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Sample records for acid transporters eaat-1

  1. Up-Regulation of the Excitatory Amino Acid Transporters EAAT1 and EAAT2 by Mammalian Target of Rapamycin

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    Abeer Abousaab

    2016-11-01

    Full Text Available Background: The excitatory amino-acid transporters EAAT1 and EAAT2 clear glutamate from the synaptic cleft and thus terminate neuronal excitation. The carriers are subject to regulation by various kinases. The EAAT3 isoform is regulated by mammalian target of rapamycin (mTOR. The present study thus explored whether mTOR influences transport by EAAT1 and/or EAAT2. Methods: cRNA encoding wild type EAAT1 (SLC1A3 or EAAT2 (SLC1A2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding mTOR. Dual electrode voltage clamp was performed in order to determine electrogenic glutamate transport (IEAAT. EAAT2 protein abundance was determined utilizing chemiluminescence. Results: Appreciable IEAAT was observed in EAAT1 or EAAT2 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of mTOR. Coexpression of mTOR increased significantly the maximal IEAAT in EAAT1 or EAAT2 expressing oocytes, without significantly modifying affinity of the carriers. Moreover, coexpression of mTOR increased significantly EAAT2 protein abundance in the cell membrane. Conclusions: The kinase mTOR up-regulates the excitatory amino acid transporters EAAT1 and EAAT2.

  2. Up-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by ß-Klotho

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    Jamshed Warsi

    2015-12-01

    Full Text Available Background/Aims: Klotho, a transmembrane protein expressed in chorioid plexus of the brain, kidney, and several other tissues, is required for inhibition of 1,25(OH2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. At least in part by exerting β-glucuronidase activity, soluble klotho regulates several ion channels and carriers. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The present study explored the effect of Klotho protein on the excitatory glutamate transporters EAAT1 (SLC1A3 and EAAT2 (SLC1A2, Na+ coupled carriers clearing excitatory amino acids from the synaptic cleft and thus participating in the regulation of neuronal excitability. Methods: cRNA encoding EAAT1 or EAAT2 was injected into Xenopus laevis oocytes and glutamate (2 mM-induced inward current (IGlu taken as measure of glutamate transport. Measurements were made without or with prior 24 h treatment with soluble ß-Klotho protein (30 ng/ml in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM. Results: IGlu was observed in EAAT1 and in EAAT2 expressing oocytes but not in water injected oocytes. In both, EAAT1 and EAAT2 expressing oocytes IGlu was significantly increased by treatment with soluble ß-Klotho protein, an effect reversed by DSAL. Treatment with ß-klotho protein increased significantly the maximal transport rate without significantly modifying the affinity of the carriers. Conclusion: ß-Klotho up-regulates the excitatory glutamate transporters EAAT1 and EAAT2 and thus participates in the regulation of neuronal excitation.

  3. Pharmacological characterization of human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 in a fluorescence-based membrane potential assay

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    Jensen, Anders A.; Bräuner-Osborne, Hans

    2004-01-01

    We have expressed the human excitatory amino acid transporters EAAT1, EAAT2 and EAAT3 stably in HEK293 cells and characterized the transporters pharmacologically in a conventional [(3) H]-d-aspartate uptake assay and in a fluorescence-based membrane potential assay, the FLIPR Membrane Potential...... (FMP) assay. The K(m) and K(i) values obtained for 12 standard EAAT ligands at EAAT1, EAAT2 and EAAT3 in the FMP assay correlated well with the K(i) values obtained in the [(3) H]-d-aspartate assay (r(2) values of 0.92, 0.92, and 0.95, respectively). Furthermore, the pharmacological characteristics...

  4. Excitatory amino acid transporters EAAT-1 and EAAT-2 in temporal lobe and hippocampus in intractable temporal lobe epilepsy

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    Sarac, Sinan; Afzal, Shoaib; Broholm, Helle

    2009-01-01

    Intractable temporal lobe epilepsy (TLE) is an invalidating disease and many patients are resistant to medical treatment. Increased glutamate concentration has been found in epileptogenic foci and may induce local over-excitation and cytotoxicity; one of the proposed mechanisms involves reduced...... extra-cellular clearance of glutamate by excitatory amino acid transporters (EAAT-1 to EAAT-5). EAAT-1 and EAAT-2 are mainly expressed on astroglial cells for the reuptake of glutamate from the extra-cellular space. We have studied the expression of EAAT-1 and EAAT-2 in the hippocampus and temporal lobe...

  5. Up-Regulation of Excitatory Amino Acid Transporters EAAT3 and EAAT4 by Lithium Sensitive Glycogen Synthase Kinase GSK3ß

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    Abeer Abousaab

    2016-12-01

    Full Text Available Background: Cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs decreases excitation and thus participates in the regulation of neuroexcitability. Kinases impacting on neuronal function include Lithium-sensitive glycogen synthase kinase GSK3ß. The present study thus explored whether the activities of EAAT3 and/or EAAT4 isoforms are sensitive to GSK3ß. Methods: cRNA encoding wild type EAAT3 (SLC1A1 or EAAT4 (SLC1A6 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild type GSK3ß or the inactive mutant K85AGSK3ß. Dual electrode voltage clamp was performed in order to determine glutamate-induced current (IEAAT. Results: Appreciable IEAAT was observed in EAAT3 or EAAT4 expressing but not in water injected oocytes. IEAAT was significantly increased by coexpression of GSK3ß but not by coexpression of K85AGSK3ß. Coexpression of GSK3ß increased significantly the maximal IEAAT in EAAT3 or EAAT4 expressing oocytes, without significantly modifying apparent affinity of the carriers. Lithium (1 mM exposure for 24 hours decreased IEAAT in EAAT3 and GSK3ß expressing oocytes to values similar to IEAAT in oocytes expressing EAAT3 alone. Lithium did not significantly modify IEAAT in oocytes expressing EAAT3 without GSK3ß. Conclusions: Lithium-sensitive GSK3ß is a powerful regulator of excitatory amino acid transporters EAAT3 and EAAT4.

  6. The importance of the excitatory amino acid transporter 3 (EAAT3)

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    E. Bjørn-Yoshimoto, Walden; Underhill, Suzanne M.

    2016-01-01

    Abstract The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post-synaptic localiza......Abstract The neuronal excitatory amino acid transporter 3 (EAAT3) is fairly ubiquitously expressed in the brain, though it does not necessarily maintain the same function everywhere. It is important in maintaining low local concentrations of glutamate, where its predominant post...

  7. A new structural class of subtype-selective inhibitor of cloned excitatory amino acid transporter, EAAT2

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    Bräuner-Osborne, Hans; Hermit, M B; Nielsen, B

    2000-01-01

    We have studied the pharmacological effects of (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and the enantiomers of (RS)-2-amino-3-(3-hydroxy-1,2, 5-thiadiazol-4-yl)propionic acid (TDPA) on cloned human excitatory amino acid transporter subtypes 1, 2 and 3 (EAAT1......-3) expressed in Cos-7 cells. Whereas AMPA and (R)-TDPA were both inactive as inhibitors of [3H]-(R)-aspartic acid uptake on all three EAAT subtypes, (S)-TDPA was shown to selectively inhibit uptake by EAAT2 with a potency equal to that of the endogenous ligand (S)-glutamic acid. (S)-TDPA thus represents a new...

  8. Differential expression of glutamate transporters EAAT1 and EAAT2 in mice deficient for PACAP-type I receptor.

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    Zink, M; Schmitt, A; Henn, F A; Gass, P

    2004-12-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) modulates glutamatergic neurotransmission and induces the expression of glutamate transporters EAAT1 and EAAT2 in newborn mouse astroglial cell cultures. Since nanomolar concentrations of PACAP exert this effect, signal transduction via the high affinity PACAP-type I-receptor PAC1 was assumed. To test this hypothesis and to assess the importance of PAC1-signalling in vivo, we analyzed glutamate transporter expression in mice with a PAC1 knockout. EAAT1 and EAAT2 expression was investigated in the hippocampus and the cerebral cortex of PAC1 mutant mice and wildtype littermates by semiquantitative in-situ-hybridization. PAC1-knockout mice show a subtle but significant reduction of EAAT1 expression in the dentate gyrus. In contrast, reduced expression levels of EAAT1 in the cerebral cortex did not reach statistical significance and EAAT2 expression was unchanged in CA3 and cerebral cortex of PAC1 mutant mice. Our data confirm the previously reported in-vitro-regulation of EAAT1 in the adult nervous system in vivo. EAAT2 expression, however, is unchanged in PAC1 knockout mice, most likely due to counterbalancing factors.

  9. The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings.

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    Suchak, Sachin K; Baloyianni, Nicoletta V; Perkinton, Michael S; Williams, Robert J; Meldrum, Brian S; Rattray, Marcus

    2003-02-01

    The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

  10. Molecular determinants of transport stimulation of EAAT2 are located at interface between the trimerization and substrate transport domains.

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    Mortensen, Ole V; Liberato, José L; Coutinho-Netto, Joaquim; Dos Santos, Wagner F; Fontana, Andréia C K

    2015-04-01

    Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications. © 2015 International Society for Neurochemistry.

  11. Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120

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    Wang Zhuying; Pekarskaya, Olga; Bencheikh, Meryem; Chao Wei; Gelbard, Harris A.; Ghorpade, Anuja; Rothstein, Jeffrey D.; Volsky, David J.

    2003-01-01

    L-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V max for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40-70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-α (TNF-α) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-α production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease

  12. OCD candidate gene SLC1A1/EAAT3 impacts basal ganglia-mediated activity and stereotypic behavior.

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    Zike, Isaac D; Chohan, Muhammad O; Kopelman, Jared M; Krasnow, Emily N; Flicker, Daniel; Nautiyal, Katherine M; Bubser, Michael; Kellendonk, Christoph; Jones, Carrie K; Stanwood, Gregg; Tanaka, Kenji Fransis; Moore, Holly; Ahmari, Susanne E; Veenstra-VanderWeele, Jeremy

    2017-05-30

    Obsessive-compulsive disorder (OCD) is a chronic, disabling condition with inadequate treatment options that leave most patients with substantial residual symptoms. Structural, neurochemical, and behavioral findings point to a significant role for basal ganglia circuits and for the glutamate system in OCD. Genetic linkage and association studies in OCD point to SLC1A1 , which encodes the neuronal glutamate/aspartate/cysteine transporter excitatory amino acid transporter 3 (EAAT3)/excitatory amino acid transporter 1 (EAAC1). However, no previous studies have investigated EAAT3 in basal ganglia circuits or in relation to OCD-related behavior. Here, we report a model of Slc1a1 loss based on an excisable STOP cassette that yields successful ablation of EAAT3 expression and function. Using amphetamine as a probe, we found that EAAT3 loss prevents expected increases in ( i ) locomotor activity, ( ii ) stereotypy, and ( iii ) immediate early gene induction in the dorsal striatum following amphetamine administration. Further, Slc1a1 -STOP mice showed diminished grooming in an SKF-38393 challenge experiment, a pharmacologic model of OCD-like grooming behavior. This reduced grooming is accompanied by reduced dopamine D 1 receptor binding in the dorsal striatum of Slc1a1 -STOP mice. Slc1a1 -STOP mice also exhibit reduced extracellular dopamine concentrations in the dorsal striatum both at baseline and following amphetamine challenge. Viral-mediated restoration of Slc1a1 /EAAT3 expression in the midbrain but not in the striatum results in partial rescue of amphetamine-induced locomotion and stereotypy in Slc1a1 -STOP mice, consistent with an impact of EAAT3 loss on presynaptic dopaminergic function. Collectively, these findings indicate that the most consistently associated OCD candidate gene impacts basal ganglia-dependent repetitive behaviors.

  13. Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism.

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    Ralphe, J Carter; Bedell, Kurt; Segar, Jeffrey L; Scholz, Thomas D

    2005-05-01

    In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.

  14. Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

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    Turgut Bastug

    Full Text Available Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+ ions, one H(+ and the counter-transport of one K(+ ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph, whose three dimensional structure is known is also coupled to three Na(+ ions but only two Na(+ ion binding sites have been observed in the crystal structure of Glt(Ph. In order to fully utilize the Glt(Ph structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph and accurately determine the number and location of Na(+ ions coupled to transport. Several sites have been proposed for the binding of a third Na(+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph and reveal a new site for the third Na(+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+ ion in Glt(Ph and EAAT1.

  15. Identification of a New Class of Selective Excitatory Amino Acid Transporter Subtype 1 (EAAT1) Inhibitors Followed by a Structure-Activity-Relationship Study

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    Hansen, Stinne Wessel; Erichsen, Mette Norman; Fu, Bingru

    2016-01-01

    in analogues with substantially improved inhibitory potencies at EAAT1 compared to that displayed by the hit, it provided a detailed insight into structural requirements for EAAT1 activity of this scaffold. The discovery of this new class of EAAT1-selective inhibitors not only supplements the currently...

  16. Discovery of the first selective inhibitor of excitatory amino acid transporter subtype 1

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    Jensen, Anders Asbjørn; Erichsen, Mette Navy; Nielsen, Christina Wøhlk

    2009-01-01

    The discovery of the first class of subtype-selective inhibitors of the human excitatory amino acid transporter subtype 1 (EAAT1) and its rat orthologue GLAST is reported. An opening structure-activity relationship of 25 analogues is presented that addresses the influence of substitutions at the 4......- and 7-positions of the parental skeleton 2-amino-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile. The most potent analogue 1o displays high nanomolar inhibitory activity at EAAT1 and a >400-fold selectivity over EAAT2 and EAAT3, making it a highly valuable pharmacological tool....

  17. The accessibility in the external part of the TM5 of the glutamate transporter EAAT1 is conformationally sensitive during the transport cycle.

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    Xiuping Zhang

    Full Text Available BACKGROUND: Excitatory amino acid transporter 1 (EAAT1 is a glutamate transporter which is a key element in the termination of the synaptic actions of glutamate. It serves to keep the extracellular glutamate concentration below neurotoxic level. However the functional significance and the change of accessibility of residues in transmembrane domain (TM 5 of the EAAT1 are not clear yet. METHODOLOGY/PRINCIPAL FINDINGS: We used cysteine mutagenesis with treatments with membrane-impermeable sulfhydryl reagent MTSET [(2-trimethylammonium methanethiosulfonate] to investigate the change of accessibility of TM5. Cysteine mutants were introduced from position 291 to 300 of the cysteine-less version of EAAT1. We checked the activity and kinetic parameters of the mutants before and after treatments with MTSET, furthermore we analyzed the effect of the substrate and blocker on the inhibition of the cysteine mutants by MTSET. Inhibition of transport by MTSET was observed in the mutants L296C, I297C and G299C, while the activity of K300C got higher after exposure to MTSET. V(max of L296C and G299C got lower while that of K300C got higher after treated by MTSET. The L296C, G299C, K300C single cysteine mutants showed a conformationally sensitive reactivity pattern. The sensitivity of L296C to MTSET was potentiated by glutamate and TBOA,but the sensitivity of G299C to MTSET was potentiated only by TBOA. CONCLUSIONS/SIGNIFICANCE: All these facts suggest that the accessibility of some positions of the external part of the TM5 is conformationally sensitive during the transport cycle. Our results indicate that some residues of TM5 take part in the transport pathway during the transport cycle.

  18. Neurochemical and behavioral characterization of neuronal glutamate transporter EAAT3 heterozygous mice

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    Luis F. González

    2017-10-01

    Full Text Available Abstract Background Obsessive–compulsive disorder (OCD is a severe neuropsychiatric condition affecting 1–3% of the worldwide population. OCD has a strong genetic component, and the SLC1A1 gene that encodes neuronal glutamate transporter EAAT3 is a strong candidate for this disorder. To evaluate the impact of reduced EAAT3 expression in vivo, we studied male EAAT3 heterozygous and wild-type littermate mice using a battery of behavioral paradigms relevant to anxiety (open field test, elevated plus maze and compulsivity (marble burying, as well as locomotor activity induced by amphetamine. Using high-performance liquid chromatography, we also determined tissue neurotransmitter levels in cortex, striatum and thalamus—brain areas that are relevant to OCD. Results Compared to wild-type littermates, EAAT3 heterozygous male mice have unaltered baseline anxiety-like, compulsive-like behavior and locomotor activity. Administration of acute amphetamine (5 mg/kg intraperitoneally increased locomotion with no differences across genotypes. Tissue levels of glutamate, GABA, dopamine and serotonin did not vary between EAAT3 heterozygous and wild-type mice. Conclusions Our results indicate that reduced EAAT3 expression does not impact neurotransmitter content in the corticostriatal circuit nor alter anxiety or compulsive-like behaviors.

  19. Excitatory amino acid transporters: recent insights into molecular mechanisms, novel modes of modulation and new therapeutic possibilities

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    Jensen, Anders A.; Fahlke, Christoph; Bjørn-Yoshimoto, Walden Emil

    2015-01-01

    The five excitatory amino acid transporters (EAAT1–5) mediating the synaptic uptake of the major excitatory neurotransmitter glutamate are differently expressed throughout the CNS and at the synaptic level. Although EAATs are crucial for normal excitatory neurotransmission, explorations into the ......The five excitatory amino acid transporters (EAAT1–5) mediating the synaptic uptake of the major excitatory neurotransmitter glutamate are differently expressed throughout the CNS and at the synaptic level. Although EAATs are crucial for normal excitatory neurotransmission, explorations...

  20. Impaired RNA splicing of 5'-regulatory sequences of the astroglial glutamate transporter EAAT2 in human astrocytoma

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    Münch, C.; Penndorf, A.; Schwalenstöcker, B.; Troost, D.; Ludolph, A. C.; Ince, P.; Meyer, T.

    2001-01-01

    A loss of the glutamate transporter EAAT2 has been reported in the neoplastic transformation of astrocytic cells and astrocytoma. The RNA expression of EAAT2 and five 5'-regulatory splice variants was investigated to identify alterations of the post-transcriptional EAAT2 gene regulation in human

  1. Designing Novel Nanoformulations Targeting Glutamate Transporter Excitatory Amino Acid Transporter 2: Implications in Treating Drug Addiction.

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    Rao, Pss; Yallapu, Murali M; Sari, Youssef; Fisher, Paul B; Kumar, Santosh

    Chronic drug abuse is associated with elevated extracellular glutamate concentration in the brain reward regions. Deficit of glutamate clearance has been identified as a contributing factor that leads to enhanced glutamate concentration following extended drug abuse. Importantly, normalization of glutamate level through induction of glutamate transporter 1 (GLT1)/ excitatory amino acid transporter 2 (EAAT2) expression has been described in several in vivo studies. GLT1 upregulators including ceftriaxone, a beta-lactam antibiotic, have been effective in attenuating drug-seeking and drug-consumption behavior in rodent models. However, potential obstacles toward clinical translation of GLT1 (EAAT2) upregulators as treatment for drug addiction might include poor gastrointestinal absorption, serious peripheral adverse effects, and/or suboptimal CNS concentrations. Given the growing success of nanotechnology in targeting CNS ailments, nanoformulating known GLT1 (EAAT2) upregulators for selective uptake across the blood brain barrier presents an ideal therapeutic approach for treating drug addiction. In this review, we summarize the results obtained with promising GLT1 (EAAT2) inducing compounds in animal models recapitulating drug addiction. Additionally, the various nanoformulations that can be employed for selectively increasing the CNS bioavailability of GLT1 (EAAT2) upregulators are discussed. Finally, the applicability of GLT1 (EAAT2) induction via central delivery of drug-loaded nanoformulations is described.

  2. Excitatory amino acid transporters as potential drug targets

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    Bunch, Lennart; Erichsen, Mette Navy; Jensen, Anders Asbjørn

    2009-01-01

    BACKGROUND: Excitatory amino acid transporters (EAATs) are transmembrane proteins responsible for the uptake of (S)-glutamate (Glu) from the synaptic cleft, thereby terminating the glutamatergic neurotransmitter signal. Today five subtypes have been identified. Except for EAAT2, their individual...

  3. Stereoselective chemoenzymatic synthesis of the four stereoisomers of l-2-(2-carboxycyclobutyl)glycine and pharmacological characterization at human excitatory amino acid transporter subtypes 1, 2, and 3

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    Faure, Sophie; Jensen, Anders A.; Maurat, Vincent

    2006-01-01

    The four stereoisomers of l-2-(2-carboxycyclobutyl)glycine, l-CBG-I, l-CBG-II, l-CBG-III, and l-CBG-IV, were synthesized in good yield and high enantiomeric excess, from the corresponding cis and trans-2-oxalylcyclobutanecarboxylic acids 5 and 6 using the enzymes aspartate aminotransferase (AAT......) and branched chain aminotransferase (BCAT) from Escherichia coli. The four stereoisomeric compounds were evaluated as potential ligands for the human excitatory amino acid transporters, subtypes 1, 2, and 3 (EAAT1, EAAT2, and EAAT3) in the FLIPR membrane potential assay. While the one trans-stereoisomer, l...

  4. Beta-Sulfonamido Functionalized Aspartate Analogs as Excitatory Amino Acid Transporter Inhibitors: Distinct Subtype-Selectivity Profiles Arising from Subtle Structural Differences

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    Hansen, Jacob Christian; Bjørn-Yoshimoto, Walden Emil; Bisballe, Niels

    2016-01-01

    In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 β-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent...

  5. Probing for improved potency and in vivo bioavailability of excitatory amino acid transporter subtype 1 inhibitors UCPH-101 and UCPH-102: Design, synthesis and pharmacological evaluation of substituted 7-biphenyl analogs

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    Erichsen, Mette Norman; Hansen, J; Artacho Ruiz, Jose

    2014-01-01

    Uptake of the major excitatory neurotransmitter in the CNS, (S)-glutamate, is mediated by a family of excitatory amino acid transporters (EAAT). Previously we have explored the structure-activity relationship (SAR) of a series of EAAT1 selective inhibitors, leading to the development of the potent...... were designed, synthesized and characterized pharmacologically at EAAT1-3 in a [(3)H]-D-aspartate uptake assay. Most notably, the potent EAAT1 inhibition displayed of UCPH-101 and UCPH-102 was retained in analog 1d in which the napht-1-yl group in the 7-position of UCPH-102 has been replaced by an o...

  6. Cellular and synaptic localization of EAAT2a in human cerebral cortex

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    Marcello eMelone

    2011-01-01

    Full Text Available We used light and electron microscopic immunocytochemical techniques to analyze the distribution, cellular and synaptic localization of EAAT2, the main glutamate transporter, in normal human neocortex. EAAT2a immunoreactivity was in all layers and consisted of small neuropilar puncta and rare cells. In white matter EAAT2a+ cells were numerous. Electron microscopic studies showed that in gray matter ∼77% of immunoreactive elements were astrocytic processes, ∼14% axon terminals, ∼2.8% dendrites, whereas ∼5% were unidentifiable. In white matter, ∼81% were astrocytic processes, ∼17% were myelinated axons and ∼2.0% were unidentified. EAAT2a immunoreactivity was never in microglial cells and oligodendrocytes. Pre-embedding electron microscopy showed that ∼67% of EAAT2a expressed at (or in the vicinity of asymmetric synapses was in astrocytes, ∼17% in axon terminals, while ∼13% was both in astrocytes and in axons. Post-embeddeding electron microscopy studies showed that in astrocytic processes contacting asymmetric synapses and in axon terminals, gold particle density was ∼25.1 and ∼2.8 particles/µm2, respectively, and was concentrated in a membrane region extending for ∼300 nm from the active zone edge. Besides representing the first detailed description of EAAT2a in human cerebral cortex, these findings may contribute to understanding its role in the pathophysiology of neuropsychiatric diseases.

  7. Glial and Neuronal Glutamate Transporters Differ in the Na+ Requirements for Activation of the Substrate-Independent Anion Conductance

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    Christopher B. Divito

    2017-05-01

    Full Text Available Excitatory amino acid transporters (EAATs are secondary active transporters of L-glutamate and L- or D-aspartate. These carriers also mediate a thermodynamically uncoupled anion conductance that is gated by Na+ and substrate binding. The activation of the anion channel by binding of Na+ alone, however, has only been demonstrated for mammalian EAAC1 (EAAT3 and EAAT4. To date, no difference has been observed for the substrate dependence of anion channel gating between the glial, EAAT1 and EAAT2, and the neuronal isoforms EAAT3, EAAT4 and EAAT5. Here we describe a difference in the Na+-dependence of anion channel gating between glial and neuronal isoforms. Chloride flux through transporters without glutamate binding has previously been described as substrate-independent or “leak” channel activity. Choline or N-methyl-D-glucamine replacement of external Na+ ions significantly reduced or abolished substrate-independent EAAT channel activity in EAAT3 and EAAT4 yet has no effect on EAAT1 or EAAT2. The interaction of Na+ with the neuronal carrier isoforms was concentration dependent, consistent with previous data. The presence of substrate and Na+-independent open states in the glial EAAT isoforms is a novel finding in the field of EAAT function. Our results reveal an important divergence in anion channel function between glial and neuronal glutamate transporters and highlight new potential roles for the EAAT-associated anion channel activity based on transporter expression and localization in the central nervous system.

  8. The glutamate transport inhibitor DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells

    DEFF Research Database (Denmark)

    Cuesta, Elena Pedraz; Christensen, Sandra; Jensen, Anders A.

    2015-01-01

    affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3 (EAAT3, EAAT1) is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability...... was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [(3)H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration...... and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point...

  9. Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2.

    Science.gov (United States)

    Zhu, Xiaoling; Yin, Jinbo; Li, Liaoliao; Ma, Lei; Tan, Hongying; Deng, Jiao; Chen, Shaoyang; Zuo, Zhiyi

    2013-10-01

    Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for five consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24h after the last electroacupuncture. Neurological outcome was assessed 2days after the MCAO. Brain tissues were harvested at 24h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2

    OpenAIRE

    Zhu, Xiaoling; Yin, Jinbo; Li, Liaoliao; Ma, Lei; Tan, Hongying; Deng, Jiao; Chen, Shaoyang; Zuo, Zhiyi

    2013-01-01

    Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for 5 consecutive days. They were subjected to a...

  11. Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

    Science.gov (United States)

    Hinson, Shannon R; Clift, Ian C; Luo, Ningling; Kryzer, Thomas J; Lennon, Vanda A

    2017-05-23

    Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

  12. Chemo-enzymatic synthesis of (2S,4R)-2-amino-4-(3-(2,2-diphenylethylamino)-3-oxopropyl)pentanedioic acid

    DEFF Research Database (Denmark)

    Sagot, Emanuelle; Jensen, Anders A.; Pickering, Darryl S

    2008-01-01

    In the mammalian central nervous system (CNS), the action of sodium dependent excitatory amino acid transporters (EAATs) is responsible for termination of glutamatergic neurotransmission by reuptake of ( S) -glutamate (Glu) from the synaptic cleft. Five EAAT subtypes have been identified, of which...

  13. Roles and regulation of brain glutamate transporters in normal and pathological brain function

    International Nuclear Information System (INIS)

    O'Shea, R.D.

    2001-01-01

    Full text: Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS. Synaptically released Glu acts on both ionotropic (iGluR) and metabotropic receptors, and excessive iGluR activation results in neuronal death (termed excitotoxicity). Removal of Glu from the synapse is thus critical for normal transmission and to prevent excitotoxicity, and is performed exclusively by a family of excitatory amino acid transporters (EAATs, also known as glutamate transporters). Disregulation of Glu transport may contribute to the pathogenesis of many neurodegenerative conditions, and altered expression or function of EAATs has been identified in a number of these pathologies. These studies investigated the functional and pathological effects of EAAT inhibitors in vitro, and developed a novel screening assay for compounds with activity at EAATs. Astrocytic EAATs are responsible for the majority of Glu uptake in brain, so preparations containing both astrocytes and neurones are required to analyse the contribution of EAATs to neuroprotection. Organotypic hippocampal cultures (OHCs), which exhibit many of the features of the intact CNS, were prepared from 11-14 day old Sprague Dawley rats (anaesthetised with halothane). Hippocampal slices (350 μm thick) were maintained on culture well inserts in chemically defined medium. After 2 weeks, cultures were treated with EAAT inhibitors for 3-7 days in the presence or absence of 300 μM Glu. Treatment with most EAAT inhibitors resulted in cell death that was proportional to the Glu concentration in the medium. In contrast, (2S,3S,4R)-2-(carboxycyclopropyl)glycine (L-CCG-III), a competitive substrate at EAATs (and possibly an antagonist at the kainate subtype of iGluR), appeared to be neuroprotective: increased Glu was not toxic in the presence of this drug. These results demonstrate the sensitivity of OHCs to inhibition of Glu uptake, highlighting the importance of EAATs in preventing excitotoxicity. Since modulation of

  14. A critical role of glutamate transporter type 3 in the learning and memory of mice.

    Science.gov (United States)

    Wang, Zhi; Park, Sang-Hon; Zhao, Huijuan; Peng, Shuling; Zuo, Zhiyi

    2014-10-01

    Hippocampus-dependent learning and memory are associated with trafficking of excitatory amino acid transporter type 3 (EAAT3) to the plasma membrane. To assess whether this trafficking is an intrinsic component of the biochemical responses underlying learning and memory, 7- to 9-week old male EAAT3 knockout mice and CD-1 wild-type mice were subjected to fear conditioning. Their hippocampal CA1 regions, amygdalae and entorhinal cortices were harvested before, or 30 min or 3 h after the fear conditioning stimulation. We found that EAAT3 knockout mice had worse contextual and tone-related learning and memory than did the wild-type mice. The expression of EAAT3, glutamate receptor (GluR)1 and GluR2 in the plasma membrane and of phospho-GluR1 (at Ser 831) and phospho-CaMKII in the hippocampus of the wild-type mice was increased at 30 min after the fear conditioning stimulation. Similar biochemical changes occurred in the amygdala. Fear conditioning also increased the expression of c-Fos and activity-regulated cytoskeleton-associated protein (Arc) in the CA1 regions and of Arc in the entorhinal cortices of the wild-type mice. These biochemical responses were attenuated in the EAAT3 knockout mice. These results suggest that EAAT3 plays a critical role in learning and memory. Our results also provide initial evidence that EAAT3 may have receptor-like functions to participate in the biochemical reactions underlying learning and memory. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia)*

    Science.gov (United States)

    Chen, Ming-xia; Li, Xiang-guang; Yang, Jun-xian; Gao, Chun-qi; Wang, Bin; Wang, Xiu-qi; Yan, Hui-chao

    2015-01-01

    The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The mRNA abundances of b0,+AT, EAAT3, y+LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b0,+AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y+LAT2 had positive correlations with body weight (0.711, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight (−0.86EAAT3, LAT4, PepT1, NHE2, NHE3, and y+LAT2 showed positive correlations with intestinal weight (0.801, CAT2, and EAAT2 showed negative correlations with intestinal weight (−0.84

  16. Growth of embryo and gene expression of nutrient transporters in the small intestine of the domestic pigeon (Columba livia).

    Science.gov (United States)

    Chen, Ming-xia; Li, Xiang-guang; Yang, Jun-xian; Gao, Chun-qi; Wang, Bin; Wang, Xiu-qi; Yan, Hui-chao

    2015-06-01

    The objective of this study was to investigate the relationship between gene expression of nutrient (amino acid, peptide, sodium and proton) transporters in the small intestine and embryonic growth in domestic pigeons (Columba livia). One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions (temperature of 38.1 °C and relative humidity of 55%). Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day (E) 9, 11, 13, 15 and day of hatch (DOH). The eggs, embryos (without yolk sac), and organs (head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax) were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The mRNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The mRNA abundances of b(0,+)AT, EAAT3, y(+)LAT2, PepT1, LAT4, NHE2, and NHE3 increased linearly with age, whereas mRNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b(0,+)AT, EAAT3, PepT1, LAT4, NHE2, NHE3, and y(+)LAT2 had positive correlations with body weight (0.711, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight (-0.86EAAT3, LAT4, PepT1, NHE2, NHE3, and y(+)LAT2 showed positive correlations with intestinal weight (0.801, CAT2, and EAAT2 showed negative correlations with intestinal weight (-0

  17. 4,4-Dimethyl- and diastereomeric 4-hydroxy-4-methyl-(2S)-glutamate analogues display distinct pharmacological profiles at ionotropic glutamate receptors and excitatory amino acid transporters

    DEFF Research Database (Denmark)

    Bunch, Lennart; Pickering, Darryl S; Gefflaut, Thierry

    2009-01-01

    this approach has provided important insight into the structure-activity relationships (SAR) for ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs), as well as the excitatory amino acid transporters (EAATs). In this work, three 4,4-disubstituted Glu analogues 1-3, which are hybrid structures......Subtype-selective ligands are of great interest to the scientific community, as they provide a tool for investigating the function of one receptor or transporter subtype when functioning in its native environment. Several 4-substituted (S)-glutamate (Glu) analogues were synthesized, and altogether...

  18. Supraspinal and spinal effects of L-trans-PDC, an inhibitor of glutamate transporter, on the micturition reflex in rats.

    Science.gov (United States)

    Honda, Masashi; Yoshimura, Naoki; Hikita, Katsuya; Hinata, Nobuyuki; Muraoka, Kuniyasu; Saito, Motoaki; Chancellor, Michael B; Takenaka, Atsushi

    2013-09-01

    Glutamate is a major excitatory transmitter in the central nervous system, controlling lower urinary tract function. Five types of glutamate transporters such as GLAST (EAAT1), GLT-1 (EAAT2), EAAC-1 (EAAT3), EAAT4, and EAAT5 have been cloned so far. In the current study we tested whether L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC), a non-selective inhibitor of glutamate transporters that increases endogenous glutamate concentration, can affect the micturition reflex in urethane anesthetized rats. Continuous cystometrograms (CMG, 0.04 ml/min infusion rate) were performed in two groups of urethane-anesthetized rats. A group of 18 rats was used for intrathecal administration of 1-10 µg of L-trans-PDC via an intrathecal catheter. In the second group of 18 rats, 1-10 µg of L-trans-PDC were administered intracerebroventricularly via a catheter inserted into the lateral ventricle. Micturition parameters were recorded and compared before and after drug administration. Intrathecal administration of L-trans-PDC at 1, 3, and 10 µg (n = 6 per dose) increased intercontraction intervals in dose dependent fashion, but did not affect postvoid residual or basal pressure at any doses tested. Intracerebroventricular administration of L-trans-PDC at 1, 3, and 10 µg (n = 6 per dose) also increased intercontraction intervals in dose dependent fashion, but did not affect postvoid residual or basal pressure at any doses tested. The current results show that, in urethane-anesthetized rats, suppression of glutamate transporters by L-trans-PDC has an inhibitory effect on the micturition reflex at supraspinal and spinal sites, possibly via activation of glutamate-mediated inhibitory pathways. Copyright © 2012 Wiley Periodicals, Inc.

  19. Mechanism of Transport Modulation by an Extracellular Loop in an Archaeal Excitatory Amino Acid Transporter (EAAT) Homolog*

    Science.gov (United States)

    Mulligan, Christopher; Mindell, Joseph A.

    2013-01-01

    Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na+-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, GltPh, suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3–4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, GltPh is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved GltPh retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process. PMID:24155238

  20. α2-Adrenergic modulation of the glutamate receptor and transporter function in a chronic ocular hypertension model.

    Science.gov (United States)

    Jung, Kyoung In; Kim, Jie Hyun; Park, Chan Kee

    2015-10-15

    Excitotoxicity, glutamate-induced toxic effects to retinal ganglion cells (RGCs), is one of several mechanisms of RGC loss suggested in glaucoma. In this study, we focused on the role of glutamate transporter of glial cells as well as N-methyl-d-aspartate (NMDA) receptor with regard to glutamate toxicity in glaucoma. We also investigated whether α2-adrenoceptor activation could modulate glutamate transporters and NMDA receptors in a chronic ocular hypertension model. Brimonidine 0.15% was administered topically to the eyes of experimental glaucoma and control animals twice daily. After 8 weeks of intraocular pressure (IOP) elevation, staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) revealed an increase in the ganglion cell layer, and the number of TUNEL-positive cells was reduced by brimonidine treatment (P<0.05). Animals with experimentally induced glaucoma exhibited an increase in retinal stress marker glial fibrillary acidic protein (GFAP) immunoreactivity; brimonidine treatment reduced GFAP. Excitatory amino acid transporter 1(EAAT1) expression remained stable throughout the period of chronic ocular hypertension. α2-Adrenergic treatment upregulated EAAT1 protein levels (P<0.05). NMDA receptor (GluN1) expression was stimulated by chronic elevation of IOP, and GluN1-positive cells in ganglion cell layer were co-localized with TUNEL staining. Brimonidine administration suppressed GluN1 levels (P<0.05). These results indicate that brimonidine decreased RGC apoptosis, upregulating EAAT1 and downregulating NMDA receptors. We suggest that topical brimonidine treatment may decrease the glutamate excitotoxicity through modulation of glutamate transporter and NMDA receptor in glaucoma. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Decreased astroglial monocarboxylate transporter 4 expression in temporal lobe epilepsy.

    Science.gov (United States)

    Liu, Bei; Niu, Le; Shen, Ming-Zhi; Gao, Lei; Wang, Chao; Li, Jie; Song, Li-Jia; Tao, Ye; Meng, Qiang; Yang, Qian-Li; Gao, Guo-Dong; Zhang, Hua

    2014-10-01

    Efflux of monocaroxylates like lactate, pyruvate, and ketone bodies from astrocytes through monocarboxylate transporter 4 (MCT4) supplies the local neuron population with metabolic intermediates to meet energy requirements under conditions of increased demand. Disruption of this astroglial-neuron metabolic coupling pathway may contribute to epileptogenesis. We measured MCT4 expression in temporal lobe epileptic foci excised from patients with intractable epilepsy and in rats injected with pilocarpine, an animal model of temporal lobe epilepsy (TLE). Cortical MCT4 expression levels were significantly lower in TLE patients compared with controls, due at least partially to MCT4 promoter methylation. Expression of MCT4 also decreased progressively in pilocarpine-treated rats from 12 h to 14 days post-administration. Underexpression of MCT4 in cultured astrocytes induced by a short hairpin RNA promoted apoptosis. Knockdown of astrocyte MCT4 also suppressed excitatory amino acid transporter 1 (EAAT1) expression. Reduced MCT4 and EAAT1 expression by astrocytes may lead to neuronal hyperexcitability and epileptogenesis in the temporal lobe by reducing the supply of metabolic intermediates and by allowing accumulation of extracellular glutamate.

  2. The effect of Eimeria maxima infection on the expression of amino acid and sugar transporters aminopeptidase, as well as the di- and tri-peptide transporter PepT1, is not solely due to decreased feed intake.

    Science.gov (United States)

    Miska, Katarzyna B; Fetterer, Raymond H

    2018-05-01

    Coccidiosis caused by Eimeria in poultry is endemic to poultry operations and results in decreased feed intake, diarrhea, and decreased weight gain. The goal was to determine the effect of Eimeria maxima infection on the expression of genes that encode peptide and amino acid transporters (AATs), and also to determine whether decreased feed intake contributes to the change in gene expression by including a pair-fed group of broilers. Three groups of male Ross broilers: 1) not infected, 2) infected, and 3) not infected pair-fed groups were used. Chicks were infected with 1,000 oocysts of E. maxima at 21 d of age. Feed consumption was obtained daily, and at d 0, 3, 5, 7, 10, and 14 post-infection (PI), 6 birds were euthanized, and a portion of the jejunum was removed for qRT-PCR. Infected birds had significantly decreased feed consumption between d 6 to 9 PI. At d 7 PI infected birds had a 45% reduction in weight gain, and pair-fed birds had a 32% reduction in weight gain. The feed conversion ratio at d 7 PI of infected birds was 2.2 while that of pair-fed birds was 1.7, compared to 1.5 in uninfected birds. Growth parameters were more affected in infected birds than in pair-fed birds. By measuring expression levels of nutrient uptake and processing genes via qRT-PCR, it was determined that genes encoding proteins located at the brush border of the gut epithelium were affected by infection as well as change in feed intake. The expression of AATs B°AT, b°,+AT, EAAT3, and PepT1 in infected birds decreased sharply at the height of infection; however, in birds that were pair fed, an increase in expression of b°,+AT, and PepT1 was observed, and little change was seen in expression of B°AT and EAAT3. In summary, the changes in expression of digestive enzymes and nutrient transporters are distinct between coccidia-infected birds compared to healthy pair-fed birds.

  3. In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate.

    Science.gov (United States)

    Helms, Hans Christian; Madelung, Rasmus; Waagepetersen, Helle Sønderby; Nielsen, Carsten Uhd; Brodin, Birger

    2012-05-01

    The concentration of the excitotoxic amino acid, L-glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood-brain barrier endothelium in brain L-glutamate homeostasis. Transendothelial transport- and accumulation studies of (3) H-L-glutamate, (3) H-L-aspartate, and (3) H-D-aspartate in an electrically tight bovine endothelial/rat astrocyte blood-brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm(2) , and (14) C-D-mannitol permeability values of 0.88 ± 0.13 × 10(-6) cm s(-1) . Unidirectional flux studies showed that L-aspartate and L-glutamate, but not D-aspartate, displayed polarized transport in the brain-to-blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a K(m) of 69 ± 15 μM and a J(max) of 44 ± 3.1 pmol min(-1) cm(-2) for L-aspartate and a K(m) of 138 ± 49 μM and J(max) of 28 ± 3.1 pmol min(-1) cm(-2) for L-glutamate. The EAAT inhibitor, DL-threo-ß-Benzyloxyaspartate, inhibited transendothelial brain-to-blood fluxes of L-glutamate and L-aspartate. Expression of EAAT-1 (Slc1a3), -2 (Slc1a2), and -3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT-1 and -3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood-brain barrier itself may participate in regulating brain L-glutamate concentrations. Copyright © 2012 Wiley Periodicals, Inc.

  4. Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment.

    Science.gov (United States)

    MacNamee, Sarah E; Liu, Kendra E; Gerhard, Stephan; Tran, Cathy T; Fetter, Richard D; Cardona, Albert; Tolbert, Leslie P; Oland, Lynne A

    2016-07-01

    Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979-1998, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Chemoenzymatic synthesis of a series of 4-substituted glutamate analogues and pharmacological characterization at human glutamate transporters subtypes 1-3

    DEFF Research Database (Denmark)

    Alaux, Sebastien; Kusk, Mie; Sagot, Emanuelle

    2005-01-01

    A series of nine L-2,4-syn-4-alkylglutamic acid analogues (1a-i) were synthesized in high yield and high enantiomeric excess (>99% ee) from their corresponding 4-substituted ketoglutaric acids (2a-i), using the enzyme aspartate aminotransferase (AAT) from pig heart or E. coli. The synthesized com...... subtypes EAAT1-3 while maintaining inhibitory activity....

  6. Effects of Glycyrrhetinic Acid on GSH Synthesis Induced by Realgar in the Mouse Hippocampus: Involvement of System [Formula: see text], System [Formula: see text], MRP-1, and Nrf2.

