In prostate cancer, particularly, glycolytic metabolism differs in androgen-responsive and nonresponsive cells. In addition, some natural compounds with antiproliferative activities are able to modify glucose entry into cells by either modulating glucose transporter (GLUT) expression or by altering glucose binding. The aim of this work was to study the regulation of some GLUTs (GLUT1 and GLUT4) in both androgen-sensitive (LNCaP) and -insensitive (PC-3) prostate cancer cells by 4 structurally different flavonoids (ie, genistein, phloretin, apigenin, and daidzein). Glucose uptake was measured using nonradiolabeled 2-deoxyglucose. The evaluation of protein levels as well as subcellular distribution of GLUT1/4 were analyzed by Western blot and immunocytochemistry, respectively. Androgen-insensitive LNCaP-R and androgen-sensitive PC-3-AR cells were used to study the effect of androgen signaling. Additionally, a docking simulation was employed to compare interactions between flavonoids and XylE, a bacterial homolog of GLUT1 to -4. Results show for the first time the presence of functionally relevant GLUT4 in prostate cancer cells. Furthermore, differences in GLUT1 and GLUT4 levels and glucose uptake were found, without differences on subcellular distribution, after incubation with flavonoids. Docking simulation showed that all compounds interact with the same location of transporters. More importantly, differences between androgen-sensitive and -insensitive prostate cancer cells were found in both GLUT protein levels and glucose uptake. Thus, phenotypic characteristics of prostate cancer cells are responsible for the different effects of these flavonoids in glucose uptake and in GLUT expression rather than their structural differences, with the most effective in reducing cell growth being the highest in modifying glucose uptake and GLUT levels.

  14. Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling.

    PubMed

    Mesev, Emily V; Miller, David S; Cannon, Ronald E

    2017-04-01

    P-glycoprotein, an ATP-driven efflux pump, regulates permeability of the blood-brain barrier (BBB). Sphingolipids, endogenous to brain tissue, influence inflammatory responses and cell survival in vitro. Our laboratory has previously shown that sphingolipid signaling by sphingosine 1-phosphate decreases basal P-glycoprotein transport activity. Here, we investigated the potential for another sphingolipid, ceramide 1-phosphate (C1P), to modulate efflux pumps at the BBB. Using confocal microscopy and measuring luminal accumulation of fluorescent substrates, we assessed the transport activity of several efflux pumps in isolated rat brain capillaries. C1P treatment induced P-glycoprotein transport activity in brain capillaries rapidly and reversibly. In contrast, C1P did not affect transport activity of two other major efflux transporters, multidrug resistance protein 2 and breast cancer resistance protein. C1P induced P-glycoprotein transport activity without changing transporter protein expression. Inhibition of the key signaling components in the cyclooxygenase-2 (COX-2)/prostaglandin E2 signaling cascade (phospholipase A2, COX-2, multidrug resistance protein 4, and G-protein-coupled prostaglandin E2 receptors 1 and 2), abolished P-glycoprotein induction by C1P. We show that COX-2 and prostaglandin E2 are required for C1P-mediated increases in P-glycoprotein activity independent of transporter protein expression. This work describes how C1P activates a signaling cascade to dynamically regulate P-glycoprotein transport at the BBB and offers potential clinical targets to modulate neuroprotection and drug delivery to the CNS.

  15. Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling

    PubMed Central

    Mesev, Emily V.; Miller, David S.

    2017-01-01

    P-glycoprotein, an ATP-driven efflux pump, regulates permeability of the blood-brain barrier (BBB). Sphingolipids, endogenous to brain tissue, influence inflammatory responses and cell survival in vitro. Our laboratory has previously shown that sphingolipid signaling by sphingosine 1-phosphate decreases basal P-glycoprotein transport activity. Here, we investigated the potential for another sphingolipid, ceramide 1-phosphate (C1P), to modulate efflux pumps at the BBB. Using confocal microscopy and measuring luminal accumulation of fluorescent substrates, we assessed the transport activity of several efflux pumps in isolated rat brain capillaries. C1P treatment induced P-glycoprotein transport activity in brain capillaries rapidly and reversibly. In contrast, C1P did not affect transport activity of two other major efflux transporters, multidrug resistance protein 2 and breast cancer resistance protein. C1P induced P-glycoprotein transport activity without changing transporter protein expression. Inhibition of the key signaling components in the cyclooxygenase-2 (COX-2)/prostaglandin E2 signaling cascade (phospholipase A2, COX-2, multidrug resistance protein 4, and G-protein–coupled prostaglandin E2 receptors 1 and 2), abolished P-glycoprotein induction by C1P. We show that COX-2 and prostaglandin E2 are required for C1P-mediated increases in P-glycoprotein activity independent of transporter protein expression. This work describes how C1P activates a signaling cascade to dynamically regulate P-glycoprotein transport at the BBB and offers potential clinical targets to modulate neuroprotection and drug delivery to the CNS. PMID:28119480

  16. Stanniocalcin-1 Controls Ion Regulation Functions of Ion-transporting Epithelium Other than Calcium Balance

    PubMed Central

    Chou, Ming-Yi; Lin, Chia-Hao; Chao, Pei-Lin; Hung, Jo-Chi; Cruz, Shelly A.; Hwang, Pung-Pung

    2015-01-01

    Stanniocalcin-1 (STC-1) was first identified to involve in Ca2+ homeostasis in teleosts, and was thought to act as a hypocalcemic hormone in vertebrate. Recent studies suggested that STC-1 exhibits broad effects on ion balance, not confines to Ca2+, but the mechanism of this regulation process remains largely unknown. Here, we used zebrafish embryos as an alternative in vivo model to investigate how STC-1 regulates transepithelial ion transport function in ion-transporting epithelium. Expression of stc-1 mRNA in zebrafish embryos was increased in high-Ca2+ environments but decreased by acidic and ion-deficient treatments while overexpression of stc-1 impaired the hypotonic acclimation by decreasing whole body Ca2+, Na+, and Cl- contents and H+ secretion ability. Injection of STC-1 mRNA also down-regulated mRNA expressions of epithelial Ca2+ channel, H+-ATPase, and Na+-Cl- cotransporter, suggesting the roles of STC-1 in regulation of ions other than Ca2+. Knockdown of STC-1 caused an increase in ionocyte progenitors (foxi3a as the marker) and mature ionocytes (ion transporters as the markers), but did not affect epithelium stem cells (p63 as the marker) in the embryonic skin. Overexpression of STC-1 had the corresponding opposite effect on ionocyte progenitors, mature ionocytes in the embryonic skin. Taken together, STC-1 negatively regulates the number of ionocytes to reduce ionocyte functions. This process is important for body fluid ionic homeostasis, which is achieved by the regulation of ion transport functions in ionocytes. The present findings provide new insights into the broader functions of STC-1, a hypocalcemic hormone. PMID:25561895

  17. Interaction of ABC transport proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line.

    PubMed

    Della Torre, Camilla; Zaja, Roko; Loncar, Jovica; Smital, Tvrtko; Focardi, Silvano; Corsi, Ilaria