    Science.gov (United States)

    Wang, Yan-Lei; Chen, Mo; Huo, Tao-Guang; Zhang, Ying-Hua; Fang, Ying; Feng, Cong; Wang, Shou-Yun; Jiang, Hong

    2017-05-01

    Realgar, a type of mineral drug-containing arsenic, exhibits neurotoxicity. Brain glutathione (GSH) is crucial to protect the nervous system and to resist arsenic toxicity. Therefore, the main aim of this study was to explore the neurotoxic mechanisms of realgar and the protective effects of glycyrrhetinic acid (GA) by observing the effects of GA on the hippocampal GSH biosynthetic pathway after exposure to realgar. Institute of Cancer Research (ICR) mice were randomly divided into five groups: a control group, a GA control group, a realgar alone group, a low-dose GA intervention group, and a high-dose GA intervention group. Cognitive ability was tested using an object recognition task (ORT). The ultrastructures of the hippocampal neurons and synapses were observed. mRNA and protein levels of EAAT1, EAAT2, EAAT3, xCT, Nrf2, HO-1, γ-GCS (GCLC, GCLM), and MRP-1 were measured, as was the cellular localization of EAAT3, xCT, MRP-1, and Nrf2. The levels of GSH in the hippocampus, the levels of glutamate (Glu) and cysteine (Cys) in the extracellular fluid of hippocampal CA1 region, and the levels of active sulfur in the brain were also investigated. The results indicate that realgar lowered hippocampal GSH levels, resulting in ultrastructural changes in hippocampal neurons and synapses and deficiencies in cognitive ability, ultimately inducing neurotoxicity. GA could trigger the expression of Nrf2, HO-1, EAAT1, EAAT2, EAAT3, xCT, MRP-1, GCLC, and GCLM. Additionally, the expression of γ-GT and the supply levels of Glu and Cys increased, ultimately causing a significant increase in hippocampal GSH to alleviate realgar-induced neurotoxicity. In conclusion, the findings from our study indicate that GA can antagonize decreased brain GSH levels induced by realgar and can lessen the neurotoxicity of realgar.

  7. Glutamate transporter type 3 knockout leads to decreased heart rate possibly via parasympathetic mechanism

    OpenAIRE

    Deng, Jiao; Li, Jiejie; Li, Liaoliao; Feng, Chenzhuo; Xiong, Lize; Zuo, Zhiyi

    2013-01-01

    Parasympathetic tone is a dominant neural regulator for basal heart rate. Glutamate transporters (EAAT) via their glutamate uptake functions regulate glutamate neurotransmission in the central nervous system. We showed that EAAT type 3 (EAAT3) knockout mice had a slower heart rate than wild-type mice when they were anesthetized. We design this study to determine whether non-anesthetized EAAT3 knockout mice have a slower heart rate and, if so, what may be the mechanism for this effect. Young a...

  8. Estrone-1-sulphate (E1S) has impact on the kinetics parameters of transporter mediated taurine and glutamate influx in Caco-2 cells

    DEFF Research Database (Denmark)

    Steffansen, Bente; El-Sayed, F

    Previously, we have suggested estrone-1-sulfate (E1S) to be intercalated into the phospholipid membrane 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC). The overall hypothesis of the present study was that E1S intercalation in the cell membrane of Caco-2 cells may changes the functionality...... of membrane transporters. The aim was therefore to investigate if addition of E1S to the growth medium of Caco-2 cells before but not during the influx study, change the kinetic parameters of transporter-mediated influx of taurine and glutamate by respective TAUT and EAAT transporters. The results show that 4...

  9. Transport of amino acids and GABA analogues via the human proton-coupled amino acid transporter, hPAT1

    DEFF Research Database (Denmark)

    Larsen, Mie; Larsen, Birger Brodin; Frølund, Bente

    2008-01-01

    The objective of this study was to investigate transepithelial amino acid transport as a function of Caco-2 cell culture time. Furthermore, the objective was to investigate apical uptake characteristics of hPAT1-mediated transport under various experimental conditions. Apical amino acid uptake......, which has been shown to function as a carboxylic acid bioisostere for substrates of the GABA receptor and transport systems....

  10. Chronic postnatal stress induces voluntary alcohol intake and modifies glutamate transporters in adolescent rats.

    Science.gov (United States)

    Odeon, María Mercedes; Andreu, Marcela; Yamauchi, Laura; Grosman, Mauricio; Acosta, Gabriela Beatriz

    2015-01-01

    Postnatal stress alters stress responses for life, with serious consequences on the central nervous system (CNS), involving glutamatergic neurotransmission and development of voluntary alcohol intake. Several drugs of abuse, including alcohol and cocaine, alter glutamate transport (GluT). Here, we evaluated effects of chronic postnatal stress (CPS) on alcohol intake and brain glutamate uptake and transporters in male adolescent Wistar rats. For CPS from postnatal day (PD) 7, pups were separated from their mothers and exposed to cold stress (4 °C) for 1 h daily for 20 days; controls remained with their mothers. Then they were exposed to either voluntary ethanol (6%) or dextrose (1%) intake for 7 days (5-7 rats per group), then killed. CPS: (1) increased voluntary ethanol intake, (2) did not affect body weight gain or produce signs of toxicity with alcohol exposure, (3) increased glutamate uptake by hippocampal synaptosomes in vitro and (4) reduced protein levels (Western measurements) in hippocampus and frontal cortex of glial glutamate transporter-1 (GLT-1) and excitatory amino-acid transporter-3 (EAAT-3) but increased glutamate aspartate transporter (GLAST) levels. We propose that CPS-induced decrements in GLT-1 and EAAT-3 expression levels are opposed by activation of a compensatory mechanism to prevent excitotoxicity. A greater role for GLAST in total glutamate uptake to prevent enlarged extracellular glutamate levels is inferred. Although CPS strongly increased intake of ethanol, this had little impact on effects of CPS on brain glutamate uptake or transporters. However, the impact of early life adverse events on glutamatergic neurotransmission may underlie increased alcohol consumption in adulthood.

  11. Effects of a series of acidic drugs on L-lactic acid transport by the monocarboxylate transporters MCT1 and MCT4.

    Science.gov (United States)

    Leung, Yat Hei; Belanger, Francois; Lu, Jennifer; Turgeon, Jacques; Michaud, Veronique

    2018-03-07

    Drug-induced myopathy is a serious side effect that often requires removal of a medication from a drug regimen. For most drugs, the underlying mechanism of drug-induced myopathy remains unclear. Monocarboxylate transporters (MCTs) mediate L-lactic acid transport, and inhibition of MCTs may potentially lead to perturbation of L-lactic acid accumulation and muscular disorders. Therefore, we hypothesized that L-lactic acid transport may be involved in the development of drug-induced myopathy. The aim of this study was to assess the inhibitory potential of 24 acidic drugs on L-lactic acid transport using breast cancer cell lines Hs578T and MDA-MB-231, which selectively express MCT1 and MCT4, respectively. The influx transport of L-lactic acid was minimally inhibited by all drugs tested. The efflux transport was next examined: loratadine (IC50: 10 and 61 µM) and atorvastatin (IC50: 78 and 41 µM) demonstrated the greatest potency for inhibition of L-lactic acid efflux by MCT1 and MCT4, respectively. Acidic drugs including fluvastatin, cerivastatin, simvastatin acid, lovastatin acid, irbesartan and losartan exhibited weak inhibitory potency on L-lactic acid efflux. Our results suggest that some acidic drugs, such as loratadine and atorvastatin, can inhibit the efflux transport of L-lactic acid. This inhibition may cause an accumulation of intracellular L-lactic acid leading to acidification and muscular disorders. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. The glutamate transport inhibitor DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells

    International Nuclear Information System (INIS)

    Pedraz-Cuesta, Elena; Christensen, Sandra; Jensen, Anders A.; Jensen, Niels Frank; Bunch, Lennart; Romer, Maria Unni; Brünner, Nils; Stenvang, Jan; Pedersen, Stine Falsig

    2015-01-01

    Colorectal cancer (CRC) is a leading cause of cancer death globally and new biomarkers and treatments are severely needed. Here, we employed HCT116 and LoVo human CRC cells made resistant to either SN38 or oxaliplatin, to investigate whether altered expression of the high affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3 (EAAT3, EAAT1) is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability and glutathione content. Results were evaluated using one- and two-way ANOVA and Students two-tailed t-test, as relevant. In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated ~60 % and up-regulated ~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [ 3 H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu 2+ -transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction. SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments

  13. Intracellular Na+ concentration influences short-term plasticity of glutamate transporter-mediated currents in neocortical astrocytes.

    Science.gov (United States)

    Unichenko, Petr; Myakhar, Olga; Kirischuk, Sergei

    2012-04-01

    Fast synaptic transmission requires a rapid clearance of the released neurotransmitter from the extracellular space. Glial glutamate transporters (excitatory amino acid transporters, EAATs) strongly contribute to glutamate removal. In this work, we investigated the paired-pulse plasticity of synaptically activated, glutamate transporter-mediated currents (STCs) in cortical layer 2/3 astrocytes. STCs were elicited by local electrical stimulation in layer 4 in the presence of ionotropic glutamate (AMPA and NMDA), GABAA, and GABAB receptor antagonists. In experiments with low [Na(+)]i (5 mM) intrapipette solution, STCs elicited by paired-pulse stimulation demonstrated paired-pulse facilitation (PPF) at short (astrocytic [Na(+)]i, reduced the mean STC amplitude, decreased PPF at short ISIs, and slowed STC kinetics. All GABA-induced changes were blocked by NO-711 and SNAP-5114, GABA transporter (GATs) antagonists. In experiments with the low intrapipette solution, GAT blockade under control conditions decreased PPF at short ISIs both at room and at near physiological temperatures. Dialysis of single astrocyte with low [Na(+)]i solution increased the amplitude and reduced PPR of evoked field potentials recorded in the vicinity of the astrocyte. We conclude that (1) endogenous GABA via GATs may influence EAAT functioning and (2) astrocytic [Na(+)]i modulates the short-term plasticity of STCs and in turn the efficacy of glutamate removal. Copyright © 2012 Wiley Periodicals, Inc.

  14. Posterior cerebellar Purkinje cells in an SCA5/SPARCA1 mouse model are especially vulnerable to the synergistic effect of loss of β-III spectrin and GLAST.

    Science.gov (United States)

    Perkins, Emma M; Suminaite, Daumante; Clarkson, Yvonne L; Lee, Sin Kwan; Lyndon, Alastair R; Rothstein, Jeffrey D; Wyllie, David J A; Tanaka, Kohichi; Jackson, Mandy

    2016-10-15

    Clinical phenotypes of spinocerebellar ataxia type-5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type-1 (SPARCA1) are mirrored in mice lacking β-III spectrin (β-III-/-). One function of β-III spectrin is the stabilization of the Purkinje cell-specific glutamate transporter EAAT4 at the plasma membrane. In β-III-/- mice EAAT4 levels are reduced from an early age. In contrast levels of the predominant cerebellar glutamate transporter GLAST, expressed in Bergmann glia, only fall progressively from 3 months onwards. Here we elucidated the roles of these two glutamate transporters in cerebellar pathogenesis mediated through loss of β-III spectrin function by studying EAAT4 and GLAST knockout mice as well as crosses of both with β-III-/- mice. Our data demonstrate that EAAT4 loss, but not abnormal AMPA receptor composition, in young β-III-/- mice underlies early Purkinje cell hyper-excitability and that subsequent loss of GLAST, superimposed on the earlier deficiency of EAAT4, is responsible for Purkinje cell loss and progression of motor deficits. Yet the loss of GLAST appears to be independent of EAAT4 loss, highlighting that other aspects of Purkinje cell dysfunction underpin the pathogenic loss of GLAST. Finally, our results demonstrate that Purkinje cells in the posterior cerebellum of β-III-/- mice are most susceptible to the combined loss of EAAT4 and GLAST, with degeneration of proximal dendrites, the site of climbing fibre innervation, most pronounced. This highlights the necessity for efficient glutamate clearance from these regions and identifies dysregulation of glutamatergic neurotransmission particularly within the posterior cerebellum as a key mechanism in SCA5 and SPARCA1 pathogenesis.

  15. Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

    Science.gov (United States)

    Su, S; Miska, K B; Fetterer, R H; Jenkins, M C; Wong, E A

    2015-03-01

    Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would

  16. Neuroprotective effects of the novel glutamate transporter inhibitor (-)-3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic acid, which preferentially inhibits reverse transport (glutamate release) compared with glutamate reuptake

    DEFF Research Database (Denmark)

    Colleoni, Simona; Jensen, Anders Asbjørn; Landucci, Elisa

    2008-01-01

    on the three hEAAT subtypes. (-)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [3H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [3H]L-glutamate uptake. New data suggest...

  17. The blood-brain barrier fatty acid transport protein 1 (FATP1/SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport.

    Science.gov (United States)

    Ochiai, Yusuke; Uchida, Yasuo; Ohtsuki, Sumio; Tachikawa, Masanori; Aizawa, Sanshiro; Terasaki, Tetsuya

    2017-05-01

    We purposed to clarify the contribution of fatty acid transport protein 1 (FATP1/SLC 27A1) to the supply of docosahexaenoic acid (DHA) to the brain across the blood-brain barrier in this study. Transport experiments showed that the uptake rate of [ 14 C]-DHA in human FATP1-expressing HEK293 cells was significantly greater than that in empty vector-transfected (mock) HEK293 cells. The steady-state intracellular DHA concentration was nearly 2-fold smaller in FATP1-expressing than in mock cells, suggesting that FATP1 works as not only an influx, but also an efflux transporter for DHA. [ 14 C]-DHA uptake by a human cerebral microvascular endothelial cell line (hCMEC/D3) increased in a time-dependent manner, and was inhibited by unlabeled DHA and a known FATP1 substrate, oleic acid. Knock-down of FATP1 in hCMEC/D3 cells with specific siRNA showed that FATP1-mediated uptake accounts for 59.2-73.0% of total [ 14 C]-DHA uptake by the cells. Insulin treatment for 30 min induced translocation of FATP1 protein to the plasma membrane in hCMEC/D3 cells and enhanced [ 14 C]-DHA uptake. Immunohistochemical analysis of mouse brain sections showed that FATP1 protein is preferentially localized at the basal membrane of brain microvessel endothelial cells. We found that two neuroprotective substances, taurine and biotin, in addition to DHA, undergo FATP1-mediated efflux. Overall, our results suggest that FATP1 localized at the basal membrane of brain microvessels contributes to the transport of DHA, taurine and biotin into the brain, and insulin rapidly increases DHA supply to the brain by promoting translocation of FATP1 to the membrane. Read the Editorial Comment for this article on page 324. © 2016 International Society for Neurochemistry.

  18. Effect of egg weight on composition, embryonic growth, and expression of amino acid transporter genes in yolk sac membranes and small intestines of the domestic pigeon (Columba livia).

    Science.gov (United States)

    Chen, M X; Li, X G; Yan, H C; Wang, X Q; Gao, C Q

    2016-06-01

    The objective of this study was to investigate the effect of egg weight on the composition of the egg, the growth of the embryo, and the expression of amino acid transporter genes in the yolk sac membranes and small intestines of the domestic pigeon (Columba livia). A total of 240 fertilized eggs were collected and divided into two groups based on the weight of the eggs, light (LE) and heavy (HE). The composition of 20 eggs from each group was measured, and the remaining eggs were weighed and placed in an incubator. On embryonic days (E) 9, 11, 13, and 15 and day of hatch (DOH), 15 embryos/hatchlings from each group were measured for embryonic growth, and samples were collected. The HE had heavier yolk and albumen weights than the LE (P < 0.01). Compared with the LE, the HE had heavier yolk-free embryonic body and yolk sac weights from E13 to DOH (P < 0.05). Additionally, the HE had larger yolk sac membrane weights from E13 to E15 (P < 0.05) and had more residual yolk sac content on DOH than those of the LE (P < 0.01). The yolk absorption was greater for the HE than for the LE from E11 to E13 (P < 0.05). Furthermore, the abundance of CAT2 and PepT1 mRNA in the yolk sac membranes was greater in the HE than in the LE on E13 (P < 0.05). Compared with the LE, the gene expression of EAAT2 in the intestine on E13 was greater in the HE, whereas the expression of EAAT3 was lower in the HE (P < 0.05). Taken together, our results suggest that egg weight influenced the composition of the eggs, embryonic development, and expression of amino acid transporter genes in the yolk sac membranes and small intestines of pigeon embryos. © 2016 Poultry Science Association Inc.

  19. Limited energy supply in Müller cells alters glutamate uptake

    DEFF Research Database (Denmark)

    Toft-Kehler, Anne Katrine; Skytt, Dorte Marie; Poulsen, Kristian Arild

    2014-01-01

    The viability of retinal ganglion cells (RGC) is essential for the maintenance of visual function. RGC homeostasis is maintained by the surrounding retinal glial cells, the Müller cells, which buffer the extracellular concentration of neurotransmitters and provide the RGCs with energy. This study...... evaluates if glucose-deprivation of Müller cells interferes with their ability to remove glutamate from the extracellular space. The human Müller glial cell line, Moorfields/Institute of Ophthalmology-Müller 1, was used to study changes in glutamate uptake. Excitatory amino acid transporter (EAAT) proteins...... were up-regulated in glucose-deprived Müller cells and glutamate uptake was significantly increased in the absence of glucose. The present findings revealed an up-regulation of EAAT1 and EAAT2 in glucose-deprived Müller cells as well as an increased ability to take up glutamate. Hence, glucose...

  20. Imaging the L-type amino acid transporter-1 (LAT1 with Zr-89 immunoPET.

    Directory of Open Access Journals (Sweden)

    Oluwatayo F Ikotun

    Full Text Available The L-type amino acid transporter-1 (LAT1, SLC7A5 is upregulated in a wide range of human cancers, positively correlated with the biological aggressiveness of tumors, and a promising target for both imaging and therapy. Radiolabeled amino acids such as O-(2-[(18F]fluoroethyl-L-tyrosine (FET that are transport substrates for system L amino acid transporters including LAT1 have met limited success for oncologic imaging outside of the brain, and thus new strategies are needed for imaging LAT1 in systemic cancers. Here, we describe the development and biological evaluation of a novel zirconium-89 labeled antibody, [(89Zr]DFO-Ab2, targeting the extracellular domain of LAT1 in a preclinical model of colorectal cancer. This tracer demonstrated specificity for LAT1 in vitro and in vivo with excellent tumor imaging properties in mice with xenograft tumors. PET imaging studies showed high tumor uptake, with optimal tumor-to-non target contrast achieved at 7 days post administration. Biodistribution studies demonstrated tumor uptake of 10.5 ± 1.8 percent injected dose per gram (%ID/g at 7 days with a tumor to muscle ratio of 13 to 1. In contrast, the peak tumor uptake of the radiolabeled amino acid [(18F]FET was 4.4 ± 0.5 %ID/g at 30 min after injection with a tumor to muscle ratio of 1.4 to 1. Blocking studies with unlabeled anti-LAT1 antibody demonstrated a 55% reduction of [(89Zr]DFO-Ab2 accumulation in the tumor at 7 days. These results are the first report of direct PET imaging of LAT1 and demonstrate the potential of immunoPET agents for imaging specific amino acid transporters.

  1. Recent advances on uric acid transporters

    Science.gov (United States)

    Xu, Liuqing; Shi, Yingfeng; Zhuang, Shougang; Liu, Na

    2017-01-01

    Uric acid is the product of purine metabolism and its increased levels result in hyperuricemia. A number of epidemiological reports link hyperuricemia with multiple disorders, such as kidney diseases, cardiovascular diseases and diabetes. Recent studies also showed that expression and functional changes of urate transporters are associated with hyperuricemia. Uric acid transporters are divided into two categories: urate reabsorption transporters, including urate anion transporter 1 (URAT1), organic anion transporter 4 (OAT4) and glucose transporter 9 (GLUT9), and urate excretion transporetrs, including OAT1, OAT3, urate transporter (UAT), multidrug resistance protein 4 (MRP4/ABCC4), ABCG-2 and sodium-dependent phosphate transport protein. In the kidney, uric acid transporters decrease the reabsorption of urate and increase its secretion. These transporters’ dysfunction would lead to hyperuricemia. As the function of urate transporters is important to control the level of serum uric acid, studies on the functional role of uric acid transporter may provide a new strategy to treat hyperuricemia associated diseases, such as gout, chronic kidney disease, hyperlipidemia, hypertension, coronary heart disease, diabetes and other disorders. This review article summarizes the physiology of urate reabsorption and excretion transporters and highlights the recent advances on their roles in hyperuricemia and various diseases. PMID:29246027

  2. Cardioprotective effect of L-glutamate in obese type 2 diabetic Zucker fatty rats

    DEFF Research Database (Denmark)

    Povlsen, Jonas Agerlund; Løfgren, Bo; Rasmussen, Lars Ege

    2009-01-01

    (Wistar-Kyoto) and diabetic (Zucker diabetic fatty (ZDF)) rats, studied at 16 weeks of age. The infarct size (IS)/area-at-risk (AAR) ratio was the primary end-point. Expression of L-glutamate excitatory amino acid transporter (EAAT) 1 (mitochondrial) and EAAT3 (sarcolemmal) was determined by quantitative...... was downregulated in hearts from ZDF rats at both the mRNA and protein levels (P diabetic hearts (P obese diabetic rats have......1. Because diabetic hearts have an increased threshold for cardioprotection by ischaemic preconditioning (IPC), we hypothesized that protection by L-glutamate during reperfusion is restricted in Type 2 diabetic hearts. Previously, we found that L-glutamate-mediated postischaemic cardioprotection...

  3. Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability

    NARCIS (Netherlands)

    Aulak, K. S.; Mishra, R.; Zhou, L.; Hyatt, S. L.; de Jonge, W.; Lamers, W.; Snider, M.; Hatzoglou, M.

    1999-01-01

    The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged

  4. LAT1 acts as a crucial transporter of amino acids in human thymic carcinoma cells

    Directory of Open Access Journals (Sweden)

    Keitaro Hayashi

    2016-11-01

    Full Text Available L-type amino acid transporter 1 (LAT1, SLC7A5 incorporates essential amino acids into cells. Recent studies have shown that LAT1 is a predominant transporter in various human cancers. However, the function of LAT1 in thymic carcinoma remains unknown. Here we demonstrate that LAT1 is a critical transporter for human thymic carcinoma cells. LAT1 was strongly expressed in human thymic carcinoma tissues. LAT1-specific inhibitor significantly suppressed leucine uptake and growth of Ty82 human thymic carcinoma cell lines, suggesting that thymic carcinoma takes advantage of LAT1 as a quality transporter and that LAT1-specific inhibitor might be clinically beneficial in therapy for thymic carcinoma.

  5. Evaluation of [1-11C]-α-aminoisobutyric acid for tumor detection and amino acid transport measurement: Spontaneous canine tumor studies

    International Nuclear Information System (INIS)

    Bigler, R.E.; Zanzonico, P.B.; Schmall, B.; Conti, P.S.; Dahl, J.R.; Rothman, L.; Sgouros, G.

    1985-01-01

    Alpha-aminoisobutyric acid (AIB) or α-methyl alanine, is a nonmetabolized amino acid treansported into cells particularly malignant cells, predominantly by the ''A'' amino acid transport system. Since it is not metabolized, [1- 11 C]-AIB can be used to quantify A-type amino acid transport into cells using a relatively simple compartmental model and quantitative imaging procedures (e.g. positron tomography). The tissue distribution of [1- 11 C]-AIB was determined in six dogs bearing spontaneous tumors, including lymphosarcoma, osteogenic sarcoma, mammary carcinoma, and adenocarcinoma. Quantitative imaging with tissue radioassay confirmation at necropsy showed poor to excellent tumor localization. However, in all cases the concentrations achieved appear adequate for amino acid transport measurement at known tumor locations. The observed low normal brain (due to blood-brain barrier exclusion) and high (relative to brain) tumor concentrations of [1- 11 C]-AIB suggest that this agent may prove effective for the early detection of human brain tumors. (orig.)

  6. Functional analysis of apf1 mutation causing defective amino acid transport in Saccharomyces cerevisiae.

    Science.gov (United States)

    Horák, J; Kotyk, A

    1993-04-01

    Mutation in the Apf1 locus causes a pleiotropic effect of H(+)-driven active amino acid transport in baker's yeast Saccharomyces cerevisiae. The uptake of other, presumably H(+)-driven, substances, e.g. of purine and pyrimidine bases, maltose and phosphate ions, is not significantly influenced by this mutation. The apf1 mutation decreases not only the initial rates of amino acid uptake but also the accumulation ratios of amino acids taken up but has virtually no effect on the membrane potential or on the delta pH which constitute the thermodynamically relevant source of energy for their transport. Similarly, no changes in intracellular ATP content, in ATP-hydrolyzing and H(+)-extruding H(+)-ATPase activities, in the efflux of intracellularly accumulated amino acids, or in rates of endogenous respiration, were observed in the apf1 mutant phenotype. Hence, all these data are in accordance with the experiments showing that the Apf1 protein, an integral protein of the endoplasmic reticulum, is required exclusively for efficient processing and translocation of transport proteins specific for amino acids from the endoplasmic reticulum to their final destination, the plasma membrane.

  7. Ascorbic acid transport and accumulation in human neutrophils

    International Nuclear Information System (INIS)

    Washko, P.; Rotrosen, D.; Levine, M.

    1989-01-01

    The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake

  8. Insights into the Structure, Function, and Ligand Discovery of the Large Neutral Amino Acid Transporter 1, LAT1

    Directory of Open Access Journals (Sweden)

    Natesh Singh

    2018-04-01

    Full Text Available The large neutral amino acid transporter 1 (LAT1, or SLC7A5 is a sodium- and pH-independent transporter, which supplies essential amino acids (e.g., leucine, phenylalanine to cells. It plays an important role at the Blood–Brain Barrier (BBB where it facilitates the transport of thyroid hormones, pharmaceuticals (e.g., l-DOPA, gabapentin, and metabolites into the brain. Moreover, its expression is highly upregulated in various types of human cancer that are characterized by an intense demand for amino acids for growth and proliferation. Therefore, LAT1 is believed to be an important drug target for cancer treatment. With the crystallization of the arginine/agmatine antiporter (AdiC from Escherichia Coli, numerous homology models of LAT1 have been built to elucidate the substrate binding site, ligand–transporter interaction, and structure–function relationship. The use of these models in combination with molecular docking and experimental testing has identified novel chemotypes of ligands of LAT1. Here, we highlight the structure, function, transport mechanism, and homology modeling of LAT1. Additionally, results from structure–function studies performed on LAT1 are addressed, which have enhanced our knowledge of the mechanism of substrate binding and translocation. This is followed by a discussion on ligand- and structure-based approaches, with an emphasis on elucidating the molecular basis of LAT1 inhibition. Finally, we provide an exhaustive summary of different LAT1 inhibitors that have been identified so far, including the recently discovered irreversible covalent inhibitors.

  9. Differential SLC1A2 Promoter Methylation in Bipolar Disorder With or Without Addiction

    Directory of Open Access Journals (Sweden)

    Yun-Fang Jia

    2017-07-01

    Full Text Available While downregulation of excitatory amino acid transporter 2 (EAAT2, the main transporter removing glutamate from the synapse, has been recognized in bipolar disorder (BD, the underlying mechanisms of downregulation have not been elucidated. BD is influenced by environmental factors, which may, via epigenetic modulation of gene expression, differentially affect illness presentation. This study thus focused on epigenetic DNA methylation regulation of SLC1A2, encoding for EAAT2, in BD with variable environmental influences of addiction. High resolution melting PCR (HRM-PCR and thymine–adenine (TA cloning with sequence analysis were conducted to examine methylation of the promoter region of the SLC1A2. DNA was isolated from blood samples drawn from BD patients (N = 150 with or without addiction to alcohol, nicotine, or food, defined as binge eating, and matched controls (N = 32. In comparison to controls, the SLC1A2 promoter region was hypermethylated in BD without addiction but was hypomethylated in BD with addiction. After adjusting for age and sex, the association of methylation levels with nicotine addiction (p = 0.0009 and binge eating (p = 0.0002 remained significant. Consistent with HRM-PCR, direct sequencing revealed increased methylation in CpG site 6 in BD, but decreased methylation in three CpG sites (6, 48, 156 in BD with alcohol and nicotine addictions. These results suggest that individual point methylation within the SLC1A2 promoter region may be modified by exogenous addiction and may have a potential for developing clinically valuable epigenetic biomarkers for BD diagnosis and monitoring.

  10. Transport of Indole-3-Butyric Acid and Indole-3-Acetic Acid in Arabidopsis Hypocotyls Using Stable Isotope Labeling1[C][W][OA

    Science.gov (United States)

    Liu, Xing; Barkawi, Lana; Gardner, Gary; Cohen, Jerry D.

    2012-01-01

    The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling. PMID:22323783

  11. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Melton, Elaina M. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Center for Cardiovascular Sciences, Albany Medical College, Albany, NY (United States); Cerny, Ronald L. [Department of Chemistry, University of Nebraska, Lincoln, NE (United States); DiRusso, Concetta C. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Black, Paul N., E-mail: pblack2@unl.edu [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States)

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  12. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    International Nuclear Information System (INIS)

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-01-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  13. Transport of phosphoric acid through supported liquid membrane

    International Nuclear Information System (INIS)

    Zayzafoon, G.; Yassine, T.; Baidoun, R.

    2003-01-01

    The transport of phosphhoric acid through liquid membranes of amylalkohol, 1-octanol and 2-octanol was studied. It was found that phosphoric acid is transfered from feed side to strip side and the transport increased with the concentration of phosphoric acid up to 5M. The permeability in each membrane was determined for 5M phosphoic acid. It was found that the permeability values are 1.45 x 10 1 0 m 2 s 1 for amylakohol and ∼ 1x10 1 0 m 2 s 1 for each of 1-octanol and 2-octanol

  14. Partial Loss of the Glutamate Transporter GLT-1 Alters Brain Akt and Insulin Signaling in a Mouse Model of Alzheimer's Disease.

    Science.gov (United States)

    Meeker, Kole D; Meabon, James S; Cook, David G

    2015-01-01

    The glutamate transporter GLT-1 (also called EAAT2 in humans) plays a critical role in regulating extracellular glutamate levels in the central nervous system (CNS). In Alzheimer's disease (AD), EAAT2 loss is associated with neuropathology and cognitive impairment. In keeping with this, we have reported that partial GLT-1 loss (GLT-1+/-) causes early-occurring cognitive deficits in mice harboring familial AD AβPPswe/PS1ΔE9 mutations. GLT-1 plays important roles in several molecular pathways that regulate brain metabolism, including Akt and insulin signaling in astrocytes. Significantly, AD pathogenesis also involves chronic Akt activation and reduced insulin signaling in the CNS. In this report we tested the hypothesis that GLT-1 heterozygosity (which reduces GLT-1 to levels that are comparable to losses in AD patients) in AβPPswe/PS1ΔE9 mice would induce sustained activation of Akt and disturb components of the CNS insulin signaling cascade. We found that partial GLT-1 loss chronically increased Akt activation (reflected by increased phosphorylation at serine 473), impaired insulin signaling (reflected by decreased IRβ phosphorylation of tyrosines 1150/1151 and increased IRS-1 phosphorylation at serines 632/635 - denoted as 636/639 in humans), and reduced insulin degrading enzyme (IDE) activity in brains of mice expressing familial AβPPswe/PS1ΔE9 AD mutations. GLT-1 loss also caused an apparent compensatory increase in IDE activity in the liver, an organ that has been shown to regulate peripheral amyloid-β levels and expresses GLT-1. Taken together, these findings demonstrate that partial GLT-1 loss can cause insulin/Akt signaling abnormalities that are in keeping with those observed in AD.

  15. Acid-base transport in pancreas-new challenges

    DEFF Research Database (Denmark)

    Novak, Ivana; Haanes, Kristian Agmund; Wang, Jing

    2013-01-01

    Along the gastrointestinal tract a number of epithelia contribute with acid or basic secretions in order to aid digestive processes. The stomach and pancreas are the most extreme examples of acid (H+) and base (HCO-3) transporters, respectively. Nevertheless, they share the same challenges...... to consider in pancreas are the proton pumps (H-K-ATPases), as well as the calcium-activated K and Cl channels, such as K3.1 and TMEM16A/ANO1. Local regulators, such as purinergic signaling, fine-tune, and coordinate pancreatic secretion. Lastly, we speculate whether dys-regulation of acid-base transport...

  16. Cardioprotective effect of L-glutamate in obese type 2 diabetic Zucker fatty rats

    DEFF Research Database (Denmark)

    Povlsen, Jonas Agerlund; Løfgren, Bo; Rasmussen, Lars Ege

    2009-01-01

    (Wistar-Kyoto) and diabetic (Zucker diabetic fatty (ZDF)) rats, studied at 16 weeks of age. The infarct size (IS)/area-at-risk (AAR) ratio was the primary end-point. Expression of L-glutamate excitatory amino acid transporter (EAAT) 1 (mitochondrial) and EAAT3 (sarcolemmal) was determined by quantitative......1. Because diabetic hearts have an increased threshold for cardioprotection by ischaemic preconditioning (IPC), we hypothesized that protection by L-glutamate during reperfusion is restricted in Type 2 diabetic hearts. Previously, we found that L-glutamate-mediated postischaemic cardioprotection...... mimics IPC. 2. Rat hearts were studied in a Langendorff preparation perfused with Krebs'-Henseleit solution and subjected to 40 min global no-flow ischaemia, followed by 120 min reperfusion. L-Glutamate (0, 15 and 30 mmol/L) was added to the perfusate during reperfusion of hearts from non-diabetic...

  17. Potential Biomarker of L type Amino Acid Transporter 1 in Breast Cancer Progression

    International Nuclear Information System (INIS)

    Liang, Zhongxing; Cho, Heidi T.; Williams, Larry; Zhu, Aizhi; Liang, Ke; Huang, Ke; Wu, Hui; Jiang, Chunsu; Hong, Samuel; Crowe, Ronald; Goodman, Mark M.; Shim, Hyunsuk

    2011-01-01

    L type amino acid transporter 1 (LAT1) is essential for the transport of large neutral amino acids. However, its role in breast cancer growth remains largely unknown. The purpose of the study is to investigate whether LAT1 is a potential biomarker for the diagnosis and treatment of breast cancer. LAT1 mRNA and protein levels in breast cancer cell lines and tissues were analyzed. In addition, the effects of targeting LAT1 for the inhibition of breast cancer cell tumorigenesis were assessed with soft agar assay. The imaging of xenograft with 1 amino 3 [ 18F ]fluorocyclo butane 1 carboxylic acid ([ 18F ]FACBC) PET was assessed for its diagnostic biomarker potential. Normal breast tissue or low malignant cell lines expressed low levels of LAT1 mRNA and protein, while highly malignant cancer cell lines and high grade breast cancer tissue expressed high levels of LAT1. In addition, higher expression levels of LAT1 in breast cancer tissues were consistent with advanced stage breast cancer. Furtermore, the blockade of LAT1 with its inhibitor, 2 amino bicyclo[2.2.1]heptane 2 carboxylic acid (BCH), or the knockdown of LAT1 with siRNA, inhibited proliferation and tumorigenesis of breast cancer cells. A leucine analog, [ 18F ]FACBC, has been demonstrated to be an excellent PET tracer for the non invasive imaging og malignant breast cancer using an orthotopic animal model. The overexpression of LAT1 is required for the progression of breast cancer. LAT1 represents a potential biomarker for therapy and diagnosis of breast cancer. [ 18F ]FACBC that correlates with LAT1 function is a potential PET tracer for malignant breast tumor imaging

  18. Acid-base transport in pancreas – new challenges

    Directory of Open Access Journals (Sweden)

    Ivana eNovak

    2013-12-01

    Full Text Available Along the gastrointestinal tract a number of epithelia contribute with acid or basic secretions in order to aid digestive processes. The stomach and pancreas are the most extreme examples of acid (H+ and base (HCO3- transporters, respectively. Nevertheless, they share the same challenges of transporting acid and bases across epithelia and effectively regulating their intracellular pH. In this review, we will make use of comparative physiology to enlighten the cellular mechanisms of pancreatic HCO3- and fluid secretion, which is still challenging physiologists. Some of the novel transporters to consider in pancreas are the proton pumps (H+-K+-ATPases, as well as the calcium-activated K+ and Cl- channels, such as KCa3.1 and TMEM16A/ANO1. Local regulators, such as purinergic signalling, fine-tune and coordinate pancreatic secretion. Lastly, we speculate whether dys-regulation of acid-base transport contributes to pancreatic diseases including cystic fibrosis, pancreatitis and cancer.

  19. DNA methylation of amino acid transporter genes in the human placenta.

    Science.gov (United States)

    Simner, C; Novakovic, B; Lillycrop, K A; Bell, C G; Harvey, N C; Cooper, C; Saffery, R; Lewis, R M; Cleal, J K

    2017-12-01

    Placental transfer of amino acids via amino acid transporters is essential for fetal growth. Little is known about the epigenetic regulation of amino acid transporters in placenta. This study investigates the DNA methylation status of amino acid transporters and their expression across gestation in human placenta. BeWo cells were treated with 5-aza-2'-deoxycytidine to inhibit methylation and assess the effects on amino acid transporter gene expression. The DNA methylation levels of amino acid transporter genes in human placenta were determined across gestation using DNA methylation array data. Placental amino acid transporter gene expression across gestation was also analysed using data from publically available Gene Expression Omnibus data sets. The expression levels of these transporters at term were established using RNA sequencing data. Inhibition of DNA methylation in BeWo cells demonstrated that expression of specific amino acid transporters can be inversely associated with DNA methylation. Amino acid transporters expressed in term placenta generally showed low levels of promoter DNA methylation. Transporters with little or no expression in term placenta tended to be more highly methylated at gene promoter regions. The transporter genes SLC1A2, SLC1A3, SLC1A4, SLC7A5, SLC7A11 and SLC7A10 had significant changes in enhancer DNA methylation across gestation, as well as gene expression changes across gestation. This study implicates DNA methylation in the regulation of amino acid transporter gene expression. However, in human placenta, DNA methylation of these genes remains low across gestation and does not always play an obvious role in regulating gene expression, despite clear evidence for differential expression as gestation proceeds. Copyright © 2017. Published by Elsevier Ltd.

  20. A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

    International Nuclear Information System (INIS)

    Fukumoto, Shinya; Hanazono, Kiwamu; Fu, Dah-Renn; Endo, Yoshifumi; Kadosawa, Tsuyoshi; Iwano, Hidetomo; Uchide, Tsuyoshi

    2013-01-01

    Highlights: •LAT1 is highly expressed in tumors but at low levels in normal tissues. •We examine LAT1 expression and function in malignant melanoma (MM). •LAT1 expression in MM tissues and cell lines is higher than those in normal tissues. •LAT1 selective inhibitors inhibit amino acid uptake and cell growth in MM cells. •New chemotherapeutic protocols including LAT1 inhibitors are effective for treatment. -- Abstract: L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P 3 H]L-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM

  1. Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2).

    Science.gov (United States)

    Edwards, Noel; Anderson, Catriona M H; Gatfield, Kelly M; Jevons, Mark P; Ganapathy, Vadivel; Thwaites, David T

    2011-01-01

    The H(+)-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[(3)H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC(50) 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2. Copyright © 2010 Elsevier B.V. All rights

  2. Characterization of simvastatin acid uptake by organic anion transporting polypeptide 3A1 (OATP3A1) and influence of drug-drug interaction.

    Science.gov (United States)

    Atilano-Roque, Amandla; Joy, Melanie S

    2017-12-01

    Human organic anion transporting polypeptide 3A1 (OATP3A1) is predominately expressed in the heart. The ability of OATP3A1 to transport statins into cardiomyocytes is unknown, although other OATPs are known to mediate the uptake of statin drugs in liver. The pleiotropic effects and uptake of simvastatin acid were analyzed in primary human cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. Treatment with simvastatin acid reduced indoxyl sulfate-mediated reactive oxygen species and modulated OATP3A1 expression in cardiomyocytes and HEK293 cells transfected with the OATP3A1 gene. We observed a pH-dependent effect on OATP3A1 uptake, with more efficient simvastatin acid uptake at pH5.5 in HEK293 cells transfected with the OATP3A1 gene. The Michaelis-Menten constant (K m ) for simvastatin acid uptake by OATP3A1 was 0.017±0.002μM and the V max was 0.995±0.027fmol/min/10 5 cells. Uptake of simvastatin acid was significantly increased by known (benzylpenicillin and estrone-3-sulfate) and potential (indoxyl sulfate and cyclosporine) substrates of OATP3A1. In conclusion, the presence of OATP3A1 in cardiomyocytes suggests that this transporter may modulate the exposure of cardiac tissue to simvastatin acid due to its enrichment in cardiomyocytes. Increases in uptake of simvastatin acid by OATP3A1 when combined with OATP substrates suggest the potential for drug-drug interactions that could influence clinical outcomes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5).

    Science.gov (United States)

    Scalise, Mariafrancesca; Pochini, Lorena; Panni, Simona; Pingitore, Piero; Hedfalk, Kristina; Indiveri, Cesare

    2014-11-01

    The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na(+)-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K(+) gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na(+). Internal Na(+) exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.

  4. A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Shinya; Hanazono, Kiwamu [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Fu, Dah-Renn; Endo, Yoshifumi; Kadosawa, Tsuyoshi [Veterinary Oncology, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Iwano, Hidetomo [Veterinary Biochemistry, Department of Basic Veterinary Medicine, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan); Uchide, Tsuyoshi, E-mail: uchide@rakuno.ac.jp [Veterinary Internal Medicine, Department of Small Animal Clinical Sciences, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501 (Japan)

    2013-09-13

    Highlights: •LAT1 is highly expressed in tumors but at low levels in normal tissues. •We examine LAT1 expression and function in malignant melanoma (MM). •LAT1 expression in MM tissues and cell lines is higher than those in normal tissues. •LAT1 selective inhibitors inhibit amino acid uptake and cell growth in MM cells. •New chemotherapeutic protocols including LAT1 inhibitors are effective for treatment. -- Abstract: L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [{sup 3}H]L-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM

  5. CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1.

    Science.gov (United States)

    Huynh, Kevin W; Jiang, Jiansen; Abuladze, Natalia; Tsirulnikov, Kirill; Kao, Liyo; Shao, Xuesi; Newman, Debra; Azimov, Rustam; Pushkin, Alexander; Zhou, Z Hong; Kurtz, Ira

    2018-03-02

    Na + -coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na + -coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

  6. 5'-azido-N-1-naphthylphthalamic acid, a photolabile analog of the auxin transport inhibitor, N-1-naphthylphthalamic acid: synthesis and binding properties

    International Nuclear Information System (INIS)

    Voet, J.G.; Howley, K.; Shumsky, J.S.