    2012-06-25

    The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. Cells were exposed for 24 h to 0.01-1 μM of CdCl(2), HgCl(2), As(2)O(3), or K(2)Cr(2)O(7) and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P-gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl(2) with 3.3-fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9- to 4-fold vs. controls) and MRP (1.3- to 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 μM HgCl(2) for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl(2). Exposure to HgCl(2) for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl(2), did not cause prolonged up-regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

  18. Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity

    PubMed Central

    Shmueli, Anat; Segal, Michal; Sapir, Tamar; Tsutsumi, Ryouhei; Noritake, Jun; Bar, Avi; Sapoznik, Sivan; Fukata, Yuko; Orr, Irit; Fukata, Masaki; Reiner, Orly

    2010-01-01

    Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine-tuning the activity of the retrograde motor cytoplasmic dynein. PMID:19927128

  19. Importin 8 regulates the transport of mature microRNAs into the cell nucleus.

    PubMed

    Wei, Yao; Li, Limin; Wang, Dong; Zhang, Chen-Yu; Zen, Ke

    2014-04-11

    Mature microRNAs (miRNAs), ∼ 22-nucleotide noncoding RNAs regulating target gene expression at the post-transcriptional level, have been recently shown to be transported into the nucleus where they modulate the biogenesis of other miRNAs or their own expression. However, the mechanism that governs the transport of mature miRNAs from cytoplasm to nucleus remains unknown. Here, we report that importin 8 (IPO8), a member of the karyopherin β (also named the protein import receptor importin β) family, plays a critical role in mediating the cytoplasm-to-nucleus transport of mature miRNAs. Specifically knocking down IPO8 but not other karyopherin β family proteins via siRNA significantly decreases the nuclear transport of various known nucleus-enriched miRNAs without affecting their total cellular levels. IPO8-mediated nuclear transport of mature miRNAs is also dependent on the association of IPO8 with the Argonaute 2 (Ago2) complex. Cross-immunoprecipitation and Western blot analysis show that IPO8 is physically associated with Ago2. Knocking down IPO8 via siRNA markedly decreases the nuclear transport of Ago2 but does not affect the total cellular Ago2 level. Furthermore, dissociating the binding of miRNAs with Ago2 by trypaflavine strongly reduces the IPO8-mediated nuclear transport of miRNAs.

  20. Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

    PubMed Central

    German, Christopher L.; Baladi, Michelle G.; McFadden, Lisa M.; Hanson, Glen R.

    2015-01-01

    Dopamine (DA) plays a well recognized role in a variety of physiologic functions such as movement, cognition, mood, and reward. Consequently, many human disorders are due, in part, to dysfunctional dopaminergic systems, including Parkinson’s disease, attention deficit hyperactivity disorder, and substance abuse. Drugs that modify the DA system are clinically effective in treating symptoms of these diseases or are involved in their manifestation, implicating DA in their etiology. DA signaling and distribution are primarily modulated by the DA transporter (DAT) and by vesicular monoamine transporter (VMAT)-2, which transport DA into presynaptic terminals and synaptic vesicles, respectively. These transporters are regulated by complex processes such as phosphorylation, protein–protein interactions, and changes in intracellular localization. This review provides an overview of 1) the current understanding of DAT and VMAT2 neurobiology, including discussion of studies ranging from those conducted in vitro to those involving human subjects; 2) the role of these transporters in disease and how these transporters are affected by disease; and 3) and how selected drugs alter the function and expression of these transporters. Understanding the regulatory processes and the pathologic consequences of DAT and VMAT2 dysfunction underlies the evolution of therapeutic development for the treatment of DA-related disorders. PMID:26408528

  1. Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells

    PubMed Central

    Jansson, Kyle; Venugopal, Jessica; Sánchez, Gladis; Magenheimer, Brenda S.; Reif, Gail A.; Wallace, Darren P.; Calvet, James P.

    2015-01-01

    Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1m1Bei mice, but had no effect on metanephroi from Pkd1m1Bei mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl− efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and upregulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na+ transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD. PMID:26289599

  2. Intronic tandem repeat in the serotonin transporter gene in Old World monkeys: a new transcriptional regulator?

    PubMed

    Paredes, Ursula M; Bubb, Vivien J; Haddley, Kate; Macho, Gabriele A; Quinn, John P

    2012-06-01

    The serotonin transporter gene (SLC6A4) is heavily involved in the regulation of social behaviour of primates. Old World monkeys (e.g. macaques, baboons) have been used to study interactions between variation in the SLC6A4 gene and behaviour. Correlations of variation at one polymorphism located in the promoter region (known as 5HTTLPR) and variation at SLC6A4 expression levels, serotonin turnover and behaviour has been widely studied. In Old World monkeys, the third intron of the SLC6A4 gene also presents a tandem repeat, which sequence varies across species by a few point substitutions. We predict that in these species, this repeated region also acts as transcriptional regulatory domain and that sequence variation at this polymorphic locus might result in differential levels of expression in gene-environment interactions. For testing these hypotheses, the tandem repeat of Mandrillus sphinx and Cercopithecus aethiops from the third intron were cloned into a reporter gene vector and delivered to either primary cultures of rat neonate frontal cortex or the human cell line (JAr) to analyse their transcriptional activities. These repeated sequences supported significantly different levels of gene expression only when delivered into frontal cortex cultures. Furthermore, we tested in silico if such substitutions could have an effect on their binding profile to RNA- and DNA-binding proteins and on splicing. Taken together our results suggest that the tandem repeat in the third intron of the SLC6A4 gene of Old World monkeys could constitute a second transcriptional regulator as suggested for the 5HTTLPR and therefore contribute to diversification of serotonin-related behaviour in these primates.

  3. Roles of auxin transport and action in the gravity-regulated morphogenesis of cucumber seedlings

    NASA Astrophysics Data System (ADS)

    Saito, Y.; Shimizu, M.; Hotta, T.; Dai-Hee, K.; Yanai, K.; Kamada, M.; Fujii, N.; Miyazawa, Y.; Takahashi, H.

    Cucumber Cucumis sativus L seedlings develop a specialized protuberance peg on the lower side of the transition zone between the hypocotyl and the root when seeds germinate in a horizontal position The peg plays an important role in pulling seedling out from the seed coat We have reported that cucumber seedlings potentially develop a peg on each side of the transition zone but peg development on the upper side is suppressed in response to gravity Auxin is the primary factor responsible for the induction or the suppression of peg formation Here we investigated the roles of auxin transport and action in the gravity-regulated formation suppression of the peg in cucumber seedlings When cucumber seedlings were treated with inhibitors of auxin efflux carrier a peg was formed not only on the lower side but also on the upper side of the gravistimulated transition zone suggesting that activation of auxin efflux carriers is required for the suppression of peg formation To identify auxin efflux carriers involved in the suppression of peg formation by graviresponse we isolated six cucumber cDNAs of PIN auxin efflux carrier genes and investigated their mRNA accumulation and protein expression Our results show that CsPIN1 and CsPIN6 could play a role in the redistribution of auxin in the transition zone To understand auxin action on peg formation suppression we next examined the transcriptional regulators for the expressions of auxin-responsive genes The results suggest that a higher level of auxin in the lower side of the