    1987-01-01

    The polar transport of the plant growth regulator, auxin (indole-3-acetic acid, IAAH), is thought to involve the participation of several proteins in the plasma membrane, including a specific, saturable, voltage independent H + /IAA - efflux carrier located preferentially at the basal end of each cell. Auxin transport is specifically inhibited by the herbicide, N-1-naphthylphthalamic acid (NPA), which binds specifically to a protein in the plasma membrane, thought to be either the IAA - efflux carrier or an allosteric effector protein. They have synthesized and characterized a photolabile analog of NPA, 5'-azido-N-1-naphthylphthalamic acid (Az-NPA). This potential photoaffinity label for the NPA binding protein competes with 3 H-NPA for binding sites on Curcurbita pepo L. (zucchini) stem cell membranes with K/sub j/ = 1.5 x 10 -7 M. The K/sub i/ for NPA under these conditions is 2 x 10 -8 M, indicating that the affinity of Az-NPA for the membranes is only 7.5 fold lower than NPA. While the binding of 4.6 x 10 -6 M Az-NPA to NPA binding sites is reversible in the dark, exposure to light results in a 30% loss in 3 H-NPA binding ability. Pretreatment with 10 -4 M NPA protects the membranes against photodestruction of 3 H-NPA binding sites by Az-NPA, supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment

  7. Induction of amino acid transporters expression by endurance exercise in rat skeletal muscle

    International Nuclear Information System (INIS)

    Murakami, Taro; Yoshinaga, Mariko

    2013-01-01

    Highlights: •Regulation of amino acid transporter expression in working muscle remains unclear. •Expression of amino acid transporters for leucine were induced by a bout of exercise. •Requirement of leucine in muscle cells might regulate expression of its transporters. •This information is beneficial for understanding the muscle remodeling by exercise. -- Abstract: We here investigated whether an acute bout of endurance exercise would induce the expression of amino acid transporters that regulate leucine transport across plasma and lysosomal membranes in rat skeletal muscle. Rats ran on a motor-driven treadmill at a speed of 28 m/min for 90 min. Immediately after the exercise, we observed that expression of mRNAs encoding L-type amino acid transporter 1 (LAT1) and CD98 was induced in the gastrocnemius, soleus, and extensor digitorum longus (EDL) muscles. Sodium-coupled neutral amino acid transporter 2 (SNAT2) mRNA was also induced by the exercise in those three muscles. Expression of proton-assisted amino acid transporter 1 (PAT1) mRNA was slightly but not significantly induced by a single bout of exercise in soleus and EDL muscles. Exercise-induced mRNA expression of these amino acid transporters appeared to be attenuated by repeated bouts of the exercise. These results suggested that the expression of amino acid transporters for leucine may be induced in response to an increase in the requirement for this amino acid in the cells of working skeletal muscles

  8. Ascorbic acid and striatal transport of [3H]1-methyl-4-phenylpyridine (MPP+) and [3H]dopamine

    International Nuclear Information System (INIS)

    Debler, E.A.; Hashim, A.; Lajtha, A.; Sershen, H.

    1988-01-01

    The inhibition of uptake of [ 3 H]dopamine and [ 3 H]1-methyl-4-phenylpyridine (MPP + ) was examined in mouse striatal synaptosomal preparations. Kinetic analysis indicated that ascorbic acid is a noncompetitive inhibitor of [ 3 H]MPP + uptake. No inhibition of [ 3 H]dopamine uptake is observed. The dopamine uptake blockers, GBR-12909, cocaine, and mazindol strongly inhibit (IC 50 3 H]dopamine and [ 3 H]MPP + transport. Nicotine, its metabolites, and other tobacco alkaloids are weak inhibitors except 4-phenylpyridine and lobeline, which are moderate inhibitors of both [ 3 H]dopamine and [ 3 H]MPP + uptake. These similarities in potencies are in agreement with the suggestion that [ 3 H]MPP + and [ 3 H] are transported by the same carrier. The differences observed in the alteration of dopaminergic transport and mazindol binding by ascorbic acid suggest that ascorbic acid's effects on [ 3 H]MPP + transport are related to translocation and/or dissociation processes occurring subsequent to the initial binding event

  9. "Facilitated" amino acid transport is upregulated in brain tumors.

    Science.gov (United States)

    Miyagawa, T; Oku, T; Uehara, H; Desai, R; Beattie, B; Tjuvajev, J; Blasberg, R

    1998-05-01

    The goal of this study was to determine the magnitude of "facilitated" amino acid transport across tumor and brain capillaries and to evaluate whether amino acid transporter expression is "upregulated" in tumor vessels compared to capillaries in contralateral brain tissue. Aminocyclopentane carboxylic acid (ACPC), a non-metabolized [14C]-labeled amino acid, and a reference molecule for passive vascular permeability, [67Ga]-gallium-diethylenetriaminepentaacetic acid (Ga-DTPA), were used in these studies. Two experimental rat gliomas were studied (C6 and RG2). Brain tissue was rapidly processed for double label quantitative autoradiography 10 minutes after intravenous injection of ACPC and Ga-DTPA. Parametric images of blood-to-brain transport (K1ACPC and K1Ga-DTPA, microL/min/g) produced from the autoradiograms and the histology were obtained from the same tissue section. These three images were registered in an image array processor; regions of interest in tumor and contralateral brain were defined on morphologic criteria (histology) and were transferred to the autoradiographic images to obtain mean values. The facilitated component of ACPC transport (deltaK1ACPC) was calculated from the K1ACPC and K1Ga-DTPA data, and paired comparisons between tumor and contralateral brain were performed. ACPC flux, K1ACPC, across normal brain capillaries (22.6 +/- 8.1 microL/g/min) was >200-fold greater than that of Ga-DTPA (0.09 +/- 0.04 microL/g/min), and this difference was largely (approximately 90%) due to facilitated ACPC transport. Substantially higher K1ACPC values compared to corresponding K1DTPA values were also measured in C6 and RG2 gliomas. The deltaK1ACPC values for C6 glioma were more than twice that of contralateral brain cortex. K1ACPC and deltaK1ACPC values for RG2 gliomas was not significantly higher than that of contralateral cortex, although a approximately 2-fold difference in facilitated transport is obtained after normalization for differences in capillary

  10. Passive water and urea permeability of a human Na(+)-glutamate cotransporter expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Macaulay, Nanna; Gether, Ulrik; Klærke, Dan Arne

    2002-01-01

    to the K(0.5) value for glutamate activation of transport. The specific inhibitor DL-threo-beta-benzyloxyaspartate (TBOA) reduced the EAAT1-specific L(p) to 72 %. EAAT1 supported passive fluxes of [(14)C]urea and [(14)C]glycerol. The [(14)C]urea flux was increased in the presence of glutamate. The data...... suggest that the permeability depends on the conformational equilibrium of the EAAT1. At positive potentials and in the presence of Na(+) and glutamate, the pore is enlarged and water and urea penetrate more readily. The L(p) was larger when measured with urea or glycerol as osmolytes as compared...... with mannitol. Apparently, the properties of the pore are not uniform along its length. The outer section may accommodate urea and glycerol in an osmotically active form, giving rise to larger water fluxes. The physiological role of EAAT1 for water homeostasis in the central nervous system is discussed....

  11. Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. Strain 6803

    International Nuclear Information System (INIS)

    Labarre, J.; Thuriaux, P.; Chauvat, F.

    1987-01-01

    The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14 C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent K/sub m/ ranging from 6 to 60 μM) and of V/sub max/

  12. Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters

    International Nuclear Information System (INIS)

    Straka, Elisabeth; Ellinger, Isabella; Balthasar, Christina; Scheinast, Matthias; Schatz, Jasmin; Szattler, Tamara; Bleichert, Sonja; Saleh, Leila; Knöfler, Martin; Zeisler, Harald; Hengstschläger, Markus; Rosner, Margit; Salzer, Hans; Gundacker, Claudia

    2016-01-01

    Highlights: • It is known that MeHg is able to pass the placenta and to affect fetal brain development. • Uptake and efflux transporters were examined in human primary trophoblast cells and BeWo cells. • Involvement in mercury transfer was assessed by measurement of cellular mercury content upon siRNA mediated gene knockdown. • Localization of transporters was determined by immunofluorescence microscopy. • LAT1 and rBAT at the apical membrane of the syncytiotrophoblast (STB) are involved in MeHg uptake. • MRP1 located at basal membrane of STB mediates mercury efflux. - Abstract: Background: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. Objectives: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. Methods: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. Results: We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b 0,+ type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P < 0.05). Conclusion: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is

  13. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

    Science.gov (United States)

    Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

    2014-01-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

  14. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

    Science.gov (United States)

    Widdows, Kate L.; Panitchob, Nuttanont; Crocker, Ian P.; Please, Colin P.; Hanson, Mark A.; Sibley, Colin P.; Johnstone, Edward D.; Sengers, Bram G.; Lewis, Rohan M.; Glazier, Jocelyn D.

    2015-01-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [14C]l-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]l-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with l-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. PMID:25761365

  15. Water transport in the brain

    DEFF Research Database (Denmark)

    MacAulay, N; Hamann, S; Zeuthen, T

    2004-01-01

    the number of cotransporters per cell and the unit water permeability are high. For example, the Na(+)-glutamate cotransporter (EAAT1) has a unit water permeability one tenth of that of aquaporin (AQP) 1. Cotransporters are widely distributed in the brain and participate in several vital functions: inorganic...

  16. Increased Bile Acid Synthesis and Impaired Bile Acid Transport in Human Obesity

    OpenAIRE

    Haeusler, Rebecca A.; Camastra, Stefania; Nannipieri, Monica; Astiarraga, Brenno; Castro-Perez, Jose; Xie, Dan; Wang, Liangsu; Chakravarthy, Manu; Ferrannini, Ele

    2015-01-01

    We measured plasma bile acids, markers of bile acid synthesis, and expression of bile acid transporters in obese and nonobese subjects. We found that obesity was associated with increased bile acid synthesis and 12-hydroxylation, blunted response of plasma bile acids to insulin infusion or a mixed meal, and decreased expression of liver bile acid transporters.

  17. Transport of acidic amino acids by human jejunal brush-border membrane vesicles

    International Nuclear Information System (INIS)

    Rajendran, V.M.; Harig, J.M.; Adams, M.B.; Ramaswamy, K.

    1987-01-01

    This study characterizes the transport of radiolabeled acidic amino acids into brush-border membrane vesicles prepared from human jejunum. The uptakes of L-glutamic, L-aspartic, and D-aspartic acids were stimulated by a Na + gradient. Concentrative uptake (resulting in an overshoot phenomenon) of these dicarboxylic amino acids occurred when there was an outward K + gradient. In addition, increasing K + gradients resulted in enhanced uptake of L-glutamic acid. This K + requirement is somewhat specific as Rb + and Cs + could enhance uptake to a limited extent, whereas Li + and choline + showed no enhancement. The presence of a K + gradient did not affect the affinity of the carrier system for L-glutamic acid but it did increase the V/sub max/. The presence of extravesicular anions having differing membrane permeabilities did not altar L-glutamic acid uptake indicating an absence of an effect of membrane potential on the transport process. Finally, the human transport system for L-glutamic acid appears to be specific for acidic amino acids as demonstrated by inhibition studies. The studies demonstrate a transport system in human jejunum specific for acidic amino acids that is energized by an inward Na + gradient and an outward K + gradient

  18. Characterisation of L-Type Amino Acid Transporter 1 (LAT1 Expression in Human Skeletal Muscle by Immunofluorescent Microscopy

    Directory of Open Access Journals (Sweden)

    Nathan Hodson

    2017-12-01

    Full Text Available The branch chain amino acid leucine is a potent stimulator of protein synthesis in skeletal muscle. Leucine rapidly enters the cell via the L-Type Amino Acid Transporter 1 (LAT1; however, little is known regarding the localisation and distribution of this transporter in human skeletal muscle. Therefore, we applied immunofluorescence staining approaches to visualise LAT1 in wild type (WT and LAT1 muscle-specific knockout (mKO mice, in addition to basal human skeletal muscle samples. LAT1 positive staining was visually greater in WT muscles compared to mKO muscle. In human skeletal muscle, positive LAT1 staining was noted close to the sarcolemmal membrane (dystrophin positive staining, with a greater staining intensity for LAT1 observed in the sarcoplasmic regions of type II fibres (those not stained positively for myosin heavy-chain 1, Type II—25.07 ± 5.93, Type I—13.71 ± 1.98, p < 0.01, suggesting a greater abundance of this protein in these fibres. Finally, we observed association with LAT1 and endothelial nitric oxide synthase (eNOS, suggesting LAT1 association close to the microvasculature. This is the first study to visualise the distribution and localisation of LAT1 in human skeletal muscle. As such, this approach provides a validated experimental platform to study the role and regulation of LAT1 in human skeletal muscle in response to various physiological and pathophysiological models.

  19. Control of amino acid transport coordinates metabolic reprogramming in T-cell malignancy.

    Science.gov (United States)

    Grzes, K M; Swamy, M; Hukelmann, J L; Emslie, E; Sinclair, L V; Cantrell, D A

    2017-12-01

    This study explores the regulation and importance of System L amino acid transport in a murine model of T-cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN -/- T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T-cell malignancies, highlighting how a single amino acid transporter can have a key role.

  20. Role of the Intestinal Bile Acid Transporters in Bile Acid and Drug Disposition

    Science.gov (United States)

    Dawson, Paul A.

    2011-01-01

    Membrane transporters expressed by the hepatocyte and enterocyte play critical roles in maintaining the enterohepatic circulation of bile acids, an effective recycling and conservation mechanism that largely restricts these potentially cytotoxic detergents to the intestinal and hepatobiliary compartments. In doing so, the hepatic and enterocyte transport systems ensure a continuous supply of bile acids to be used repeatedly during the digestion of multiple meals throughout the day. Absorption of bile acids from the intestinal lumen and export into the portal circulation is mediated by a series of transporters expressed on the enterocyte apical and basolateral membranes. The ileal apical sodium-dependent bile acid cotransporter (abbreviated ASBT; gene symbol, SLC10A2) is responsible for the initial uptake of bile acids across the enterocyte brush border membrane. The bile acids are then efficiently shuttled across the cell and exported across the basolateral membrane by the heteromeric Organic Solute Transporter, OSTα-OSTβ. This chapter briefly reviews the tissue expression, physiology, genetics, pathophysiology, and transport properties of the ASBT and OSTα-OSTα. In addition, the chapter discusses the relationship between the intestinal bile acid transporters and drug metabolism, including development of ASBT inhibitors as novel hypocholesterolemic or hepatoprotective agents, prodrug targeting of the ASBT to increase oral bioavailability, and involvement of the intestinal bile acid transporters in drug absorption and drug-drug interactions. PMID:21103970

  1. Detecting Electron Transport of Amino Acids by Using Conductance Measurement

    Directory of Open Access Journals (Sweden)

    Wei-Qiong Li

    2017-04-01

    Full Text Available The single molecular conductance of amino acids was measured by a scanning tunneling microscope (STM break junction. Conductance measurement of alanine gives out two conductance values at 10−1.85 G0 (1095 nS and 10−3.7 G0 (15.5 nS, while similar conductance values are also observed for aspartic acid and glutamic acid, which have one more carboxylic acid group compared with alanine. This may show that the backbone of NH2–C–COOH is the primary means of electron transport in the molecular junction of aspartic acid and glutamic acid. However, NH2–C–COOH is not the primary means of electron transport in the methionine junction, which may be caused by the strong interaction of the Au–SMe (methyl sulfide bond for the methionine junction. The current work reveals the important role of the anchoring group in the electron transport in different amino acids junctions.

  2. Retinal glutamate transporter changes in experimental glaucoma and after optic nerve transection in the rat.

    Science.gov (United States)

    Martin, Keith R G; Levkovitch-Verbin, Hana; Valenta, Danielle; Baumrind, Lisa; Pease, Mary Ellen; Quigley, Harry A

    2002-07-01

    High levels of glutamate can be toxic to retinal ganglion cells. Effective buffering of extracellular glutamate by retinal glutamate transporters is therefore important. This study was conducted to investigate whether glutamate transporter changes occur with two models of optic nerve injury in the rat. Glaucoma was induced in one eye of 35 adult Wistar rats by translimbal diode laser treatment to the trabecular meshwork. Twenty-five more rats underwent unilateral optic nerve transection. Two glutamate transporters, GLAST (EAAT-1) and GLT-1 (EAAT-2), were studied by immunohistochemistry and quantitative Western blot analysis. Treated and control eyes were compared 3 days and 1, 4, and 6 weeks after injury. Optic nerve damage was assessed semiquantitatively in epoxy-embedded optic nerve cross sections. Trabecular laser treatment resulted in moderate intraocular pressure (IOP) elevation in all animals. After 1 to 6 weeks of experimental glaucoma, all treated eyes had significant optic nerve damage. Glutamate transporter changes were not detected by immunohistochemistry. Western blot analysis demonstrated significantly reduced GLT-1 in glaucomatous eyes compared with control eyes at 3 days (29.3% +/- 6.7%, P = 0.01), 1 week (55.5% +/- 13.6%, P = 0.02), 4 weeks (27.2% +/- 10.1%, P = 0.05), and 6 weeks (38.1% +/- 7.9%, P = 0.01; mean reduction +/- SEM, paired t-tests, n = 5 animals per group, four duplicate Western blot analyses per eye). The magnitude of the reduction in GLT-1 correlated significantly with mean IOP in the glaucomatous eye (r(2) = 0.31, P = 0.01, linear regression). GLAST was significantly reduced (33.8% +/- 8.1%, mean +/- SEM) after 4 weeks of elevated IOP (P = 0.01, paired t-test, n = 5 animals per group). In contrast to glaucoma, optic nerve transection resulted in an increase in GLT-1 compared with the control eye (P = 0.01, paired t-test, n = 15 animals). There was no significant change in GLAST after transection. GLT-1 and GLAST were significantly

  3. Discovery and Validation of Pyridoxic Acid and Homovanillic Acid as Novel Endogenous Plasma Biomarkers of Organic Anion Transporter (OAT) 1 and OAT3 in Cynomolgus Monkeys.

    Science.gov (United States)

    Shen, Hong; Nelson, David M; Oliveira, Regina V; Zhang, Yueping; Mcnaney, Colleen A; Gu, Xiaomei; Chen, Weiqi; Su, Ching; Reily, Michael D; Shipkova, Petia A; Gan, Jinping; Lai, Yurong; Marathe, Punit; Humphreys, W Griffith

    2018-02-01

    Perturbation of organic anion transporter (OAT) 1- and OAT3-mediated transport can alter the exposure, efficacy, and safety of drugs. Although there have been reports of the endogenous biomarkers for OAT1/3, none of these have all of the characteristics required for a clinical useful biomarker. Cynomolgus monkeys were treated with intravenous probenecid (PROB) at a dose of 40 mg/kg in this study. As expected, PROB increased the area under the plasma concentration-time curve (AUC) of coadministered furosemide, a known substrate of OAT1 and OAT3, by 4.1-fold, consistent with the values reported in humans (3.1- to 3.7-fold). Of the 233 plasma metabolites analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics method, 29 metabolites, including pyridoxic acid (PDA) and homovanillic acid (HVA), were significantly increased after either 1 or 3 hours in plasma from the monkeys pretreated with PROB compared with the treated animals. The plasma of animals was then subjected to targeted LC-MS/MS analysis, which confirmed that the PDA and HVA AUCs increased by approximately 2- to 3-fold by PROB pretreatments. PROB also increased the plasma concentrations of hexadecanedioic acid (HDA) and tetradecanedioic acid (TDA), although the increases were not statistically significant. Moreover, transporter profiling assessed using stable cell lines constitutively expressing transporters demonstrated that PDA and HVA are substrates for human OAT1, OAT3, OAT2 (HVA), and OAT4 (PDA), but not OCT2, MATE1, MATE2K, OATP1B1, OATP1B3, and sodium taurocholate cotransporting polypeptide. Collectively, these findings suggest that PDA and HVA might serve as blood-based endogenous probes of cynomolgus monkey OAT1 and OAT3, and investigation of PDA and HVA as circulating endogenous biomarkers of human OAT1 and OAT3 function is warranted. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  4. Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

    International Nuclear Information System (INIS)

    Lasry, Inbal; Berman, Bluma; Glaser, Fabian; Jansen, Gerrit; Assaraf, Yehuda G.

    2009-01-01

    The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.

  5. The Implication of PGC-1α on Fatty Acid Transport across Plasma and Mitochondrial Membranes in the Insulin Sensitive Tissues

    Directory of Open Access Journals (Sweden)

    Elżbieta Supruniuk

    2017-11-01

    Full Text Available PGC-1α coactivator plays a decisive role in the maintenance of lipid balance via engagement in numerous metabolic processes (i.e., Krebs cycle, β-oxidation, oxidative phosphorylation and electron transport chain. It constitutes a link between fatty acids import and their complete oxidation or conversion into bioactive fractions through the coordination of both the expression and subcellular relocation of the proteins involved in fatty acid transmembrane movement. Studies on cell lines and/or animal models highlighted the existence of an upregulation of the total and mitochondrial FAT/CD36, FABPpm and FATPs content in skeletal muscle in response to PGC-1α stimulation. On the other hand, the association between PGC-1α level or activity and the fatty acids transport in the heart and adipocytes is still elusive. So far, the effects of PGC-1α on the total and sarcolemmal expression of FAT/CD36, FATP1, and FABPpm in cardiomyocytes have been shown to vary in relation to the type of PPAR that was coactivated. In brown adipose tissue (BAT PGC-1α knockdown was linked with a decreased level of lipid metabolizing enzymes and fatty acid transporters (FAT/CD36, FABP3, whereas the results obtained for white adipose tissue (WAT remain contradictory. Furthermore, dysregulation in lipid turnover is often associated with insulin intolerance, which suggests the coactivator's potential role as a therapeutic target.

  6. Acidic and uncharged polar residues in the consensus motifs of the yeast Ca2+ transporter Gdt1p are required for calcium transport.

    Science.gov (United States)

    Colinet, Anne-Sophie; Thines, Louise; Deschamps, Antoine; Flémal, Gaëlle; Demaegd, Didier; Morsomme, Pierre

    2017-07-01

    The UPF0016 family is a recently identified group of poorly characterized membrane proteins whose function is conserved through evolution and that are defined by the presence of 1 or 2 copies of the E-φ-G-D-[KR]-[TS] consensus motif in their transmembrane domain. We showed that 2 members of this family, the human TMEM165 and the budding yeast Gdt1p, are functionally related and are likely to form a new group of Ca 2+ transporters. Mutations in TMEM165 have been demonstrated to cause a new type of rare human genetic diseases denominated as Congenital Disorders of Glycosylation. Using site-directed mutagenesis, we generated 17 mutations in the yeast Golgi-localized Ca 2+ transporter Gdt1p. Single alanine substitutions were targeted to the highly conserved consensus motifs, 4 acidic residues localized in the central cytosolic loop, and the arginine at position 71. The mutants were screened in a yeast strain devoid of both the endogenous Gdt1p exchanger and Pmr1p, the Ca 2+ -ATPase of the Golgi apparatus. We show here that acidic and polar uncharged residues of the consensus motifs play a crucial role in calcium tolerance and calcium transport activity and are therefore likely to be architectural components of the cation binding site of Gdt1p. Importantly, we confirm the essential role of the E53 residue whose mutation in humans triggers congenital disorders of glycosylation. © 2017 John Wiley & Sons Ltd.

  7. Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Khaleda, Laila; Park, Hee Jin; Yun, Dae-Jin; Jeon, Jong-Rok; Kim, Min Gab; Cha, Joon-Yung; Kim, Woe-Yeon

    2017-12-31

    Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

  8. Intestinal drug transport via the proton-coupled amino acid transporter PAT1 (SLC36A1) is inhibited by Gly-X(aa) dipeptides

    DEFF Research Database (Denmark)

    Frølund, Sidsel; Langthaler, Louise; Kall, Morten A

    2012-01-01

    -Sar as substrates of the amino acid transporter PAT1. The aim of the present study is to investigate if other Gly-containing dipeptides interact with PAT1, and whether they can inhibit PAT1 mediated drug absorption, in vitro and in vivo. The in vitro methods included two-electrode voltage clamp measurements on h...... of different dipeptides. The in vivo part consisted of a pharmacokinetic study in rats following oral administration of gaboxadol and preadministration of 200 mg/kg dipeptide. The results showed that in hPAT1 expressing oocytes Gly-Tyr, Gly-Pro, and Gly-Phe inhibited currents induced by drug substances......, the present study identifies selected dipeptides as inhibitors of PAT1 mediated drug absorption in various in vitro models....

  9. Inhibitors of glutamate dehydrogenase block sodium-dependent glutamate uptake in rat brain membranes

    Directory of Open Access Journals (Sweden)

    Brendan S Whitelaw

    2013-09-01

    Full Text Available We recently found evidence for anatomic and physical linkages between the astroglial Na+-dependent glutamate transporters (GLT-1/EAAT2 and GLAST/EAAT1 and mitochondria. In these same studies, we found that the glutamate dehydrogenase (GDH inhibitor, epigallocatechin-monogallate (EGCG, inhibits both glutamate oxidation and Na+-dependent glutamate uptake in astrocytes. In the present study, we extend this finding by exploring the effects of EGCG on Na+-dependent L-[3H]-glutamate (Glu uptake in crude membranes (P2 prepared from rat brain cortex. In this preparation, uptake is almost exclusively mediated by GLT-1. EGCG inhibited L-[3H]-Glu uptake in cortical membranes with an IC50 value of 230 µM. We also studied the effects of two additional inhibitors of GDH, hexachlorophene (HCP and bithionol (BTH. Both of these compounds also caused concentration-dependent inhibition of glutamate uptake in cortical membranes. Pre-incubating with HCP for up to 15 min had no greater effect than that observed with no pre-incubation, showing that the effects occur rapidly. HCP decreased the Vmax for glutamate uptake without changing the Km, consistent with a non-competitive mechanism of action. EGCG, HCP, and BTH also inhibited Na+-dependent transport of D-[3H]-aspartate (Asp, a non-metabolizable substrate, and [3H]-γ-aminobutyric acid (GABA. In contrast to the forebrain, glutamate uptake in crude cerebellar membranes (P2 is likely mediated by GLAST (EAAT1. Therefore, the effects of these compounds were examined in cerebellar membranes. In this region, none of these compounds had any effect on uptake of either L-[3H]-Glu or D-[3H]-Asp, but they all inhibited [3H]-GABA uptake. Together these studies suggest that GDH is preferentially required for glutamate uptake in forebrain as compared to cerebellum, and GDH may be required for GABA uptake as well. They also provide further evidence for a functional linkage between glutamate transport and mitochondria.

  10. Metabolic reprogramming through fatty acid transport protein 1 (FATP1 regulates macrophage inflammatory potential and adipose inflammation

    Directory of Open Access Journals (Sweden)

    Amy R. Johnson

    2016-07-01

    Full Text Available Objective: A novel approach to regulate obesity-associated adipose inflammation may be through metabolic reprogramming of macrophages (MΦs. Broadly speaking, MΦs dependent on glucose are pro-inflammatory, classically activated MΦs (CAM, which contribute to adipose inflammation and insulin resistance. In contrast, MΦs that primarily metabolize fatty acids are alternatively activated MΦs (AAM and maintain tissue insulin sensitivity. In actuality, there is much flexibility and overlap in the CAM-AAM spectrum in vivo dependent upon various stimuli in the microenvironment. We hypothesized that specific lipid trafficking proteins, e.g. fatty acid transport protein 1 (FATP1, would direct MΦ fatty acid transport and metabolism to limit inflammation and contribute to the maintenance of adipose tissue homeostasis. Methods: Bone marrow derived MΦs (BMDMs from Fatp1−/− and Fatp1+/+ mice were used to investigate FATP1-dependent substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. We also generated C57BL/6J chimeric mice by bone marrow transplant specifically lacking hematopoetic FATP1 (Fatp1B−/− and controls Fatp1B+/+. Mice were challenged by high fat diet (HFD or low fat diet (LFD and analyses including MRI, glucose and insulin tolerance tests, flow cytometric, histologic, and protein quantification assays were conducted. Finally, an FATP1-overexpressing RAW 264.7 MΦ cell line (FATP1-OE and empty vector control (FATP1-EV were developed as a gain of function model to test effects on substrate metabolism, bioenergetics, metabolomics, and inflammatory responses. Results: Fatp1 is downregulated with pro-inflammatory stimulation of MΦs. Fatp1−/− BMDMs and FATP1-OE RAW 264.7 MΦs demonstrated that FATP1 reciprocally controled metabolic flexibility, i.e. lipid and glucose metabolism, which was associated with inflammatory response. Supporting our previous work demonstrating the positive relationship between glucose

  11. Transportation impact analysis for the shipment of low specific activity nitric acid. Revisison 1

    International Nuclear Information System (INIS)

    Green, J.R.

    1995-01-01

    This is in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes potential toxicological and radiological risks associated with transportation of PUREX Facility LSA Nitric Acid from the Hanford Site to Portsmouth VA, Baltimore MD, and Port Elizabeth NJ

  12. Transportation impact analysis for the shipment of low specific activity nitric acid. Revisison 1

    Energy Technology Data Exchange (ETDEWEB)

    Green, J.R.

    1995-05-16

    This is in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes potential toxicological and radiological risks associated with transportation of PUREX Facility LSA Nitric Acid from the Hanford Site to Portsmouth VA, Baltimore MD, and Port Elizabeth NJ.

  13. Effect of common polymorphisms of the farnesoid X receptor and bile acid transporters on the pharmacokinetics of ursodeoxycholic acid.

    Science.gov (United States)

    Hu, Miao; Fok, Benny S P; Wo, Siu-Kwan; Lee, Vincent H L; Zuo, Zhong; Tomlinson, Brian

    2016-01-01

    Ursodeoxycholic acid (UDCA), a natural, dihydroxy bile acid, promotes gallstone dissolution and has been attributed with several other beneficial effects. The farnesoid X receptor (FXR) may influence the pharmacokinetics of UDCA by modulating the expression of bile acid transporters. This exploratory study examined whether common functional polymorphisms in FXR and in bile acid transporter genes affect the pharmacokinetics of exogenous UDCA. Polymorphisms in genes for transporters involved in bile acid transport, solute carrier organic anion 1B1 (SLCO1B1) 388A>G and 521T>C, solute carrier 10A1 (SLC10A1) 800 C>T and ATP-binding cassette B11 (ABCB11) 1331T>C, and the FXR -1G>T polymorphism were genotyped in 26 male Chinese subjects who ingested single oral 500-mg doses of UDCA. Plasma concentrations of UDCA and its major conjugate metabolite glycoursodeoxycholic acid (GUDCA) were determined. The mean systemic exposure of UDCA was higher in the five subjects with one copy of the FXR -1G>T variant allele than in those homozygous for the wild-type allele (n = 21) (AUC0-24 h : 38.5 ± 28.2 vs. 20.9 ± 8.0 μg h/mL, P = 0.021), but this difference appeared mainly due to one outlier with the -1GT genotype and elevated baseline and post-treatment UDCA concentrations. After excluding the outlier, body weight was the only factor associated with plasma concentrations of UDCA and there were no significant associations with the other polymorphisms examined. None of the polymorphisms affected the pharmacokinetics of GUDCA. This study showed that the common polymorphisms in bile acid transporters had no significant effect on the pharmacokinetics of exogenous UDCA but an effect of the FXR polymorphism cannot be excluded. © 2015 Wiley Publishing Asia Pty Ltd.

  14. Extensive neuroadaptive changes in cortical gene-transcript expressions of the glutamate system in response to repeated intermittent MDMA administration in adolescent rats

    Directory of Open Access Journals (Sweden)

    Malki Rana

    2008-04-01

    Full Text Available Abstract Background Many studies have focused on the implication of the serotonin and dopamine systems in neuroadaptive responses to the recreational drug 3,4-methylenedioxy-metamphetamine (MDMA. Less attention has been given to the major excitatory neurotransmitter glutamate known to be implicated in schizophrenia and drug addiction. The aim of the present study was to investigate the effect of repeated intermittent MDMA administration upon gene-transcript expression of the glutamate transporters (EAAT1, EAAT2-1, EAAT2-2, the glutamate receptor subunits of AMPA (GluR1, GluR2, GluR3, the glutamate receptor subunits of NMDA (NR1, NR2A and NR2B, as well as metabotropic glutamate receptors (mGluR1, mGluR2, mGluR3, mGluR5 in six different brain regions. Adolescent male Sprague Dawley rats received MDMA at the doses of 3 × 1 and 3 × 5 mg/kg/day, or 3× vehicle 3 hours apart, every 7th day for 4 weeks. The gene-transcript levels were assessed using real-time PCR validated with a range of housekeeping genes. Results The findings showed pronounced enhancements in gene-transcript expression of GluR2, mGluR1, mGluR5, NR1, NR2A, NR2B, EAAT1, and EAAT2-2 in the cortex at bregma +1.6. In the caudate putamen, mRNA levels of GluR3, NR2A, and NR2B receptor subunits were significantly increased. In contrast, the gene-transcript expression of GluR1 was reduced in the hippocampus. In the hypothalamus, there was a significant increase of GluR1, GluR3, mGluR1, and mGluR3 gene-transcript expressions. Conclusion Repeated intermittent MDMA administration induces neuroadaptive changes in gene-transcript expressions of glutamatergic NMDA and AMPA receptor subunits, metabotropic receptors and transporters in regions of the brain regulating reward-related associative learning, cognition, and memory and neuro-endocrine functions.

  15. Intracellular pH regulation by acid-base transporters in mammalian neurons

    Science.gov (United States)

    Ruffin, Vernon A.; Salameh, Ahlam I.; Boron, Walter F.; Parker, Mark D.

    2014-01-01

    Intracellular pH (pHi) regulation in the brain is important in both physiological and physiopathological conditions because changes in pHi generally result in altered neuronal excitability. In this review, we will cover 4 major areas: (1) The effect of pHi on cellular processes in the brain, including channel activity and neuronal excitability. (2) pHi homeostasis and how it is determined by the balance between rates of acid loading (JL) and extrusion (JE). The balance between JE and JL determine steady-state pHi, as well as the ability of the cell to defend pHi in the face of extracellular acid-base disturbances (e.g., metabolic acidosis). (3) The properties and importance of members of the SLC4 and SLC9 families of acid-base transporters expressed in the brain that contribute to JL (namely the Cl-HCO3 exchanger AE3) and JE (the Na-H exchangers NHE1, NHE3, and NHE5 as well as the Na+- coupled HCO3− transporters NBCe1, NBCn1, NDCBE, and NBCn2). (4) The effect of acid-base disturbances on neuronal function and the roles of acid-base transporters in defending neuronal pHi under physiopathologic conditions. PMID:24592239

  16. Transport in Halobacterium Halobium: Light-Induced Cation-Gradients, Amino Acid Transport Kinetics, and Properties of Transport Carriers

    Science.gov (United States)

    Lanyi, Janos K.

    1977-01-01

    Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na(+). Measurements of Na-22 flux, exterior pH change, and membrane potential, Delta(psi) (with the dye 3,3'-dipentyloxadicarbocyanine) indicate that the means of Na(+) transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H(+)/Na(++ greater than 1). The resulting large chemical gradient for Na(+) (outside much greater than inside), as well as the membrane potential, will drive the transport of 18 amino acids. The I9th, glutamate, is unique in that its accumulation is indifferent to Delta(psi): this amino acid is transported only when a chemical gradient for Na(+) is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H+() collapses within 1 min, while the large Na(+) gradient and glutamate transporting activity persists for 10- 15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na(+), arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V(sub max) and K(sub m) comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na(+), in an electrically neutral fashion, so that only the chemical component of the Na(+) gradient is a driving force.

  17. Characterization of a novel sialic acid transporter of the sodium solute symporter (SSS) family and in vivo comparison with known bacterial sialic acid transporters.

    Science.gov (United States)

    Severi, Emmanuele; Hosie, Arthur H F; Hawkhead, Judith A; Thomas, Gavin H

    2010-03-01

    The function of sialic acids in the biology of bacterial pathogens is reflected by the diverse range of solute transporters that can recognize these sugar acids. Here, we use an Escherichia coliDeltananT strain to characterize the function of known and proposed bacterial sialic acid transporters. We discover that the STM1128 gene from Salmonella enterica serovar Typhimurium, which encodes a member of the sodium solute symporter family, is able to restore growth on sialic acid to the DeltananT strain and is able to transport [(14)C]-sialic acid. Using the DeltananT genetic background, we performed a direct in vivo comparison of the transport properties of the STM1128 protein with those of sialic acid transporters of the major facilitator superfamily and tripartite ATP-independent periplasmic families, E. coli NanT and Haemophilus influenzae SiaPQM, respectively. This revealed that both STM1128 and SiaPQM are sodium-dependent and, unlike SiaPQM, both STM1128 and NanT are reversible secondary carriers, demonstrating qualitative functional differences in the properties of sialic acid transporters used by bacteria that colonize humans.

  18. Breed and species comparison of amino acid transport variation in equine erythrocytes.

    Science.gov (United States)

    Fincham, D A; Young, J D; Mason, D K; Collins, E A; Snow, D H

    1985-05-01

    The amino acid permeability of red blood cells from Equus caballus (thoroughbred, Arab, shire and pony), E przewalskii (Przewalski's horse), E asinus (donkey and mule) and E burchelli (common or plains zebra) was measured. Individual animals exhibited stable but widely differing rates of L-[U-14C]alanine uptake in the range 5 to 1554 mumol (litre cells)-1 h-1 (0.2 mM extracellular L-alanine, 37 degrees C). Of the thoroughbreds tested, 30 per cent had red blood cells which were essentially impermeable to L-alanine (5 to 10 mumol (litre cells)-1 h-1, giving transport rates similar to those found previously in amino acid transport-deficient sheep erythrocytes. In contrast, only 3 per cent of the ponies tested had red blood cells impermeable to L-alanine. No cases of erythrocyte amino acid transport deficiency were found in the other horse breeds and species tested.

  19. Fatty acid transport protein 1 regulates retinoid metabolism and photoreceptor development in mouse retina.

    Directory of Open Access Journals (Sweden)

    Aurélie Cubizolle

    Full Text Available In retinal pigment epithelium (RPE, RPE65 catalyzes the isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol in the visual cycle and controls the rhodopsin regeneration rate. However, the mechanisms by which these processes are regulated are still unclear. Fatty Acid Transport Protein 1 (FATP1 is involved in fatty acid uptake and lipid metabolism in a variety of cell types. FATP1 co-localizes with RPE65 in RPE and inhibits its isomerase activity in vitro. Here, we further investigated the role of FATP1 in the visual cycle using transgenic mice that overexpress human FATP1 specifically in the RPE (hFATP1TG mice. The mice displayed no delay in the kinetics of regeneration of the visual chromophore 11-cis-retinal after photobleaching and had no defects in light sensitivity. However, the total retinoid content was higher in the hFATP1TG mice than in wild type mice, and the transgenic mice also displayed an age-related accumulation (up to 40% of all-trans-retinal and retinyl esters that was not observed in control mice. Consistent with these results, hFATP1TG mice were more susceptible to light-induced photoreceptor degeneration. hFATP1 overexpression also induced an ~3.5-fold increase in retinosome autofluorescence, as measured by two-photon microscopy. Interestingly, hFATP1TG retina contained ~25% more photoreceptor cells and ~35% longer outer segments than wild type mice, revealing a non-cell-autonomous effect of hFATP1 expressed in the RPE. These data are the first to show that FATP1-mediated fatty acid uptake in the RPE controls both retinoid metabolism in the outer retina and photoreceptor development.

  20. Enhanced absorption and growth inhibition with amino acid monoester prodrugs of floxuridine by targeting hPEPT1 transporters.

    Science.gov (United States)

    Tsume, Yasuhiro; Vig, Balvinder S; Sun, Jing; Landowski, Christopher P; Hilfinger, John M; Ramachandran, Chandrasekharan; Amidon, Gordon L

    2008-06-28

    A series of amino acid monoester prodrugs of floxuridine was synthesized and evaluated for the improvement of oral bioavailability and the feasibility of target drug delivery via oligopeptide transporters. All floxuridine 5'-amino acid monoester prodrugs exhibited PEPT1 affinity, with inhibition coefficients of Gly-Sar uptake (IC50) ranging from 0.7 - 2.3 mM in Caco-2 and 2.0 - 4.8 mM in AsPC-1 cells, while that of floxuridine was 7.3 mM and 6.3 mM, respectively. Caco-2 membrane permeabilities of floxuridine prodrugs (1.01 - 5.31 x 10(-6 )cm/sec) and floxuridine (0.48 x 10(-6 )cm/sec) were much higher than that of 5-FU (0.038 x 10(-6) cm/sec). MDCK cells stably transfected with the human oligopeptide transporter PEPT1 (MDCK/hPEPT1) exhibited enhanced cell growth inhibition in the presence of the prodrugs. This prodrug strategy offers great potential, not only for increased drug absorption but also for improved tumor selectivity and drug efficacy.

  1. Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Uemura, Satoshi; Mochizuki, Takahiro; Kurosaka, Goyu; Hashimoto, Takanori; Masukawa, Yuki; Abe, Fumiyoshi

    2017-10-01

    Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Nature of the elements transporting long-chain fatty acids through the red cell membrane

    DEFF Research Database (Denmark)

    Bojesen, Inge Norby; Bojesen, Eigil

    1998-01-01

    Docosahexaenoic acid, linoleic acid, red cell membrane, transporting elements, transport kinetics, fatty acid transport......Docosahexaenoic acid, linoleic acid, red cell membrane, transporting elements, transport kinetics, fatty acid transport...

  3. Specific lysosomal transport of small neutral amino acids

    International Nuclear Information System (INIS)

    Pisoni, R.L.; Flickinger, K.S.; Thoene, J.G.; Christensen, H.N.

    1986-01-01

    Studies of amino acid exodus from lysosomes have allowed us previously to describe transport systems specific for cystine and another for cationic amino acids in fibroblast lysosomes. They are now able to study amino acid uptake into highly purified fibroblast lysosomes obtained by separating crude granular fraction on gradients formed by centrifugation in 35% isoosmotic Percoll solutions. Analog inhibition and saturation studies indicate that L-[ 14 C]proline (50 μM) uptake by fibroblast lysosomes at 37 0 C in 50 mM citrate/tris pH 7.0 buffer containing 0.25 M sucrose is mediated by two transport systems, one largely specific for L-proline and the other for which transport is shared with small neutral amino acids such as alanine, serine and threonine. At 7 mM, L-proline inhibits L-[ 14 C]proline uptake almost completely, whereas ala, ser, val, thr, gly, N-methylalanine and sarcosine inhibit proline uptake by 50-65%. The system shared by alanine, serine and threonine is further characterized by these amino acids strongly inhibiting the uptakes of each other. Lysosomal proline transport is selective for the L-isomer of the amino acid, and is scarcely inhibited by 7 mM arg, glu, asp, leu, phe, his, met, (methylamino) isobutyrate, betaine or N,N-dimethylglycine. Cis or trans-4-hydroxy-L-proline inhibit proline uptake only slightly. In sharp contrast to the fibroblast plasma membrane in which Na + is required for most proline and alanine transport, lysosomal uptake of these amino acids occurs independently of Na +

  4. Regional amino acid transport into brain during diabetes: Effect of plasma amino acids

    International Nuclear Information System (INIS)

    Mans, A.M.; DeJoseph, M.R.; Davis, D.W.; Hawkins, R.A.