  4. Biosynthesis and PBAN-regulated transport of pheromone polyenes in the winter moth, Operophtera brumata.

    PubMed

    Wang, Hong-Lei; Zhao, Cheng-Hua; Szöcs, Gabor; Chinta, Satya Prabhakar; Schulz, Stefan; Löfstedt, Christer

    2013-06-01

    The trienoic and tetraenoic polyenes, (3Z,6Z,9Z)-3,6,9-nonadecatriene, (3Z,6Z,9Z)-3,6,9-henicosatriene, and (3Z,6Z,9Z)-1,3,6,9-henicosatetraene were found in the abdominal cuticle and pheromone gland of the winter moth Operophtera brumata L. (Lepidoptera: Geometridae), in addition to the previously identified single component sex pheromone (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene. The pheromone biosynthesis activating neuropeptide (PBAN) is involved in the regulation of polyene transport from abdominal cuticle to the pheromone gland. In vivo deuterium labeling experiments showed that (11Z,14Z,17Z)-11,14,17-icosatrienoic acid, the malonate elongation product of linolenic acid, (9Z,12Z,15Z)-9,12,15-octadecatrienoic acid, is used to produce (3Z,6Z,9Z)-3,6,9-nonadecatriene and (3Z,6Z,9Z)-1,3,6,9-nonadecatetraene.

  5. Feedback Regulation of Glucose Transporter Gene Transcription in Kluyveromyces lactis by Glucose Uptake

    PubMed Central

    Milkowski, C.; Krampe, S.; Weirich, J.; Hasse, V.; Boles, E.; Breunig, K. D.

    2001-01-01

    In the respirofermentative yeast Kluyveromyces lactis, only a single genetic locus encodes glucose transporters that can support fermentative growth. This locus is polymorphic in wild-type isolates carrying either KHT1 and KHT2, two tandemly arranged HXT-like genes, or RAG1, a low-affinity transporter gene that arose by recombination between KHT1 and KHT2. Here we show that KHT1 is a glucose-induced gene encoding a low-affinity transporter very similar to Rag1p. Kht2p has a lower Km (3.7 mM) and a more complex regulation. Transcription is high in the absence of glucose, further induced by low glucose concentrations, and repressed at higher glucose concentrations. The response of KHT1 and KHT2 gene regulation to high but not to low concentrations of glucose depends on glucose transport. The function of either Kht1p or Kht2p is sufficient to mediate the characteristic response to high glucose, which is impaired in a kht1 kht2 deletion mutant. Thus, the KHT genes are subject to mutual feedback regulation. Moreover, glucose repression of the endogenous β-galactosidase (LAC4) promoter and glucose induction of pyruvate decarboxylase were abolished in the kht1 kht2 mutant. These phenotypes could be partially restored by HXT gene family members from Saccharomyces cerevisiae. The results indicate that the specific responses to high but not to low glucose concentrations require a high rate of glucose uptake. PMID:11514503

  6. [Effect of nitrates on active transport of iodine].

    PubMed

    Szökeová, E; Tajtáková, M; Mirossay, L; Mojzis, J; Langer, P; Marcinová, E; Petrovicová, J; Zemberová, E; Bodnár, J

    2001-11-01

    Active iodine transport into the thyrocyte is catalyzed by the transmembrane transport protein Na+/J- symport (NIS) Nitrates can expel iodine from the bond with this transport protein which was found not only in the thyrocyte membrane but also in the cell membrane of the gastric mucosa. The weight of the thyroid gland in mg was significantly greater even when calculated in relation to body weight in the NIT group of rats who were given for 6 days nitrate by gastric tube (100 mg/kg/day) as compared with controls (CON) 17.56 +/- 8.4, 0.07 +/- 0.03/12.10 +/- 9.57, 0.05 +/- 0.03, P < or = 0.01. A lower thyroid activity in per cent calculated per 1 mg of its weight (1.39 +/- 1.0/2.22 +/- 0.9, P < or = 0.01), a higher activity in blood before removal of the thyroid gland (8.54 +/- 4.09/5.45 +/- 2.78) and a lower one after removal of the thyroid gland (1.09 +/- 0.05/0.21 +/- 0.10) before oral administration of I131 in group NIT, suggests a negative effect of nitrates on active iodine transport not only at the level of the thyrocyte but also possible interaction with iodine at the level of the digestive tract. A significantly higher serum level of TT3 in group NIT (0.66 +/- 0.27/0.44 +/- 0.21, P < or = 0.01 regardless of the TSH serum level (2.31 +/- 1.83/2.64 +/- 1.52) and T4 (22.72 +/- 8.2/25 +/- 11.0) suggests a qualitative change in thyroid hormone production in favour of T3 caused even by short-term nitrate administration.

  7. The Asymmetric Active Coupler: Stable Nonlinear Supermodes and Directed Transport

    PubMed Central

    Kominis, Yannis; Bountis, Tassos; Flach, Sergej

    2016-01-01

    We consider the asymmetric active coupler (AAC) consisting of two coupled dissimilar waveguides with gain and loss. We show that under generic conditions, not restricted by parity-time symmetry, there exist finite-power, constant-intensity nonlinear supermodes (NS), resulting from the balance between gain, loss, nonlinearity, coupling and dissimilarity. The system is shown to possess non-reciprocal dynamics enabling directed power transport functionality. PMID:27640818

  8. 77 FR 71430 - New Agency Information Collection Activity Under OMB Review: Public Transportation Baseline...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-30

    ... SECURITY Transportation Security Administration New Agency Information Collection Activity Under OMB Review: Public Transportation Baseline Assessment for Security Enhancement (BASE) Program AGENCY: Transportation Security Administration, DHS. ACTION: 30-day notice. SUMMARY: This notice announces that the...

  9. Regulation of Parkin E3 ubiquitin ligase activity.

    PubMed

    Walden, Helen; Martinez-Torres, R Julio

    2012-09-01

    Parkin is an E3 ubiquitin ligase mutated in autosomal recessive juvenile Parkinson's disease. In addition, it is a putative tumour suppressor, and has roles outside its enzymatic activity. It is critical for mitochondrial clearance through mitophagy, and is an essential protein in most eukaryotes. As such, it is a tightly controlled protein, regulated through an array of external interactions with multiple proteins, posttranslational modifications including phosphorylation and S-nitrosylation, and self-regulation through internal associations. In this review, we highlight some of the recent studies into Parkin regulation and discuss future challenges for gaining a full molecular understanding of the regulation of Parkin E3 ligase activity.