    1987-01-01

    Transport of phenylalanine and lysine into the brain was measured in 4-wk streptozotocin-diabetic rats to assess the effect on the neutral and basic amino acid transport systems at the blood-brain barrier. Amino acid concentrations in plasma and brain were also measured. Regional permeability-times-surface area (PS) products and influx were determined using a continuous infusion method and quantitative autoradiography. The PS of phenylalanine was decreased by an average of 40% throughout the entire brain. Influx was depressed by 35%. The PS of lysine was increased by an average of 44%, but the influx was decreased by 27%. Several plasma neutral amino acids (branched chain) were increased, whereas all basic amino acids were decreased. Brain tryptophan, phenylalanine, tyrosine, methionine, and lysine contents were markedly decreased. The transport changes were almost entirely accounted for by the alterations in the concentrations of the plasma amino acids that compete for the neutral and basic amino acid carriers. The reduced influx could be responsible for the low brain content of some essential amino acids, with possibly deleterious consequences for brain functions

  5. Proton transport properties in zwitterion blends with Brønsted acids.

    Science.gov (United States)

    Yoshizawa-Fujita, Masahiro; Byrne, Nolene; Forsyth, Maria; MacFarlane, Douglas R; Ohno, Hiroyuki

    2010-12-16

    We describe zwitterion, 3-(1-butyl-1H-imidazol-3-ium-3-yl)propane-1-sulfonate (Bimps), mixtures with 1,1,1-trifluoro-N-(trifluoromethylsulfonyl)methanesulfoneamide (HN(Tf)(2)) as new proton transport electrolytes. We report proton transport mechanisms in the mixtures based on results from several methods including thermal analyses, the complex-impedance method, and the pulsed field gradient spin echo NMR (pfg-NMR) method. The glass transition temperature (Tg) of the mixtures decreased with increasing HN(Tf)(2) concentration up to 50 mol %. The Tg remained constant at -55 °C with further acid doping. The ionic conductivity of HN(Tf)(2) mixtures increased with the HN(Tf)(2) content up to 50 mol %. Beyond that ratio, the mixtures showed no increase in ionic conductivity (10(-4) S cm(-1) at room temperature). This tendency agrees well with that of Tg. However, the self-diffusion coefficients obtained from the pfg-NMR method increased with HN(Tf)(2) content even above 50 mol % for all component ions. At HN(Tf)(2) 50 mol %, the proton diffusion of HN(Tf)(2) was the fastest in the mixture. These results suggest that Bimps cannot dissociate excess HN(Tf)(2), that is, the excess HN(Tf)(2) exists as molecular HN(Tf)(2) in the mixtures. The zwitterion, Bimps, forms a 1:1 complex with HN(Tf)(2) and the proton transport property in this mixture is superior to those of other mixing ratios. Furthermore, CH(3)SO(3)H and CF(3)SO(3)H were mixed with Bimps for comparison. Both systems showed a similar tendency, which differed from that of the HN(Tf)(2) system. The Tg decreased linearly with increasing acid content for every mixing ratio, while the ionic conductivity increased linearly. Proton transport properties in zwitterion/acid mixtures were strongly affected by the acid species added.

  6. Bibliography for acid-rock drainage and selected acid-mine drainage issues related to acid-rock drainage from transportation activities

    Science.gov (United States)

    Bradley, Michael W.; Worland, Scott C.

    2015-01-01

    Acid-rock drainage occurs through the interaction of rainfall on pyrite-bearing formations. When pyrite (FeS2) is exposed to oxygen and water in mine workings or roadcuts, the mineral decomposes and sulfur may react to form sulfuric acid, which often results in environmental problems and potential damage to the transportation infrastructure. The accelerated oxidation of pyrite and other sulfidic minerals generates low pH water with potentially high concentrations of trace metals. Much attention has been given to contamination arising from acid mine drainage, but studies related to acid-rock drainage from road construction are relatively limited. The U.S. Geological Survey, in cooperation with the Tennessee Department of Transportation, is conducting an investigation to evaluate the occurrence and processes controlling acid-rock drainage and contaminant transport from roadcuts in Tennessee. The basic components of acid-rock drainage resulting from transportation activities are described and a bibliography, organized by relevant categories (remediation, geochemical, microbial, biological impact, and secondary mineralization) is presented.

  7. Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system

    Directory of Open Access Journals (Sweden)

    García-Lobo Juan M

    2010-04-01

    Full Text Available Abstract Background Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown. Results Re-examination of the ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium. Conclusions Based on these results, we concluded that the operon ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.

  8. Novel Properties of the Wheat Aluminum Tolerance Organic Acid Transporter (TaALMT1) Revealed by Electrophysiological Characterization in Xenopus Oocytes: Functional and Structural Implications1[OA

    Science.gov (United States)

    Piñeros, Miguel A.; Cançado, Geraldo M.A.; Kochian, Leon V.

    2008-01-01

    Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding dicarboxylic and tricarboxylic acids that chelate and immobilize Al3+ at the root surface, thus preventing it from entering root cells. Several novel genes that encode membrane transporters from the ALMT and MATE families recently were cloned and implicated in mediating the organic acid transport underlying this Al tolerance response. Given our limited understanding of the functional properties of ALMTs, in this study a detailed characterization of the transport properties of TaALMT1 (formerly named ALMT1) from wheat (Triticum aestivum) expressed in Xenopus laevis oocytes was conducted. The electrophysiological findings are as follows. Although the activity of TaALMT1 is highly dependent on the presence of extracellular Al3+ (Km1/2 of approximately 5 μm Al3+ activity), TaALMT1 is functionally active and can mediate ion transport in the absence of extracellular Al3+. The lack of change in the reversal potential (Erev) upon exposure to Al3+ suggests that the “enhancement” of TaALMT1 malate transport by Al is not due to alteration in the transporter's selectivity properties but is solely due to increases in its anion permeability. The consistent shift in the direction of the Erev as the intracellular malate activity increases indicates that TaALMT1 is selective for the transport of malate over other anions. The estimated permeability ratio between malate and chloride varied between 1 and 30. However, the complex behavior of the Erev as the extracellular Cl− activity was varied indicates that this estimate can only be used as a general guide to understanding the relative affinity of TaALMT1 for malate, representing only an approximation of those expected under physiologically relevant ionic conditions. TaALMT1 can also mediate a large anion influx (i.e. outward currents). TaALMT1 is permeable not only to malate but also to other physiologically relevant anions such as Cl−, NO3−, and

  9. Molecular cloning and characterization of glucose transporter 1 ...

    African Journals Online (AJOL)

    Glucose transporter type-1 (glut1) and citrate synthase plays crucial role in glucose transport and regulation of tricarboxylic acid cycle (TCA) cycle in mammalian energy metabolism. The present study was aimed to clone and characterize glut1 and citrate synthase cDNA in water buffalo (Bubalus bubalis). Total of 90 ...

  10. Renal transport and metabolism of nicotinic acid

    International Nuclear Information System (INIS)

    Schuette, S.; Rose, R.C.

    1986-01-01

    Renal metabolism and brush-border transport of nicotinic acid were studied in renal cortical slices and brush-border membrane vesicles exposed to a physiological concentration of vitamin (2.2-3.5 microM). Vesicle transport of [ 3 H]nicotinic acid was found to be Na+ dependent and concentrative. The presence of a Na+ gradient resulted in a fivefold increase in the rate of nicotinic acid uptake over that observed with mannitol and caused a transient nicotinic acid accumulation two- to fourfold above the equilibrium value. The effects of membrane potential, pH, and elimination of Na+-H+ exchange were also studied. Cortical slices and isolated tubules exposed to 2.2 microM [ 14 C]nicotinic acid took up vitamin and rapidly metabolized most of it to intermediates in the Preiss-Handler pathway for NAD biosynthesis; little free nicotinic acid was detectable intracellularly. The replacement of Na+ with Li+ in the bathing medium reduced total accumulation of 14 C label primarily as a result of reduced nicotinic acid uptake. Cortical tissue concentrated free nicotinic acid only when the involved metabolic pathways were saturated by levels of nicotinic acid far in excess of what occurs in vivo

  11. Hypoxia and the anticoagulants dalteparin and acetylsalicylic acid affect human placental amino acid transport.

    Directory of Open Access Journals (Sweden)

    Marc-Jens Kleppa

    Full Text Available BACKGROUND: Anticoagulants, e.g. low-molecular weight heparins (LMWHs and acetylsalicylic acid (ASA are prescribed to women at risk for pregnancy complications that are associated with impaired placentation and placental hypoxia. Beyond their role as anticoagulants these compounds exhibit direct effects on trophoblast but their impact on placental function is unknown. The amino acid transport systems A and L, which preferably transfer essential amino acids, are well-described models to study placental nutrient transport. We aimed to examine the effect of hypoxia, LMWHs and ASA on the activity of the placental amino acid transport systems A and L and associated signalling mechanisms. METHODS: The uptake of C14-MeAIB (system A or H3-leucin (system L was investigated after incubation of primary villous fragments isolated from term placentas. Villous tissue was incubated at 2% O2 (hypoxia, 8% O2 and standard culture conditions (21% O2 or at 2% O2 and 21% O2 with dalteparin or ASA. Activation of the JAK/STAT or mTOR signalling pathways was determined by Western analysis of total and phosphorylated STAT3 or Raptor. RESULTS: Hypoxia decreased system A mediated MeAIB uptake and increased system L mediated leucine uptake compared to standard culture conditions (21% O2. This was accompanied by an impairment of STAT3 and a stimulation of Raptor signalling. System L activity increased at 8% O2. Dalteparin treatment reduced system A and system L activity under normoxic conditions and ASA (1 mM decreased system A and L transporter activity under normoxic and hypoxic conditions. CONCLUSIONS: Our data underline the dependency of placental function on oxygen supply. LMWHs and ASA are not able to reverse the effects of hypoxia on placental amino acid transport. These findings and the uncovering of the signalling mechanisms in more detail will help to understand the impact of LMWHs and ASA on placental function and fetal growth.

  12. Tissue-specific amino acid transporter partners ACE2 and collectrin differentially interact with hartnup mutations

    DEFF Research Database (Denmark)

    Camargo, Simone M R; Singer, Dustin; Makrides, Victoria

    2008-01-01

    BACKGROUND & AIMS: Hartnup amino acid transporter B(0)AT1 (SLC6A19) is the major luminal sodium-dependent neutral amino acid transporter of small intestine and kidney proximal tubule. The expression of B(0)AT1 in kidney was recently shown to depend on its association with collectrin (Tmem27...

  13. Rate and Regulation of Copper Transport by Human Copper Transporter 1 (hCTR1)*

    Science.gov (United States)

    Maryon, Edward B.; Molloy, Shannon A.; Ivy, Kristin; Yu, Huijun; Kaplan, Jack H.

    2013-01-01

    Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using 64Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu+ first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry. PMID:23658018

  14. Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-06-01

    The aim of the present study was to investigate the transport of γ-aminobutyric acid (GABA) across the basolateral membrane of intestinal cells. The proton-coupled amino acid transporter, hPAT1, mediates the influx of GABA and GABA mimetic drug substances such as vigabatrin and gaboxadol and the anticancer prodrug δ-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290 μM and V(max) of 75 pmol cm(-2) min(-1) and a low affinity system with a K(m) of approximately 64 mM and V(max) of 1.6 nmol cm(-2) min(-1). The high-affinity transporter is Na(+) and Cl(-) dependent. The substrate specificity of the high-affinity transporter was further studied and Gly-Sar, Leucine, gaboxadol, sarcosine, lysine, betaine, 5-hydroxythryptophan, proline and glycine reduced the GABA uptake to approximately 44-70% of the GABA uptake in the absence of inhibitor. Other substances such as β-alanine, GABA, 5-aminovaleric acid, taurine and δ-aminolevulinic acid reduced the basolateral GABA uptake to 6-25% of the uptake in the absence of inhibitor. Our results indicate that the distance between the charged amino- and acid-groups is particular important for inhibition of basolateral GABA uptake. Thus, there seems to be a partial substrate overlap between the basolateral GABA transporter and hPAT1, which may prove important for understanding drug interactions at the level of intestinal transport. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

    Science.gov (United States)

    Cao, Wei; Kayama, Hisako; Chen, Mei Lan; Delmas, Amber; Sun, Amy; Kim, Sang Yong; Rangarajan, Erumbi S; McKevitt, Kelly; Beck, Amanda P; Jackson, Cody B; Crynen, Gogce; Oikonomopoulos, Angelos; Lacey, Precious N; Martinez, Gustavo J; Izard, Tina; Lorenz, Robin G; Rodriguez-Palacios, Alex; Cominelli, Fabio; Abreu, Maria T; Hommes, Daniel W; Koralov, Sergei B; Takeda, Kiyoshi; Sundrud, Mark S

    2017-12-19

    CD4 + T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4 + T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1 -/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1 -/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Na--dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

    International Nuclear Information System (INIS)

    Van Winkle, L.J.; Christensen, H.N.; Campione, A.L.

    1985-01-01

    Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na - -dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na - dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na - -dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid and L-valine appeared to inhibit Na - -dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, the authors conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system

  17. The proton-coupled amino acid transporter hPAT1 is the main transporter involved in vigabatrin uptake in intestinal Caco-2 cells

    DEFF Research Database (Denmark)

    Nøhr, Martha Kampp; Hansen, Steen Honore'; Brodin, Birger

    2012-01-01

    transporter hPAT1. The aim of the project was to identify if transporters are involved in cellular uptake of vigabatrin in Caco-2 cells. Methods: The uptake rate of vigabatrin was measured in Caco-2 cells at pH 6.0 or 7.4 for 15 min after application of 0.1 – 25.0 mM vigabatrin. The inhibitory effect...... of selected amino acids and -derivatives on the apical vigabatrin uptake in Caco-2 cells was investigated. Vigabatrin samples were analyzed using liquid chromatography (LC) coupled to a mass selective detector (MSD). Results: The uptake rate of vigabatrin in Caco-2 cells was pH-dependent. The uptake...... of vigabatrin was saturable at pH 6.0 with a Michaelis constant, Km of 12.7 ± 3.7 mM and a maximal flux, Jmax of 3.7 ± 0.5 nmol•min-1•cm-2. The presences of hPAT1 ligands significantly inhibited the uptake of vigabatrin in Caco-2 cells at pH 6.0, whereas hPAT1 non-ligands did not. Discussion: The saturability...

  18. Enhanced Absorption and Growth Inhibition with Amino Acid Monoester Prodrugs of Floxuridine by Targeting hPEPT1 Transporters

    Directory of Open Access Journals (Sweden)

    Gordon L. Amidon

    2008-06-01

    Full Text Available A series of amino acid monoester prodrugs of floxuridine was synthesized and evaluated for the improvement of oral bioavailability and the feasibility of target drug delivery via oligopeptide transporters. All floxuridine 5′-amino acid monoester prodrugs exhibited PEPT1 affinity, with inhibition coefficients of Gly-Sar uptake (IC50 ranging from 0.7 – 2.3 mM in Caco-2 and 2.0 – 4.8 mM in AsPC-1 cells, while that of floxuridine was 7.3 mM and 6.3 mM, respectively. Caco-2 membrane permeabilities of floxuridine prodrugs (1.01 – 5.31 x 10-6 cm/sec and floxuridine (0.48 x 10-6 cm/sec were much higher than that of 5-FU (0.038 x 10-6 cm/sec. MDCK cells stably transfected with the human oligopeptide transporter PEPT1 (MDCK/hPEPT1 exhibited enhanced cell growth inhibition in the presence of the prodrugs. This prodrug strategy offers great potential, not only for increased drug absorption but also for improved tumor selectivity and drug efficacy.

  19. Role of sialic acid in synaptosomal transport of amino acid transmitters

    International Nuclear Information System (INIS)

    Zaleska, M.M.; Erecinska, M.

    1987-01-01

    Active, high-affinity, sodium-dependent uptake of [ 14 C]-aminobutyric acid and of the acidic amino acid D-[ 3 H]-aspartate was inhibited by pretreatment of synaptosomes with neuraminidase from Vibrio cholerae. Inhibition was of a noncompetitive type and was related to the amount of sialic acid released. The maximum accumulation ratios of both amino acids (intracellular [amino acid]/extracellular [amino acid]) remained largely unaltered. Treatment with neuraminidase affected neither the synaptosomal energy levels nor the concentration of internal potassium. It is suggested that the γ-aminobutyric acid and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of the carrier proteins directly and not through modification of driving forces responsible for amino acid uptake

  20. Quantum-mechanical analysis of amino acid residues function in the proton transport during F0F1-ATP synthase catalytic cycle

    Science.gov (United States)

    Ivontsin, L. A.; Mashkovtseva, E. V.; Nartsissov, Ya R.

    2017-11-01

    Implications of quantum-mechanical approach to the description of proton transport in biological systems are a tempting subject for an overlapping of fundamental physics and biology. The model of proton transport through the integrated membrane enzyme FoF1-ATP synthase responsible for ATP synthesis was developed. The estimation of the mathematical expectation of the proton transfer time through the half-channel was performed. Observed set of proton pathways through the inlet half-channel showed the nanosecond timescale highly dependable of some amino acid residues. There were proposed two types of crucial amino acids: critically localized (His245) and being a part of energy conserving system (Asp119).

  1. L-type amino-acid transporter 1 (LAT1): a therapeutic target supporting growth and survival of T-cell lymphoblastic lymphoma/T-cell acute lymphoblastic leukemia

    NARCIS (Netherlands)

    Rosilio, C.; Nebout, M.; Imbert, V.; Griessinger, E.; Neffati, Z.; Benadiba, J.; Hagenbeek, T.; Spits, H.; Reverso, J.; Ambrosetti, D.; Michiels, J.-F.; Bailly-Maitre, B.; Endou, H.; Wempe, M. F.; Peyron, J.-F.

    2015-01-01

    The altered metabolism of cancer cells is a treasure trove to discover new antitumoral strategies. The gene (SLC7A5) encoding system L amino-acid transporter 1 (LAT1) is overexpressed in murine lymphoma cells generated via T-cell deletion of the pten tumor suppressor, and also in human T-cell acute

  2. Sodium-bicarbonate cotransporter NBCn1 in the kidney medullary thick ascending limb cell line is upregulated under acidic conditions and enhances ammonium transport.

    Science.gov (United States)

    Lee, Soojung; Lee, Hye Jeong; Yang, Han Soo; Thornell, Ian M; Bevensee, Mark O; Choi, Inyeong

    2010-09-01

    In this study, we examined the effect of bicarbonate transporters on ammonium/ammonia uptake in the medullary thick ascending limb cell line ST-1. Cells were treated with 1 mm ouabain and 0.2 mM bumetanide to minimize carrier-mediated NH(4)(+) transport, and the intracellular accumulation of (14)C-methylammonium/methylammonia ((14)C-MA) was determined. In CO(2)/HCO(3)(-)-free solution, cells at normal pH briefly accumulated (14)C-MA over 7 min and reached a plateau. In CO(2)/HCO(3)(-) solution, however, cells markedly accumulated (14)C-MA over the experimental period of 30 min. This CO(2)/HCO(3)(-)-dependent accumulation was reduced by the bicarbonate transporter blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonate (DIDS; 0.5 mM). Replacing Cl(-) with gluconate reduced the accumulation, but the reduction was more substantial in the presence of DIDS. Incubation of cells at pH 6.8 (adjusted with NaHCO(3) in 5% CO(2)) for 24 h lowered the mean steady-state intracellular pH to 6.96, significantly lower than 7.28 for control cells. The presence of DIDS reduced (14)C-MA accumulation in control conditions but had no effect after acidic incubation. Immunoblotting showed that NBCn1 was upregulated after acidic incubation and in NH(4)Cl-containing media. The Cl(-)-HCO(3)(-) exchanger AE2 was present, but its expression remained unaffected by acidic incubation. Expressed in Xenopus oocytes, NBCn1 increased carrier-mediated (14)C-MA transport, which was abolished by replacing Na(+). Two-electrode voltage clamp of oocytes exhibited negligible current after NH(4)Cl application. These results suggest that DIDS-sensitive HCO(3)(-) extrusion normally governs NH(4)(+)/NH(3) uptake in the medullary thick ascending limb cells. We propose that, in acidic conditions, DIDS-sensitive HCO(3)(-) extrusion is inactivated, while NBCn1 is upregulated to stimulate NH(4)(+) transport.

  3. Function and expression of the proton-coupled amino acid transporter Slc36a1 along the rat gastrointestinal tract

    DEFF Research Database (Denmark)

    Broberg, M. L.; Holm, Rasmus Koldborg; Tønsberg, H

    2012-01-01

    BACKGROUND AND PURPOSE: Intestinal absorption via membrane transporters may determine the pharmacokinetics of drug compounds. The hypothesis is that oral absorption of gaboxadol (4, 5, 6, 7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol) in rats occurs via the proton-coupled amino acid transporter, r....... The intestinal expression of rSlc36a1 mRNA was measured by quantitative real-time PCR (q-RT-PCR). Furthermore, the hPAT1-/rPAT1-mediated transport of gaboxadol or L-proline was studied in hPAT1-expressing X. laevis oocytes, Caco-2 cell monolayers and excised segments of the rat intestine. KEY RESULTS......). The in vitro carrier-mediated uptake rate of L-proline in the excised intestinal segments was highest in the mid jejunum and low in the colon. The in vitro uptake and the in vivo absorption correlated with the expression of rSlc36a1 mRNA along the rat intestine. CONCLUSIONS AND IMPLICATIONS: The results...

  4. Electron transport chains of lactic acid bacteria

    NARCIS (Netherlands)

    Brooijmans, R.J.W.

    2008-01-01

    Lactic acid bacteria are generally considered facultative anaerobic obligate fermentative bacteria. They are unable to synthesize heme. Some lactic acid bacteria are unable to form menaquinone as well. Both these components are cofactors of respiratory (electron transport) chains of prokaryotic

  5. Possible site-specific reagent for the general amino acid transport system of Saccharomyces cerevisiae.

    Science.gov (United States)

    Larimore, F S; Roon, R J

    1978-02-07

    The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids. The amino acid analogue N-delta-chloroacetyl-L-ornithine (NCAO) has been tested as potential site specific reagent for this system. L-Tryptophan, which is transported exclusively by the general transport system, was used as a substrate. In the presence of glucose as an energy source, NCAO inhibited tryptophan transport competitively (Ki = 80 micrometer) during short time intervals (1-2 min), but adding 100 micrometer NCAO to a yeast cell suspension resulted in a time-dependent activation of tryptophan transport during the first 15 min of treatment. Following the activation a time-dependent decay of tryptophan transport activity occurred. Approximately 80% inactivation of the system was observed after 90 min. When a yeast cell suspension was treated with NCAO in the absence of an energy source, an 80% inactivation of tryptophan transport occurred in 90 min. The inactivation was noncompetitive (Ki congruent to 60 micrometer) and could not be reversed by the removal of the NCAO. Addition of a five-fold excess of L-lysine during NCAO treatment or prevented inactivation of tryptophan transport. Under parallel conditions of incubation, other closely related transport systems were not inhibited by NCAO.

  6. GLAST But Not Least—Distribution, Function, Genetics and Epigenetics of l-Glutamate Transport in Brain—Focus on GLAST/EAAT1

    Czech Academy of Sciences Publication Activity Database

    Šerý, Omar; Sultana, N.; Kashem, M. A.; Pow, D. W.; Balcar, V. J.

    2015-01-01

    Roč. 40, č. 12 (2015), s. 2461-2472 ISSN 0364-3190 R&D Projects: GA MZd NT14504 Institutional support: RVO:67985904 Keywords : alcoholism * glutamate transport * polymorphism Subject RIV: ED - Physiology Impact factor: 2.472, year: 2015

  7. Accumulation, selection and covariation of amino acids in sieve tube sap of tansy (Tanacetum vulgare) and castor bean (Ricinus communis): evidence for the function of a basic amino acid transporter and the absence of a γ-amino butyric acid transporter.

    Science.gov (United States)

    Bauer, Susanne N; Nowak, Heike; Keller, Frank; Kallarackal, Jose; Hajirezaei, Mohamad-Reza; Komor, Ewald

    2014-09-01

    Sieve tube sap was obtained from Tanacetum by aphid stylectomy and from Ricinus after apical bud decapitation. The amino acids in sieve tube sap were analyzed and compared with those from leaves. Arginine and lysine accumulated in the sieve tube sap of Tanacetum more than 10-fold compared to the leaf extracts and they were, together with asparagine and serine, preferably selected into the sieve tube sap, whereas glycine, methionine/tryptophan and γ-amino butyric acid were partially or completely excluded. The two basic amino acids also showed a close covariation in sieve tube sap. The acidic amino acids also grouped together, but antagonistic to the other amino acids. The accumulation ratios between sieve tube sap and leaf extracts were smaller in Ricinus than in Tanacetum. Arginine, histidine, lysine and glutamine were enriched and preferentially loaded into the phloem, together with isoleucine and valine. In contrast, glycine and methionine/tryptophan were partially and γ-amino butyric acid almost completely excluded from sieve tube sap. The covariation analysis grouped arginine together with several neutral amino acids. The acidic amino acids were loaded under competition with neutral amino acids. It is concluded from comparison with the substrate specificities of already characterized plant amino acid transporters, that an AtCAT1-like transporter functions in phloem loading of basic amino acids, whereas a transporter like AtGAT1 is absent in phloem. Although Tanacetum and Ricinus have different minor vein architecture, their phloem loading specificities for amino acids are relatively similar. © 2014 Scandinavian Plant Physiology Society.

  8. Enteroendocrine-derived glucagon-like peptide-2 controls intestinal amino acid transport.

    Science.gov (United States)

    Lee, Jennifer; Koehler, Jacqueline; Yusta, Bernardo; Bahrami, Jasmine; Matthews, Dianne; Rafii, Mahroukh; Pencharz, Paul B; Drucker, Daniel J

    2017-03-01

    Glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 from gut endocrine cells, and both peptides act as growth factors to expand the surface area of the mucosal epithelium. Notably, GLP-2 also enhances glucose and lipid transport in enterocytes; however, its actions on control of amino acid (AA) transport remain unclear. Here we examined the mechanisms linking gain and loss of GLP-2 receptor (GLP-2R) signaling to control of intestinal amino acid absorption in mice. Absorption, transport, and clearance of essential AAs, specifically lysine, were measured in vivo by Liquid Chromatography triple quadrupole Mass Spectrometry (LC-MS/MS) and ex vivo with Ussing chambers using intestinal preparations from Glp2 r +/+ and Glp2r - / - mice. Immunoblotting determined jejunal levels of protein components of signaling pathways (PI3K-AKT, and mTORC1-pS6-p4E-BP1) following administration of GLP-2, protein gavage, and rapamycin to fasted Glp2 r +/+ and Glp2r - / - mice. Expression of AA transporters from full thickness jejunum and 4F2hc from brush border membrane vesicles (BBMVs) was measured by real-time PCR and immunoblotting, respectively. Acute administration of GLP-2 increased basal AA absorption in vivo and augmented basal lysine transport ex vivo . GLP-2-stimulated lysine transport was attenuated by co-incubation with wortmannin, rapamycin, or tetrodotoxin ex vivo . Phosphorylation of mTORC1 effector proteins S6 and 4E-BP1 was significantly increased in wild-type mice in response to GLP-2 alone, or when co-administered with protein gavage, and abolished following oral gavage of rapamycin. In contrast, activation of GLP-1R signaling did not enhance S6 phosphorylation. Disruption of GLP-2 action in Glp2r -/- mice reduced lysine transport ex vivo and attenuated the phosphorylation of S6 and 4E-BP1 in response to oral protein. Moreover, the expression of cationic AA transporter slc7a9 in response to refeeding, and the abundance of 4F2hc in BBMVs following protein

  9. MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

    Science.gov (United States)

    Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

    2014-08-05

    Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

  10. Biodistribution of [11C] methylaminoisobutyric acid, a tracer for PET studies on system A amino acid transport in vivo

    International Nuclear Information System (INIS)

    Sutinen, E.; Jyrkkioe, S.; Groenroos, T.; Haaparanta, M.; Lehikoinen, P.; Naagren, K.

    2001-01-01

    [N-methyl- 11 C]α-Methylaminoisobutyric acid ( 11 C-MeAIB) is a potentially useful tracer for positron emission tomography (PET) studies on hormonally regulated system A amino acid transport. 11 C-MeAIB is a metabolically stable amino acid analogue specific for system A amino acid transport. We evaluated the biodistribution of 11 C-MeAIB in rats and humans to estimate the usefulness of the tracer for in vivo human PET studies, for example, on regulation of system A amino acid transport and on tumour imaging. Healthy Sprague-Dawley rats (n=14) were killed 5, 20, 40 or 60 min after the injection of 11 C-MeAIB, and the tissue samples were weighed and counted for 11 C radioactivity. Ten lymphoma patients with relatively limited tumour burden underwent whole-body (WB) PET imaging with 11 C-MeAIB. In addition, three other patients had dynamic PET scanning of the head and neck area, and the tracer uptake was quantitated by calculating the kinetic influx constants (K i values) for the tracer. In animal studies, the highest activity was detected in the kidney, pancreas, adrenal gland and intestines. In humans, the highest activity was found in the salivary glands, and after that in the kidney and pancreas, similar to the results in animal studies. Rapid uptake was also detected in the skeletal muscle. In the graphical analysis, linear plots were obtained, and the mean fractional tracer uptake values (K i ) of the parotid glands (n=3) and cervical muscles (n=3) were 0.039±0.008 min -1 and 0.013±0.006 min -1 , respectively. The K i value of the tumour (n=1) was 0.064 min -1 . Higher uptake of 11 C-MeAIB into the tumour tissue was encountered. These results encourage further 11 C-MeAIB PET studies in humans on the physiology and pathology of system A amino acid transport and on tumour detection. (orig.)

  11. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120

    Directory of Open Access Journals (Sweden)

    Rafael Pernil

    2015-04-01

    Full Text Available Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion.

  12. Potential for food-drug interactions by dietary phenolic acids on human organic anion transporters 1 (SLC22A6), 3 (SLC22A8), and 4 (SLC22A11).

    Science.gov (United States)

    Wang, Li; Sweet, Douglas H

    2012-10-15

    Phenolic acids exert beneficial health effects such as anti-oxidant, anti-carcinogenic, and anti-inflammatory activities and show systemic exposure after consumption of common fruits, vegetables, and beverages. However, knowledge regarding which components convey therapeutic benefits and the mechanism(s) by which they cross cell membranes is extremely limited. Therefore, we determined the inhibitory effects of nine food-derived phenolic acids, p-coumaric acid, ferulic acid, gallic acid, gentisic acid, 4-hydroxybenzoic acid, protocatechuic acid, sinapinic acid, syringic acid, and vanillic acid, on human organic anion transporter 1 (hOAT1), hOAT3, and hOAT4. In the present study, inhibition of OAT-mediated transport of prototypical substrates (1 μM) by phenolic acids (100 μM) was examined in stably expressing cell lines. All compounds significantly inhibited hOAT3 transport, while just ferulic, gallic, protocatechuic, sinapinic, and vanillic acid significantly blocked hOAT1 activity. Only sinapinic acid inhibited hOAT4 (~35%). For compounds exhibiting inhibition > ~60%, known clinical plasma concentration levels and plasma protein binding in humans were examined to select compounds to evaluate further with dose-response curves (IC(50) values) and drug-drug interaction (DDI) index determinations. IC(50) values ranged from 1.24 to 18.08 μM for hOAT1 and from 7.35 to 87.36 μM for hOAT3. Maximum DDI indices for gallic and gentisic acid (≫0.1) indicated a very strong potential for DDIs on hOAT1 and/or hOAT3. This study indicates that gallic acid from foods or supplements, or gentisic acid from salicylate-based drug metabolism, may significantly alter the pharmacokinetics (efficacy and toxicity) of concomitant therapeutics that are hOAT1 and/or hOAT3 substrates. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Characterization of vacuolar amino acid transporter from Fusarium oxysporum in Saccharomyces cerevisiae.

    Science.gov (United States)

    Lunprom, Siriporn; Pongcharoen, Pongsanat; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi; Akiyama, Koichi

    2015-01-01

    Fusarium oxysporum causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in Schizosaccharomyces pombe and Saccharomyces cerevisiae. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the FOXG_11334 (FoAVT3) gene from F. oxysporum was isolated and its function was characterized. Transcription of FoAVT3 was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both S. cerevisiae and F. oxysporum. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from S. cerevisiae cells heterologously expressing FoAVT3 revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the FoAVT3 gene encodes a vacuolar neutral amino acid transporter.

  14. gamma-Glutamyl amino acids. Transport and conversion to 5-oxoproline in the kidney

    International Nuclear Information System (INIS)

    Bridges, R.J.; Meister, A.

    1985-01-01

    Transport of gamma-glutamyl amino acids, a step in the proposed glutathione-gamma-glutamyl transpeptidase-mediated amino acid transport pathway, was examined in mouse kidney. The transport of gamma-glutamyl amino acids was demonstrated in vitro in studies on kidney slices. Transport was followed by measuring uptake of 35 S after incubation of the slices in media containing gamma-glutamyl methionine [ 35 S]sulfone. The experimental complication associated with extracellular conversion of the gamma-glutamyl amino acid to amino acid and uptake of the latter by slices was overcome by using 5-oxoproline formation (catalyzed by intracellular gamma-glutamyl-cyclotransferase) as an indicator of gamma-glutamyl amino acid transport. This method was also successfully applied to studies on transport of gamma-glutamyl amino acids in vivo. Transport of gamma-glutamyl amino acids in vitro and in vivo is inhibited by several inhibitors of gamma-glutamyl transpeptidase and also by high extracellular levels of glutathione. This seems to explain urinary excretion of gamma-glutamylcystine by humans with gamma-glutamyl transpeptidase deficiency and by mice treated with inhibitors of this enzyme. Mice depleted of glutathione by treatment with buthionine sulfoximine (which inhibits glutathione synthesis) or by treatment with 2,6-dimethyl-2,5-heptadiene-4-one (which effectively interacts with tissue glutathione) exhibited significantly less transport of gamma-glutamyl amino acids than did untreated controls. The findings suggest that intracellular glutathione functions in transport of gamma-glutamyl amino acids. Evidence was also obtained for transport of gamma-glutamyl gamma-glutamylphenylalanine into kidney slices

  15. Perfluoroalkyl Acid Concentrations in Blood Samples Subjected to Transportation and Processing Delay.

    Science.gov (United States)

    Bach, Cathrine Carlsen; Henriksen, Tine Brink; Bossi, Rossana; Bech, Bodil Hammer; Fuglsang, Jens; Olsen, Jørn; Nohr, Ellen Aagaard

    2015-01-01

    In studies of perfluoroalkyl acids, the validity and comparability of measured concentrations may be affected by differences in the handling of biospecimens. We aimed to investigate whether measured plasma levels of perfluoroalkyl acids differed between blood samples subjected to delay and transportation prior to processing and samples with immediate processing and freezing. Pregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification. For samples taken in the winter, relative differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate there was no difference between the two setups [corresponding estimate 1% (0, 3)]. Differences were negligible in the summer for all compounds. Transport of blood samples and processing delay, similar to conditions applied in some large, population-based studies, may affect measured perfluoroalkyl acid concentrations, mainly when outdoor temperatures are low. Attention to processing conditions is needed in studies of perfluoroalkyl acid exposure in humans.

  16. Disposition and transportation of surplus radioactive low specific activity nitric acid. Volume 1, Environmental Assessment

    International Nuclear Information System (INIS)

    1995-05-01

    DOE is deactivating the PUREX plant at Hanford; this will involve the disposition of about 692,000 liters (183,000 gallons) of surplus nitric acid contaminated with low levels of U and other radionuclides. The nitric acid, designated as low specific activity, is stored in 4 storage tanks at PUREX. Five principal alternatives were evaluated: transfer for reuse (sale to BNF plc), no action, continued storage in Hanford upgraded or new facility, consolidation of DOE surplus acid, and processing the LSA nitric acid as waste. The transfer to BNF plc is the preferred alternative. From the analysis, it is concluded that the proposed disposition and transportation of the acid does not constitute a major federal action significantly affecting the quality of the human environment within the meaning of NEPA; therefore an environmental impact statement is not required

  17. Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1)

    Energy Technology Data Exchange (ETDEWEB)

    Popovic, Marta; Zaja, Roko [Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute, Bijenicka 54, 10 000 Zagreb (Croatia); Fent, Karl [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology (ETH Zürich), Department of Environmental System Sciences, Institute of Biogeochemistry and Pollution Dynamics, CH-8092 Zürich (Switzerland); Smital, Tvrtko, E-mail: smital@irb.hr [Laboratory for Molecular Ecotoxicology, Division for Marine and Environmental Research, Rudjer Boskovic Institute, Bijenicka 54, 10 000 Zagreb (Croatia)

    2014-10-01

    Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towards perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17β-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species. - Highlights: • We optimized a novel assay for determination of Oatp1d1 interactors • Oatp1d1 is the first SLC characterized fish xenobiotic transporter • PFOS, nonylphenol, diclofenac, EE2, caffeine are high affinity Oatp1d1substrates • PFOA, chlorpyrifos

  18. Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1)

    International Nuclear Information System (INIS)

    Popovic, Marta; Zaja, Roko; Fent, Karl; Smital, Tvrtko

    2014-01-01

    Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towards perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17β-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species. - Highlights: • We optimized a novel assay for determination of Oatp1d1 interactors • Oatp1d1 is the first SLC characterized fish xenobiotic transporter • PFOS, nonylphenol, diclofenac, EE2, caffeine are high affinity Oatp1d1substrates • PFOA, chlorpyrifos

  19. Conformational basis for the Li(+)-induced leak current in the rat gamma-aminobutyric acid (GABA) transporter-1

    DEFF Research Database (Denmark)

    MacAulay, Nanna; Zeuthen, Thomas; Gether, Ulrik

    2002-01-01

    The rat gamma-aminobutyric acid transporter-1 (GAT-1) was expressed in Xenopus laevis oocytes and the substrate-independent Li(+)-induced leak current was examined using two-electrode voltage clamp. The leak current was not affected by the addition of GABA and was not due to H(+) permeation. The Li......(+)-bound conformation of the protein displayed a lower passive water permeability than that of the Na(+)- and choline (Ch(+))-bound conformations and the leak current did not saturate with increasing amounts of Li(+) in the test solution. The mechanism that gives rise to the leak current did not support active water...... transport in contrast to the mechanism responsible for GABA translocation (approximately 330 water molecules per charge). Altogether, these data support the distinct nature of the leak conductance in relation to the substrate translocation process. It was observed that the leak current was inhibited by low...

  20. Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

    Energy Technology Data Exchange (ETDEWEB)

    Ohkawa, Mayumi [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Ohno, Yoshiya [Laboratory of Immunobiology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe-shi, Hyogo 650-8530 (Japan); Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan); Tanaka, Toshiyuki [Laboratory of Immunobiology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Kobe-shi, Hyogo 650-8530 (Japan); Saya, Hideyuki [Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8502 (Japan); Seki, Masayuki; Enomoto, Takemi [Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8578 (Japan); Yagi, Hideki [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan); Hashimoto, Yoshiyuki [Tohoku University, Sendai (Japan); Masuko, Takashi, E-mail: masuko@phar.kindai.ac.jp [Cell Biology Laboratory, Department of Pharmaceutical Sciences, School of Pharmacy, Kinki University, 4-1 Kowakae 3-chome, Higashiosaka-shi, Osaka 577-8502 (Japan)

    2011-03-25

    Highlights: {yields} We established LAT1 amino-acid transporter-disrupted DT40 cells. {yields} LAT1-disrupted cells showed slow growth and lost the oncogenicity. {yields} siRNA and mAb inhibited human tumor growth in vitro and in vivo. {yields} LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1{sup -/-}) cell clones, derived from a heterozygous LAT1{sup +/-} clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1{sup -/-} DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1{sup -/-} cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1{sup -/-} DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1{sup -/-} DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1{sup +/-} DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

  1. Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

    International Nuclear Information System (INIS)

    Ohkawa, Mayumi; Ohno, Yoshiya; Masuko, Kazue; Takeuchi, Akiko; Suda, Kentaro; Kubo, Akihiro; Kawahara, Rieko; Okazaki, Shogo; Tanaka, Toshiyuki; Saya, Hideyuki; Seki, Masayuki; Enomoto, Takemi; Yagi, Hideki; Hashimoto, Yoshiyuki; Masuko, Takashi

    2011-01-01

    Highlights: → We established LAT1 amino-acid transporter-disrupted DT40 cells. → LAT1-disrupted cells showed slow growth and lost the oncogenicity. → siRNA and mAb inhibited human tumor growth in vitro and in vivo. → LAT1 is a promising target molecule for cancer therapy. -- Abstract: L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1 -/- ) cell clones, derived from a heterozygous LAT1 +/- clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1 -/- DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1 -/- cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1 -/- DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1 -/- DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1 +/- DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

  2. Acid-base transport by the renal proximal tubule.

    Science.gov (United States)

    Skelton, Lara A; Boron, Walter F; Zhou, Yuehan

    2010-01-01

    Each day, the kidneys filter 180 L of blood plasma, equating to some 4,300 mmol of the major blood buffer, bicarbonate (HCO3-). The glomerular filtrate enters the lumen of the proximal tubule (PT), and the majority of filtered HCO3- is reclaimed along the early (S1) and convoluted (S2) portions of the PT in a manner coupled to the secretion of H+ into the lumen. The PT also uses the secreted H+ to titrate non-HCO3- buffers in the lumen, in the process creating "new HCO3-" for transport into the blood. Thus, the PT - along with more distal renal segments - is largely responsible for regulating plasma [HCO3-]. In this review we first focus on the milestone discoveries over the past 50+ years that define the mechanism and regulation of acid-base transport by the proximal tubule. Further on in the review, we will summarize research still in progress from our laboratory, work that addresses the problem of how the PT is able to finely adapt to acid-base disturbances by rapidly sensing changes in basolateral levels of HCO3- and CO2 (but not pH), and thereby to exert tight control over the acid-base composition of the blood plasma.

  3. Action of Abscisic Acid on Auxin Transport and its Relation to Phototropism

    DEFF Research Database (Denmark)

    Naqvi, S. M.; Engvild, Kjeld Christensen

    1974-01-01

    The action of abscisic acid on the kinetics of auxin transport through Zea mays L. (cv. Goudster) coleoptiles has been investigated. Abscisic acid applied simultaneously with indoleacetic acid-2-14C in the donor block reduced the transport intensity without materially affecting the basipetal...... velocity or the uptake. No effect on acropetal transport was observed. The data have been used to discuss the similarities in effects of abscisic acid and visible radiation and a hypothesis is proposed to explain the phenomena of phototropism....

  4. Potent human uric acid transporter 1 inhibitors: in vitro and in vivo metabolism and pharmacokinetic studies

    Directory of Open Access Journals (Sweden)

    Wempe MF

    2012-11-01

    Full Text Available Michael F Wempe,1 Janet W Lightner,2 Bettina Miller,1 Timothy J Iwen,1 Peter J Rice,1 Shin Wakui,3 Naohiko Anzai,4 Promsuk Jutabha,4 Hitoshi Endou51Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Anschutz Medical Campus, Aurora, CO, USA; 2Department of Pharmacology, East Tennessee State University, Johnson City, TN, USA; 3Department of Toxicology, Azabu University School of Veterinary Medicine, Chuo Sagamihara, Kanagawa, Japan; 4Department of Pharmacology and Toxicology, Dokkyo Medical University School of Medicine, Mibu, Shimotsuga, Tochigi, Japan; 5Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo, JapanAbstract: Human uric acid transporter 1 (hURAT1; SLC22A12 is a very important urate anion exchanger. Elevated urate levels are known to play a pivotal role in cardiovascular diseases, chronic renal disease, diabetes, and hypertension. Therefore, the development of potent uric acid transport inhibitors may lead to novel therapeutic agents to combat these human diseases. The current study investigates small molecular weight compounds and their ability to inhibit 14C-urate uptake in oocytes expressing hURAT1. Using the most promising drug candidates generated from our structure–activity relationship findings, we subsequently conducted in vitro hepatic metabolism and pharmacokinetic (PK studies in male Sprague-Dawley rats. Compounds were incubated with rat liver microsomes containing cofactors nicotinamide adenine dinucleotide phosphate and uridine 5'-diphosphoglucuronic acid. In vitro metabolism and PK samples were analyzed using liquid chromatography/mass spectrometry-mass spectrometry methods. Independently, six different inhibitors were orally (capsule dosing or intravenously (orbital sinus administered to fasting male Sprague-Dawley rats. Blood samples were collected and analyzed; these data were used to compare in vitro and in vivo metabolism and to

  5. Disturbed mitochondrial function restricts glutamate uptake in the human Müller glia cell line, MIO-M1

    DEFF Research Database (Denmark)

    Vohra, Rupali; Gurubaran, Iswariyaraja Sridevi; Henriksen, Ulrik

    2017-01-01

    Using the human Müller cell line, MIO-M1, the aim was to study the impact of mitochondrial inhibition in Müller glia through antimycin A treatment. MIO-M1 cell survival, levels of released lactate, mitochondrial function, and glutamate uptake were studied in response to mitochondrial inhibition...... and glucose restriction. Lactate release decreased in response to glucose restriction. Combined glucose restriction and blocked mitochondrial activity decreased survival and caused collapse of the respiratory chain measured by oxygen consumption rate and extracellular acidification rate. Mitochondrial...... inhibition caused impaired glutamate uptake and decreased mRNA expression of the glutamate transporter, EAAT1. Over all, we show important roles of mitochondrial activity in MIO-M1 cell function and survival....

  6. Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue

    Science.gov (United States)

    Salameh, Ahmad; Daquinag, Alexes C.; Staquicini, Daniela I.; An, Zhiqiang; Pasqualini, Renata; Kolonin, Mikhail G.

    2016-01-01

    We have previously identified prohibitin (PHB) and annexin A2 (ANX2) as proteins interacting on the surface of vascular endothelial cells in white adipose tissue (WAT) of humans and mice. Here, we demonstrate that ANX2 and PHB also interact in adipocytes. Mice lacking ANX2 have normal WAT vascularization, adipogenesis, and glucose metabolism but display WAT hypotrophy due to reduced fatty acid uptake by WAT endothelium and adipocytes. By using cell culture systems in which ANX2/PHB binding is disrupted either genetically or through treatment with a blocking peptide, we show that fatty acid transport efficiency relies on this protein complex. We also provide evidence that the interaction between ANX2 and PHB mediates fatty acid transport from the endothelium into adipocytes. Moreover, we demonstrate that ANX2 and PHB form a complex with the fatty acid transporter CD36. Finally, we show that the colocalization of PHB and CD36 on adipocyte surface is induced by extracellular fatty acids. Together, our results suggest that an unrecognized biochemical interaction between ANX2 and PHB regulates CD36-mediated fatty acid transport in WAT, thus revealing a new potential pathway for intervention in metabolic diseases. PMID:27468426

  7. Reactive solute transport in acidic streams

    Science.gov (United States)

    Broshears, R.E.

    1996-01-01

    Spatial and temporal profiles of Ph and concentrations of toxic metals in streams affected by acid mine drainage are the result of the interplay of physical and biogeochemical processes. This paper describes a reactive solute transport model that provides a physically and thermodynamically quantitative interpretation of these profiles. The model combines a transport module that includes advection-dispersion and transient storage with a geochemical speciation module based on MINTEQA2. Input to the model includes stream hydrologic properties derived from tracer-dilution experiments, headwater and lateral inflow concentrations analyzed in field samples, and a thermodynamic database. Simulations reproduced the general features of steady-state patterns of observed pH and concentrations of aluminum and sulfate in St. Kevin Gulch, an acid mine drainage stream near Leadville, Colorado. These patterns were altered temporarily by injection of sodium carbonate into the stream. A transient simulation reproduced the observed effects of the base injection.

  8. Nano and Mesoscale Ion and Water Transport in Perfluorosulfonic AcidMembranes

    Science.gov (United States)

    2017-10-01

    Nano- and Mesoscale Ion and Water Transport in Perfluorosulfonic-Acid Membranes A. R. Crothers a,b , C. J. Radke a,b , A. Z. Weber a a...Berkeley, CA 94720, USA Water and aqueous cations transport along multiple length scales in perfluorosulfonic-acid membranes. Molecular interactions...as a function of hydration. A resistor network upscales the nanoscale properties to predict effective membrane ion and water transport and their

  9. Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus Oocytes: functional and structural implications.

    Science.gov (United States)

    Piñeros, Miguel A; Cançado, Geraldo M A; Kochian, Leon V

    2008-08-01

    Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding dicarboxylic and tricarboxylic acids that chelate and immobilize Al(3+) at the root surface, thus preventing it from entering root cells. Several novel genes that encode membrane transporters from the ALMT and MATE families recently were cloned and implicated in mediating the organic acid transport underlying this Al tolerance response. Given our limited understanding of the functional properties of ALMTs, in this study a detailed characterization of the transport properties of TaALMT1 (formerly named ALMT1) from wheat (Triticum aestivum) expressed in Xenopus laevis oocytes was conducted. The electrophysiological findings are as follows. Although the activity of TaALMT1 is highly dependent on the presence of extracellular Al(3+) (K(m1/2) of approximately 5 microm Al(3+) activity), TaALMT1 is functionally active and can mediate ion transport in the absence of extracellular Al(3+). The lack of change in the reversal potential (E(rev)) upon exposure to Al(3+) suggests that the "enhancement" of TaALMT1 malate transport by Al is not due to alteration in the transporter's selectivity properties but is solely due to increases in its anion permeability. The consistent shift in the direction of the E(rev) as the intracellular malate activity increases indicates that TaALMT1 is selective for the transport of malate over other anions. The estimated permeability ratio between malate and chloride varied between 1 and 30. However, the complex behavior of the E(rev) as the extracellular Cl(-) activity was varied indicates that this estimate can only be used as a general guide to understanding the relative affinity of TaALMT1 for malate, representing only an approximation of those expected under physiologically relevant ionic conditions. TaALMT1 can also mediate a large anion influx (i.e. outward currents). TaALMT1 is permeable not only to malate but also to other physiologically relevant anions such as Cl

  10. Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression1[W][OPEN

    Science.gov (United States)

    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. PMID:24272701

  11. Functional characterization of folic acid transport in the intestine of the laying hen using the everted intestinal sac model.

    Science.gov (United States)

    Tactacan, G B; Rodriguez-Lecompte, J C; Karmin, O; House, J D

    2011-01-01

    Absorption at the level of the intestine is likely a primary regulatory mechanism for the deposition of dietary supplemented folic acid into the chicken egg. Therefore, factors affecting the intestinal transport of folic acid in the laying hen may influence the level of egg folate concentrations. To this end, a series of experiments using intestinal everted sacs were conducted to characterize intestinal folic acid absorption processes in laying hens. Effects of naturally occurring folate derivatives (5-methyl and 10-formyltetrahydrofolate) as well as heme on folic acid absorption were also investigated. Folic acid absorption was measured based on the rate of uptake of (3)H-labeled folic acid in the everted sac from various segments of the small and large intestines. Folic acid concentration, incubation length, and pH condition were optimized before the performance of uptake experiments. The distribution profile of folic acid transport along the intestine was highest in the upper half of the small intestine. Maximum uptake rate (nmol·100 g tissue(-1)·min(-1)) was observed in the duodenum (20.6 ± 1.9) and jejunum (22.3 ± 2.0) and decreased significantly in the ileum (15.3 ± 1.1) and cecum (9.3 ± 0.9). Transport increased proportionately (P methyl and 10-formyltetrahydrofolate as well as heme impeded folic acid uptake, reducing intestinal folic acid absorption when added at concentrations ranging from 0 to 100 µM. Overall, these data indicated the presence of a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folate.

  12. Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

    Science.gov (United States)

    Fincham, D A; Ellory, J C; Young, J D

    1992-08-01

    In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target

  13. Acidic pH and short-chain fatty acids activate Na+ transport but differentially modulate expression of Na+/H+ exchanger isoforms 1, 2, and 3 in omasal epithelium.

    Science.gov (United States)

    Lu, Zhongyan; Yao, Lei; Jiang, Zhengqian; Aschenbach, Jörg R; Martens, Holger; Shen, Zanming

    2016-01-01

    Low sodium content in feed and large amounts of salivary sodium secretion are essential requirements to efficient sodium reabsorption in the dairy cow. It is already known that Na(+)/H(+) exchange (NHE) of the ruminal epithelium plays a key role in Na(+) absorption, and its function is influenced by the presence of short-chain fatty acids (SCFA) and mucosal pH. By contrast, the functional role and regulation of NHE in omasal epithelium have not been completely understood. In the present study, we used model studies in small ruminants (sheep and goats) to investigate NHE-mediated Na(+) transport and the effects of pH and SCFA on NHE activity in omasal epithelium and on the expression of NHE isoform in omasal epithelial cells. Conventional Ussing chamber technique, primary cell culture, quantitative PCR, and Western blot were used. In native omasal epithelium of sheep, the Na(+) transport was electroneutral, and it was inhibited by the specific NHE3 inhibitor 3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopropylidene-2-methyl-acrylamide dihydrochloride, which decreased mucosal-to-serosal, serosal-to-mucosal, and net flux rates of Na(+) by 80% each. The application of low mucosal pH (6.4 or 5.8) in the presence of SCFA activated the Na(+) transport across omasal epithelium of sheep compared with that at pH 7.4. In cultured omasal epithelial cells of goats, mRNA and protein of NHE1, NHE2, and NHE3 were detected. The application of SCFA increased NHE1 mRNA and protein expression, which was most prominent when the culture medium pH decreased from 7.4 to 6.8. At variance, the mRNA and protein expression of NHE2 and NHE3 were decreased with low pH and SCFA, which was contrary to the published data from ruminal epithelial studies. In conclusion, this paper shows that (1) NHE1, NHE2, and NHE3 are expressed in omasal epithelium; (2) NHE3 mediates the major portion of transepithelial Na(+) transport in omasal epithelium; and (3) SCFA and acidic pH acutely

  14. Transport of the photodynamic therapy agent 5-aminolevulinic acid by distinct H+-coupled nutrient carriers coexpressed in the small intestine.

    Science.gov (United States)

    Anderson, Catriona M H; Jevons, Mark; Thangaraju, Muthusamy; Edwards, Noel; Conlon, Nichola J; Woods, Steven; Ganapathy, Vadivel; Thwaites, David T

    2010-01-01

    5-Aminolevulinic acid (ALA) is a prodrug used in photodynamic therapy, fluorescent diagnosis, and fluorescent-guided resection because it leads to accumulation of the photosensitizer protoporphyrin IX (PpIX) in tumor tissues. ALA has good oral bioavailability, but high oral doses are required to obtain selective PpIX accumulation in colonic tumors because accumulation is also observed in normal gut mucosa. Structural similarities between ALA and GABA led us to test the hypothesis that the H(+)-coupled amino acid transporter PAT1 (SLC36A1) will contribute to luminal ALA uptake. Radiolabel uptake and electrophysiological measurements identified PAT1-mediated H(+)-coupled ALA symport after heterologous expression in Xenopus oocytes. The selectivity of the nontransported inhibitors 5-hydroxytryptophan and 4-aminomethylbenzoic acid for, respectively, PAT1 and the H(+)-coupled di/tripeptide transporter PepT1 (SLC15A1) were examined. 5-Hydroxytryptophan selectively inhibited PAT1-mediated amino acid uptake across the brush-border membrane of the human intestinal (Caco-2) epithelium whereas 4-aminomethylbenzoic acid selectively inhibited PepT1-mediated dipeptide uptake. The inhibitory effects of 5-hydroxytryptophan and 4-aminomethylbenzoic acid were additive, demonstrating that both PAT1 and PepT1 contribute to intestinal transport of ALA. This is the first demonstration of overlap in substrate specificity between these distinct transporters for amino acids and dipeptides. PAT1 and PepT1 expression was monitored by reverse transcriptase-polymerase chain reaction using paired samples of normal and cancer tissue from human colon. mRNA for both transporters was detected. PepT1 mRNA was increased 2.3-fold in cancer tissues. Thus, increased PepT1 expression in colonic cancer could contribute to the increased PpIX accumulation observed. Selective inhibition of PAT1 could enhance PpIX loading in tumor tissue relative to that in normal tissue.

  15. Radioiodinated 4-iodo-L-meta-tyrosine, a system L selective artificial amino acid: molecular design and transport characterization in Chinese hamster ovary cells (CHO-K1 cells)

    Energy Technology Data Exchange (ETDEWEB)

    Shikano, Naoto, E-mail: sikano@ipu.ac.j [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kotani, Takashi; Nakajima, Syuichi; Ogura, Masato; Nakazawa, Shinya [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Sagara, Jun-ichi [Center for Humanities and Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kobayashi, Masato [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200-942 (Japan); Baba, Takeshi; Yamaguchi, Naoto [Center for Medical Science, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kubota, Nobuo [Department of Radiological Sciences, Ibaraki Prefectural University of Health Sciences, 4669-2 Ami, Ami-machi, Inashiki-gun, Ibaraki 300-0394 (Japan); Kawai, Keiichi [Division of Health Science, Graduate School of Health Sciences, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 9200-942 (Japan)

    2010-11-15

    Introduction: High expression of the system L amino acid transporter has been observed in clinically important tissues including tumors and the blood-brain barrier. We examined amino acid transport system L selectivity of {sup 14}C(U)-L-tyrosine ({sup 14}C-Tyr), {sup 125}I-4-iodo-L-meta-tyrosine (4-{sup 125}I-mTyr), {sup 125}I-6-iodo-L-meta-tyrosine (6-{sup 125}I-mTyr), {sup 125}I-3-iodo-{alpha}-methyl-L-tyrosine ({sup 125}I-IMT) and {sup 125}I-3-iodo-L-tyrosine (3-{sup 125}I-Tyr) using Chinese hamster ovary cells (CHO-K1). Methods: Cells in the exponential growth phase were incubated with 18.5 kBq of labeled amino acid in 2 mL of phosphate-buffered saline-based uptake solution and an uptake solution with/without Na{sup +} at 37{sup o}C or 4{sup o}C. We examined the effects of the following compounds (1.0 mM) on transport: 2-(methylamino)isobutyric acid (a specific inhibitor of system A, in Na{sup +}-containing uptake solution); 2-amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L, in Na{sup +}-free uptake solution); sodium azide and 2,4-dinitrophenol (NaN{sub 3} and DNP, inhibitors of the generation of adenosine triphosphate); p-aminohippurate and tetraethylammonium (PAH and TEA, inhibitors of organic anion and cation transporters); and L- and D-isomers of natural amino acids. Results: {sup 14}C-Tyr exhibited affinity for systems L, A and ASC. 4-{sup 125}I-mTyr and 3-{sup 125}I-Tyr exhibited high specificity for system L, whereas 6-{sup 125}I-mTyr and {sup 125}I-IMT exhibited affinity for both systems L and ASC. Uptake of 4-{sup 125}I-mTyr was markedly reduced by incubation at 4 {sup o}C, and was not significantly inhibited by NaN{sub 3}, DNP, PAH or TEA. The inhibition profiles of the L- and D-isomers of natural amino acids indicated that system L mediates the transport of 4-{sup 125}I-mTyr. Conclusions: 4-{sup 125}I-mTyr exhibited the greatest system L specificity (93.46{+-}0.13%) of all of the tested amino acids.

  16. Chemical Transport Knockout for Oxidized Vitamin C, Dehydroascorbic Acid, Reveals Its Functions in vivo

    Directory of Open Access Journals (Sweden)

    Hongbin Tu

    2017-09-01

    Full Text Available Despite its transport by glucose transporters (GLUTs in vitro, it is unknown whether dehydroascorbic acid (oxidized vitamin C, DHA has any in vivo function. To investigate, we created a chemical transport knockout model using the vitamin C analog 6-bromo-ascorbate. This analog is transported on sodium-dependent vitamin C transporters but its oxidized form, 6-bromo-dehydroascorbic acid, is not transported by GLUTs. Mice (gulo−/− unable to synthesize ascorbate (vitamin C were raised on 6-bromo-ascorbate. Despite normal survival, centrifugation of blood produced hemolysis secondary to near absence of red blood cell (RBC ascorbate/6-bromo-ascorbate. Key findings with clinical implications were that RBCs in vitro transported dehydroascorbic acid but not bromo-dehydroascorbic acid; RBC ascorbate in vivo was obtained only via DHA transport; ascorbate via DHA transport in vivo was necessary for RBC structural integrity; and internal RBC ascorbate was essential to maintain ascorbate plasma concentrations in vitro/in vivo.

  17. Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

    Science.gov (United States)

    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. Copyright © 2015. Published by Elsevier B.V.

  18. Interleukin-1β inhibits insulin signaling and prevents insulin-stimulated system A amino acid transport in primary human trophoblasts.

    Science.gov (United States)

    Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

    2013-12-05

    Interleukin-1β (IL-1β) promotes insulin resistance in tissues such as liver and skeletal muscle; however the influence of IL-1β on placental insulin signaling is unknown. We recently reported increased IL-1β protein expression in placentas of obese mothers, which could contribute to insulin resistance. In this study, we tested the hypothesis that IL-1β inhibits insulin signaling and prevents insulin-stimulated amino acid transport in cultured primary human trophoblast (PHT) cells. Cultured trophoblasts isolated from term placentas were treated with physiological concentrations of IL-1β (10pg/ml) for 24h. IL-1β increased the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser307 (inhibitory) and decreased total IRS-1 protein abundance but did not affect insulin receptor β expression. Furthermore, IL-1β inhibited insulin-stimulated phosphorylation of IRS-1 (Tyr612, activation site) and Akt (Thr308) and prevented insulin-stimulated increase in PI3K/p85 and Grb2 protein expression. IL-1β alone stimulated cRaf (Ser338), MEK (Ser221) and Erk1/2 (Thr202/Tyr204) phosphorylation. The inflammatory pathways nuclear factor kappa B and c-Jun N-terminal kinase, which are involved in insulin resistance, were also activated by IL-1β treatment. Moreover, IL-1β inhibited insulin-stimulated System A, but not System L amino acid uptake, indicating functional impairment of insulin signaling. In conclusion, IL-1β inhibited the insulin signaling pathway by inhibiting IRS-1 signaling and prevented insulin-stimulated System A transport, thereby promoting insulin resistance in cultured PHT cells. These findings indicate that conditions which lead to increased systemic maternal or placental IL-1β levels may attenuate the effects of maternal insulin on placental function and consequently fetal growth. Published by Elsevier Ireland Ltd.

  19. Defective canalicular transport and toxicity of dietary ursodeoxycholic acid in the abcb11-/- mouse: transport and gene expression studies.

    Science.gov (United States)

    Wang, Renxue; Liu, Lin; Sheps, Jonathan A; Forrest, Dana; Hofmann, Alan F; Hagey, Lee R; Ling, Victor

    2013-08-15

    The bile salt export pump (BSEP), encoded by the abcb11 gene, is the major canalicular transporter of bile acids from the hepatocyte. BSEP malfunction in humans causes bile acid retention and progressive liver injury, ultimately leading to end-stage liver failure. The natural, hydrophilic, bile acid ursodeoxycholic acid (UDCA) is efficacious in the treatment of cholestatic conditions, such as primary biliary cirrhosis and cholestasis of pregnancy. The beneficial effects of UDCA include promoting bile flow, reducing hepatic inflammation, preventing apoptosis, and maintaining mitochondrial integrity in hepatocytes. However, the role of BSEP in mediating UDCA efficacy is not known. Here, we used abcb11 knockout mice (abcb11-/-) to test the effects of acute and chronic UDCA administration on biliary secretion, bile acid composition, liver histology, and liver gene expression. Acutely infused UDCA, or its taurine conjugate (TUDC), was taken up by the liver but retained, with negligible biliary output, in abcb11-/- mice. Feeding UDCA to abcb11-/- mice led to weight loss, retention of bile acids, elevated liver enzymes, and histological damage to the liver. Semiquantitative RT-PCR showed that genes encoding Mdr1a and Mdr1b (canalicular) as well as Mrp4 (basolateral) transporters were upregulated in abcb11-/- mice. We concluded that infusion of UDCA and TUDC failed to induce bile flow in abcb11-/- mice. UDCA fed to abcb11-/- mice caused liver damage and the appearance of biliary tetra- and penta-hydroxy bile acids. Supplementation with UDCA in the absence of Bsep caused adverse effects in abcb11-/- mice.

  20. Prostaglandin transporter (OATP2A1/SLCO2A1) contributes to local disposition of eicosapentaenoic acid-derived PGE3.

    Science.gov (United States)

    Gose, Tomoka; Nakanishi, Takeo; Kamo, Shunsuke; Shimada, Hiroaki; Otake, Katsumasa; Tamai, Ikumi

    2016-01-01

    Eicosapentaenoic acid (EPA)-derived prostaglandin E3 (PGE3) possesses an anti-inflammatory effect; however, information for transporters that regulate its peri-cellular concentration is limited. The present study, therefore, aimed to clarify transporters involved in local disposition of PGE3. PGE3 uptake was assessed in HEK293 cells transfected with OATP2A1/SLCO2A1, OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, OAT1/SLC22A6, OCT1/SLC22A1 or OCT2/SLC22A2 genes, compared with HEK293 cells transfected with plasmid vector alone (Mock). PGE3 uptake by OATP2A1-expressing HEK293 cells (HEK/2A1) was the highest and followed by HEK/1B1, while no significantly higher uptake of PGE3 than Mock cells was detected by other transporters. Saturation kinetics in PGE3 uptake by HEK/2A1 estimated the Km as 7.202 ± 0.595 μM, which was 22 times higher than that of PGE2 (Km=0.331 ± 0.131 μM). Furthermore, tissue disposition of PGE3 was examined in wild-type (WT) and Slco2a1-deficient (Slco2a1(-/-)) mice after oral administration of EPA ethyl ester (EPA-E) when they underwent intraperitoneal injection of endotoxin (e.g., lipopolysaccharide). PGE3 concentration was significantly higher in the lung, and tended to increase in the colon, stomach, and kidney of Slco2a1(-/-), compared to WT mice. Ratio of PGE2 metabolite 15-keto PGE2 over PGE2 concentration was significantly lower in the lung and colon of Slco2a1(-/-) than that of WT mice, suggesting that PGE3 metabolism is downregulated in Slco2a1(-/-) mice. In conclusion, PGE3 was found to be a substrate of OATP2A1, and local disposition of PGE3 could be regulated by OATP2A1 at least in the lung. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Spin-dependent transport properties of oleic acid molecule self-assembled La0.7Sr0.3MnO3 nanoparticles

    International Nuclear Information System (INIS)

    Xi, L.; Du, J.H.; Ma, J.H.; Wang, Z.; Zuo, Y.L.; Xue, D.S.

    2013-01-01

    Highlights: ► Spin-dependent transport property of LSMO/oleic acid nanoparticles is investigated. ► Transport properties and MR measured by Cu/nanoparticle assembly/elargol device. ► Non-linear I–V curve indicates a tunneling type transport properties. ► Tunnel barrier height around 1.3 ± 0.15 eV was obtained by fitting I–V curves. ► LFMR of LSMO/oleic acid molecules value reaches −18% with current of 0.1 μA at 10 K. - Abstract: Spin-dependent transport property through molecules is investigated using a monolayer of oleic acid molecule self-assembled half metallic La 0.7 Sr 0.3 MnO 3 (LSMO) nanoparticles, which was synthesized using a coprecipitation method. Fourier transform infrared spectroscopy was used to confirm that one-monolayer oleic acid molecules chemically bond to the LSMO nanoparticles. The transport properties and magnetoresistance (MR) effect of the oleic acid molecule coated LSMO nanoparticles were measured by a direct current four probes method using a Cu/nanoparticle assembly/elargol electrode sandwich device with various temperatures and bias voltages. The non-linear I–V curve indicates a tunneling type transport properties. The tunnel barrier height around 1.3 ± 0.15 eV was obtained by fitting the I–V curve according to the Simmons equation. The magnetoresistance curves can be divided to high-field MR and low-field MR (LFMR) parts. The former is ascribed to the influence of spin disorder or canting within the LSMO nanoparticle surface and the latter one with strong bias dependence is attributed to the spin-dependent tunneling effect through the insulating surface layer of LSMO and oleic acid molecules. The enhanced LFMR effect for oleic acid coated LSMO with respect to the bare LSMO was attributed to the enhanced tunneling transport and weak spin scattering in oleic acid molecule barrier.

  2. AmSUT1, a Sucrose Transporter in Collection and Transport Phloem of the Putative Symplastic Phloem Loader Alonsoa meridionalis1

    Science.gov (United States)

    Knop, Christian; Stadler, Ruth; Sauer, Norbert; Lohaus, Gertrud

    2004-01-01

    A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading. PMID:14730068

  3. Intestinal transport and metabolism of bile acids

    Science.gov (United States)

    Dawson, Paul A.; Karpen, Saul J.

    2015-01-01

    In addition to their classical roles as detergents to aid in the process of digestion, bile acids have been identified as important signaling molecules that function through various nuclear and G protein-coupled receptors to regulate a myriad of cellular and molecular functions across both metabolic and nonmetabolic pathways. Signaling via these pathways will vary depending on the tissue and the concentration and chemical structure of the bile acid species. Important determinants of the size and composition of the bile acid pool are their efficient enterohepatic recirculation, their host and microbial metabolism, and the homeostatic feedback mechanisms connecting hepatocytes, enterocytes, and the luminal microbiota. This review focuses on the mammalian intestine, discussing the physiology of bile acid transport, the metabolism of bile acids in the gut, and new developments in our understanding of how intestinal metabolism, particularly by the gut microbiota, affects bile acid signaling. PMID:25210150

  4. Evaluating Hepatobiliary Transport with 18F-Labeled Bile Acids: The Effect of Radiolabel Position and Bile Acid Structure on Radiosynthesis and In Vitro and In Vivo Performance

    Directory of Open Access Journals (Sweden)

    Stef De Lombaerde

    2018-01-01

    Full Text Available Introduction. An in vivo determination of bile acid hepatobiliary transport efficiency can be of use in liver disease and preclinical drug development. Given the increased interest in bile acid Positron Emission Tomography- (PET- imaging, a further understanding of the impact of 18-fluorine substitution on bile acid handling in vitro and in vivo can be of significance. Methods. A number of bile acid analogues were conceived for nucleophilic substitution with [18F]fluoride: cholic acid analogues of which the 3-, 7-, or 12-OH function is substituted with a fluorine atom (3α-[18F]FCA; 7β-[18F]FCA; 12β-[18F]FCA; a glycocholic and chenodeoxycholic acid analogue, substituted on the 3-position (3β-[18F]FGCA and 3β-[18F]FCDCA, resp.. Uptake by the bile acid transporters NTCP and OATP1B1 was evaluated with competition assays in transfected CHO and HEK cell lines and efflux by BSEP in membrane vesicles. PET-scans with the tracers were performed in wild-type mice (n=3 per group: hepatobiliary transport was monitored and compared to a reference tracer, namely, 3β-[18F]FCA. Results. Compounds 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA were synthesized in moderate radiochemical yields (4–10% n.d.c. and high radiochemical purity (>99%; 7β-[18F]FCA and 12β-[18F]FCA could not be synthesized and included further in this study. In vitro evaluation showed that 3α-FCA, 3β-FGCA, and 3β-FCDCA all had a low micromolar Ki-value for NTCP, OATP1B1, and BSEP. In vivo, 3α-[18F]FCA, 3β-[18F]FGCA, and 3β-[18F]FCDCA displayed hepatobiliary transport with varying efficiency. A slight yet significant difference in uptake and efflux rate was noticed between the 3α-[18F]FCA and 3β-[18F]FCA epimers. Conjugation of 3β-[18F]FCA with glycine had no significant effect in vivo. Compound 3β-[18F]FCDCA showed a significantly slower hepatic uptake and efflux towards gallbladder and intestines. Conclusion. A set of 18F labeled bile acids was synthesized that are

  5. Weekly intra-amniotic IGF-1 treatment increases growth of growth-restricted ovine fetuses and up-regulates placental amino acid transporters.

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    Jibran A Wali

    Full Text Available Frequent treatment of the growth-restricted (IUGR ovine fetus with intra-amniotic IGF-1 increases fetal growth. We aimed to determine whether increased growth was maintained with an extended dosing interval and to examine possible mechanisms. Pregnant ewes were allocated to three groups: Control, and two IUGR groups (induced by placental embolization treated with weekly intra-amniotic injections of either saline (IUGR or 360 µg IGF-1 (IGF1. IUGR fetuses were hypoxic, hyperuremic, hypoglycemic, and grew more slowly than controls. Placental glucose uptake and SLC2A1 (GLUT2 mRNA levels decreased in IUGR fetuses, but SLC2A3 (GLUT3 and SLC2A4 (GLUT4 levels were unaffected. IGF-1 treatment increased fetal growth rate, did not alter uterine blood flow or placental glucose uptake, and increased placental SLC2A1 and SLC2A4 (but not SLC2A3 mRNA levels compared with saline-treated IUGR animals. Following IGF-1 treatment, placental mRNA levels of isoforms of the system A, y(+, and L amino acid transporters increased 1.3 to 5.0 fold, while the ratio of phosphorylated-mTOR to total mTOR also tended to increase. Weekly intra-amniotic IGF-1 treatment provides a promising avenue for intra-uterine treatment of IUGR babies, and may act via increased fetal substrate supply, up-regulating placental transporters for neutral, cationic, and branched-chain amino acids, possibly via increased activation of the mTOR pathway.

  6. Influence of Humic Acid on the Transport and Deposition of Colloidal Silica under Different Hydrogeochemical Conditions

    Directory of Open Access Journals (Sweden)

    Jingjing Zhou

    2016-12-01

    Full Text Available The transport and deposition of colloids in aquifers plays an important role in managed aquifer recharge (MAR schemes. Here, the processes of colloidal silica transport and deposition were studied by displacing groundwater with recharge water. The results showed that significant amounts of colloidal silica transport occurred when native groundwater was displaced by HA solution. Solution contains varying conditions of ionic strength and ion valence. The presence of humic acid could affect the zeta potential and size of the colloidal silica, which led to obvious colloidal silica aggregation in the divalent ion solution. Humic acid increased colloidal silica transport by formation of non-adsorbing aqueous phase silica–HA complexes. The experimental and modeling results showed good agreement, indicating that the essential physics were accurately captured by the model. The deposition rates were less than 10−8 s−1 in deionized water and monovalent ion solution. Moreover, the addition of Ca2+ and increase of IS resulted in the deposition rates increasing by five orders of magnitude to 10−4 s−1. In all experiments, the deposition rates decreased in the presence of humic acid. Overall, the promotion of humic acid in colloidal silica was strongly associated with changes in water quality, indicating that they should receive greater attention during MAR.

  7. Role of cholangiocyte bile Acid transporters in large bile duct injury after rat liver transplantation.

    Science.gov (United States)

    Cheng, Long; Zhao, Lijin; Li, Dajiang; Liu, Zipei; Chen, Geng; Tian, Feng; Li, Xiaowu; Wang, Shuguang

    2010-07-27

    The pathogenesis of nonanastomotic strictures with a patent hepatic artery remains to be investigated. This study focuses on the role of cholangiocyte bile acid transporters in bile duct injury after liver transplantation. Sprague-Dawley rats were divided into three groups (n=20 for each): the sham-operated group (Sham), the transplant group with 1-hr donor liver cold preservation (CP-1h), and the transplant group with 12-hr donor liver cold preservation (CP-12h). Bile was collected for biochemical analysis. The histopathologic evaluation of bile duct injury was performed and the cholangiocyte bile acid transporters apical sodium-dependent bile acid transporter (ASBT), ileal lipid binding protein (ILBP), and Ostalpha/Ostbeta were investigated. RESULTS.: The immunohistochemical assay suggested that ASBT and ILBP were expressed exclusively on large bile duct epithelial cells, whereas Ostalpha and Ostbeta were expressed on both small and large bile ducts. Western blot and quantitative polymerase chain reaction analysis showed that the expression levels of these transporters dramatically decreased after transplantation. It took seven to 14 days for ILBP, Ostalpha, and Ostbeta to recover, whereas ASBT recovered within 3 days and even reached a peak above the normal level seven days after operation. In the CP-12h group, the ratios of the ASBT/ILBP, ASBT/Ostalpha and ASBT/Ostbeta expression levels were correlated with the injury severity scores of large but not small bile ducts. The results suggest that the unparallel alteration of cholangiocyte bile acid transporters may play a potential role in large bile duct injury after liver transplantation with prolonged donor liver preservation.

  8. Down-Regulation of Placental Transport of Amino Acids Precedes the Development of Intrauterine Growth Restriction in Maternal Nutrient Restricted Baboons.

    Science.gov (United States)

    Pantham, Priyadarshini; Rosario, Fredrick J; Weintraub, Susan T; Nathanielsz, Peter W; Powell, Theresa L; Li, Cun; Jansson, Thomas

    2016-11-01

    Intrauterine growth restriction (IUGR) is an important risk factor for perinatal complications and adult disease. IUGR is associated with down-regulation of placental amino acid transporter expression and activity at birth. It is unknown whether these changes are a cause or a consequence of human IUGR. We hypothesized that placental amino acid transport capacity is reduced prior to onset of reduced fetal growth in baboons with maternal nutrient restriction (MNR). Pregnant baboons were fed either a control (n = 8) or MNR diet (70% of control diet, n = 9) from Gestational Day 30. At Gestational Day 120 (0.65 of gestation), fetuses and placentas were collected. Microvillous (MVM) and basal (BM) plasma membrane vesicles were isolated. System A and system L transport activity was determined in MVM, and leucine transporter activity was assessed in BM using radiolabeled substrates. MVM amino acid transporter isoform expression (SNAT1, SNAT2, and SNAT4 and LAT1 and LAT2) was measured using Western blots. LAT1 and LAT2 expression were also determined in BM. Maternal and fetal plasma amino acids concentrations were determined using mass spectrometry. Fetal and placental weights were unaffected by MNR. MVM system A activity was decreased by 37% in MNR baboon placentas (P = 0.03); however MVM system A amino acid transporter protein expression was unchanged. MVM system L activity and BM leucine transporter activity were not altered by MNR. Fetal plasma concentrations of essential amino acids isoleucine and leucine were reduced, while citrulline increased (P growth trajectory. The reduction in plasma leucine and isoleucine in MNR fetuses may be caused by reduced activity of MVM system A, which is strongly coupled with system L essential amino acid uptake. Our findings indicate that reduced placental amino acid transport may be a cause rather than a consequence of IUGR due to inadequate maternal nutrition. © 2016 by the Society for the Study of Reproduction, Inc.

  9. Gamma aminobutyric acid transporter subtype 1 gene knockout mice: a new model for attention deficit/hyperactivity disorder

    Institute of Scientific and Technical Information of China (English)

    Ping Yang; Guoqiang Cai; Youqing Cai; Jian Fei; Guoxiang Liu

    2013-01-01

    Attention deficit/hyperactivity disorder (ADHD) is characterized by hyperactivity,impaired sustained attention,impulsivity,and is usually accompanied by varying degrees of learning difficulties and lack of motor coordination.However,the pathophysiology and etiology of ADHD remain inconclusive so far.Our previous studies have demonstrated that the gamma aminobutyric acid transporter subtype 1 (GAT1) gene knockout (ko) mouse (gat1-/-)is hyperactive and exhibited impaired memory performance in the Morris water maze.In the current study,we found that the gat1-/-mice showed low levels of attentional focusing and increased impulsivity.In addition,the gat1-/-mice displayed ataxia characterized by defects in motor coordination and balance skills.The hyperactivity in the ko mice was reduced by both methylphenidate and amphetamine.Collectively,these results suggest that GAT1 ko mouse is a new animal model for ADHD studying and GAT1 may be a new target to treat ADHD.

  10. How the intestinal peptide transporter PEPT-1 contributes to an obesity phenotype in Caenorhabditits elegans.

    Directory of Open Access Journals (Sweden)

    Britta Spanier

    Full Text Available BACKGROUND: Amino acid absorption in the form of di- and tripeptides is mediated by the intestinal proton-coupled peptide transporter PEPT-1 (formally OPT-2 in Caenorhabditits elegans. Transporter-deficient animals (pept-1(lg601 show impaired growth, slowed postembryonal development and major changes in amino acid status. PRINCIPAL FINDINGS: Here we demonstrate that abolished intestinal peptide transport also leads to major metabolic alterations that culminate in a two fold increase in total body fat content. Feeding of C. elegans with [U-(13C]-labelled E. coli revealed a decreased de novo synthesis of long-chain fatty acids in pept-1(lg601 and reduced levels of polyunsaturated fatty acids. mRNA profiling revealed increased transcript levels of enzymes/transporters needed for peroxisomal beta-oxidation and decreased levels for those required for fatty acid synthesis, elongation and desaturation. As a prime and most fundamental process that may account for the increased fat content in pept-1(lg601 we identified a highly accelerated absorption of free fatty acids from the bacterial food in the intestine. CONCLUSIONS: The influx of free fatty acids into intestinal epithelial cells is strongly dependent on alterations in intracellular pH which is regulated by the interplay of PEPT-1 and the sodium-proton exchanger NHX-2. We here provide evidence for a central mechanism by which the PEPT-1/NHX-2 system strongly influences the in vivo fat content of C. elegans. Loss of PEPT-1 decreases intestinal proton influx leading to a higher uptake of free fatty acids with fat accumulation whereas loss of NHX-2 causes intracellular acidification by the PEPT-1 mediated proton/dipeptide symport with an almost abolished uptake of fatty acids and a lean phenotype.

  11. L-aspartic acid transport by cat erythrocytes

    International Nuclear Information System (INIS)

    Chen, C.W.; Preston, R.L.

    1986-01-01

    Cat and dog red cells are unusual in that they have no Na/K ATPase and contain low K and high Na intracellularly. They also show significant Na dependent L-aspartate (L-asp) transport. The authors have characterized this system in cat RBCs. The influx of 3 H-L-asp (typically 2μM) was measured in washed RBCs incubated for 60 s at 37 0 C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl 2 , 15 mM MOPS pH 7.4, 5 mM glucose, and 14 C-PEG as a space marker. The cells were washed 3 times in the medium immediately before incubation which was terminated by centrifuging the RBCs through a layer of dibutylphthalate. Over an L-asp concentration range of 0.5-1000μM, influx obeyed Michaelis-Menten kinetics with a small added linear diffusion component. The Kt and Jmax of the saturable component were 5.40 +/- 0.34 μM and 148.8 +/- 7.2 μmol 1. cell -1 h -1 respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4 + M L-asp, 40 + M inhibitor), the following sequence of inhibition was observed (range 80% to 40% inhib.): L-glutamate > L-cysteine sulfonate > D-asp > L-cysteic acid > D-glutamate. Other amino acids such as L-alanine, L-proline, L-lysine, L-cysteine, and taurine showed no inhibition (<5%). These data suggest that cat red cells contain a high-affinity Na dependent transport system for L-asp, glutamate, and closely related analogues which resembles that found in the RBCs of other carnivores and in neural tissues

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

    Science.gov (United States)

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

    2013-11-01

    Plants have evolved to develop an efficient system of boron uptake and transport using a range of efflux carriers named BOR proteins. In this work we isolated and characterized a boron transporter of citrus (Citrus macrophylla), which was named CmBOR1 for its high homology to AtBOR1. CmBOR1 has 4403 bp and 12 exons. Its coding region has 2145 bp and encodes for a protein of 714 amino acids. CmBOR1 possesses the molecular features of BORs such as an anion exchanger domain and the presence of 10 transmembrane domains. Functional analysis in yeast indicated that CmBOR1 has an efflux boron transporter activity, and transformants have increased tolerance to excess boron. CmBOR1 is expressed in leaves, stem and flowers and shows the greatest accumulation in roots. The transcript accumulation was significantly increased under boron deficiency conditions in shoots. In contrast, the accumulation of the transcript did not change in boron toxicity conditions. Finally, we observed that constitutive expression of CmBOR1 was able to increase tolerance to boron deficiency conditions in Arabidopsis thaliana, suggesting that CmBOR1 is a xylem loading boron transporter. Based on these results, it was determined that CmBOR1 encodes a boric acid/borate transporter involved in tolerance to boron deficiency in plants. © 2013 Scandinavian Plant Physiology Society.

  13. Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides across the Caco-2 Cell Monolayer.

    Science.gov (United States)

    Ding, Long; Wang, Liying; Yu, Zhipeng; Ma, Sitong; Du, Zhiyang; Zhang, Ting; Liu, Jingbo

    2017-09-06

    The objective of this paper was to investigate the effects of terminal amino acids on the transport of oligopeptides across the Caco-2 cell monolayer. Ala-based tetra- and pentapeptides were designed, and the N- or C-terminal amino acid residues were replaced by different amino acids. The results showed that the oligopeptides had a wide range of transport permeability across the Caco-2 cell monolayer and could be divided into four categories: non-/poor permeability, low permeability, intermediate permeability, and good permeability. Tetrapeptides with N-terminal Leu, Pro, Ile, Cys, Met, and Val or C-terminal Val showed the highest permeability, with apparent permeability coefficient (P app ) values over 10 × 10 -6 cm/s (p transport of tetrapeptides. Pentapeptides with N- or C-terminal Tyr also showed high permeability levels, with P app values of about 10 × 10 -6 cm/s. The amino acids Glu, Asn, and Thr at the N terminus or Lys, Asp, and Arg at the C terminus were also beneficial for the transport of tetra- and pentapeptides, with P app values ranging from 1 × 10 -6 to 10 × 10 -6 cm/s. In addition, peptides with amino acids replaced at the N terminus generally showed higher permeability than those with amino acids replaced at the C terminus (p transport of oligopeptides across the Caco-2 cell monolayer.

  14. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors

    DEFF Research Database (Denmark)

    Brighton, Cheryl A.; Rievaj, Juraj; Kuhre, Rune E.

    2015-01-01

    Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium......-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L...... to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms....

  15. Transport of perfluoroalkyl acids in a water-saturated sediment column investigated under near-natural conditions

    International Nuclear Information System (INIS)

    Vierke, Lena; Möller, Axel; Klitzke, Sondra

    2014-01-01

    The aim of this study was to gain an understanding of the transport of C 4–10 perfluoroalkyl carboxylic acids (PFCAs) and C 4,6,8 perfluoroalkyl sulfonic acids (PFSAs) in a water-saturated sediment column representing a riverbank filtration scenario under near-natural conditions. Short-chain PFCAs and PFSAs with up to six C-atoms showed complete tracer-like breakthrough. Longer chain ones were retarded due to sorption to the sediment or due to other processes in the aqueous phase. The study reports the first column derived sediment–water partition coefficients ranging from 0.01 cm 3 g −1 to 0.41 cm 3 g −1 for C 4,6 PFSAs and from 0.0 cm 3 g −1 to 6.5 cm 3 g −1 for C 4,5,6,8,9 PFCAs. The results clearly indicate that short-chain PFCAs and PFSAs may pose a problem if contaminated surface waters are used for drinking water production via riverbank filtration. Highlights: • Transport of per- and polyfluorinated compounds in a riverbank filtration scenario. • Investigations under near-natural conditions with a water-saturated sediment column. • Processes in water and sediment control the transport of analytes. • Short chain PFCAs and PFSAs are not retarded in the water-saturated sediment column. • First column derived sediment–water partition coefficients. -- Quantification of breakthrough of perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkyl sulfonic acids (PFSAs) under conditions simulating a riverbank filtration scenario

  16. Mechanisms Regulating Acid-Base Transporter Expression in Breast- and Pancreatic Cancer

    DEFF Research Database (Denmark)

    Gorbatenko, Andrej

    , characteristics of which are a shift towards glycolytic metabolism and increased acid production. HER2 receptor overexpression in breast cancer leads to further increased glycolysis, invasion and metastasis, drug resistance and poor prognosis. Increased tumor glycolysis requires acquisition of mechanisms...... for dealing with excess acid production. In this light, evidence accumulates on the importance of pH regulatory proteins to cancer cell survival and motility. Our group previously demonstrated upregulation of the Na+/HCO3 - co-transporter NBCn1 (SLC4A7) by a constitutively active form of HER2 receptor (p95HER...