  10. Maltose transport in Lactobacillus casei and its regulation by inducer exclusion.

    PubMed

    Monedero, Vicente; Yebra, María Jesús; Poncet, Sandrine; Deutscher, Josef

    2008-03-01

    Transport of maltose in Lactobacillus casei BL23 is subject to regulation by inducer exclusion. The presence of glucose or other rapidly metabolized carbon sources blocks maltose transport by a control mechanism that depends on the phosphorylation of the HPr protein at serine residue 46. We have identified the L. casei gene cluster for maltose/maltodextrin utilization by sequence analysis and mutagenesis. It is composed of genes coding for a transcriptional regulator, oligosaccharide hydrolytic enzymes, an ABC transporter (MalEFGK2) and the enzymes for the metabolism of maltose or the degradation products of maltodextrins: maltose phosphorylase and beta-phospho-glucomutase. These genes are induced by maltose and repressed by the presence of glucose via the catabolite control protein A (CcpA). A mutant strain was constructed which expressed the hprKV267F allele and therefore formed large amounts of P-Ser-HPr even in the absence of a repressive carbon source. In this mutant, transport of maltose was severely impaired, whereas transport of sugars not subject to inducer exclusion was not changed. These results strengthen the idea that P-Ser-HPr controls inducer exclusion and make the maltose system of L. casei a suitable model for studying this process in Firmicutes.

  11. Regulation of vitamin C transporter in the type 1 diabetic mouse bone and bone marrow.

    PubMed

    Sangani, Rajnikumar; Naime, Mohammad; Zakhary, Ibrahim; Ahmad, Saif; Chutkan, Norman; Zhu, Andy; Ha, Yonju; Hamrick, Mark; Isales, Carlos; Elsalanty, Mohammed; Smith, Sylvia; Liou, Gregory I; Fulzele, Sadanand

    2013-12-01

    A number of studies have revealed that Type I diabetes (T1D) is associated with bone loss and an increased risk of fractures. T1D induces oxidative stress in various tissues and organs. Vitamin C plays an important role in the attenuation of oxidative stress; however, little is known about the effect of T1D induced oxidative stress on the regulation of vitamin C transporter in bone and bone marrow cells. To investigate this, T1D was induced in mice by multiple low dose injections of streptozotocin. We have demonstrated that endogenous antioxidants, glutathione peroxidase (GPx) and superoxide dismutase (SOD) are down-regulated in the bone and bone marrow of T1D. The vitamin C transporter isoform SVCT2, the only known transporter expressed in bone and bone marrow stromal cells (BMSCs), is negatively regulated in the bone and bone marrow of T1D. The μCT imaging of the bone showed significantly lower bone quality in the 8 week T1D mouse. The in-vitro study in BMSCS showed that the knockdown of SVCT2 transporter decreases ascorbic acid (AA) uptake, and increases oxidative stress. The significant reversing effect of antioxidant vitamin C is only possible in control cells, not in knockdown cells. This study suggested that T1D induces oxidative stress and decreases SVCT2 expression in the bone and bone marrow environment. Furthermore, this study confirms that T1D increases bone resorption, decreases bone formation and changes the microstructure of bones. This study has provided evidence that the regulation of the SVCT2 transporter plays an important role not only in T1D osteoporosis but also in other oxidative stress-related musculoskeletal complications.

  12. Effect of multi-ions on active flow regulation in plants

    NASA Astrophysics Data System (ADS)

    Ryu, Jeongeun; Ahn, Sungsook; Kim, Seung-Gon; Oh, Hwasuk; Kim, Taejoo; Lee, Sang Joon

    2012-11-01

    Plants have been known to regulate ion-mediated flows actively in xylem vessels. Pits, the porous structures in xylem vessels, are presumed to play a key role in the ion-mediated flow regulation based on dynamic swelling and shrinking of their pectic matrix. However, the autonomous flow regulation in plants has not been elucidated yet and the pectin-swelling hypothesis seems to be simply applied to account for dynamic modulation of xylem conductance. In this study, the effects of multiple ions and their concentration on the water transport in plants were experimentally investigated. In addition, the active regulation mechanism of xylem water flow was also examined with considering the ionic effect.

  13. Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana

    PubMed Central

    Negi, Sangeeta; Ivanchenko, Maria G; Muday, Gloria K

    2008-01-01

    Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5, enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation. PMID:18363780

  14. Summary report on transportation of nuclear fuel materials in Japan : transportation infrastructure, threats identified in open literature, and physical protection regulations.

    SciTech Connect

    Cochran, John Russell; Ouchi, Yuichiro; Furaus, James Phillip; Marincel, Michelle K.

    2008-03-01

    This report summarizes the results of three detailed studies of the physical protection systems for the protection of nuclear materials transport in Japan, with an emphasis on the transportation of mixed oxide fuel materials1. The Japanese infrastructure for transporting nuclear fuel materials is addressed in the first section. The second section of this report presents a summary of baseline data from the open literature on the threats of sabotage and theft during the transport of nuclear fuel materials in Japan. The third section summarizes a review of current International Atomic Energy Agency, Japanese and United States guidelines and regulations concerning the physical protection for the transportation of nuclear fuel materials.

  15. Non-ionic diffusion and carrier-mediated transport drive extracellullar pH regulation of mouse colonic crypts.

    PubMed Central

    Chu, S; Montrose, M H

    1996-01-01

    1. Extracellular pH (pHo) regulation within mouse colonic crypt lumens is stimulated by transepithelial gradients of short-chain fatty acids (SCFAs). Current work assesses underlying mechanisms contributing to pHo regulation. 2. Crypt luminal alkalinization was saturable by apical SCFA (substrate concentration activating half-maximal transport (KT) of isobutyrate = 45 mM). However, saturation was consistent with either carrier-mediated SCFA flux or non-ionic diffusion, because the non-ionized form was titrated by luminal alkalinization. Direct acidification of apical perfusates increased the magnitude of SCFA-induced luminal alkalinization, roughly in the same proportion to the increased concentration of non-ionized SCFA in the crypt lumen. 3. Transepithelial gradients of an alternative weak acid (CO2) produce pHo changes similar to SCFA. In contrast, a weak base (NH3) changes pHo with reverse dependence on the orientation of the transepithelial gradient compared with SCFA. Results implicate non-ionic diffusion in pHo regulation, and suggest that pHo changes may underly SCFA-stimulated bicarbonate secretion and ammonium absorption. 4. SCFA metabolism plays a minor role in extracellular pH regulation. An avidly metabolized SCFA (N-butyrate) augments crypt luminal alkalinization only slightly (0.08 pH units) versus a poorly metabolized SCFA (isobutyrate). 5. Apical addition of 1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) partially inhibits luminal alkalinization caused by apical SCFA. DIDS has no effect on luminal alkalinization caused by transepithelial CO2 gradients. Probenecid (1 mM), alpha-cyano-4-hydroxycinnamic acid (4 mM) or basolateral DIDS (1 mM) do not affect pHo regulation. Results suggest that DIDS-sensitive, SCFA-dependent transport in the colonocyte apical membrane contributes to pHo regulation. Images Figure 7 PMID:8865074

  16. Molecular Theory for Electrokinetic Transport in pH-Regulated Nanochannels.

    PubMed

    Kong, Xian; Jiang, Jian; Lu, Diannan; Liu, Zheng; Wu, Jianzhong

    2014-09-04

    Ion transport through nanochannels depends on various external driving forces as well as the structural and hydrodynamic inhomogeneity of the confined fluid inside of the pore. Conventional models of electrokinetic transport neglect the discrete nature of ionic species and electrostatic correlations important at the boundary and often lead to inconsistent predictions of the surface potential and the surface charge density. Here, we demonstrate that the electrokinetic phenomena can be successfully described by the classical density functional theory in conjunction with the Navier-Stokes equation for the fluid flow. The new theoretical procedure predicts ion conductivity in various pH-regulated nanochannels under different driving forces, in excellent agreement with experimental data.