  17. Sodium-coupled neutral amino acid (System N/A) transporters of the SLC38 gene family.

    Science.gov (United States)

    Mackenzie, Bryan; Erickson, Jeffrey D

    2004-02-01

    The sodium-coupled neutral amino acid transporters (SNAT) of the SLC38 gene family resemble the classically-described System A and System N transport activities in terms of their functional properties and patterns of regulation. Transport of small, aliphatic amino acids by System A subtypes (SNAT1, SNAT2, and SNAT4) is rheogenic and pH sensitive. The System N subtypes SNAT3 and SNAT5 also countertransport H(+), which may be key to their operation in reverse, and have narrower substrate profiles than do the System A subtypes. Glutamine emerges as a favored substrate throughout the family, except for SNAT4. The SLC38 transporters undoubtedly play many physiological roles including the transfer of glutamine from astrocyte to neuron in the CNS, ammonia detoxification and gluconeogenesis in the liver, and the renal response to acidosis. Probing their regulation has revealed additional roles, and recent work has considered SLC38 transporters as therapeutic targets in neoplasia.

  18. TNF-α stimulates System A amino acid transport in primary human trophoblast cells mediated by p38 MAPK signaling.

    Science.gov (United States)

    Aye, Irving L M H; Jansson, Thomas; Powell, Theresa L

    2015-10-01

    Maternal obesity and gestational diabetes mellitus (GDM) increase the risk of delivering infants that are large for gestational age with greater adiposity, who are prone to the development of metabolic disease in childhood and beyond. These maternal conditions are also associated with increased levels of the proinflammatory cytokine TNF-α in maternal tissues and the placenta. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. TNF-α was previously shown to stimulate System A amino acid transport in primary human trophoblasts (PHTs), however the molecular mechanisms remain unknown. In this study, we tested the hypothesis that TNF-α regulates amino acid uptake in cultured PHTs by a mitogen-activated protein kinase (MAPK)-dependent mechanism. Treatment of PHTs with TNF-α significantly increased System A amino acid transport, as well as Erk and p38 MAPK signaling. Pharmacological antagonism of p38, but not Erk MAPK activity, inhibited TNF-α stimulated System A activity. Silencing of p38 MAPK using siRNA transfections prevented TNF-α stimulated System A transport in PHTs. TNF-α significantly increased the protein expression of System A transporters SNAT1 and SNAT2, but did not affect their mRNA expression. The effects of TNF-α on SNAT1 and SNAT2 protein expression were reversed by p38 MAPK siRNA silencing. In conclusion, TNF-α regulates System A activity through increased SNAT1 and SNAT2 transporter protein expression in PHTs. These findings suggest that p38 MAPK may represent a critical mechanistic link between elevated proinflammatory cytokines and increased placental amino acid transport in obese and GDM pregnancies associated with fetal overgrowth. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  19. A traffic signal for heterodimeric amino acid transporters to transfer from the ER to the Golgi.

    Science.gov (United States)

    Ganapathy, Vadivel

    2009-01-15

    Heterodimeric amino acid transporters represent a unique class of transport systems that consist of a light chain that serves as the 'transporter proper' and a heavy chain that is necessary for targeting the complex to the plasma membrane. The currently prevailing paradigm assigns no role for the light chains in the cellular processing of these transporters. In this issue of the Biochemical Journal, Sakamoto et al. provide evidence contrary to this paradigm. Their studies with the rBAT -b(0,+)AT (related to b(0,+) amino acid transporter-b(0,+)-type amino acid transporter) heterodimeric amino acid transporter show that the C-terminus of the light chain b(0,+)AT contains a sequence motif that serves as the traffic signal for the transfer of the heterodimeric complex from the endoplasmic reticulum to the Golgi. This is a novel function for the light chain in addition to its already established role as the subunit responsible for the transport activity. These new findings also seem to be applicable to other heterodimeric amino acid transporters as well.

  20. "1"8F-FBPA as a tumor-specific probe of L-type amino acid transporter 1 (LAT1): a comparison study with "1"8F-FDG and "1"1C-Methionine PET

    International Nuclear Information System (INIS)

    Watabe, Tadashi; Ikeda, Hayato; Aoki, Masanao; Nagamori, Shushi; Wiriyasermkul, Pattama; Tanaka, Yoko; Hagiwara, Kohei; Kanai, Yoshikatsu; Naka, Sadahiro; Kanai, Yasukazu; Shimosegawa, Eku; Hatazawa, Jun

    2017-01-01

    The purpose of this study was to evaluate the usefulness of L-4-borono-2-"1"8F-fluoro-phenylalanine ("1"8F-FBPA) as a tumor-specific probe, in comparison to "1"8F-FDG and "1"1C-methionine (Met), focusing on its transport selectivity by L-type amino acid transporter 1 (LAT1), which is highly upregulated in cancers. Cellular analyses of FBPA were performed to evaluate the transportability and K_m value. PET studies were performed in rat xenograft models of C6 glioma (n = 12) and in rat models of turpentine oil-induced subcutaneous inflammation (n = 9). The kinetic parameters and uptake values on static PET images were compared using the one-tissue compartment model (K_1, k_2) and maximum standardized uptake value (SUVmax). The cellular analyses showed that FBPA had a lower affinity to a normal cell-type transporter LAT2 and induced less efflux through LAT2 among FBPA, Met, and BPA, while the efflux through LAT1 induced by FBPA was similar among the three compounds. The K_m value of "1"8F-FBPA for LAT1 (196.8 ± 11.4 μM) was dramatically lower than that for LAT2 (2813.8 ± 574.5 μM), suggesting the higher selectivity of "1"8F-FBPA for LAT1. K_1 and k_2 values were significantly smaller in "1"8F-FBPA PET (K_1 = 0.04 ± 0.01 ml/ccm/min and k_2 = 0.07 ± 0.01 /min) as compared to "1"1C-Met PET (0.22 ± 0.09 and 0.52 ± 0.10, respectively) in inflammatory lesions. Static PET analysis based on the SUVmax showed significantly higher accumulation of "1"8F-FDG in the tumor and inflammatory lesions (7.2 ± 2.1 and 4.6 ± 0.63, respectively) as compared to both "1"8F-FBPA (3.2 ± 0.40 and 1.9 ± 0.19) and "1"1C-Met (3.4 ± 0.43 and 1.6 ± 0.11). No significant difference was observed between "1"8F-FBPA and "1"1C-Met in the static PET images. This study shows the utility of "1"8F-FBPA as a tumor-specific probe of LAT1 with low accumulation in the inflammatory lesions. (orig.)

  1. Role of stimulated amino acid transport in promoting glycogenesis in the irradiated rat

    International Nuclear Information System (INIS)

    Kilberg, M.S.; Neuhaus, O.W.

    1976-01-01

    Whole-body irradiation of rats stimulates an amino acid transport system in the liver. Another phenomenon observed after exposure to ionizing radiations is the accumulation of hepatic glycogen. The data presented here relate the increased hepatic uptake of amino acids to glycogenesis. Male rats were exposed to two doses of γ rays, 2500 and 1500 R. Following exposure to 2500 R, the hepatic free amino acids were elevated during the first 48 hr accompanied by a decline in serum levels. At 72 hr the hepatic amino acids diminished to the control levels while the serum increased abruptly. By contrast, 72 hr after exposure to 1500 R the serum amino acid levels increased only 27 percent and the hepatic amino acid values increased 52 percent. These results are explained on the basis of the changes in AIB transport previously reported. The incorporation of 14 C from labeled L-alanine into hepatic glycogen was maximal 48 hr postexposure to 2500 R but declined to below control values at 72 hr. On the other hand, exposure to 1500 R resulted in maximal incorporation of 14 C at both 48 and 72 hr. We propose that transport of amino acids into liver cells is stimulated by the elevated blood levels of amino acids released from the degradation of protein. The transport increases the levels of hepatic free amino acids, and therefore, is a key factor in regulating postirradiation glycogenesis

  2. Structure-affinity relationship in the interactions of human organic anion transporter 1 with caffeine, theophylline, theobromine and their metabolites.

    Science.gov (United States)

    Sugawara, Mitsuru; Mochizuki, Takahiro; Takekuma, Yoh; Miyazaki, Katsumi

    2005-08-15

    It is well known that human organic anion transporter 1 (hOAT1) transports many kinds of drugs, endogenous compounds, and toxins. However, little is known about the structure-affinity relationship. The aim of this study was to elucidate the structure-affinity relationship using a series of structurally related compounds that interact with hOAT1. Inhibitory effects of xanthine- and uric acid-related compounds on the transport of p-aminohippuric acid were examined using CHO-K1 cells stably expressing hOAT1. The order of potency for the inhibitory effects of xanthine-related compounds on PAH uptake was 1-methyl derivative>7-methyl derivative>3-methyl derivative falling dotsxanthine>1,3,7-trimethyl derivative (caffeine). The order of potency of the inhibition was 1,3,7-trimethyluric acid>1,3-dimethyluric acid>1,7-dimethyluric acid>1-methyluric acid>uric acid. A significant correlation between inhibitory potency and lipophilicity of the tested uric acid-related compounds was observed. The main determinant of the affinity of xanthine-related compounds is the position of the methyl group. On the other hand, lipophilicity is the main determinant of the affinity of uric acid-related compounds.

  3. Transport of indoleacetic acid in intact corn coleoptiles

    International Nuclear Information System (INIS)

    Parker, K.E.; Briggs, W.R.

    1990-01-01

    We have characterized the transport of [ 3 H]indoleacetic acid (IAA) in intact corn (Zea mays L.) coleoptiles. We have used a wide range of concentrations of added IAA (28 femtomoles to 100 picomoles taken up over 60 minutes). The shape of the transport curve varies with the concentration of added IAA, although the rate of movement of the observed front of tracer is invariant with concentration. At the lowest concentration of tracer used, the labeled IAA in the transport stream is not detectably metabolized or immobilized, curvature does not develop as a result of tracer application, and normal phototropic and gravitropic responsiveness are not affected. Therefore we believe we are observing the transport of true tracer quantities of labeled auxin at this lowest concentration

  4. Fatty acid profile of maternal and fetal erythrocytes and placental expression of fatty acid transport proteins in normal and intrauterine growth restriction pregnancies.

    Science.gov (United States)

    Assumpção, Renata P; Mucci, Daniela B; Fonseca, Fernanda C P; Marcondes, Henrique; Sardinha, Fátima L C; Citelli, Marta; Tavares do Carmo, Maria G

    2017-10-01

    Long-chain polyunsaturated fatty acids (LC-PUFA), mainly docosahexaenoic (DHA) and arachidonic acids (AA), are critical for adequate fetal growth and development. We investigated mRNA expression of proteins involved in hydrolysis, uptake and/or transport of fatty acids in placenta of fifteen full term normal pregnancies and eleven pregnancies complicated by intrauterine growth restriction (IUGR) with normal umbilical blood flows. The mRNA expression of LPL, FATPs (-1, -2 and -4) and FABPs (-1 and -3) was increased in IUGR placentas, however, tissue profile of LC-PUFA was not different between groups. Erythrocytes from both mothers and fetuses of the IUGR group showed lower concentrations of AA and DHA and inferior DHA/ALA ratio compared to normal pregnancies (P < 0.05). We hypothesize that reduced circulating levels of AA and DHA could up-regulate mRNA expression of placental fatty acids transporters, as a compensatory mechanism, however this failed to sustain normal LC-PUFA supply to the fetus in IUGR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Tubular urate transporter gene polymorphisms differentiate patients with gout who have normal and decreased urinary uric acid excretion.

    Science.gov (United States)

    Torres, Rosa J; de Miguel, Eugenio; Bailén, Rebeca; Banegas, José R; Puig, Juan G

    2014-09-01

    Primary gout has been associated with single-nucleotide polymorphisms (SNP) in several tubular urate transporter genes. No study has assessed the association of reabsorption and secretion urate transporter gene SNP with gout in a single cohort of documented primary patients with gout carefully subclassified as normoexcretors or underexcretors. Three reabsorption SNP (SLC22A12/URAT1, SLC2A9/GLUT9, and SLC22A11/OAT4) and 2 secretion transporter SNP (SLC17A1/NPT1 and ABCG2/BRCP) were studied in 104 patients with primary gout and in 300 control subjects. The patients were subclassified into normoexcretors and underexcretors according to their serum and 24-h urinary uric acid levels under strict conditions of dietary control. Compared with control subjects, patients with gout showed different allele distributions of the 5 SNP analyzed. However, the diagnosis of underexcretor was only positively associated with the presence of the T allele of URAT1 rs11231825, the G allele of GLUT9 rs16890979, and the A allele of ABCG2 rs2231142. The association of the A allele of ABCG2 rs2231142 in normoexcretors was 10 times higher than in underexcretors. The C allele of NPT1 rs1165196 was only significantly associated with gout in patients with normal uric acid excretion. Gout with uric acid underexcretion is associated with transporter gene SNP related mainly to tubular reabsorption, whereas uric acid normoexcretion is associated only with tubular secretion SNP. This finding supports the concept of distinctive mechanisms to account for hyperuricemia in patients with gout with reduced or normal uric acid excretion.

  6. Transport of monocarboxylic acids at the blood-brain barrier: Studies with monolayers of primary cultured bovine brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Terasaki, T.; Takakuwa, S.; Moritani, S.; Tsuji, A.

    1991-01-01

    The kinetics and mechanism of the transport of monocarboxylic acids (MCAs) were studied by using primary cultured bovine brain capillary endothelial cells. Concentration-dependent uptake of acetic acid was observed, and the kinetic parameters were estimated as follows: the Michaelis constant, Kt, was 3.41 ± 1.87 mM, the maximum uptake rate, Jmax, was 144.7 ± 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 ± 1.98 microliters/mg of protein/min. At medium pH below 7.0, the uptake rate of [3H]acetic acid increased markedly with decreasing medium pH, whereas pH-independent uptake was observed in the presence of 10 mM acetic acid. An energy requirement for [3H]acetic acid uptake was also demonstrated, because metabolic inhibitors (2,4-dinitrophenol and rotenone) reduced significantly the uptake rate (P less than .05). Carbonylcyanide-p-trifluoro-methoxyphenylhydrazone, a protonophore, inhibited significantly the uptake of [3H]acetic acid at medium pH of 5.0 and 6.0, whereas 4,4'-diisothiocyanostilben-2,2'-disulfonic acid did not. Several MCAs inhibited significantly the uptake rate of [3H]acetic acid, whereas di- and tricarboxylic acids did not. The uptake of [3H]acetic acid was competitively inhibited by salicylic acid, with an inhibition constant, Ki, of 3.60 mM, suggesting a common transport system between acetic acid and salicylic acid. Moreover, at the medium pH of 7.4, salicylic acid and valproic acid inhibited significantly the uptake of [3H]acetic acid, demonstrating that the transport of MCA drugs could also be ascribed to the MCA transport system at the physiologic pH

  7. Role of NH3 and NH4+ transporters in renal acid-base transport.

    Science.gov (United States)

    Weiner, I David; Verlander, Jill W

    2011-01-01

    Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henle's loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.

  8. Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

    Science.gov (United States)

    Slijepcevic, Davor; Roscam Abbing, Reinout L P; Katafuchi, Takeshi; Blank, Antje; Donkers, Joanne M; van Hoppe, Stéphanie; de Waart, Dirk R; Tolenaars, Dagmar; van der Meer, Jonathan H M; Wildenberg, Manon; Beuers, Ulrich; Oude Elferink, Ronald P J; Schinkel, Alfred H; van de Graaf, Stan F J

    2017-11-01

    The Na + -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).

  9. Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

    Science.gov (United States)

    Hosomi, Atsushi; Nakanishi, Takeo; Fujita, Takuya; Tamai, Ikumi

    2012-01-01

    Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats. PMID:22348008

  10. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    Science.gov (United States)

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Carnitine transport and fatty acid oxidation.

    Science.gov (United States)

    Longo, Nicola; Frigeni, Marta; Pasquali, Marzia

    2016-10-01

    Carnitine is essential for the transfer of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. It can be synthesized by the body or assumed with the diet from meat and dairy products. Defects in carnitine biosynthesis do not routinely result in low plasma carnitine levels. Carnitine is accumulated by the cells and retained by kidneys using OCTN2, a high affinity organic cation transporter specific for carnitine. Defects in the OCTN2 carnitine transporter results in autosomal recessive primary carnitine deficiency characterized by decreased intracellular carnitine accumulation, increased losses of carnitine in the urine, and low serum carnitine levels. Patients can present early in life with hypoketotic hypoglycemia and hepatic encephalopathy, or later in life with skeletal and cardiac myopathy or sudden death from cardiac arrhythmia, usually triggered by fasting or catabolic state. This disease responds to oral carnitine that, in pharmacological doses, enters cells using the amino acid transporter B(0,+). Primary carnitine deficiency can be suspected from the clinical presentation or identified by low levels of free carnitine (C0) in the newborn screening. Some adult patients have been diagnosed following the birth of an unaffected child with very low carnitine levels in the newborn screening. The diagnosis is confirmed by measuring low carnitine uptake in the patients' fibroblasts or by DNA sequencing of the SLC22A5 gene encoding the OCTN2 carnitine transporter. Some mutations are specific for certain ethnic backgrounds, but the majority are private and identified only in individual families. Although the genotype usually does not correlate with metabolic or cardiac involvement in primary carnitine deficiency, patients presenting as adults tend to have at least one missense mutation retaining residual activity. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler

  12. Preparation of Citric Acid Crosslinked Chitosan/Poly(Vinyl Alcohol Blend Membranes for Creatinine Transport

    Directory of Open Access Journals (Sweden)

    Retno Ariadi Lusiana

    2016-08-01

    Full Text Available Preparation of membrane using crosslinking reaction between chitosan and citric acid showed that functional group modification increased the number of active carrier groups which lead to better transport capacity of the membrane. In addition, the substitution of the carboxyl group increased creatinine permeation of chitosan membrane. The transport capacity of citric acid crosslinked chitosan membrane for creatinine was found to be 6.3 mg/L. The presence of cyanocobalamin slightly hindered the transport of creatinine although compounds did not able to pass through citric acid crosslinked chitosan/poly(vinyl alcohol blend membrane, as compounds no found in the acceptor phase.

  13. Carrier-mediated ¿-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-01-01

    and the anticancer prodrug d-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least...... two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290µM and V(max) of 75pmolcm(-2)min(-1) and a low affinity system with a K(m) of approximately 64mM and V(max) of 1.6nmolcm(-2)min(-1). The high...

  14. Uptake of 3-[125I]iodo-α-methyl-L-tyrosine into colon cancer DLD-1 cells: characterization and inhibitory effect of natural amino acids and amino acid-like drugs

    International Nuclear Information System (INIS)

    Shikano, Naoto; Ogura, Masato; Okudaira, Hiroyuki; Nakajima, Syuichi; Kotani, Takashi; Kobayashi, Masato; Nakazawa, Shinya; Baba, Takeshi; Yamaguchi, Naoto; Kubota, Nobuo; Iwamura, Yukio; Kawai, Keiichi

    2010-01-01

    Introduction: We examined 3-[ 123 I]iodo-α-methyl-L-tyrosine ([ 123 I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure. Methods: Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [ 125 I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [ 125 I]IMT uptake were examined. Results: Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B 0 AT was not detected. [ 125 I]IMT uptake in DLD-1 cells involved Na + -independent system L primarily and Na + -dependent system(s). Uptake of [ 125 I]IMT in Na + -free buffer followed Michaelis-Menten kinetics, with a K m of 78 μM and V max of 333 pmol/10 6 cells per minute. Neutral D- and L-amino acids with branched or aromatic large side chains inhibited [ 125 I]IMT uptake. Tyrosine analogues, tryptophan analogues, L-phenylalanine and p-halogeno-L-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-L-phenylalanine (L-DOPA), DL-threo-β-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-L-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [ 125 I]IMT uptake, but L-tyrosine methyl ester and R(+)/S(-)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [ 125 I]IMT uptake except L-serine and D/L-cysteine. Conclusions: [ 125 I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid

  15. Serotonin transporter activity of imidazolidine-2,4-dione and imidazo[2,1-f]purine-2,4-dione derivatives in aspect of their acid-base properties.

    Science.gov (United States)

    Zagórska, Agnieszka; Czopek, Anna; Pawłowski, Maciej; Dybała, Małgorzata; Siwek, Agata; Nowak, Gabriel

    2012-11-01

    Affinities of arylpiperazinylalkyl derivatives of imidazo[2,1-f]purine-2,4-dione and imidazolidine-2,4-dione for serotonin transporter and their acid-base properties were evaluated. The dissociation constant (pK(a)) of compounds 1-22 were determinated by potentiometric titration and calculated using pKalc 3.1 module of the Pallas system. The data from experimental methods and computational calculations were compared and suitable conclusions were reached.

  16. Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating

    International Nuclear Information System (INIS)

    Dantzig, A.H.; Slayman, C.W.; Adelberg, E.A.

    1982-01-01

    A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [ 3 H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment

  17. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nagao, Yuki, E-mail: ynagao@jaist.ac.jp; Kubo, Takahiro

    2014-12-30

    Graphical abstract: - Highlights: • Proton transport of fully protonated poly(aspartic acid) thin film was investigated. • The thin film structure differed greatly from the partially protonated one. • Proton transport occurs on the surface, not inside of the thin film. • This result contributes to biological transport systems such as bacteriorhodopsin. - Abstract: Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120–670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system.

  18. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    International Nuclear Information System (INIS)

    Nagao, Yuki; Kubo, Takahiro

    2014-01-01

    Graphical abstract: - Highlights: • Proton transport of fully protonated poly(aspartic acid) thin film was investigated. • The thin film structure differed greatly from the partially protonated one. • Proton transport occurs on the surface, not inside of the thin film. • This result contributes to biological transport systems such as bacteriorhodopsin. - Abstract: Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120–670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system

  19. Targeting tumor highly-expressed LAT1 transporter with amino acid-modified nanoparticles: Toward a novel active targeting strategy in breast cancer therapy.

    Science.gov (United States)

    Li, Lin; Di, Xingsheng; Wu, Mingrui; Sun, Zhisu; Zhong, Lu; Wang, Yongjun; Fu, Qiang; Kan, Qiming; Sun, Jin; He, Zhonggui

    2017-04-01

    Designing active targeting nanocarriers with increased cellular accumulation of chemotherapeutic agents is a promising strategy in cancer therapy. Herein, we report a novel active targeting strategy based on the large amino acid transporter 1 (LAT1) overexpressed in a variety of cancers. Glutamate was conjugated to polyoxyethylene stearate as a targeting ligand to achieve LAT1-targeting PLGA nanoparticles. The targeting efficiency of nanoparticles was investigated in HeLa and MCF-7 cells. Significant increase in cellular uptake and cytotoxicity was observed in LAT1-targeting nanoparticles compared to the unmodified ones. More interestingly, the internalized LAT1 together with targeting nanoparticles could recycle back to the cell membrane within 3 h, guaranteeing sufficient transporters on cell membrane for continuous cellular uptake. The LAT1 targeting nanoparticles exhibited better tumor accumulation and antitumor effects. These results suggested that the overexpressed LAT1 on cancer cells holds a great potential to be a high-efficiency target for the rational design of active-targeting nanosystems. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Hydrogen-rich saline protects retina against glutamate-induced excitotoxic injury in guinea pig.

    Science.gov (United States)

    Wei, Lihua; Ge, Li; Qin, Shucun; Shi, Yunzhi; Du, Changqing; Du, Hui; Liu, Liwei; Yu, Yang; Sun, Xuejun

    2012-01-01

    Molecular hydrogen (H(2)) is an efficient antioxidant that can selectively reduce hydroxyl radicals and inhibit oxidative stress-induced injuries. We investigated the protective effects and mechanism of hydrogen-rich saline in a glutamate-induced retinal injury model. Retinal excitotoxicity was induced in healthy guinea pigs by injecting glutamate into the vitreous cavity. After 30 min, hydrogen-rich saline was injected into the vitreous cavity, the peritoneal cavity or both. Seven days later, the retinal stress response was evaluated by examining the stress biomarkers, inducible nitric-oxide synthase (iNOS) and glucose-regulated protein 78 (GRP78). The impaired glutamate uptake was assessed by the expression of the excitatory amino acid transporter 1(EAAT-1). The retinal histopathological changes were investigated, focusing on the thicknesses of the entire retina and its inner layer, the number of cells in the retinal ganglion cell layer (GCL) and the ultrastructure of the retinal ganglion cells (RGCs) and glial cells. Compared with the glutamate-induced injury group, the hydrogen-rich saline treatment reduced the loss of cells in the GCL and thinning of the retina and attenuated cellular morphological damage. These improvements were greatest in animals that received H(2) injections into both the vitreous and the peritoneal cavities. The hydrogen-rich saline also inhibited the expression of glial fibrillary acidic protein (GFAP) in Müller cells, CD11b in microglia, and iNOS and GRP78 in glial cells. Moreover, the hydrogen-rich saline increased the expression of EAAT-1. In conclusion, the administration of hydrogen-rich saline through the intravitreal or/and intraperitoneal routes could reduce the retinal excitotoxic injury and promote retinal recovery. This result likely occurs by inhibiting the activation of glial cells, decreasing the production of the iNOS and GRP78 and promoting glutamate clearance. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity

    International Nuclear Information System (INIS)

    Oppedisano, Francesca; Catto, Marco; Koutentis, Panayiotis A.; Nicolotti, Orazio; Pochini, Lorena; Koyioni, Maria; Introcaso, Antonellina; Michaelidou, Sophia S.; Carotti, Angelo; Indiveri, Cesare

    2012-01-01

    The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20 μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose–response analysis of the most active compounds gave IC 50 values in the range of 3–30 μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein. -- Highlights: ► Non‐competitive inhibition of ASCT2 by 1,2,3-dithiazoles was studied in proteoliposomes. ► Different 1,2,3-dithiazoles were synthesized and evaluated as transporter inhibitors. ► Many compounds potently inhibited the glutamine/glutamine antiport catalyzed by ASCT2. ► The inhibition was reversed by DTE indicating reaction with protein Cys. ► The most active compounds gave IC 50 in the range of 3–30 μM.

  2. Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity

    Energy Technology Data Exchange (ETDEWEB)

    Oppedisano, Francesca [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy); Catto, Marco [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Koutentis, Panayiotis A. [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Nicolotti, Orazio [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Pochini, Lorena [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy); Koyioni, Maria [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Introcaso, Antonellina [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Michaelidou, Sophia S. [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Carotti, Angelo, E-mail: carotti@farmchim.uniba.it [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Indiveri, Cesare, E-mail: indiveri@unical.it [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy)

    2012-11-15

    The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20 μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose–response analysis of the most active compounds gave IC{sub 50} values in the range of 3–30 μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein. -- Highlights: ► Non‐competitive inhibition of ASCT2 by 1,2,3-dithiazoles was studied in proteoliposomes. ► Different 1,2,3-dithiazoles were synthesized and evaluated as transporter inhibitors. ► Many compounds potently inhibited the glutamine/glutamine antiport catalyzed by ASCT2. ► The inhibition was reversed by DTE indicating reaction with protein Cys. ► The most active compounds gave IC{sub 50} in the range of 3–30 μM.

  3. Simultaneous determination of gallic acid and gentisic acid in organic anion transporter expressing cells by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Wang, Li; Halquist, Matthew S; Sweet, Douglas H

    2013-10-15

    In order to elucidate the role of organic anion transporters (OATs) in the renal elimination of gallic acid and gentisic acid, a new, rapid, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous determination of gallic acid and gentisic acid in cell lysate, using Danshensu as the internal standard (IS). After a simple liquid-liquid extraction, the analytes were detected in negative ESI mode using selected reaction monitoring. The precursor-to-product ion transitions (m/z) were 169.0→125.0, 153.1→108.0, and 196.8→135.2 for gallic acid, gentisic acid, and the IS, respectively. Chromatographic separation was achieved on a C18 column using mobile phases consisting of water with 0.1% acetic acid (A) and acetonitrile with 0.05% formic acid. (B) The total run time was 3min and calibration curves were linear over the concentrations of 0.33-2400ng/mL for both compounds (r(2)>0.995). Good precision (between 3.11% and 14.1% RSD) and accuracy (between -12.7% and 11% bias) was observed for quality controls at concentrations of 0.33 (lower limit of quantification), 1, 50, and 2000ng/mL. The mean extraction recovery of gallic acid and gentisic acid was 80.7% and 83.5%, respectively. Results from post-column infusion and post-extraction methods indicated that the analytical method exhibited negligible matrix effects. Finally, this validated assay was successfully applied in a cellular uptake study to determine the intracellular concentrations of gallic acid and gentisic acid in OAT expressing cells. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Graphene for amino acid biosensing: Theoretical study of the electronic transport

    Science.gov (United States)

    Rodríguez, S. J.; Makinistian, L.; Albanesi, E. A.

    2017-10-01

    The study of biosensors based on graphene has increased in the last years, the combination of excellent electrical properties and low noise makes graphene a material for next generation electronic devices. This work discusses the application of a graphene-based biosensor for the detection of amino acids histidine (His), alanine (Ala), aspartic acid (Asp), and tyrosine (Tyr). First, we present the results of modeling from first principles the adsorption of the four amino acids on a graphene sheet, we calculate adsorption energy, substrate-adsorbate distance, equilibrium geometrical configurations (upon relaxation) and densities of states (DOS) for each biomolecule adsorbed. Furthermore, in order to evaluate the effects of amino acid adsorption on the electronic transport of graphene, we modeled a device using first-principles calculations with a combination of Density Functional Theory (DFT) and Nonequilibrium Greens Functions (NEGF). We provide with a detailed discussion in terms of transmission, current-voltage curves, and charge transfer. We found evidence of differences in the electronic transport through the graphene sheet due to amino acid adsorption, reinforcing the possibility of graphene-based sensors for amino acid sequencing of proteins.

  5. Arg188 in rice sucrose transporter OsSUT1 is crucial for substrate transport

    Directory of Open Access Journals (Sweden)

    Sun Ye

    2012-11-01

    Full Text Available Abstract Background Plant sucrose uptake transporters (SUTs are H+/sucrose symporters related to the major facilitator superfamily (MFS. SUTs are essential for plant growth but little is known about their transport mechanism. Recent work identified several conserved, charged amino acids within transmembrane spans (TMS in SUTs that are essential for transport activity. Here we further evaluated the role of one of these positions, R188 in the fourth TMS of OsSUT1, a type II SUT. Results The OsSUT1(R188K mutant, studied by expression in plants, yeast, and Xenopus oocytes, did not transport sucrose but showed a H+ leak that was blocked by sucrose. The H+ leak was also blocked by β-phenyl glucoside which is not translocated by OsSUT1. Replacing the corresponding Arg in type I and type III SUTs, AtSUC1(R163K and LjSUT4(R169K, respectively, also resulted in loss of sucrose transport activity. Fluorination at the glucosyl 3 and 4 positions of α-phenyl glucoside greatly decreased transport by wild type OsSUT1 but did not affect the ability to block H+ leak in the R188K mutant. Conclusion OsSUT1 R188 appears to be essential for sucrose translocation but not for substrate interaction that blocks H+ leak. Therefore, we propose that an additional binding site functions in the initial recognition of substrates. The corresponding Arg in type I and III SUTs are equally important. We propose that R188 interacts with glucosyl 3-OH and 4-OH during translocation.

  6. Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

    Science.gov (United States)

    Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

    2008-01-01

    The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792

  7. The Monocarboxylate Transporter Inhibitor α-Cyano-4-Hydroxycinnamic Acid Disrupts Rat Lung Branching

    Directory of Open Access Journals (Sweden)

    Sara Granja

    2013-12-01

    Full Text Available Background/Aims: The human embryo develops in a hypoxic environment. In this way, cells have to rely on the glycolytic pathway for energy supply, leading to an intracellular accumulation of monocarboxylates such as lactate and pyruvate. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for the maintenance of intracellular pH homeostasis. This transport is mediated by a family of transporters, designated by monocarboxylate transporters (MCTs, namely isoforms 1 and 4. MCT1/4 expression is regulated by the ancillary protein CD147.The general aim of this study was to characterize the expression pattern of MCT1/4, CD147 and the glucose transporter GLUT1 during human fetal lung development and elucidate the role of MCTs in lung development. Methods: The expression pattern of MCT1/4 and GLUT1 was characterized by immunohistochemistry and fetal lung viability and branching were evaluated by exposing rat fetal lung explants to CHC, an inhibitor of MCT activity. Results: Our findings show that all the biomarkers are differently expressed during fetal lung development and that CHC appears to have an inhibitory effect on lung branching and viability, in a dose dependent way. Conclusion: We provide evidence for the role of MCTs in embryo lung development, however to prove the dependence of MCT activity further studies are waranted.

  8. ρ0 Cells Feature De-Ubiquitination of SLC Transporters and Increased Levels and Fluxes of Amino Acids

    Directory of Open Access Journals (Sweden)

    André Bordinassi Medina

    2017-04-01

    Full Text Available Solute carrier (SLC transporters are a diverse group of membrane transporter proteins that regulate the cellular flux and distribution of endogenous and xenobiotic compounds. Post-translational modifications (PTMs, such as ubiquitination, have recently emerged as one of the major regulatory mechanisms in protein function and localization. Previously, we showed that SLC amino acid transporters were on average 6-fold de-ubiquitinated and increased amino acid levels were detected in ρ0 cells (lacking mitochondrial DNA, mtDNA compared to parental cells. Here, we elucidated the altered functionality of SLC transporters and their dynamic ubiquitination status by measuring the uptake of several isotopically labeled amino acids in both human osteosarcoma 143B.TK- and ρ0 cells. Our pulse chase analysis indicated that de-ubiquitinated amino acid transporters in ρ0 cells were accompanied by an increased transport rate, which leads to higher levels of amino acids in the cell. Finding SLC transport enhancers is an aim of the pharmaceutical industry in order to compensate for loss of function mutations in these genes. Thus, the ubiquitination status of SLC transporters could be an indicator for their functionality, but evidence for a direct connection between de-ubiquitination and transporter activity has to be further elucidated.

  9. Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide

    Directory of Open Access Journals (Sweden)

    Jeong HU

    2015-01-01

    Full Text Available Hyeon-Uk Jeong,1 Mihwa Kwon,2 Yongnam Lee,3 Ji Seok Yoo,3 Dae Hee Shin,3 Im-Sook Song,2 Hye Suk Lee1 1College of Pharmacy, The Catholic University of Korea, Bucheon 420-743, Korea; 2College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Korea; 3Central R&D Institute, Yungjin Pharm Co., Ltd., Suwon 443-270, Korea Abstract: We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1, OAT3, organic anion transporting polypeptide 1B1 (OATP1B1, OATP1B3, organic cation transporter 1 (OCT1, OCT2, P-glycoprotein (P-gp, and breast cancer resistance protein (BCRP. The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3 and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3. The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (Km =41.5 µM, maximum uptake rate (Vmax =46.2 pmol/minute, and intrinsic clearance (CLint =1.11 µL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CLint values of 0.035 and 0.034 µL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 µ

  10. Silicon in vascular plants: uptake, transport and its influence on mineral stress under acidic conditions.

    Science.gov (United States)

    Pontigo, Sofía; Ribera, Alejandra; Gianfreda, Liliana; de la Luz Mora, María; Nikolic, Miroslav; Cartes, Paula

    2015-07-01

    So far, considerable advances have been achieved in understanding the mechanisms of Si uptake and transport in vascular plants. This review presents a comprehensive update about this issue, but also provides the new insights into the role of Si against mineral stresses that occur in acid soils. Such information could be helpful to understand both the differential Si uptake ability as well as the benefits of this mineral element on plants grown under acidic conditions. Silicon (Si) has been widely recognized as a beneficial element for many plant species, especially under stress conditions. In the last few years, great efforts have been made to elucidate the mechanisms involved in uptake and transport of Si by vascular plants and recently, different Si transporters have been identified. Several researches indicate that Si can alleviate various mineral stresses in plants growing under acidic conditions, including aluminium (Al) and manganese (Mn) toxicities as well as phosphorus (P) deficiency all of which are highly detrimental to crop production. This review presents recent findings concerning the influence of uptake and transport of Si on mineral stress under acidic conditions because a knowledge of this interaction provides the basis for understanding the role of Si in mitigating mineral stress in acid soils. Currently, only four Si transporters have been identified and there is little information concerning the response of Si transporters under stress conditions. More investigations are therefore needed to establish whether there is a relationship between Si transporters and the benefits of Si to plants subjected to mineral stress. Evidence presented suggests that Si supply and its subsequent accumulation in plant tissues could be exploited as a strategy to improve crop productivity on acid soils.

  11. Strigolactone Inhibition of Branching Independent of Polar Auxin Transport1[OPEN

    Science.gov (United States)

    Mason, Michael G.; Beveridge, Christine A.

    2015-01-01

    The outgrowth of axillary buds into branches is regulated systemically via plant hormones and the demand of growing shoot tips for sugars. The plant hormone auxin is thought to act via two mechanisms. One mechanism involves auxin regulation of systemic signals, cytokinins and strigolactones, which can move into axillary buds. The other involves suppression of auxin transport/canalization from axillary buds into the main stem and is enhanced by a low sink for auxin in the stem. In this theory, the relative ability of the buds and stem to transport auxin controls bud outgrowth. Here, we evaluate whether auxin transport is required or regulated during bud outgrowth in pea (Pisum sativum). The profound, systemic, and long-term effects of the auxin transport inhibitor N-1-naphthylphthalamic acid had very little inhibitory effect on bud outgrowth in strigolactone-deficient mutants. Strigolactones can also inhibit bud outgrowth in N-1-naphthylphthalamic acid-treated shoots that have greatly diminished auxin transport. Moreover, strigolactones can inhibit bud outgrowth despite a much diminished auxin supply in in vitro or decapitated plants. These findings demonstrate that auxin sink strength in the stem is not important for bud outgrowth in pea. Consistent with alternative mechanisms of auxin regulation of systemic signals, enhanced auxin biosynthesis in Arabidopsis (Arabidopsis thaliana) can suppress branching in yucca1D plants compared with wild-type plants, but has no effect on bud outgrowth in a strigolactone-deficient mutant background. PMID:26111543

  12. The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

    Science.gov (United States)

    Martin, Audrey; Daniel, Jaiyanth

    2018-02-05

    Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Acanthopanax trifoliatus inhibits lipopolysaccharide-induced inflammatory response in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Tzu-Mei Chien

    2015-10-01

    Full Text Available Acanthopanax trifoliatus is a well-known herb that is used for the treatment of bruising, neuralgia, impotence, and gout in Taiwan. This herb exhibits multifunctional activities, including anticancer, anti-inflammation, and antioxidant effects. This paper investigated the in vitro and in vivo anti-inflammatory effect of A. trifoliatus. High-performance liquid chromatography analysis established the fingerprint chromatogram of the ethyl acetate fraction of A. trifoliatus (EAAT. The anti-inflammatory effect of EAAT was detected using lipopolysaccharide (LPS stimulation of the mouse macrophage cell line RAW264.7 in vitro and LPS-induced lung injury in vivo. The effects of EAAT on LPS-induced production of inflammatory mediators in RAW264.7 murine macrophages and the mouse model were measured using enzyme-linked immunosorbent assay and Western blot. EAAT attenuated the production of LPS-induced nitric oxide (NO, tumor necrosis factor-alpha, interleukin-1β (IL-1β, and IL-6 in vitro and in vivo. Pretreatment with EAAT markedly reduced LPS-induced histological alterations in lung tissues. Furthermore, EAAT significantly reduced the number of total cells and protein concentration levels in the bronchoalveolar lavage fluid. Western blotting test results revealed that EAAT blocked protein expression of inducible NO synthase, cyclooxygenase-2, phosphorylation of Nuclear factor-kappa-B Inhibitor alpha (IκB-α protein, and mitogen-activated protein kinases in LPS-stimulated RAW264.7 cells as well as LPS-induced lung injury. This study suggests that A. trifoliatus may be a potential therapeutic candidate for the treatment of inflammatory diseases.

  14. Supervanadophile: Complexation, preconcentration and transport studies of vanadium by octa functionalized calix [4] resorcinarene - hydroxamic acid

    International Nuclear Information System (INIS)

    Jain, V. K.; Pillai, S. G.; Gupte, H. S.

    2008-01-01

    A new octa functionalized calix[4] resorcinarene bearing eight hydroxamic acid groups has been synthesized and its analytical properties have been investigated. To elucidate the structure of the compound, elemental analysis, fourier transform infrared and 1 H NMR spectral data have been used. The compound showed high affinity and selectivity for vanadium(V) in presence of large quantities of associated metal ions. The complexation of vanadium(V) with octa functionalized calix[4] resorcinarene bearing eight hydroxamic acid has a 4:1 metal: ligand stoichiometry as evaluated by Job's plot. A spectrophotometric method is proposed for the extractive determination of vanadium(V) in an acidic medium in the presence of diversified matrix, and verified by inductively coupled plasma-atomic emission spectrometry. Under the optimum condition of acidity, solvent, interfering ions and octa functionalized calix[4] resorcinarene bearing eight hydroxamic acid concentration, the molar absorptivity of the complex is 5630 1 mol -1 cm -1 at 495 nm. The system obeys Beer's law over the range 0.125-8.75 μg ml -1 of vanadium(V) with Sandell sensitivity 0.009 μg cm -2 . The preconcentration factor and overall stability constant evaluated at 25 d eg C were 142 and 14.18, respectively. The complexation is characterized by favorable enthalpy and entropy changes. Liquid membrane transport studies of vanadium(V) were carried out from source to the receiving phase under controlled conditions and a mechanism for transport is suggested. To check the validity of the proposed method, vanadium is determined in environmental, biological samples and some standard reference materials from NIST and BCS

  15. Transport of uranium by supported liquid membrane containing bis(2-ethylhexyl) hydrogenphosphate and 1-octanol

    International Nuclear Information System (INIS)

    Akiba, Kenichi; Kanno, Takuji; Takahashi, Toshihiko.

    1984-01-01

    Carrier-mediated transport of uranium(VI) has been studied by means of liquid membranes impregnated in a microporous polymer. Liquid membranes containing bis(2-ethylhexyl) hydrogenphosphate (DEHPA) alone yielded inadequate stripping of uranium. The addition of 1-octanol to DEHPA solutions resulted in a decrease in extractability, and made it possible to control the distribution ratio of uranium. Uranium in the feed solution was sufficiently transported across the liquid membrane containing this DEHPA-1-octanol mixture into the product solution. The apparent rate constant (ksub(obs)) of transport increased slightly with an increase in carrier concentrations. Variations in acid concentrations of the feed solution (pH 2.5--3.2) and the product solution (0.1--1.0 M H 2 SO 4 ) had little effect on the transport rate. A large excess of uranium, more than the carrier content in the liquid membrane, was finally concentrated in the stripping acid. (author)

  16. Diagnostic usefulness of 18F-FAMT PET and L-type amino acid transporter 1 (LAT1) expression in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Nobusawa, Aiko; Kim, Mai; Kaira, Kyoichi; Miyashita, Go; Negishi, Akihide; Yokoo, Satoshi; Oriuchi, Noboru; Higuchi, Tetsuya; Tsushima, Yoshito; Kanai, Yoshikatsu; Oyama, Tetsunari

    2013-01-01

    l-[3- 18 F]-α-Methyltyrosine ( 18 F-FAMT) was developed as an amino acid tracer for PET imaging to provide better specificity than 2-[ 18 F]fluoro-2-deoxy-d-glucose ( 18 F-FDG) PET for cancer diagnosis. We investigated the diagnostic usefulness of 18 F-FAMT in oral squamous cell carcinoma (OSCC). The correlation between tumour uptake of 18 F-FAMT and L-type amino acid transporter 1 (LAT1) expression was determined. The study group comprised 68 OSCC patients who underwent both 18 F-FAMT and 18 F-FDG PET. Resected tumour sections were stained by immunohistochemistry for LAT1, CD98 and Ki-67, and microvessel density was determined in terms of CD34 and p53 expression. The sensitivity of primary tumour detection by 18 F-FAMT and 18 F-FDG PET was 98 % and 100 %, respectively. The sensitivity, specificity and accuracy of 18 F-FAMT PET for detecting malignant lymph nodes were 68 %, 99 % and 97 %, respectively, and equivalent values for 18 F-FDG PET were 84 %, 94 % and 94 %, respectively. The specificity and accuracy of 18 F-FAMT were significantly higher than those of 18 F-FDG. The uptake of 18 F-FAMT was significantly correlated with LAT1 expression, cell proliferation and advanced stage. The expression of LAT1 in OSCC cells was closely correlated with CD98 levels, cell proliferation and angiogenesis. 18 F-FAMT PET showed higher specificity for detecting malignant lesions than 18 F-FDG PET. The uptake of 18 F-FAMT by OSCC cells can be determined by the presence of LAT1 expression and tumour cell proliferation. (orig.)

  17. Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor

    DEFF Research Database (Denmark)

    Poulsen, Peter; Gaber, Richard F.; Kielland-Brandt, Morten

    2008-01-01

    The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structural...

  18. Proliferation-dependent changes in amino acid transport and glucose metabolism in glioma cell lines

    International Nuclear Information System (INIS)

    Sasajima, Toshio; Miyagawa, Tadashi; Oku, Takamitsu; Gelovani, Juri G.; Finn, Ronald; Blasberg, Ronald

    2004-01-01

    Amino acid imaging is increasingly being used for assessment of brain tumor malignancy, extent of disease, and prognosis. This study explores the relationship between proliferative activity, amino acid transport, and glucose metabolism in three glioma cell lines (U87, Hs683, C6) at different phases of growth in culture. Growth phase was characterized by direct cell counting, proliferation index determined by flow cytometry, and [ 3 H]thymidine (TdR) accumulation, and was compared with the uptake of two non-metabolized amino acids ([ 14 C]aminocyclopentane carboxylic acid (ACPC) and [ 14 C]aminoisobutyric acid (AIB)), and [ 18 F]fluorodeoxyglucose (FDG). Highly significant relationships between cell number (density), proliferation index, and TdR accumulation rate were observed in all cell lines (r>0.99). Influx (K 1 ) of both ACPC and AIB was directly related to cell density, and inversely related to the proliferation index and TdR accumulation in all cell lines. The volume of distribution (V d ) for ACPC and AIB was lowest during rapid growth and highest during the near-plateau growth phase in all cell lines. FDG accumulation in Hs683 and C6 cells was unaffected by proliferation rate, growth phase, and cell density, whereas FDG accumulation was correlated with TdR accumulation, growth phase, and cell density in U87 cells. This study demonstrates that proliferation rate and glucose metabolism are not necessarily co-related in all glioma cell lines. The values of K 1 and V d for ACPC and AIB under different growth conditions suggest that these tumor cell lines can up-regulate amino acid transporters in their cell membranes when their growth conditions become adverse and less than optimal. (orig.)

  19. High expression of L-type amino acid transporter 1 as a prognostic marker in bile duct adenocarcinomas

    International Nuclear Information System (INIS)

    Yanagisawa, Nobuyuki; Hana, Kiyomi; Nakada, Norihiro; Ichinoe, Masaaki; Koizumi, Wasaburo; Endou, Hitoshi; Okayasu, Isao; Murakumo, Yoshiki

    2014-01-01

    Oncocytic L-type amino acid transporter (LAT) 1 may be a prognostic indicator and target of new molecular therapeutic agents against malignancies. To investigate whether LAT1 expression influence the outcomes of patients with bile duct cancer, the expression of LAT1, LAT2, CD98, and Ki-67 was investigated immunohistochemically in 134 surgically resected bile duct adenocarcinomas, including 84 distal extrahepatic bile duct adenocarcinomas, 21 hilar cholangiocarcinomas, 15 intrahepatic cholangiocarcinomas, and 14 ampullary adenocarcinomas. LAT1 expression was weakly correlated with CD98 expression and Ki-67 labeling index (LI). Kaplan–Meier analysis showed a significant difference in prognosis between patients with bile duct adenocarcinomas having LAT1-high and -low scores, whereas LAT2 and CD98 expression and Ki-67 LI were not predictive of poor prognosis. Prognosis tended to be worse in patients having tumors with LAT1-high/LAT2-low than LAT1-low/LAT2-high scores (P = 0.0686). Multivariable analyses revealed that LAT1 expression, surgical margin, pT stage were independent prognostic factors. In conclusion, aberrant overexpression of LAT1 in bile duct adenocarcinoma predicts poor prognosis, suggesting that LAT1 may be a potential target of anticancer therapy

  20. A coupled hydrodynamic-hydrochemical modeling for predicting mineral transport in a natural acid drainage system.

    Science.gov (United States)

    Zegers Risopatron, G., Sr.; Navarro, L.; Montserrat, S., Sr.; McPhee, J. P.; Niño, Y.

    2017-12-01

    The geochemistry of water and sediments, coupled with hydrodynamic transport in mountainous channels, is of particular interest in central Chilean Andes due to natural occurrence of acid waters. In this paper, we present a coupled transport and geochemical model to estimate and understand transport processes and fate of minerals at the Yerba Loca Basin, located near Santiago, Chile. In the upper zone, water presentes low pH ( 3) and high concentrations of iron, aluminum, copper, manganese and zinc. Acidity and minerals are the consequence of water-rock interactions in hydrothermal alteration zones, rich in sulphides and sulphates, covered by seasonal snow and glaciers. Downstream, as a consequence of neutral to alkaline lateral water contributions (pH >7) along the river, pH increases and concentration of solutes decreases. The mineral transport model has three components: (i) a hydrodynamic model, where we use HEC-RAS to solve 1D Saint-Venant equations, (ii) a sediment transport model to estimate erosion and sedimentation rates, which quantify minerals transference between water and riverbed and (iii) a solute transport model, based on the 1D OTIS model which takes into account the temporal delay in solutes transport that typically is observed in natural channels (transient storage). Hydrochemistry is solved using PHREEQC, a software for speciation and batch reaction. Our results show that correlation between mineral precipitation and dissolution according to pH values changes along the river. Based on pH measurements (and according to literature) we inferred that main minerals in the water system are brochantite, ferrihydrite, hydrobasaluminite and schwertmannite. Results show that our model can predict the transport and fate of minerals and metals in the Yerba Loca Basin. Mineral dissolution and precipitation process occur for limited ranges of pH values. When pH values are increased, iron minerals (schwertmannite) are the first to precipitate ( 2.5

  1. Membrane topology of rat sodium-coupled neutral amino acid transporter 2 (SNAT2).

    Science.gov (United States)

    Ge, Yudan; Gu, Yanting; Wang, Jiahong; Zhang, Zhou

    2018-07-01

    Sodium-coupled neutral amino acid transporter 2 (SNAT2) is a subtype of the amino acid transport system A that is widely expressed in mammalian tissues. It plays critical roles in glutamic acid-glutamine circulation, liver gluconeogenesis and other biological pathway. However, the topology of the SNAT2 amino acid transporter is unknown. Here we identified the topological structure of SNAT2 using bioinformatics analysis, Methoxy-polyethylene glycol maleimide (mPEG-Mal) chemical modification, protease cleavage assays, immunofluorescence and examination of glycosylation. Our results show that SNAT2 contains 11 transmembrane domains (TMDs) with an intracellular N terminus and an extracellular C terminus. Three N-glycosylation sites were verified at the largest extracellular loop. This model is consistent with the previous model of SNAT2 with the exception of a difference in number of glycosylation sites. This is the first time to confirm the SNAT2 membrane topology using experimental methods. Our study on SNAT2 topology provides valuable structural information of one of the solute carrier family 38 (SLC38) members. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Low brain ascorbic acid increases susceptibility to seizures in mouse models of decreased brain ascorbic acid transport and Alzheimer's disease.

    Science.gov (United States)

    Warner, Timothy A; Kang, Jing-Qiong; Kennard, John A; Harrison, Fiona E

    2015-02-01

    Seizures are a known co-occurring symptom of Alzheimer's disease, and they can accelerate cognitive and neuropathological dysfunction. Sub-optimal vitamin C (ascorbic acid) deficiency, that is low levels that do not lead the sufferer to present with clinical signs of scurvy (e.g. lethargy, hemorrhage, hyperkeratosis), are easily obtainable with insufficient dietary intake, and may contribute to the oxidative stress environment of both Alzheimer's disease and epilepsy. The purpose of this study was to test whether mice that have diminished brain ascorbic acid in addition to carrying human Alzheimer's disease mutations in the amyloid precursor protein (APP) and presenilin 1 (PSEN1) genes, had altered electrical activity in the brain (electroencephalography; EEG), and were more susceptible to pharmacologically induced seizures. Brain ascorbic acid was decreased in APP/PSEN1 mice by crossing them with sodium vitamin C transporter 2 (SVCT2) heterozygous knockout mice. These mice have an approximately 30% decrease in brain ascorbic acid due to lower levels of SVCT2 that supplies the brain with ASC. SVCT2+/-APP/PSEN1 mice had decreased ascorbic acid and increased oxidative stress in brain, increased mortality, faster seizure onset latency following treatment with kainic acid (10 mg/kg i.p.), and more ictal events following pentylenetetrazol (50 mg/kg i.p.) treatment. Furthermore, we report the entirely novel phenomenon that ascorbic acid deficiency alone increased the severity of kainic acid- and pentylenetetrazol-induced seizures. These data suggest that avoiding ascorbic acid deficiency may be particularly important in populations at increased risk for epilepsy and seizures, such as Alzheimer's disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Ammonium ion transport by the AMT/Rh homolog TaAMT1;1 is stimulated by acidic pH

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Alsterfjord, Magnus; Macaulay, Nanna

    2009-01-01

    It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH (4......) (+) or methylammonium ions (MeA(+)). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH (4) (+) or MeA(+) transport. This was confirmed by the close correlation between the uptake of radio-labeled Me......A(+) and MeA(+)-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH (4) (+) and MeA(+). We suggest a model...

  4. Impact of Microbial Growth on Subsurface Perfluoroalkyl Acid Transport

    Science.gov (United States)

    Weathers, T. S.; Higgins, C. P.; Sharp, J.

    2014-12-01

    The fate and transport of poly and perfluoroalkyl substances (PFASs) in the presence of active microbial communities has not been widely investigated. These emerging contaminants are commonly utilized in aqueous film-forming foams (AFFF) and have often been detected in groundwater. This study explores the transport of a suite of perfluorocarboxylic acids and perfluoroalkylsulfonates, including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), in microbially active settings. Single point organic carbon normalized sorption coefficients derived by exposing inactive cellular material to PFASs result in more than an order of magnitude increase in sorption compared to soil organic carbon sorption coefficients found in literature. For example, the sorption coefficients for PFOS are 4.05±0.07 L/kg and 2.80±0.08 L/kg for cellular organic carbon and soil organic carbon respectively. This increase in sorption, coupled with enhanced extracellular polymeric substance production observed during growth of a common hydrocarbon degrading soil microbe exposed to source-level concentrations of PFASs (10 mg/L of 11 analytes, 110 mg/L total) may result in PFAS retardation in situ. To address the upscaling of this phenomenon, flow-through columns packed with low-organic carbon sediment and biostimulated with 10 mg/L glucose were exposed to PFAS concentrations from 15 μg/L to 10 mg/L of each 11 analytes. Breakthrough and tailing of each analyte was measured and modeled with Hydrus-1D to explore sorption coefficients over time for microbially active columns.

  5. Acid-extrusion from tissue: the interplay between membrane transporters and pH buffers.

    Science.gov (United States)

    Hulikova, Alzbeta; Harris, Adrian L; Vaughan-Jones, Richard D; Swietach, Pawel

    2012-01-01

    The acid-base balance of cells is related to the concentration of free H⁺ ions. These are highly reactive, and their intracellular concentration must be regulated to avoid detrimental effects to the cell. H⁺ ion dynamics are influenced by binding to chelator substances ('buffering'), and by the production, diffusion and membrane-transport of free H⁺ ions or of the H⁺-bound chelators. Intracellular pH (pHi) regulation aims to balance this system of diffusion-reaction-transport processes at a favourable steady-state pHi. The ability of cells to regulate pHi may set a limit to tissue growth and can be subject to selection pressures. Cancer cells have been postulated to respond favourably to such selection pressures by evolving a better means of pHi regulation. A particularly important feature of tumour pHi regulation is acid-extrusion, which involves H⁺-extrusion and HCO₃⁻-uptake by membrane-bound transporter-proteins. Extracellular CO₂/HCO₃⁻ buffer facilitates these membrane-transport processes. As a mobile pH-buffer, CO₂/HCO₃⁻ protects the extracellular space from excessive acidification that could otherwise inhibit further acid-extrusion. CO₂/HCO₃⁻ also provides substrate for HCO₃⁻-transporters. However, the inherently slow reaction kinetics of CO₂/HCO₃⁻ can be rate-limiting for acid-extrusion. To circumvent this, cells can express extracellular-facing carbonic anhydrase enzymes to accelerate the attainment of equilibrium between CO₂, HCO₃⁻ and H⁺. The acid-extrusion apparatus has been proposed as a target for anti-cancer therapy. The major targets include H⁺ pumps, Na⁺/H⁺ exchangers and carbonic anhydrases. The effectiveness of such therapy will depend on the correct identification of rate-limiting steps in pHi regulation in a specific type of cancer.

  6. Experimental Study and Reactive Transport Modeling of Boric Acid Leaching of Concrete

    Directory of Open Access Journals (Sweden)

    Chiang K.-T. K.

    2013-07-01

    Full Text Available Borated water leakage through spent fuel pools (SFPs at pressurized water reactors is a concern because it could cause corrosion of reinforcement steel in the concrete structure, compromise the integrity of the structure, or cause unmonitored releases of contaminated water to the environment. Experimental data indicate that pH is a critical parameter that determines the corrosion susceptibility of rebar in borated water and the degree of concrete degradation by boric acid leaching. In this study, reactive transport modeling of concrete leaching by borated water was performed to provide information on the solution pH in the concrete crack or matrix and the degree of concrete degradation at different locations of an SFP concrete structure exposed to borated water. Simulations up to 100 years were performed using different boric acid concentrations, crack apertures, and solution flow rates. Concrete cylinders were immersed in boric acid solutions for several months and the mineralogical changes and boric acid penetration in the concrete cylinder were evaluated as a function of time. The depths of concrete leaching by boric acid solution derived from the reactive transport simulations were compared with the measured boric acid penetration depth.

  7. Transport and degradation of 2-14C abscicine acid in the coleus rehneltianus berger sprout

    International Nuclear Information System (INIS)

    Klaska, A.

    1979-01-01

    1 μg ABA-2- 14 C aqueous solution was injected into the youngest or into a fully grown leaf of young and older coleus plants. The activity quantity in the various sprout parts is investigated after 2, 24 and 72 h; as well as which labelled substances other than abscisic acid (ABA) occur. The activity in the ethanol extracts was detected with the help of liquid scintillation measurements. Thin layer chromatography and gas chromatography were used to characterize the radioactive substances. The results show that ABA is degraded into three metabolites which are characterized by their relative Rsub(F) values using chromatography with LM 2 as substance 0.2, 0.5a, 0.5b and 0.8. Comparing with the literature shows that it could be 6'-hydroxy methyl ABA, ABA glucoside, phaseic acid and dihydrophaseic acid. Young and old leaves in older plants have the same ability to degrade ABA taking the occurence of the 0.8 metabolite as standard. The degradation in younger plants is firstly very slight. However, within 72 h the ability of ABA degradation is induced in older leaves of younger plants, so that finally the degradation rate is as big as in older plants. Activity is essentially exported by adult leaves of older plants. The basipetal transport is considerably greater than the acropetal one. Results show that mainly ABA is transported in the acropetal test, whereas ABA and fraction 0.5 are transported in the basipetal direction. A reversed transport direction is observed after applying ABA to a young leaf. The transport basipetal is polarized in the first two hours, after 72 hours of transport there is a definite acropetal polarity. (orig./MG) [de

  8. Amino acid transporter genes are essential for FLO11-dependent and FLO11-independent biofilm formation and invasive growth in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Rasmus Torbensen

    Full Text Available Amino acids can induce yeast cell adhesion but how amino acids are sensed and signal the modulation of the FLO adhesion genes is not clear. We discovered that the budding yeast Saccharomyces cerevisiae CEN.PK evolved invasive growth ability under prolonged nitrogen limitation. Such invasive mutants were used to identify amino acid transporters as regulators of FLO11 and invasive growth. One invasive mutant had elevated levels of FLO11 mRNA and a Q320STOP mutation in the SFL1 gene that encodes a protein kinase A pathway regulated repressor of FLO11. Glutamine-transporter genes DIP5 and GNP1 were essential for FLO11 expression, invasive growth and biofilm formation in this mutant. Invasive growth relied on known regulators of FLO11 and the Ssy1-Ptr3-Ssy5 complex that controls DIP5 and GNP1, suggesting that Dip5 and Gnp1 operates downstream of the Ssy1-Ptr3-Ssy5 complex for regulation of FLO11 expression in a protein kinase A dependent manner. The role of Dip5 and Gnp1 appears to be conserved in the S. cerevisiae strain ∑1278b since the dip5 gnp1 ∑1278b mutant showed no invasive phenotype. Secondly, the amino acid transporter gene GAP1 was found to influence invasive growth through FLO11 as well as other FLO genes. Cells carrying a dominant loss-of-function PTR3(647::CWNKNPLSSIN allele had increased transcription of the adhesion genes FLO1, 5, 9, 10, 11 and the amino acid transporter gene GAP1. Deletion of GAP1 caused loss of FLO11 expression and invasive growth. However, deletions of FLO11 and genes encoding components of the mitogen-activated protein kinase pathway or the protein kinase A pathway were not sufficient to abolish invasive growth, suggesting involvement of other FLO genes and alternative pathways. Increased intracellular amino acid pools in the PTR3(647::CWNKNPLSSIN-containing strain opens the possibility that Gap1 regulates the FLO genes through alteration of the amino acid pool sizes.

  9. Androgen receptor and nutrient signaling pathways coordinate the demand for increased amino acid transport during prostate cancer progression

    DEFF Research Database (Denmark)

    Wang, Qian; Bailey, Charles G; Ng, Cynthia

    2011-01-01

    was sufficient to decrease cell growth and mTORC1 signaling in prostate cancer cells. These cells maintained levels of amino acid influx through androgen receptor-mediated regulation of LAT3 expression and ATF4 regulation of LAT1 expression after amino acid deprivation. These responses remained intact in primary......L-Type amino acid transporters such as LAT1 and LAT3 mediate the uptake of essential amino acids. Here, we report that prostate cancer cells coordinate the expression of LAT1 and LAT3 to maintain sufficient levels of leucine needed for mTORC1 signaling and cell growth. Inhibiting LAT function...... prostate cancer, as indicated by high levels of LAT3 in primary disease, and by increased levels of LAT1 after hormone ablation and in metastatic lesions. Taken together, our results show how prostate cancer cells respond to demands for increased essential amino acids by coordinately activating amino acid...

  10. Insulin acutely upregulates protein expression of the fatty acid transporter CD36 in human skeletal muscle in vivo

    NARCIS (Netherlands)

    Corpeleijn, E.; Pelsers, M.M.A.L.; Soenen, S.; Mensink, M.; Bouwman, F.G.; Kooi, M.E.; Saris, W.H.M.; Glatz, J.F.C.; Blaak, E.E.

    2008-01-01

    Enhanced fatty acid uptake may lead to the accumulation of lipid intermediates. This is related to insulin resistance and type 2 diabetes mellitus. Rodent studies suggest that fatty acid transporters are acutely regulated by insulin. We investigated differences in fatty acid transporter content

  11. Fasting induces basolateral uptake transporters of the SLC family in the liver via HNF4alpha and PGC1alpha.

    Science.gov (United States)

    Dietrich, Christoph G; Martin, Ina V; Porn, Anne C; Voigt, Sebastian; Gartung, Carsten; Trautwein, Christian; Geier, Andreas

    2007-09-01

    Fasting induces numerous adaptive changes in metabolism by several central signaling pathways, the most important represented by the HNF4alpha/PGC-1alpha-pathway. Because HNF4alpha has been identified as central regulator of basolateral bile acid transporters and a previous study reports increased basolateral bile acid uptake into the liver during fasting, we hypothesized that HNF4alpha is involved in fasting-induced bile acid uptake via upregulation of basolateral bile acid transporters. In rats, mRNA of Ntcp, Oatp1, and Oatp2 were significantly increased after 48 h of fasting. Protein expression as determined by Western blot showed significant increases for all three transporters 72 h after the onset of fasting. Whereas binding activity of HNF1alpha in electrophoretic mobility shift assays remained unchanged, HNF4alpha binding activity to the Ntcp promoter was increased significantly. In line with this result, we found significantly increased mRNA expression of HNF4alpha and PGC-1alpha. Functional studies in HepG2 cells revealed an increased endogenous NTCP mRNA expression upon cotransfection with either HNF4alpha, PGC-1alpha, or a combination of both. We conclude that upregulation of the basolateral bile acid transporters Ntcp, Oatp1, and Oatp2 in fasted rats is mediated via the HNF4alpha/PGC-1alpha pathway.

  12. NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense1

    Science.gov (United States)

    Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

    2005-01-01

    Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family. PMID:16126865

  13. Induction of Heavy-Metal-Transporting CPX-Type ATPases during Acid Adaptation in Lactobacillus bulgaricus▿

    Science.gov (United States)

    Penaud, S.; Fernandez, A.; Boudebbouze, S.; Ehrlich, S. D.; Maguin, E.; van de Guchte, M.

    2006-01-01

    Lactobacillus bulgaricus is a lactic acid bacteria (LAB) that, through the production of lactic acid, gradually acidifies its environment during growth. In the course of this process, L. bulgaricus acquires an improved tolerance to acidity. A survey of the recently established genome sequence shows that this bacterium possesses few of the pH control functions that have been described in other LAB and raises the question of what other mechanisms could be involved in its adaptation to the decreasing environmental pH. In some bacteria other than LAB, ion transport systems have been implicated in acid adaptation. We therefore studied the expression of this type of transport system during acid adaptation in L. bulgaricus by reverse transcription and real-time quantitative PCR and mapped transcription start sites. Intriguingly, the most significantly induced were three ATPases carrying the CPX signature of heavy-metal transporters. Protein homology and the presence of a conserved sequence motif in the promoter regions of the genes encoding these proteins strongly suggest that they are involved in copper homeostasis. Induction of this system is thought to assist in avoiding indirect damage that could result from medium acidification. PMID:16997986

  14. Surface proton transport of fully protonated poly(aspartic acid) thin films on quartz substrates

    Science.gov (United States)

    Nagao, Yuki; Kubo, Takahiro

    2014-12-01

    Thin film structure and the proton transport property of fully protonated poly(aspartic acid) (P-Asp100) have been investigated. An earlier study assessed partially protonated poly(aspartic acid), highly oriented thin film structure and enhancement of the internal proton transport. In this study of P-Asp100, IR p-polarized multiple-angle incidence resolution (P-MAIR) spectra were measured to investigate the thin film structure. The obtained thin films, with thicknesses of 120-670 nm, had no oriented structure. Relative humidity dependence of the resistance, proton conductivity, and normalized resistance were examined to ascertain the proton transport property of P-Asp100 thin films. The obtained data showed that the proton transport of P-Asp100 thin films might occur on the surface, not inside of the thin film. This phenomenon might be related with the proton transport of the biological system.

  15. Transportation impact analysis for the shipment of Low Specific Activity Nitric Acid

    International Nuclear Information System (INIS)

    Green, J.R.

    1994-01-01

    This document was written in support of the Plutonium-Uranium Extraction (PUREX) Facility Low Specific Activity (LSA) Nitric Acid Shipment Environmental Assessment. It analyzes the potential toxicological and radiological risks associated with the transportation of PUREX Facility LSA Nitric Acid from the Hanford Site in Washington State to three Eastern ports

  16. Perfluoroalkyl Acid Concentrations in Blood Samples Subjected to Transportation and Processing Delay

    DEFF Research Database (Denmark)

    Bach, Cathrine Carlsen; Henriksen, Tine Brink; Bossi, Rossana

    2015-01-01

    and transportation prior to processing and samples with immediate processing and freezing. METHODS: Pregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed...... and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification. RESULTS: For samples taken in the winter, relative...... differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate...

  17. The actions of exogenous leucine on mTOR signalling and amino acid transporters in human myotubes

    Directory of Open Access Journals (Sweden)

    Cameron-Smith David

    2011-06-01

    Full Text Available Abstract Background The branched-chain amino acid (BCAA leucine has been identified to be a key regulator of skeletal muscle anabolism. Activation of anabolic signalling occurs via the mammalian target of rapamycin (mTOR through an undefined mechanism. System A and L solute carriers transport essential amino acids across plasma membranes; however it remains unknown whether an exogenous supply of leucine regulates their gene expression. The aim of the present study was to investigate the effects of acute and chronic leucine stimulation of anabolic signalling and specific amino acid transporters, using cultured primary human skeletal muscle cells. Results Human myotubes were treated with leucine, insulin or co-treated with leucine and insulin for 30 min, 3 h or 24 h. Activation of mTOR signalling kinases were examined, together with putative nutrient sensor human vacuolar protein sorting 34 (hVps34 and gene expression of selected amino acid transporters. Phosphorylation of mTOR and p70S6K was transiently increased following leucine exposure, independently to insulin. hVps34 protein expression was also significantly increased. However, genes encoding amino acid transporters were differentially regulated by insulin and not leucine. Conclusions mTOR signalling is transiently activated by leucine within human myotubes independently of insulin stimulation. While this occurred in the absence of changes in gene expression of amino acid transporters, protein expression of hVps34 increased.

  18. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

    Science.gov (United States)

    McGinley, Cian; Bishop, David J

    2016-12-01

    McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βm in vitro ), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βm in vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O 2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

  19. Supplementation of branched-chain amino acids in protein-restricted diets modulates the expression levels of amino acid transporters and energy metabolism associated regulators in the adipose tissue of growing pigs

    Directory of Open Access Journals (Sweden)

    Yinghui Li

    2016-03-01

    Full Text Available This experiment was conducted to investigate the effects of branched-chain amino acids (BCAA supplemented in protein-restricted diets on the growth performance and the expression profile of amino acid transporters and energy metabolism related regulators in the white adipose tissue (WAT of different regional depots including dorsal subcutaneous adipose (DSA and abdominal subcutaneous adipose (ASA. A total of 24 crossbred barrows (7.40 ± 0.70 kg were randomly divided into 4 groups and were fed the following isocaloric diets for 33 days: 1 a recommended adequate protein diet (AP, 20% CP, as a positive control; 2 a low protein diet (LP, 17% CP; 3 the LP diet supplemented with BCAA (LP + B, 17% CP to reach the same level of the AP diet group; 4 the LP diet supplemented with 2 times the amount of BCAA (LP + 2B, 17% CP. The daily gain and daily feed intake of the LP diet group were the lowest among all the treatments (P  0.05. Moreover, BCAA supplementation down-regulated the expression levels of amino acid transporters including L-type amino acid transporter 1 and sodium-coupled neutral amino acid transporter 2 in DSA, but up-regulated the expression level of L-type amino acid transporter 4 in ASA (P < 0.05. Meanwhile, the energy sensor AMP-activated protein kinase α was activated in the DSA of pigs fed LP diet and in the ASA of the pigs fed AP or LP + 2B diets (P < 0.05. The mRNA expression profile of the selected mitochondrial component and mitochondrial biogenesis associated regulators in DSA and ASA also responded differently to dietary BCAA supplementation. These results suggested that the growth performance of growing pigs fed protein restricted diets supplemented with BCAA could catch up to that of the pigs fed AP diets. The results also partly demonstrated that the regulation mechanisms of BCAA are different in the adipose tissues of different depots.

  20. Amino acid transport across the tonoplast of vacuoles isolated from barley mesophyll protoplasts: Uptake of alanine, leucine, and glutamine

    International Nuclear Information System (INIS)

    Dietz, K.J.; Jaeger, R.; Kaiser, G.; Martinoia, E.

    1990-01-01

    Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using 14 C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors

  1. Biosynthesis of l-Ascorbic Acid and Conversion of Carbons 1 and 2 of l-Ascorbic Acid to Oxalic Acid Occurs within Individual Calcium Oxalate Crystal Idioblasts1

    Science.gov (United States)

    Kostman, Todd A.; Tarlyn, Nathan M.; Loewus, Frank A.; Franceschi, Vincent R.

    2001-01-01

    l-Ascorbic acid (AsA) and its metabolic precursors give rise to oxalic acid (OxA) found in calcium oxalate crystals in specialized crystal idioblast cells in plants; however, it is not known if AsA and OxA are synthesized within the crystal idioblast cell or transported in from surrounding mesophyll cells. Isolated developing crystal idioblasts from Pistia stratiotes were used to study the pathway of OxA biosynthesis and to determine if idioblasts contain the entire path and are essentially independent in OxA synthesis. Idioblasts were supplied with various 14C-labeled compounds and examined by micro-autoradiography for incorporation of 14C into calcium oxalate crystals. [14C]OxA gave heavy labeling of crystals, indicating the isolated idioblasts are functional in crystal formation. Incubation with [1-14C]AsA also gave heavy labeling of crystals, whereas [6-14C]AsA gave no labeling. Labeled precursors of AsA (l-[1-14C]galactose; d-[1-14C]mannose) also resulted in crystal labeling, as did the ascorbic acid analog, d-[1-14C]erythorbic acid. Intensity of labeling of isolated idioblasts followed the pattern OxA > AsA (erythorbic acid) > l-galactose > d-mannose. Our results demonstrate that P. stratiotes crystal idioblasts synthesize the OxA used for crystal formation, the OxA is derived from the number 1 and 2 carbons of AsA, and the proposed pathway of ascorbic acid synthesis via d-mannose and l-galactose is operational in individual P. stratiotes crystal idioblasts. These results are discussed with respect to fine control of calcium oxalate precipitation and the concept of crystal idioblasts as independent physiological compartments. PMID:11161021

  2. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report...... autoradiography on sections of pig brain was performed using [(3)H]HOCPCA. In vivo evaluation of [(11)C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [(11)C]HOCPCA....

  3. Diagnostic usefulness of {sup 18}F-FAMT PET and L-type amino acid transporter 1 (LAT1) expression in oral squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Nobusawa, Aiko [Gunma University Graduate School of Medicine, Department of Stomatology and Maxillofacial Surgery, Maebashi, Gunma (Japan); Gunma University Graduate School of Medicine, Department of Diagnostic Pathology, Maebashi, Gunma (Japan); Kim, Mai [Gunma University Graduate School of Medicine, Department of Stomatology and Maxillofacial Surgery, Maebashi, Gunma (Japan); Gunma University Graduate School of Medicine, Department of Diagnostic Radiology and Nuclear Medicine, Maebashi, Gunma (Japan); Kaira, Kyoichi [Gunma University Graduate School of Medicine, Department of Diagnostic Pathology, Maebashi, Gunma (Japan); Gunma University Hospital, Oncology Center, Maebashi, Gunma (Japan); Miyashita, Go; Negishi, Akihide; Yokoo, Satoshi [Gunma University Graduate School of Medicine, Department of Stomatology and Maxillofacial Surgery, Maebashi, Gunma (Japan); Oriuchi, Noboru; Higuchi, Tetsuya; Tsushima, Yoshito [Gunma University Graduate School of Medicine, Department of Diagnostic Radiology and Nuclear Medicine, Maebashi, Gunma (Japan); Kanai, Yoshikatsu [Osaka University, Division of Bio-system Pharmacology, Graduate School of Medicine, Osaka (Japan); Oyama, Tetsunari [Gunma University Graduate School of Medicine, Department of Diagnostic Pathology, Maebashi, Gunma (Japan)

    2013-10-15

    l-[3-{sup 18}F]-{alpha}-Methyltyrosine ({sup 18}F-FAMT) was developed as an amino acid tracer for PET imaging to provide better specificity than 2-[{sup 18}F]fluoro-2-deoxy-d-glucose ({sup 18}F-FDG) PET for cancer diagnosis. We investigated the diagnostic usefulness of {sup 18}F-FAMT in oral squamous cell carcinoma (OSCC). The correlation between tumour uptake of {sup 18}F-FAMT and L-type amino acid transporter 1 (LAT1) expression was determined. The study group comprised 68 OSCC patients who underwent both {sup 18}F-FAMT and {sup 18}F-FDG PET. Resected tumour sections were stained by immunohistochemistry for LAT1, CD98 and Ki-67, and microvessel density was determined in terms of CD34 and p53 expression. The sensitivity of primary tumour detection by {sup 18}F-FAMT and {sup 18}F-FDG PET was 98 % and 100 %, respectively. The sensitivity, specificity and accuracy of {sup 18}F-FAMT PET for detecting malignant lymph nodes were 68 %, 99 % and 97 %, respectively, and equivalent values for {sup 18}F-FDG PET were 84 %, 94 % and 94 %, respectively. The specificity and accuracy of {sup 18}F-FAMT were significantly higher than those of {sup 18}F-FDG. The uptake of {sup 18}F-FAMT was significantly correlated with LAT1 expression, cell proliferation and advanced stage. The expression of LAT1 in OSCC cells was closely correlated with CD98 levels, cell proliferation and angiogenesis. {sup 18}F-FAMT PET showed higher specificity for detecting malignant lesions than {sup 18}F-FDG PET. The uptake of {sup 18}F-FAMT by OSCC cells can be determined by the presence of LAT1 expression and tumour cell proliferation. (orig.)

  4. Elevated ammonium levels

    DEFF Research Database (Denmark)

    Søgaard, Rikke; Novak, Ivana; MacAulay, Nanna

    2012-01-01

    was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte...

  5. Cationic amino acid transporter 2 enhances innate immunity during Helicobacter pylori infection.

    Directory of Open Access Journals (Sweden)

    Daniel P Barry

    Full Text Available Once acquired, Helicobacter pylori infection is lifelong due to an inadequate innate and adaptive immune response. Our previous studies indicate that interactions among the various pathways of arginine metabolism in the host are critical determinants of outcomes following infection. Cationic amino acid transporter 2 (CAT2 is essential for transport of L-arginine (L-Arg into monocytic immune cells during H. pylori infection. Once within the cell, this amino acid is utilized by opposing pathways that lead to elaboration of either bactericidal nitric oxide (NO produced from inducible NO synthase (iNOS, or hydrogen peroxide, which causes macrophage apoptosis, via arginase and the polyamine pathway. Because of its central role in controlling L-Arg availability in macrophages, we investigated the importance of CAT2 in vivo during H. pylori infection. CAT2(-/- mice infected for 4 months exhibited decreased gastritis and increased levels of colonization compared to wild type mice. We observed suppression of gastric macrophage levels, macrophage expression of iNOS, dendritic cell activation, and expression of granulocyte-colony stimulating factor in CAT2(-/- mice suggesting that CAT2 is involved in enhancing the innate immune response. In addition, cytokine expression in CAT2(-/- mice was altered from an antimicrobial Th1 response to a Th2 response, indicating that the transporter has downstream effects on adaptive immunity as well. These findings demonstrate that CAT2 is an important regulator of the immune response during H. pylori infection.

  6. The mRNA expression of amino acid and sugar transporters, aminopeptidase, as well as the di- and tri-peptide transporter PepT1 in the intestines of Eimeria infected broiler chickens.

    Science.gov (United States)

    Miska, K B; Fetterer, R H

    2017-02-01

    Coccidiosis in chickens is caused by infection of gut epithelial cells with protozoan parasites of the genus Eimeria This disease causes losses to the poultry industry since infected birds fail to gain weight as rapidly as non-infected birds and efficiency of feed conversion is compromised. For the present study the effect of Eimeria on expression of components of amino acid and sugar uptake mechanisms was determined. Broiler chicks were infected with Eimeria maxima, which infects the jejunum; Eimeria acervulina, which infects the duodenum; or Eimeria tenella, which infects the ceca. Sections of the jejunum, duodenum, and ceca (depending on species of Eimeria) were taken at several time points between d zero and 14 post infection (PI) for mRNA expression analysis. Genes examined included one digestive enzyme, 7 peptide and amino acid transporters located on the brush border, 8 transporters located at the basolateral surface of the gut epithelium, and 5 sugar transporters. All 3 Eimeria species examined caused decrease in expression of brush border transporters particularly at d 5 to 7 PI, which corresponds to the time when pathology is greatest. The same pattern was seen in expression of sugar transporters. However, the expression of basolateral transporters differed among species. Eimeria tenella infection resulted in decreased expression of all basolateral transporters, while E. maxima infection caused increased expression of 2 genes and slight decrease in expression of the remaining 5 genes. Infection with E. acervulina resulted in increased expression at the height of infection of all but one basolateral transporter. In conclusion, Eimeria infection causes a general decrease in gene expression of sugar transporter and brush border AATs at the height of infection. However the expression of basolateral transporters is increased in E. maxima and E. acervulina infected birds. It is possible that decreased expression of brush border transporters in combination with

  7. Active Hydrophilic Components of the Medicinal Herb Salvia miltiorrhiza (Danshen Potently Inhibit Organic Anion Transporters 1 (Slc22a6 and 3 (Slc22a8

    Directory of Open Access Journals (Sweden)

    Li Wang

    2012-01-01

    Full Text Available Many active components of herbal products are small organic anions, and organic anion transporters were previously demonstrated to be a potential site of drug-drug interactions. In this study, we assessed the inhibitory effects of six hydrophilic components of the herbal medicine Danshen, lithospermic acid, protocatechuic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and tanshinol, on the function of the murine organic anion transporters, mOat1 and mOat3. All of Danshen components significantly inhibited mOat1- and mOat3-mediated substrate uptake (<0.001 with lithospermic acid (LSA, protocatechuic acid, rosmarinic acid (RMA, and salvianolic acid A (SAA producing virtually complete inhibition under test conditions. Kinetic analysis demonstrated that LSA, RMA, and SAA were competitive inhibitors. As such, values were estimated as 14.9±4.9 μM for LSA, 5.5±2.2 μM for RMA, and 4.9±2.2 μM for SAA on mOat1-mediated transport, and as 31.1±7.0 μM for LSA, 4.3±0.2 μM for RMA, and 21.3±7.7 μM for SAA on mOat3-mediated transport. These data suggest that herb-drug interactions may occur in vivo on the human orthologs of these transporters in situations of polypharmacy involving Danshen and clinical therapeutics known to be organic anion transporter substrates.

  8. Long-range atmospheric transport of volatile monocarboxylic acids with Asian dust over a high mountain snow site, central Japan

    Directory of Open Access Journals (Sweden)

    T. Mochizuki

    2016-11-01

    Full Text Available To understand the long-range transport of monocarboxylic acids from the Asian continent to the Japanese islands, we collected snowpack samples from a pit sequence (depth ca. 6 m at the Murodo-Daira snowfield near the summit of Mt. Tateyama, central Japan, in 2009 and 2011. Snow samples (n = 16 were analyzed for normal (C1–C10, branched chain (iC4–iC6, aromatic (benzoic and toluic acid isomers, and hydroxyl (glycolic and lactic monocarboxylic acids, together with inorganic ions and dissolved organic carbon (DOC. Acetic acid (C2 was found to be a dominant species (average 125 ng g−1, followed by formic acid (C1 (85.7 ng g−1 and isopentanoic acid (iC5 (20.0 ng g−1. We found a strong correlation (r =  0.88 between formic plus acetic acids and non-sea-salt Ca2+ that is a proxy of Asian dust. Contributions of total monocarboxylic acids to DOC in 2009 (21.2 ± 11.6 % were higher than that in 2011 (3.75 ± 2.62 %, being consistent with higher intensity of Asian dust in 2009 than in 2011. Formic plus acetic acids also showed a positive correlation (r =  0.90 with benzoic acid that is a tracer of automobile exhaust, indicating that monocarboxylic acids and their precursors are largely emitted from anthropogenic sources in China and/or secondarily produced in the atmosphere by photochemical processing. In addition, the ratio of formic plus acetic acids to nss–Ca2+ (0.27 was significantly higher than those (0.00036–0.0018 obtained for reference dust materials of Chinese loess deposits from the Tengger and Gobi deserts. This result suggests that volatile and semi-volatile organic acids are adsorbed on the alkaline dust particles during long-range atmospheric transport. Entrainment of organic acids by dusts is supported by a good correlation (r = 0.87 between formic plus acetic acids and pH of melt snow samples. Our study suggests that Asian alkaline dusts may be a carrier of volatile monocarboxylic

  9. Environmental, health, and safety issues of fuel cells in transportation. Volume 1: Phosphoric acid fuel-cell buses

    Energy Technology Data Exchange (ETDEWEB)

    Ring, S

    1994-12-01

    The U.S. Department of Energy (DOE) chartered the Phosphoric Acid Fuel-Cell (PAFC) Bus Program to demonstrate the feasibility of fuel cells in heavy-duty transportation systems. As part of this program, PAFC- powered buses are being built to meet transit industry design and performance standards. Test-bed bus-1 (TBB-1) was designed in 1993 and integrated in March 1994. TBB-2 and TBB-3 are under construction and should be integrated in early 1995. In 1987 Phase I of the program began with the development and testing of two conceptual system designs- liquid- and air-cooled systems. The liquid-cooled PAFC system was chosen to continue, through a competitive award, into Phase H, beginning in 1991. Three hybrid buses, which combine fuel-cell and battery technologies, were designed during Phase III. After completing Phase II, DOE plans a comprehensive performance testing program (Phase HI) to verify that the buses meet stringent transit industry requirements. The Phase III study will evaluate the PAFC bus and compare it to a conventional diesel bus. This NREL study assesses the environmental, health, and safety (EH&S) issues that may affect the commercialization of the PAFC bus. Because safety is a critical factor for consumer acceptance of new transportation-based technologies the study focuses on these issues. The study examines health and safety together because they are integrally related. In addition, this report briefly discusses two environmental issues that are of concern to the Environmental Protection Agency (EPA). The first issue involves a surge battery used by the PAFC bus that contains hazardous constituents. The second issue concerns the regulated air emissions produced during operation of the PAFC bus.