  17. Sudden clearing of estuarine waters upon crossing the threshold from transport to supply regulation of sediment transport as an erodible sediment pool is depleted: San Francisco Bay, 1999

    USGS Publications Warehouse

    Schoellhamer, David H.

    2011-01-01

    The quantity of suspended sediment in an estuary is regulated either by transport, where energy or time needed to suspend sediment is limiting, or by supply, where the quantity of erodible sediment is limiting. This paper presents a hypothesis that suspended-sediment concentration (SSC) in estuaries can suddenly decrease when the threshold from transport to supply regulation is crossed as an erodible sediment pool is depleted. This study was motivated by a statistically significant 36% step decrease in SSC in San Francisco Bay from water years 1991–1998 to 1999–2007. A quantitative conceptual model of an estuary with an erodible sediment pool and transport or supply regulation of sediment transport is developed. Model results confirm that, if the regulation threshold was crossed in 1999, SSC would decrease rapidly after water year 1999 as observed. Estuaries with a similar history of a depositional sediment pulse followed by erosion may experience sudden clearing.

  18. Regulation of Drug Disposition Gene Expression in Pregnant Mice with Car Receptor Activation

    PubMed Central

    Bright, Amanda S.; Herrera-Garcia, Guadalupe; Moscovitz, Jamie E.; You, Dahea; Guo, Grace L.; Aleksunes, Lauren M.

    2016-01-01

    More than half of pregnant women use prescription medications in order to maintain both maternal and fetal health. The constitutive androstane receptor (Car) critically affects the disposition of chemicals by regulating the transcription of genes encoding metabolic enzymes and transporters. However, the effects of Car activation on chemical disposition during pregnancy are unclear. This study aims to determine the degree to which pregnancy alters the expression of drug metabolizing enzymes and transporters in response to the pharmacological activation of Car. To test this, pregnant C57BL/6 mice were administered IP doses of vehicle, or a potent Car agonist, TCPOBOP, on gestation days 14, 15 and 16. Hepatic mRNA and protein expression of Car target genes (phase I, II and transporters) were quantified on gestation day 17. Pregnancy-related changes, such as induction of Cyp2b10, Ugt1a1 and Sult1a1 and repression of Ugt1a6, Gsta1, Gsta2 and Mrp6, were observed. Interestingly, the induction of Cyp2b10, Gsta1, Gsta2 and Mrp2-4 mRNAs by TCPOBOP was attenuated in maternal livers suggesting that Car activation is impeded by the biochemical and/or physiological changes that occur during gestation. Taken together, these findings suggest that pregnancy and pharmacological activation of Car can differentially regulate the expression of drug metabolism and transport genes. PMID:27818994

  19. The TORC1 effector kinase Npr1 fine tunes the inherent activity of the Mep2 ammonium transport protein.

    PubMed

    Boeckstaens, Mélanie; Llinares, Elisa; Van Vooren, Pascale; Marini, Anna Maria

    2014-01-01

    The TORC1 complex controls cell growth upon integrating nutritional signals including amino-acid availability. TORC1 notably adapts the plasma membrane protein content by regulating arrestin-mediated endocytosis of amino-acid transporters. Here we demonstrate that TORC1 further fine tunes the inherent activity of the ammonium transport protein, Mep2, a yeast homologue of mammalian Rhesus factors, independently of arrestin-mediated endocytosis. The TORC1 effector kinase Npr1 and the upstream TORC1 regulator Npr2 control Mep2 transport activity by phospho-silencing a carboxy-terminal autoinhibitory domain. Under poor nitrogen supply, Npr1 enables Mep2 S457 phosphorylation and thus ammonium transport activity. Supplementation of the preferred nitrogen source glutamine leads to Mep2 inactivation and instant S457 dephosphorylation via plasma membrane Psr1 and Psr2 redundant phosphatases. This study underscores that TORC1 also adjusts nutrient permeability to regulate cell growth in a fast and flexible response to environmental perturbation, establishing a hierarchy in the transporters to be degraded, inactivated or maintained active at the plasma membrane.

  20. Kinesin-Binding Protein Controls Microtubule Dynamics and Cargo Trafficking by Regulating Kinesin Motor Activity.

    PubMed

    Kevenaar, Josta T; Bianchi, Sarah; van Spronsen, Myrrhe; Olieric, Natacha; Lipka, Joanna; Frias, Cátia P; Mikhaylova, Marina; Harterink, Martin; Keijzer, Nanda; Wulf, Phebe S; Hilbert, Manuel; Kapitein, Lukas C; de Graaff, Esther; Ahkmanova, Anna; Steinmetz, Michel O; Hoogenraad, Casper C

    2016-04-04

    Kinesin motor proteins play a fundamental role for normal neuronal development by controlling intracellular cargo transport and microtubule (MT) cytoskeleton organization. Regulating kinesin activity is important to ensure their proper functioning, and their misregulation often leads to severe human neurological disorders. Homozygous nonsense mutations in kinesin-binding protein (KBP)/KIAA1279 cause the neurological disorder Goldberg-Shprintzen syndrome (GOSHS), which is characterized by intellectual disability, microcephaly, and axonal neuropathy. Here, we show that KBP regulates kinesin activity by interacting with the motor domains of a specific subset of kinesins to prevent their association with the MT cytoskeleton. The KBP-interacting kinesins include cargo-transporting motors such as kinesin-3/KIF1A and MT-depolymerizing motor kinesin-8/KIF18A. We found that KBP blocks KIF1A/UNC-104-mediated synaptic vesicle transport in cultured hippocampal neurons and in C. elegans PVD sensory neurons. In contrast, depletion of KBP results in the accumulation of KIF1A motors and synaptic vesicles in the axonal growth cone. We also show that KBP regulates neuronal MT dynamics by controlling KIF18A activity. Our data suggest that KBP functions as a kinesin inhibitor that modulates MT-based cargo motility and depolymerizing activity of a subset of kinesin motors. We propose that misregulation of KBP-controlled kinesin motors may represent the underlying molecular mechanism that contributes to the neuropathological defects observed in GOSHS patients.

  1. A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

    PubMed

    Chen, Lin; Duan, Kangmin

    2016-05-01

    Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell.

  2. Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

    SciTech Connect

    Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting

    2014-12-12

    Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

  3. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

    PubMed

    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

  4. Role of reactive oxygen species in regulation of glucose transport in skeletal muscle during exercise.

    PubMed

    Katz, Abram

    2016-06-01

    Glucose derived from extracellular sources serves as an energy source in virtually all eukaryotic cells, including skeletal muscle. Its contribution to energy turnover increases with exercise intensity up to moderately heavy workloads. However, at very high workloads, the contribution of extracellular glucose to energy turnover is negligible, despite the high rate of glucose transport. Reactive oxygen species (ROS) are involved in the stimulation of glucose transport in isolated skeletal muscle preparations during intense repeated contractions. Consistent with this observation, heavy exercise is associated with significant production of ROS. However, during more mild to moderate stimulation or exercise conditions (in vitro, in situ and in vivo) antioxidants do not affect glucose transport. It is noteworthy that the production of ROS is limited or not observed under these conditions and that the concentration of the antioxidant used was extremely low. The results to date suggest that ROS involvement in activation of glucose transport occurs primarily during intense short-term exercise and that other mechanisms are involved during mild to moderate exercise. What remains puzzling is why ROS-mediated activation of glucose transport would occur under conditions where glucose transport is highest and utilization (i.e. phosphorylation of glucose by hexokinase) is low. Possibly ROS production is involved in priming glucose transport during heavy exercise to accelerate glycogen biogenesis during the initial recovery period after exercise, as well as altering other aspects of intracellular metabolism.

  5. Cysteine cathepsin activity regulation by glycosaminoglycans.

    PubMed

    Novinec, Marko; Lenarčič, Brigita; Turk, Boris

    2014-01-01

    Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail.

  6. Examining Changes in Radioxenon Isotope Activity Ratios during Subsurface Transport

    NASA Astrophysics Data System (ADS)

    Annewandter, Robert

    2014-05-01

    The Non-Proliferation Experiment (NPE) has demonstrated and modelled the usefulness of barometric pumping induced gas transport and subsequent soil gas sampling during On-Site inspections. Generally, gas transport has been widely studied with different numerical codes. However, gas transport of radioxenons and radioiodines in the post-detonation regime and their possible fractionation is still neglected in the open peer-reviewed literature. Atmospheric concentrations of the radioxenons Xe-135, Xe-133m, Xe-133 and Xe-131m can be used to discriminate between civilian releases (nuclear power plants or medical isotope facilities), and nuclear explosion sources. It is based on the multiple isotopic activity ratio method. Yet it is not clear whether subsurface migration of the radionuclides, with eventual release into the atmosphere, can affect the activity ratios due to fractionation. Fractionation can be caused by different mass diffusivities due to mass differences between the radionuclides. Cyclical changes in atmospheric pressure can drive subsurface gas transport. This barometric pumping phenomenon causes an oscillatoric flow in upward trending fractures or highly conductive faults which, combined with diffusion into the porous matrix, leads to a net transport of gaseous components - a so-called ratcheting effect. We use a general purpose reservoir simulator (Complex System Modelling Platform, CSMP++) which is recognized by the oil industry as leading in Discrete Fracture-Matrix (DFM) simulations. It has been applied in a range of fields such as deep geothermal systems, three-phase black oil simulations, fracture propagation in fractured, porous media, and Navier-Stokes pore-scale modelling among others. It is specifically designed to account for structurally complex geologic situation of fractured, porous media. Parabolic differential equations are solved by a continuous Galerkin finite-element method, hyperbolic differential equations by a complementary finite

  7. Regulation of myostatin activity and muscle growth.

    PubMed

    Lee, S J; McPherron, A C

    2001-07-31

    Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may have applications for promoting muscle growth, we investigated the regulation of myostatin signaling. Myostatin protein purified from mammalian cells consisted of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal myostatin dimer was capable of binding the activin type II receptors, Act RIIB and, to a lesser extent, Act RIIA. Binding of myostatin to Act RIIB could be inhibited by the activin-binding protein follistatin and, at higher concentrations, by the myostatin propeptide. To determine the functional significance of these interactions in vivo, we generated transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB by using a skeletal muscle-specific promoter. Independent transgenic mouse lines for each construct exhibited dramatic increases in muscle mass comparable to those seen in myostatin knockout mice. Our findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful agents for enhancing muscle growth for both human therapeutic and agricultural applications.

  8. Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

    PubMed

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V; Bevan, Michael W

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

  9. Differences in associations between active transportation and built environmental exposures when expressed using different components of individual activity spaces.

    PubMed

    van Heeswijck, Torbjorn; Paquet, Catherine; Kestens, Yan; Thierry, Benoit; Morency, Catherine; Daniel, Mark

    2015-05-01

    This study assessed relationships between built environmental exposures measured within components of individual activity spaces (i.e., travel origins, destinations and paths in-between), and use of active transportation in a metropolitan setting. Individuals (n=37,165) were categorised as using active or sedentary transportation based on travel survey data. Generalised Estimating Equations analysis was used to test relationships with active transportation. Strength and significance of relationships between exposures and active transportation varied for different components of the activity space. Associations were strongest when including travel paths in expression of the built environment. Land use mix and greenness were negatively related to active transportation.

  10. Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation.

    PubMed

    Kessler, E C; Gross, J J; Bruckmaier, R M; Albrecht, C

    2014-09-01

    The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and

  11. Diversity and activity of sugar transporters in nematode-induced root syncytia

    PubMed Central

    Hofmann, Julia; Hess, Paul H.; Szakasits, Dagmar; Blöchl, Andreas; Wieczorek, Krzysztof; Daxböck-Horvath, Sabine; Bohlmann, Holger; van Bel, Aart J. E.; Grundler, Florian M. W.

    2009-01-01

    The plant-parasitic nematode Heterodera schachtii stimulates plant root cells to form syncytial feeding structures which synthesize all nutrients required for successful nematode development. Cellular re-arrangements and modified metabolism of the syncytia are accompanied by massive intra- and intercellular solute allocations. In this study the expression of all genes annotated as sugar transporters in the Arabidopsis Membrane Protein Library was investigated by Affymetrix gene chip analysis in young and fully developed syncytia compared with non-infected Arabidopsis thaliana roots. The expression of three highly up-regulated (STP12, MEX1, and GTP2) and three highly down-regulated genes (SFP1, STP7, and STP4) was analysed by quantitative RT-PCR (qRT-PCR). The most up-regulated gene (STP12) was chosen for further in-depth studies using in situ RT-PCR and a nematode development assay with a T-DNA insertion line revealing a significant reduction of male nematode development. The specific role of STP12 expression in syncytia of male juveniles compared with those of female juveniles was further shown by qRT-PCR. In order to provide evidence for sugar transporter activity across the plasma membrane of syncytia, fluorescence-labelled glucose was used and membrane potential recordings following the application of several sugars were performed. Analyses of soluble sugar pools revealed a highly specific composition in syncytia. The presented work demonstrates that sugar transporters are specifically expressed and active in syncytia, indicating a profound role in inter- and intracelluar transport processes. PMID:19487386

  12. Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation

    PubMed Central

    Vitreschak, Alexey G.; Rodionov, Dmitry A.; Mironov, Andrey A.; Gelfand, Mikhail S.

    2002-01-01

    The riboflavin biosynthesis in bacteria was analyzed using comparative analysis of genes, operons and regulatory elements. A model for regulation based on formation of alternative RNA structures involving the RFN elements is suggested. In Gram-positive bacteria including actinomycetes, Thermotoga, Thermus and Deinococcus, the riboflavin metabolism and transport genes are predicted to be regulated by transcriptional attenuation, whereas in most Gram-negative bacteria, the riboflavin biosynthesis genes seem to be regulated on the level of translation initiation. Several new candidate riboflavin transporters were identified (impX in Desulfitobacterium halfniense and Fusobacterium nucleatum; pnuX in several actinomycetes, including some Corynebacterium species and Strepto myces coelicolor; rfnT in Rhizobiaceae). Traces of a number of likely horizontal transfer events were found: the complete riboflavin operon with the upstream regulatory element was transferred to Haemophilus influenzae and Actinobacillus pleuropneumoniae from some Gram-positive bacterium; non-regulated riboflavin operon in Pyrococcus furiousus was likely transferred from Thermotoga; and the RFN element was inserted into the riboflavin operon of Pseudomonas aeruginosa from some other Pseudomonas species, where it had regulated the ribH2 gene. PMID:12136096

  13. Simulation of Cl− Secretion in Epithelial Tissues: New Methodology Estimating Activity of Electro-Neutral Cl− Transporter

    PubMed Central

    Sasamoto, Kouhei; Niisato, Naomi; Taruno, Akiyuki; Marunaka, Yoshinori

    2015-01-01

    Transcellular Cl− secretion is, in general, mediated by two steps; (1) the entry step of Cl− into the cytosolic space from the basolateral space across the basolateral membrane by Cl− transporters, such as Na+-K+-2Cl− cotransporter (NKCC1, an isoform of NKCC), and (2) the releasing step of Cl− from the cytosolic space into the luminal (air) space across the apical membrane via Cl− channels, such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Transcellular Cl− secretion has been characterized by using various experimental techniques. For example, measurements of short-circuit currents in the Ussing chamber and patch clamp techniques provide us information on transepithelial ion movements via transcellular pathway, transepithelial conductance, activity (open probability) of single channel, and whole cell currents. Although many investigators have tried to clarify roles of Cl− channels and transporters located at the apical and basolateral membranes in transcellular Cl− secretion, it is still unclear how Cl− channels/transporters contribute to transcellular Cl− secretion and are regulated by various stimuli such as Ca2+ and cAMP. In the present study, we simulate transcellular Cl− secretion using mathematical models combined with electrophysiological measurements, providing information on contribution of Cl− channels/transporters to transcellular Cl− secretion, activity of electro-neutral ion transporters and how Cl− channels/transporters are regulated. PMID:26779025

  14. Active and passive transport of drugs in the human placenta.

    PubMed

    Włoch, Stanisław; Pałasz, Artur; Kamiński, Marcin

    2009-10-01

    The human placenta, characterized by the processes of passive transport and facilitated diffusion, contains numerous active transport proteins, usually located in the microvilli of the syncytiotrophoblast or in the endothelium of the capillaries of the villi. These proteins use either the energy from ATP hydrolysis or other mechanisms resulting, among others, from the formation of the maternofetal ion gradient, which facilitates the transfer of various endogenous substances or xenobiotics across the body membranes. The proteins either trigger the efflux of these substances from the fetal tissues via the placenta into the maternal bloodstream, or conversely they accumulate them in the fetal tissues. Both the placenta and the fetus are equipped with independent systems of enzymes of 1st and 2nd phase of substrate metabolism, such as CYP450, glucuronyltransferase or sulphatase. An active therapy with a wide range of drugs, often at high toxicity levels, either shortly before or during pregnancy, has naturally posed a question concerning the degree of impermeability of the placental barrier and how effectively it can be crossed, including any possible negative embryotoxic or teratogenic consequences. Such hazards seem to be quite real, as many drugs are substrates for ABC transporters. Also the placenta itself, including its structure, is subject to vast transformations during pregnancy which may be observed as the thinning of the barrier separating the maternal blood from the fetal one, from 20-30 microm in the first trimester of gestation down to 2-4 microm in the third trimester of gestation.

  15. Dietary Methanol Regulates Human Gene Activity

    PubMed Central

    Komarova, Tatiana V.; Sheshukova, Ekaterina V.; Kosorukov, Vyacheslav S.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

    2014-01-01

    Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH to formaldehyde (FA), which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling. PMID:25033451

  16. 15 CFR 922.102 - Prohibited or otherwise regulated activities.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 15 Commerce and Foreign Trade 3 2012-01-01 2012-01-01 false Prohibited or otherwise regulated activities. 922.102 Section 922.102 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE OCEAN...

  17. Regulation of MDM2 Activity by Nucleolin

    DTIC Science & Technology

    2007-06-01

    p53 (1 ml produced in a wheat germ transcription-coupled in vitro translation system (Pro- mega, Madison, WI, USA)), GST-Mdm2 (400 ng) and 10mg...interaction of the p53 Box V region with the acid domain of Hdm2, activating the Hdm2 ubiquitin ligase activity towards p53. Nucleolin contains three...is located in the N-terminal half of nucleolin, adjacent to the 4th acidic domain. While not extensive, we will test these sequences in future

  18. [PLASMALEMMAL ION TRANSPORT IN POLLEN TUBES IS REGULATED BY HYDROGEN PEROXIDE].

    PubMed

    Maksimov, N M; Breygina, M A; Yermakov, I P

    2015-01-01

    Pollen tube growth is a key step in the life cycle of seed plants, which defines the success of sexual reproduction. One of the most important contributions to this process is made by ion transport through plasmalemma, which is tightly coordinated in time and space. Different classes of signaling molecules are involved in the regulation of transmembrane ion transport including reactive oxygen species as it has been recently demonstrated. Here, using subprotoplasts isolated from pollen tubes, we have demonstrated a connection between hydrogen peroxide, on one side, and two groups of targets on the plasma membrane, on the other side: nifedipine-sensitive Ca(2+)-permeable channels and transport systems controlling membrane potential. H2O2 interaction with these targets causes the increase in cytoplasmic Ca2+ concentration and plasmalemma hyperpolarization. One of the consequences of target modification was acceleration of cell wall regeneration.

  19. Influence of different host associations on glutamine synthetase activity and ammonium transporter in Santalumalbum L.

    PubMed

    Deepa, P; Yusuf, A

    2016-07-01

    The present study was aimed at understanding the role of different hosts in ammonium transporter1;2 expressions and glutamine synthetase(GS) activity and their effects on the growth parameters in the sandal. Sandal plant associated with leguminous host expressed better growth parameters. GS activity of leguminous hosts alone and in host associated sandals was analyzed using GS transferase assay. Highest GS activity was expressed in Mimosa pudica-sandal association compared to other leguminous and non-leguminous host associations. The association of N2 fixing host with sandal enhanced C and N levels in order to maintain the C/N value. The role of ammonium transporters in N nutrition of sandal-host association was elucidated by cloning AMT1;2 from the leaves, haustoria and roots of host associated sandal and quantifying the relative expression by the [Formula: see text] method. SaAMT1;2 was strongly up-regulated in leaves, roots and haustoria of leguminous host associated sandal compared to non-leguminous host associations. The relative increase in SaAMT1;2 expressions and up-regulated GS activity positively affected the growth parameters in sandal when associated with leguminous hosts.

  20. Regulation of hepatic EAAT-2 glutamate transporter expression in human liver cholestasis

    PubMed Central

    Najimi, Mustapha; Stéphenne, Xavier; Sempoux, Christine; Sokal, Etienne

    2014-01-01

    AIM: To investigate the activity and expression of EAAT2 glutamate transporter in both in vitro and in vivo models of cholestasis. METHODS: This study was conducted on human hepatoblastoma HepG2 cell cultures, the liver of bile duct ligated rats and human specimens from cholestatic patients. EAAT2 glutamate transporter activity and expression were analyzed using a substrate uptake assay, immunofluorescence, reverse transcription-polymerase chain reaction, and immunohistochemistry, respectively. RESULTS: In HepG2 cells, cholestasis was mimicked by treating cells with the protein kinase C activator, phorbol 12-myristate 13-acetate. Under such conditions, EAAT2 transporter activity was decreased both at the level of substrate affinity and maximal transport velocity. The decreased uptake was correlated with intracellular translocation of EAAT2 molecules as demonstrated using immunofluorescence. In the liver of bile duct ligated rats, an increase in EAAT2 transporter protein expression in hepatocytes was demonstrated using immunohistochemistry. The same findings were observed in human liver specimens of cholestasis in which high levels of γ-glutamyl transpeptidase were documented in patients with biliary atresia and progressive familial intrahepatic cholestasis type 3. CONCLUSION: This study demonstrates the alteration in glutamate handling by hepatocytes in liver cholestasis and suggests a potential cross-talk between glutamatergic and bile systems. PMID:24587631

  1. Curcumin directly inhibits the transport activity of GLUT1.

    PubMed

    Gunnink, Leesha K; Alabi, Ola D; Kuiper, Benjamin D; Gunnink, Stephen M; Schuiteman, Sam J; Strohbehn, Lauren E; Hamilton, Kathryn E; Wrobel, Kathryn E; Louters, Larry L

    2016-06-01

    Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin's inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin.

  2. Chloride transport in functionally active phagosomes isolated from Human neutrophils

    PubMed Central

    Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

    2012-01-01

    Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport

  3. Curcumin directly inhibits the transport activity of GLUT1

    PubMed Central

    Gunnink, Leesha K.; Alabi, Ola D.; Kuiper, Benjamin D.; Gunnink, Stephen M.; Schuiteman, Sam J.; Strohbehn, Lauren E.; Hamilton, Kathryn E.; Wrobel, Kathryn E.; Louters, Larry L.

    2016-01-01

    Curcumin, a major ingredient in turmeric, has a long history of medicinal applications in a wide array of maladies including treatment for diabetes and cancer. Seemingly counterintuitive to the documented hypoglycemic effects of curcumin, however, a recent report indicates that curcumin directly inhibits glucose uptake in adipocytes. The major glucose transporter in adipocytes is GLUT4. Therefore, this study investigates the effects of curcumin in cell lines where the major transporter is GLUT1. We report that curcumin has an immediate inhibitory effect on basal glucose uptake in L929 fibroblast cells with a maximum inhibition of 80% achieved at 75 μM curcumin. Curcumin also blocks activation of glucose uptake by azide, glucose deprivation, hydroxylamine, or phenylarsine oxide. Inhibition does not increase with exposure time and the inhibitory effects reverse within an hour. Inhibition does not appear to involve a reaction between curcumin and the thiol side chain of a cysteine residue since neither prior treatment of cells with iodoacetamide nor curcumin with cysteine alters curcumin’s inhibitory effects. Curcumin is a mixed inhibitor reducing the Vmax of 2DG transport by about half with little effect on the Km. The inhibitory effects of curcumin are not additive to the effects of cytochalasin B and 75 μM curcumin actually reduces specific cytochalasin B binding by 80%. Taken together, the data suggest that curcumin binds directly to GLUT1 at a site that overlaps with the cytochalasin B binding site and thereby inhibits glucose transport. A direct inhibition of GLUT proteins in intestinal epithelial cells would likely reduce absorption of dietary glucose and contribute to a hypoglycemic effect of curcumin. Also, inhibition of GLUT1 activity might compromise cancer cells that overexpress GLUT1 and be another possible mechanism for the documented anticancer effects of curcumin. PMID:27039889

  4. Evaluation of the physical activity biography: sport and transport.

    PubMed

    Rogen, Sandra; Hofmann, Peter; Bauernhofer, Thomas; Müller, Wolfram

    2014-05-01

    Beside the genetic disposition, physical activity (PA) is one of the major health factors and can play a large role in the prevention and therapy of many diseases (cardiovascular diseases, cancer, obesity-related diseases etc.). In contrast to the genetic disposition, PA can be deliberately influenced by lifestyle. Therefore, it is of high importance to assess PA patterns. In order to assess PA reliably and validly, a new questionnaire (Physical Activity Biography, PAB) was created. The PAB assesses recreational PA (sport and transport) and enables to distinguish between endurance intensity levels and considers strength and high speed activity patterns throughout life. This study aims to evaluate the PAB by means of item analysis, retest-reliability and validity (criteria were physical fitness assessed by the questionnaire FFB-mot and by exercise tests). 141 participants answered the PAB. For deriving retest-reliability, 81 participants completed the PAB after a retest-interval of one month again. 55 participated in exercise tests and answered the FFB-mot to determine construct validity. Retest-reliability (ICC) above 0.7 was found for most items. For the items assessing recent PA, the criteria of convergent and discriminant validity were given. Despite the complexity of the question under study, the results fulfilled the expectations concerning reliability and validity. The PAB enables to assess the amount of sport and locomotion a person has accomplished during different life time frames and, because of the protective effects of PA on various diseases, may become an important tool for risk assessment. Key pointsThe risk of chronic diseases depends largely on physical activity biography.A new questionnaire (PAB) assessing recent and lifetime physical activity was created.The PAB assesses physical activity during sports and transport.The results of the evaluation of the PAB fulfilled the expectations.The PAB enables to determine a person's amount of recreational

  5. Evaluation of the Physical Activity Biography: Sport and Transport

    PubMed Central

    Rogen, Sandra; Hofmann, Peter; Bauernhofer, Thomas; Müller, Wolfram

    2014-01-01

    Beside the genetic disposition, physical activity (PA) is one of the major health factors and can play a large role in the prevention and therapy of many diseases (cardiovascular diseases, cancer, obesity-related diseases etc.). In contrast to the genetic disposition, PA can be deliberately influenced by lifestyle. Therefore, it is of high importance to assess PA patterns. In order to assess PA reliably and validly, a new questionnaire (Physical Activity Biography, PAB) was created. The PAB assesses recreational PA (sport and transport) and enables to distinguish between endurance intensity levels and considers strength and high speed activity patterns throughout life. This study aims to evaluate the PAB by means of item analysis, retest-reliability and validity (criteria were physical fitness assessed by the questionnaire FFB-mot and by exercise tests). 141 participants answered the PAB. For deriving retest-reliability, 81 participants completed the PAB after a retest-interval of one month again. 55 participated in exercise tests and answered the FFB-mot to determine construct validity. Retest-reliability (ICC) above 0.7 was found for most items. For the items assessing recent PA,