  10. Differential substrate specificities of human ABCD1 and ABCD2 in peroxisomal fatty acid β-oxidation

    NARCIS (Netherlands)

    van Roermund, Carlo W. T.; Visser, Wouter F.; Ijlst, Lodewijk; Waterham, Hans R.; Wanders, Ronald J. A.

    2011-01-01

    The gene mutated in X-linked adrenoleukodystrophy (X-ALD) codes for the HsABCD1 protein, also named ALDP, which is a member of the superfamily of ATP-binding cassette (ABC) transporters and required for fatty acid transport across the peroxisomal membrane. Although a defective HsABCD1 results in the

  11. Vigna subterranea ammonium transporter gene (VsAMT1: Some bioinformatics insights

    Directory of Open Access Journals (Sweden)

    Adewole T. Adetunji

    2015-12-01

    Full Text Available Ammonium transporters (AMTs play a role in the uptake of ammonium, the form in which nitrogen is preferentially absorbed by plants. Vigna subterranea (VsAMT1 and Solanum tuberosum (StAMT1 AMT1s were characterized using molecular biology and bioinformatics methods. AMT1-specific primers were designed and used to amplify the AMT1 internal regions. Nucleotide sequencing, alignment and phylogenetic analysis assigned VsAMT1 and StAMT1 to the AMT1 family. The deduced amino acid sequences showed that VsAMT1 is 92% and 89% similar to Phaseolus vulgaris PvAMT1.1 and Glycine max AMT1 respectively, while StAMT1 is 92% similar to Solanum lycopersicum LeAMT1.1, and correspond to the 5th–10th trans-membrane domains. Residues VsAMT1 D23 and StAMT1 D15 are predicted to be essential for ammonium transport, while mutations of VsAMT1 W1A-L and S87A and StAMT1 S76A may further enhance ammonium transport. In addition to nitrogen uptake from the roots, VsAMT1 may also contribute to interactions with rhizobia.

  12. Hepatobiliary transport kinetics of the conjugated bile acid tracer 11C-CSar quantified in healthy humans and patients by positron emission tomography.

    Science.gov (United States)

    Ørntoft, Nikolaj Worm; Munk, Ole Lajord; Frisch, Kim; Ott, Peter; Keiding, Susanne; Sørensen, Michael

    2017-08-01

    Hepatobiliary secretion of bile acids is an important liver function. Here, we quantified the hepatic transport kinetics of conjugated bile acids using the bile acid tracer [N-methyl- 11 C]cholylsarcosine ( 11 C-CSar) and positron emission tomography (PET). Nine healthy participants and eight patients with varying degrees of cholestasis were examined with 11 C-CSar PET and measurement of arterial and hepatic venous blood concentrations of 11 C-CSar. Results are presented as median (range). The hepatic intrinsic clearance was 1.50 (1.20-1.76) ml blood/min/ml liver tissue in healthy participants and 0.46 (0.13-0.91) in patients. In healthy participants, the rate constant for secretion of 11 C-CSar from hepatocytes to bile was 0.36 (0.30-0.62)min -1 , 20 times higher than the rate constant for backflux from hepatocytes to blood (0.02, 0.005-0.07min -1 ). In the patients, rate constant for transport from hepatocyte to bile was reduced to 0.12 (0.006-0.27)min -1 , 2.3times higher than the rate constant for backflux to blood (0.05, 0.04-0.09). The increased backflux did not fully normalize exposure of the hepatocyte to bile acids as mean hepatocyte residence time of 11 C-CSar was 2.5 (1.6-3.1)min in healthy participants and 6.4 (3.1-23.7)min in patients. The rate constant for transport of 11 C-CSar from intrahepatic to extrahepatic bile was 0.057 (0.023-0.11)min -1 in healthy participants and only slightly reduced in patients 0.039 (0.017-0.066). This first in vivo quantification of individual steps involved in the hepatobiliary secretion of a conjugated bile acid in humans provided new insight into cholestatic disease. Positron emission tomography (PET) using the radiolabelled bile acid ( 11 C-CSar) enabled quantification of the individual steps of the hepatic transport of bile acids from blood to bile in man. Cholestasis reduced uptake and secretion and increased backflux to blood. These findings improve our understanding of cholestatic liver diseases and may support

  13. Evidence connecting old, new and neglected glucose-lowering drugs to bile acid-induced GLP-1 secretion

    DEFF Research Database (Denmark)

    Kårhus, Martin L; Brønden, Andreas; Sonne, David P

    2017-01-01

    Bile acids are amphipathic water-soluble steroid-based molecules best known for their important lipid-solubilizing role in the assimilation of fat. Recently, bile acids have emerged as metabolic integrators with glucose-lowering potential. Among a variety of gluco-metabolic effects, bile acids have...... current evidence connecting established glucose-lowering drugs to bile acid-induced GLP-1 secretion and discusses whether bile acid-induced GLP-1 secretion may constitute a new basis for understanding how metformin, inhibitors of the apical sodium-dependent bile acids transporter, and bile acid...... sequestrants - old, new and neglected glucose-lowering drugs - improve glucose metabolism....

  14. Regulation of the glutamine transporter SN1 by extracellular pH and intracellular sodium ions

    International Nuclear Information System (INIS)

    Broeer, A.; Broeer, S.; Setiawan, I.; Lang, F.

    2001-01-01

    Full text: SN1 has recently been identified as one of the major glutamine transporters in hepatocytes and brain astrocytes. It appears to be the molecular correlate of the system N amino acid transporter. Two different transport mechanisms have been proposed for this transporter. Either an electroneutral mechanism, in which glutamine uptake is coupled to an exchange of 1Na + and 1H + , or an electrogenic mechanism coupled to the exchange of 2Na + against 1H + . This study was performed to solve the discrepancies and to investigate the reversibility of the transporter. When expressed in Xenopus laevis oocytes glutamine uptake activity increased strongly with increasing pH. In agreement with the pH-dependence we found that uptake of glutamine was accompanied by an alkalization of the cytosol, indicating that SN1 mediates Glutamine/H + -Antiport. Uptake of glutamine into oocytes was Na + -dependent. Analysis of the Na + -dependence of glutamine transport and Flux studies using 22 Na + indicated that two or more sodium ions were cotransported together with glutamine. However, at the same time intracellular Na + was exchanged against extracellular Na + . Taken together with the results of the pH-dependence it is proposed that SN1 mediates a Na + /Na + -exchange and a Na + /H + -exchange, both being coupled to the transport of glutamine. In agreement with this mechanism we found that acidic pH caused a reversal of the transporter. To investigate the source of the glutamine-induced inward currents, we compared inward currents generated by the 1Na + /glutamine cotransporter ATA1 with those generated by SN1. Currents induced by glutamine uptake in SN1 expressing oocytes were only a fraction of the currents induced by glutamine in ATA1 expressing oocytes, indicating that they were not generated by a stoichiometric uptake of ions. It is concluded that SN1 is tightly regulated by pH and intracellular Na + -ions and is capable of mediating glutamine uptake and release

  15. Transport of C-13-labelled linoleic and C-13-labelled caprylic acid in rat plasma after administration of specific structured triacylglycerols

    DEFF Research Database (Denmark)

    Vistisen, Bodil; Høy, Carl-Erik

    2004-01-01

    the transport of dietary C-13-labelled fatty acids in rat plasma to compare the chylomicron fatty acid metabolism after administration of specific structured, long chain and medium chain triacylglycerols. Rats were fed ML*M, M*LM*, L*L*L* or M*M*M* (L=linoleic acid, 18:2n-6, M=caprylic acid, 8:0, * = C-13......-labelled fatty acid) by gavage. A maximum transport of 0.5% of the administered C-13-labelled 18:2n-6 was observed in 1mL rat plasma both after administration of L*L*L* and ML*M, while approximately 0.04% of the administered C-13-labelled 8:0 was detected in 1mL plasma following administration of M......*M*M* or M*LM*. After L*L*L* administration C-13-labelled 20:4n-6 was observed in plasma, probably formed by elongation and desaturation of 18:2n-6 in the enterocyte or liver cells. Furthermore, C-13-labelled 16:0, 48:0, 18: 1n-9 and 20:4n-6 were observed in plasma of rats fed M*M*M* and M*LM* due...

  16. Amino Acid Transport in the Thermophilic Anaerobe Clostridium fervidus Is Driven by an Electrochemical Sodium Gradient

    NARCIS (Netherlands)

    SPEELMANS, G; POOLMAN, B; KONINGS, WN

    Amino acid transport was studied in membranes of the peptidolytic, thermophitic, anaerobic bacterium Clostridium fervidus. Uptake of the negatively charged amino acid L-glutamate, the neutral amino acid L-serine, and the positively charged amino acid L-arginine was examined in membrane vesicles

  17. Low-protein diet supplemented with keto acids is associated with suppression of small-solute peritoneal transport rate in peritoneal dialysis patients.

    Science.gov (United States)

    Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

    2011-01-01

    Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6-0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6-0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0-1.2 g/kg/d) protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/P(cr) (dialysate-to-plasma concentration ratio for creatinine at 4 hour) and D/D0(glu) (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio) were similar, D/P(cr) in group sLP was lower, and D/D0(glu) was higher than those in the other two groups (P diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate.

  18. The long and winding road: transport pathways for amino acids in Arabidopsis seeds.

    Science.gov (United States)

    Karmann, Julia; Müller, Benedikt; Hammes, Ulrich Z

    2018-03-16

    certain plants, e.g., legumes as a resource to support the growth of the seedling after germination. The support of the embryo depends on transport processes that occur between the mother plant and the seed tissues including the embryo. In this review, we will focus on the processes of unloading amino acids from the phloem and their post-phloem transport. We will further highlight similarities between amino acid transport and the transport of the main assimilate and osmolyte, sucrose. Finally, we will discuss similarities and differences between different plant species in terms of structural aspects but for the molecular aspects we are almost exclusively focusing on Arabidopsis. Fig. 1 Vascularization of the Arabidopsis ovule and seed. Plants expressing ER-localized mCherry under control of the companion cell-specific SUC2 promoter and ER-localized GFP under control of the sieve element marker PD1 as described (Müller et al. 2015) are shown to visualize the phloem in the funiculus and the chalazal regions. a Overview over an ovule. FG: female gametophyte. b A magnification of the region marked by a square in panel a. c Overview over a seed. ES: endosperm; E: embryo. d A magnification of the region marked by a square in panel c. The arrows in b and d point to the terminal companion cell and arrowheads to terminal sieve elements.

  19. Resistance to diet-induced obesity and associated metabolic perturbations in haploinsufficient monocarboxylate transporter 1 mice.

    OpenAIRE

    Lengacher Sylvain; Nehiri-Sitayeb Touria; Steiner Nadia; Carneiro Lionel; Favrod Céline; Preitner Frédéric; Thorens Bernard; Stehle Jean-Christophe; Dix Laure; Pralong François; Magistretti Pierre J; Pellerin Luc

    2013-01-01

    The monocarboxylate transporter 1 (MCT1 or SLC16A1) is a carrier of short-chain fatty acids, ketone bodies, and lactate in several tissues. Genetically modified C57BL/6J mice were produced by targeted disruption of the mct1 gene in order to understand the role of this transporter in energy homeostasis. Null mutation was embryonically lethal, but MCT1(+/-) mice developed normally. However, when fed high fat diet (HFD), MCT1(+/-) mice displayed resistance to development of diet-induced obesity ...

  20. Effects of kainic acid lesions in lateral geniculate nucleus: activity dependence of retrograde axonal transport of fluorescent dyes.

    Science.gov (United States)

    Woodward, W R; Coull, B M

    1988-06-28

    Kainic acid lesions in the dorsal lateral geniculate nucleus of rats block the retrograde axonal transport of fluorescent dyes in corticogeniculate neurons without affecting the retrograde transport of D-aspartate or the orthograde transport of radiolabelled proteins in these neurons. This blocking of dye transport does not appear to be a consequence of kainic acid-induced damage to axon terminals in the geniculate since retinal ganglion cells are still able to transport dyes retrograde. A more likely explanation for these results is that fluorescent dye transport requires electrical activity in neurons, and elimination of the geniculate afferents to visual cortex reduces impulse traffic in cortical output fibers to a level below that required to support detectable dye transport. This interpretation is supported by the observation that kainic acid lesions also reduce retrograde transport of dyes in cortical neurons which project to the superior colliculus. Electrical stimulation in the subcortical white matter restores the transport of dye compounds in corticogeniculate neurons: evidence consistent with an activity-dependent mechanism of retrograde transport for these substances. These results provide evidence that axon terminals of retinal ganglion cells and corticogeniculate neurons survive in kainate-lesioned geniculates and are capable of normal neuronal function.

  1. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

    Science.gov (United States)

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-01-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

  2. Cloning, Expression, and Functional Characterization of Secondary Amino Acid Transporters of Lactococcus lactis

    NARCIS (Netherlands)

    Trip, Hein; Mulder, Niels L.; Lolkema, Juke S.

    Fourteen genes encoding putative secondary amino acid transporters were identified in the genomes of Lactococcus lactis subsp. cremoris strains MG1363 and SK11 and L. lactis subsp. lactis strains IL1403 and KF147, 12 of which were common to all four strains. Amino acid uptake in L. lactis cells

  3. Synthesis, in vitro and in vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered) hepatic transporter function

    International Nuclear Information System (INIS)

    Neyt, Sara; Vliegen, Maarten; Verreet, Bjorn; De Lombaerde, Stef; Braeckman, Kim; Vanhove, Christian; Huisman, Maarten Thomas; Dumolyn, Caroline; Kersemans, Ken; Hulpia, Fabian; Van Calenbergh, Serge; Mannens, Geert; De Vos, Filip

    2016-01-01

    Introduction: Hepatobiliary transport mechanisms are crucial for the excretion of substrate toxic compounds. Drugs can inhibit these transporters, which can lead to drug–drug interactions causing toxicity. Therefore, it is important to assess this early during the development of new drug candidates. The aim of the current study is the (radio)synthesis, in vitro and in vivo evaluation of a technetium labeled chenodeoxycholic and cholic acid analogue: [ 99m Tc]-DTPA-CDCA and [ 99m ]Tc-DTPA-CA, respectively, as biomarker for disturbed transporter functionality. Methods: [99mTc]-DTPA-CDCA([ 99m Tc]-3a) and [99mTc]-DTPA-CA ([ 99m Tc]-3b) were synthesized and evaluated in vitro and in vivo. Uptake of both tracers was investigated in NTCP, OCT1, OATP1B1, OATP1B3 transfected cell lines. K m and V max values were determined and compared to [ 99m Tc]-mebrofenin ([ 99m Tc]-MEB). Efflux was investigated by means of CTRL, MRP2 and BSEP transfected inside-out vesicles. Metabolite analysis was performed using pooled human liver S9. Wild type (n = 3) and rifampicin treated (n = 3) mice were intravenously injected with 37 MBq of tracer. After dynamic small-animal SPECT and short CT acquisitions, time–activity curves of heart, liver, gallbladder and intestines were obtained. Results: We demonstrated that OATP1B1 and OATP1B3 are the involved uptake transporters of both compounds. Both tracers show a higher affinity compared to [ 99m Tc]-MEB, but are in a similar range as endogenous bile acids for OATP1B1 and OATP1B3. [ 99m Tc]-3a shows higher affinities compared to [ 99m Tc]-3b. V max values were lower compared to [ 99m Tc]-MEB, but in the same range as endogenous bile acids. MRP2 was identified as efflux transporter. Less than 7% of both radiotracers was metabolized in the liver. In vitro results were confirmed by in vivo results. Uptake in the liver and efflux to gallbladder + intestines and urinary bladder of both tracers was observed. Transport was inhibited by rifampicin

  4. Temporal alteration of spreading depression by the glycine transporter type-1 inhibitors NFPS and Org-24461 in chicken retina.

    Science.gov (United States)

    Kertesz, Szabolcs; Szabo, Geza; Udvari, Szabolcs; Levay, Gyorgy; Matyus, Peter; Harsing, Laszlo G

    2013-01-25

    We used isolated chicken retina to induce spreading depression by the glutamate receptor agonist N-methyl-d-aspartate. The N-methyl-d-aspartate-induced latency time of spreading depression was extended by the glycine(B) binding site competitive antagonist 7-chlorokynurenic acid. Addition of the glycine transporter type-1 inhibitors NFPS and Org-24461 reversed the inhibitory effect of 7-chlorokynurenic acid on N-methyl-d-aspartate-evoked spreading depression. The glycine uptake inhibitory activity of Org-24461, NFPS, and some newly synthesized analogs of NFPS was determined in CHO cells stably expressing human glycine transporter type-1b isoform. Compounds, which failed to inhibit glycine transporter type-1, also did not have effect on retinal spreading depression. These experiments indicate that the spreading depression model in chicken retina is a useful in vitro test to determine activity of glycine transporter type-1 inhibitors. In addition, our data serve further evidence for the role of glycine transporter type-1 in retinal neurotransmission and light processing. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Keap1-knockdown decreases fasting-induced fatty liver via altered lipid metabolism and decreased fatty acid mobilization from adipose tissue.

    Directory of Open Access Journals (Sweden)

    Jialin Xu

    Full Text Available AIMS: The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD, regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting. METHODS AND RESULTS: Male C57BL/6 (WT and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36 and Fatty acid transport protein (FATP 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters--CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle. CONCLUSION: Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.

  6. Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion

    Directory of Open Access Journals (Sweden)

    Nathanson Lubov

    2011-09-01

    Full Text Available Abstract Background A major goal of molecular evolutionary biology is to understand the fate and consequences of duplicated genes. In this context, aphids are intriguing because the newly sequenced pea aphid genome harbors an extraordinary number of lineage-specific gene duplications relative to other insect genomes. Though many of their duplicated genes may be involved in their complex life cycle, duplications in nutrient amino acid transporters appear to be associated rather with their essential amino acid poor diet and the intracellular symbiosis aphids rely on to compensate for dietary deficits. Past work has shown that some duplicated amino acid transporters are highly expressed in the specialized cells housing the symbionts, including a paralog of an aphid-specific expansion homologous to the Drosophila gene slimfast. Previous data provide evidence that these bacteriocyte-expressed transporters mediate amino acid exchange between aphids and their symbionts. Results We report that some nutrient amino acid transporters show male-biased expression. Male-biased expression characterizes three paralogs in the aphid-specific slimfast expansion, and the male-biased expression is conserved across two aphid species for at least two paralogs. One of the male-biased paralogs has additionally experienced an accelerated rate of non-synonymous substitutions. Conclusions This is the first study to document male-biased slimfast expression. Our data suggest that the male-biased aphid slimfast paralogs diverged from their ancestral function to fill a functional role in males. Furthermore, our results provide evidence that members of the slimfast expansion are maintained in the aphid genome not only for the previously hypothesized role in mediating amino acid exchange between the symbiotic partners, but also for sex-specific roles.

  7. Synthesis and characterization of [3H]-5'azido-N-1-naphthylphthlamic acid, a photolabile N-1-naphthylphthalamic acid analog

    International Nuclear Information System (INIS)

    Voet, J.G.; Dodge, B.; Harris, K.; Jacobs, M.; Larkin, L.; Bader, S.; Schnitzler, G.; Sutherland, J.

    1990-01-01

    The NPA (N-1-naphthylphthalamic acid) receptor is an important protein involved in the regulation of transport of indole-3-acetic acid (IAA). In our attempt to isolate and characterize this protein we have previously synthesized and characterized a photolabile analog of NPA, 5'-azido-NPA (Az-NPA) and shown it to be a competitor of NPA for binding sites on the NPA receptor as well as an inhibitor of auxin transport. We have now synthesized and characterized [ 3 H]-Az-NPA. The precursor, 2,3,4,5-Br-5'-amino-NPA was dehydrohalogenated with tritium gas by Research Products International. The amino group was converted to an azido group and the product purified by HPLC. [ 3 H]-Az-NPA was found to be photolabile and to co-chromatograph with our synthetic unlabeled Az-NPA. Furthermore, the tritiated material was found to bind to zucchini hypocotyl cell membranes in a manner competitive with NPA as well as unlabeled Az-NPA. Photolysis of zucchini phase-partitioned plasma membranes in the presence of [ 3 H]-Az-NPA resulted in covalent association of tritium with the membranes. Much of this covalent association could be prevented by prior treatment of the membranes with excess NPA

  8. In Vivo Performance of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transport

    Science.gov (United States)

    2015-01-01

    A novel trifluorinated cholic acid derivative, CA-lys-TFA, was designed and synthesized for use as a tool to measure bile acid transport noninvasively using magnetic resonance imaging (MRI). In the present study, the in vivo performance of CA-lys-TFA for measuring bile acid transport by MRI was investigated in mice. Gallbladder CA-lys-TFA content was quantified using MRI and liquid chromatography/tandem mass spectrometry. Results in wild-type (WT) C57BL/6J mice were compared to those in mice lacking expression of Asbt, the ileal bile acid transporter. 19F signals emanating from the gallbladders of WT mice 7 h after oral gavage with 150 mg/kg CA-lys-TFA were reproducibly detected by MRI. Asbt-deficient mice administered the same dose had undetectable 19F signals by MRI, and gallbladder bile CA-lys-TFA levels were 30-fold lower compared to WT animals. To our knowledge, this represents the first report of in vivo imaging of an orally absorbed drug using 19F MRI. Fluorinated bile acid analogues have potential as tools to measure and detect abnormal bile acid transport by MRI. PMID:24708306

  9. Dynamin-dependent amino acid endocytosis activates mechanistic target of rapamycin complex 1 (mTORC1).

    Science.gov (United States)

    Shibutani, Shusaku; Okazaki, Hana; Iwata, Hiroyuki

    2017-11-03

    The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis and potential target for modifying cellular metabolism in various conditions, including cancer and aging. mTORC1 activity is tightly regulated by the availability of extracellular amino acids, and previous studies have revealed that amino acids in the extracellular fluid are transported to the lysosomal lumen. There, amino acids induce recruitment of cytoplasmic mTORC1 to the lysosome by the Rag GTPases, followed by mTORC1 activation by the small GTPase Ras homolog enriched in brain (Rheb). However, how the extracellular amino acids reach the lysosomal lumen and activate mTORC1 remains unclear. Here, we show that amino acid uptake by dynamin-dependent endocytosis plays a critical role in mTORC1 activation. We found that mTORC1 is inactivated when endocytosis is inhibited by overexpression of a dominant-negative form of dynamin 2 or by pharmacological inhibition of dynamin or clathrin. Consistently, the recruitment of mTORC1 to the lysosome was suppressed by the dynamin inhibition. The activity and lysosomal recruitment of mTORC1 were rescued by increasing intracellular amino acids via cycloheximide exposure or by Rag overexpression, indicating that amino acid deprivation is the main cause of mTORC1 inactivation via the dynamin inhibition. We further show that endocytosis inhibition does not induce autophagy even though mTORC1 inactivation is known to strongly induce autophagy. These findings open new perspectives for the use of endocytosis inhibitors as potential agents that can effectively inhibit nutrient utilization and shut down the upstream signals that activate mTORC1. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Stimulation of apical sodium-dependent bile acid transporter expands the bile acid pool and generates bile acids with positive feedback properties.

    Science.gov (United States)

    Rudling, Mats; Bonde, Ylva

    2015-01-01

    Bile acid synthesis has been considered a prototype for how a physiological process is controlled by end product feedback inhibition. By this feedback inhibition, bile acid concentrations are kept within safe ranges. However, careful examination of published rodent data strongly suggests that bile acid synthesis is also under potent positive feedback control by hydrophilic bile acids. Current concepts on the regulation of bile acid synthesis are derived from mouse models. Recent data have shown that mice have farnesoid X receptor (FXR) antagonistic bile acids capable of quenching responses elicited by FXR agonistic bile acids. This is important to recognize to understand the regulation of bile acid synthesis in the mouse, and in particular to clarify if mouse model findings are valid also in the human situation. In addition to classic end product feedback inhibition, regulation of bile acid synthesis in the mouse largely appears also to be driven by changes in hepatic levels of murine bile acids such as α- and β-muricholic acids. This has not been previously recognized. Stimulated bile acid synthesis or induction of the apical sodium-dependent bile acid transporter in the intestine, increase the availability of chenodeoxycholic acid in the liver, thereby promoting hepatic conversion of this bile acid into muricholic acids. Recognition of these mechanisms is essential for understanding the regulation of bile acid synthesis in the mouse, and for our awareness of important species differences in the regulation of bile acid synthesis in mice and humans. 2015 S. Karger AG, Basel.

  11. Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Qiang; Chen, Xin-li; Wang, Chang-yuan; Liu, Qi; Sun, Hui-jun; Sun, Peng-yuan; Huo, Xiao-kui; Liu, Zhi-hao; Yao, Ji-hong; Liu, Ke-xin, E-mail: kexinliu@dlmedu.edu.cn

    2015-03-15

    Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes. - Highlights: • AB23A has at least three roles in protection against ANIT-induced liver injury. • AB23A decreases Ntcp, and increases Bsep, Mrp2 and Mdr2 expression. • AB23A represses Cyp7a1 and Cyp8b1 through inducing Shp and Fgf15 expression. • AB23A increases bile acid metabolism through inducing Sult2a1 expression. • FXR activation is involved

  12. Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

    International Nuclear Information System (INIS)

    Meng, Qiang; Chen, Xin-li; Wang, Chang-yuan; Liu, Qi; Sun, Hui-jun; Sun, Peng-yuan; Huo, Xiao-kui; Liu, Zhi-hao; Yao, Ji-hong; Liu, Ke-xin

    2015-01-01

    Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes. - Highlights: • AB23A has at least three roles in protection against ANIT-induced liver injury. • AB23A decreases Ntcp, and increases Bsep, Mrp2 and Mdr2 expression. • AB23A represses Cyp7a1 and Cyp8b1 through inducing Shp and Fgf15 expression. • AB23A increases bile acid metabolism through inducing Sult2a1 expression. • FXR activation is involved

  13. Individual bile acids have differential effects on bile acid signaling in mice

    International Nuclear Information System (INIS)

    Song, Peizhen; Rockwell, Cheryl E.; Cui, Julia Yue; Klaassen, Curtis D.

    2015-01-01

    Bile acids (BAs) are known to regulate BA synthesis and transport by the farnesoid X receptor in the liver (FXR-SHP) and intestine (FXR-Fgf15). However, the relative importance of individual BAs in regulating these processes is not known. Therefore, mice were fed various doses of five individual BAs, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxoycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) in their diets at various concentrations for one week to increase the concentration of one BA in the enterohepatic circulation. The mRNA of BA synthesis and transporting genes in liver and ileum were quantified. In the liver, the mRNA of SHP, which is the prototypical target gene of FXR, increased in mice fed all concentrations of BAs. In the ileum, the mRNA of the intestinal FXR target gene Fgf15 was increased at lower doses and to a higher extent by CA and DCA than by CDCA and LCA. Cyp7a1, the rate-limiting enzyme in BA synthesis, was decreased more by CA and DCA than CDCA and LCA. Cyp8b1, the enzyme that 12-hydroxylates BAs and is thus responsible for the synthesis of CA, was decreased much more by CA and DCA than CDCA and LCA. Surprisingly, neither a decrease in the conjugated BA uptake transporter (Ntcp) nor increase in BA efflux transporter (Bsep) was observed by FXR activation, but an increase in the cholesterol efflux transporter (Abcg5/Abcg8) was observed with FXR activation. Thus in conclusion, CA and DCA are more potent FXR activators than CDCA and LCA when fed to mice, and thus they are more effective in decreasing the expression of the rate limiting gene in BA synthesis Cyp7a1 and the 12-hydroxylation of BAs Cyp8b1, and are also more effective in increasing the expression of Abcg5/Abcg8, which is responsible for biliary cholesterol excretion. However, feeding BAs do not alter the mRNA or protein levels of Ntcp or Bsep, suggesting that the uptake or efflux of BAs is not regulated by FXR at physiological and

  14. Individual bile acids have differential effects on bile acid signaling in mice

    Energy Technology Data Exchange (ETDEWEB)

    Song, Peizhen, E-mail: songacad@gmail.com; Rockwell, Cheryl E., E-mail: rockwelc@msu.edu; Cui, Julia Yue, E-mail: juliacui@uw.edu; Klaassen, Curtis D., E-mail: curtisklaassenphd@gmail.com

    2015-02-15

    Bile acids (BAs) are known to regulate BA synthesis and transport by the farnesoid X receptor in the liver (FXR-SHP) and intestine (FXR-Fgf15). However, the relative importance of individual BAs in regulating these processes is not known. Therefore, mice were fed various doses of five individual BAs, including cholic acid (CA), chenodeoxycholic acid (CDCA), deoxoycholic acid (DCA), lithocholic acid (LCA), and ursodeoxycholic acid (UDCA) in their diets at various concentrations for one week to increase the concentration of one BA in the enterohepatic circulation. The mRNA of BA synthesis and transporting genes in liver and ileum were quantified. In the liver, the mRNA of SHP, which is the prototypical target gene of FXR, increased in mice fed all concentrations of BAs. In the ileum, the mRNA of the intestinal FXR target gene Fgf15 was increased at lower doses and to a higher extent by CA and DCA than by CDCA and LCA. Cyp7a1, the rate-limiting enzyme in BA synthesis, was decreased more by CA and DCA than CDCA and LCA. Cyp8b1, the enzyme that 12-hydroxylates BAs and is thus responsible for the synthesis of CA, was decreased much more by CA and DCA than CDCA and LCA. Surprisingly, neither a decrease in the conjugated BA uptake transporter (Ntcp) nor increase in BA efflux transporter (Bsep) was observed by FXR activation, but an increase in the cholesterol efflux transporter (Abcg5/Abcg8) was observed with FXR activation. Thus in conclusion, CA and DCA are more potent FXR activators than CDCA and LCA when fed to mice, and thus they are more effective in decreasing the expression of the rate limiting gene in BA synthesis Cyp7a1 and the 12-hydroxylation of BAs Cyp8b1, and are also more effective in increasing the expression of Abcg5/Abcg8, which is responsible for biliary cholesterol excretion. However, feeding BAs do not alter the mRNA or protein levels of Ntcp or Bsep, suggesting that the uptake or efflux of BAs is not regulated by FXR at physiological and

  15. Cloning and expression of a b(0,+)-like amino acid transporter functioning as a heterodimer with 4F2hc instead of rBAT. A new candidate gene for cystinuria.

    Science.gov (United States)

    Rajan, D P; Kekuda, R; Huang, W; Wang, H; Devoe, L D; Leibach, F H; Prasad, P D; Ganapathy, V

    1999-10-08

    We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b(0,+)-like amino acid transport activity. This protein (4F2-lc6 for the sixth member of the 4F2 light chain family) consists of 487 amino acids and has 12 putative transmembrane domains. At the level of amino acid sequence, 4F2-lc6 shows significant homology (44% identity) to the other five known members of the 4F2 light chain family, namely LAT1 (4F2-lc1), y(+)LAT1 (4F2-lc2), y(+)LAT2 (4F2-lc3), xCT (4F2-lc4), and LAT2 (4F2-lc5). The 4F2hc/4F2-lc6 complex-mediated transport process is Na(+)-independent and exhibits high affinity for neutral and cationic amino acids and cystine. These characteristics are similar to those of the b(0,+)-like amino acid transport activity previously shown to be associated with rBAT (protein related to b(0,+) amino acid transport system). However, the newly cloned 4F2-lc6 does not interact with rBAT. This is the first report of the existence of a b(0,+)-like amino acid transport process that is independent of rBAT. 4F2-lc6 is expressed predominantly in the small intestine and kidney. Based on the characteristics of the transport process mediated by the 4F2hc/4F2-lc6 complex and the expression pattern of 4F2-lc6 in mammalian tissues, we suggest that 4F2-lc6 is a new candidate gene for cystinuria.

  16. The net acid extruders NHE1, NBCn1 and MCT4 promote mammary tumor growth through distinct but overlapping mechanisms

    DEFF Research Database (Denmark)

    Andersen, Anne Poder; Samsøe-Petersen, Jacob; Oernbo, Eva Kjer

    2018-01-01

    High metabolic and proliferative rates in cancer cells lead to production of large amounts of H+ and CO2 , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na+ /H+ exchanger NHE1, the Na+ - HCO3......- cotransporter NBCn1, and the lactate-H+ cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone...

  17. Use of 3h-γ-aminobutyric acid for transport studies with isolated nerve-terminals from rat brain

    International Nuclear Information System (INIS)

    Halvarsson, G.B.; Karlsson, I.; Sellstroem, A.

    1985-01-01

    Isolated synaptosomes were used to study the problem of net accumulation of neurotransmitters. The time-course and the kinetics of exogenous and endogenous GABA transport were studied by liquid-scintillation counting and HPLC-amino acid analysis respectively. Different pools of GABA were suggested by a 6-fold difference in tissue-to-medium-ratio of endogenous vs. exogenous GABA. Net accumulation, exchange and net efflux of GABA was found to be a function of the GABA concentration in the incubation medium. The K/sub m/s for net accumulation and for 3 H-GABA accumulation were 2.68 +/- 1.16 and 6.19 +/- 1.26 μM respectively, whereas the V/sub max/s were 5.9 +/- 4.9 and 134 +/- 13 pmol/mg w.w min respectively. This means that the transport studies which use exogenous substances (e.g. 3 H-GABA) considerably overestimate the transport by overlooking the magnitude of the counter transport. 22 references, 5 figures, 2 tables

  18. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C., E-mail: cdirusso2@unl.edu

    2015-09-25

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC{sub 50} 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC{sub 50} 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of {sup 13}C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata.

  19. Fatty acid transport protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    International Nuclear Information System (INIS)

    Saini, Nipun; Black, Paul N.; Montefusco, David; DiRusso, Concetta C.

    2015-01-01

    The inhibition of the fatty acid uptake into non-adipose tissues provides an attractive target for prevention of lipotoxicity leading to obesity-associated non-alcoholic fatty liver disease and type 2 diabetes. Fatty acid transport proteins (FATPs) are bifunctional proteins involved in the uptake and activation of fatty acids by esterification with coenzyme A. Here we characterize Grassofermata/CB5, previously identified as a fatty acid uptake inhibitor directed against HsFATP2. The compound was effective in inhibiting the uptake of fatty acids in the low micro-molar range (IC 50 8–11 μM) and prevented palmitate-mediated lipid accumulation and cell death in cell lines that are models for intestines, liver, muscle and pancreas. In adipocytes, uptake inhibition was less effective (IC 50 58 μM). Inhibition was specific for long chain fatty acids and was ineffective toward medium chain fatty acids, which are transported by diffusion. Kinetic analysis of Grassofermata-dependent FA transport inhibition verified a non-competitive mechanism. By comparison with Grassofermata, several atypical antipsychotic drugs previously implicated as inhibitors of FA uptake were ineffectual. In mice Grassofermata decreased absorption of 13 C-oleate demonstrating its potential as a therapeutic agent. - Highlights: • Grassofermata is a small compound inhibitor of FATP2. • Uptake inhibition is specific for long chain fatty acids. • Uptake kinetics shows low specificity for adipocytes compared to other cell types. • Inhibition is by a non-competitive mechanism. • Atypical antipsychotics do not inhibit FA uptake by comparison with Grassofermata

  20. TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

    Science.gov (United States)

    Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Ge, Pei; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H.; Pollmann, Stephan; Azzarello, Elisa; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland

    2016-01-01

    Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

  1. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    International Nuclear Information System (INIS)

    Ito, Tatsuo; Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko; Itoh, Kohji; Kuwahara, Jun

    2010-01-01

    Research highlights: → Sp1 zinc fingers themselves interact with importin α. → Sp1 zinc finger domains play an essential role as a nuclear localization signal. → Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin α in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin α including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin α. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  2. Hepatic disposition of the acyl glucuronide 1-O-gemfibrozil-beta-D-glucuronide: effects of clofibric acid, acetaminophen, and acetaminophen glucuronide.

    Science.gov (United States)

    Sabordo, L; Sallustio, B C; Evans, A M; Nation, R L

    2000-10-01

    Glucuronidation of carboxylic acid compounds results in the formation of electrophilic acyl glucuronides. Because of their polarity, carrier-mediated hepatic transport systems play an important role in determining both intra- and extrahepatic exposure to these reactive conjugates. We have previously shown that the hepatic membrane transport of 1-O-gemfibrozil-beta-D-glucuronide (GG) is carrier-mediated and inhibited by the organic anion dibromosulfophthalein. In this study, we examined the influence of 200 microM acetaminophen, acetaminophen glucuronide, and clofibric acid on the disposition of GG (3 microM) in the recirculating isolated perfused rat liver preparation. GG was taken up by the liver, excreted into bile, and hydrolyzed within the liver to gemfibrozil, which appeared in perfusate but not in bile. Mean +/- S. D. hepatic clearance, apparent intrinsic clearance, hepatic extraction ratio, and biliary excretion half-life of GG were 10.4 +/- 1.4 ml/min, 94.1 +/- 17.9 ml/min, 0.346 +/- 0.046, and 30.9 +/- 4.9 min, respectively, and approximately 73% of GG was excreted into bile. At the termination of the experiment (t = 90 min), the ratio of GG concentrations in perfusate, liver, and bile was 1:35:3136. Acetaminophen and acetaminophen glucuronide had no effect on the hepatic disposition of GG, suggesting relatively low affinities of acetaminophen conjugates for hepatic transport systems or the involvement of multiple transport systems for glucuronide conjugates. In contrast, clofibric acid increased the hepatic clearance, extraction ratio, and apparent intrinsic clearance of GG (P clofibric acid glucuronide at the level of hepatic transport. However, the transporter protein(s) involved remains to be identified.

  3. Effects of Long-Term Protein Restriction on Meat Quality, Muscle Amino Acids, and Amino Acid Transporters in Pigs.

    Science.gov (United States)

    Yin, Jie; Li, Yuying; Zhu, Xiaotong; Han, Hui; Ren, Wenkai; Chen, Shuai; Bin, Peng; Liu, Gang; Huang, Xingguo; Fang, Rejun; Wang, Bin; Wang, Kai; Sun, Liping; Li, Tiejun; Yin, Yulong

    2017-10-25

    This study aimed to investigate the long-term effects of protein restriction from piglets to finishing pigs for 16 weeks on meat quality, muscle amino acids, and amino acid transporters. Thirty-nine piglets were randomly divided into three groups: a control (20-18-16% crude protein, CP) and two protein restricted groups (17-15-13% CP and 14-12-10% CP). The results showed that severe protein restriction (14-12-10% CP) inhibited feed intake and body weight, while moderate protein restriction (17-15-13% CP) had little effect on growth performance in pigs. Meat quality (i.e., pH, color traits, marbling, water-holding capacity, and shearing force) were tested, and the results exhibited that 14-12-10% CP treatment markedly improved muscle marbling score and increased yellowness (b*). pH value (45 min) was significantly higher in 17-15-13% CP group than that in other groups. In addition, protein restriction reduced muscle histone, arginine, valine, and isoleucine abundances and enhanced glycine and lysine concentrations compared with the control group, while the RT-PCR results showed that protein restriction downregulated amino acids transporters. Mechanistic target of rapamycin (mTOR) signaling pathway was inactivated in the moderate protein restricted group (17-15-13% CP), while severe protein restriction with dietary 14-12-10% CP markedly enhanced mTOR phosphorylation. In conclusion, long-term protein restriction affected meat quality and muscle amino acid metabolism in pigs, which might be associated with mTOR signaling pathway.

  4. Interactions between organic anions on multiple transporters in Caco-2 cells

    DEFF Research Database (Denmark)

    Grandvuinet, Anne Sophie; Steffansen, Bente

    2011-01-01

    Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E1S) were used as model substrates. Possible inhibitors studied were TCA, E1S, taurolithocholic acid, fluvastatin, and glipizide......-dependent bile acid transporter and the organic solute transporter α/β, and to less extent by the organic anion transporting polypeptide 2B1. However, interactions on efflux transporters were not detected, although they were expected from the literature on the investigated compounds. Biosimulation methods may...

  5. CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Jie; Krausz, Kristopher W. [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Li, Feng; Ma, Xiaochao [Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, 4089 KLSIC, MS 1018, 3901 Rainbow Boulevard, Kansas City, KS 66160 (United States); Gonzalez, Frank J., E-mail: fjgonz@helix.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-01-15

    Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.

  6. CYP2E1-dependent elevation of serum cholesterol, triglycerides, and hepatic bile acids by isoniazid

    International Nuclear Information System (INIS)

    Cheng, Jie; Krausz, Kristopher W.; Li, Feng; Ma, Xiaochao; Gonzalez, Frank J.

    2013-01-01

    Isoniazid is the first-line medication in the prevention and treatment of tuberculosis. Isoniazid is known to have a biphasic effect on the inhibition–induction of CYP2E1 and is also considered to be involved in isoniazid-induced hepatotoxicity. However, the full extent and mechanism of involvement of CYP2E1 in isoniazid-induced hepatotoxicity remain to be thoroughly investigated. In the current study, isoniazid was administered to wild-type and Cyp2e1-null mice to investigate the potential toxicity of isoniazid in vivo. The results revealed that isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice, but produced elevated serum cholesterol and triglycerides, and hepatic bile acids in wild-type mice, as well as decreased abundance of free fatty acids in wild-type mice and not in Cyp2e1-null mice. Metabolomic analysis demonstrated that production of isoniazid metabolites was elevated in wild-type mice along with a higher abundance of bile acids, bile acid metabolites, carnitine and carnitine derivatives; these were not observed in Cyp2e1-null mice. In addition, the enzymes responsible for bile acid synthesis were decreased and proteins involved in bile acid transport were significantly increased in wild-type mice. Lastly, treatment of targeted isoniazid metabolites to wild-type mice led to similar changes in cholesterol, triglycerides and free fatty acids. These findings suggest that while CYP2E1 is not involved in isoniazid-induced hepatotoxicity, while an isoniazid metabolite might play a role in isoniazid-induced cholestasis through enhancement of bile acid accumulation and mitochondria β-oxidation. -- Highlights: ► Isoniazid metabolites were elevated only in wild-type mice. ► Isoniazid caused no hepatotoxicity in wild-type and Cyp2e1-null mice. ► Isoniazid elevated serum cholesterol and triglycerides, and hepatic bile acids. ► Bile acid transporters were significantly decreased in isoniazid-treated mice.

  7. Low-Protein Diet Supplemented with Keto Acids Is Associated with Suppression of Small-Solute Peritoneal Transport Rate in Peritoneal Dialysis Patients

    Directory of Open Access Journals (Sweden)

    Na Jiang

    2011-01-01

    Full Text Available Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6–0.8 g/kg/d, keto-acid-supplemented low- (sLP: 0.6–0.8 g/kg/d with 0.12 g/kg/d of keto acids, or high- (HP: 1.0–1.2 g/kg/d protein diet and lasted for one year. In this study, the variations of peritoneal transport rate were assessed. Results. While baseline D/Pcr (dialysate-to-plasma concentration ratio for creatinine at 4 hour and D/D0glu (dialysate glucose at 4 hour to baseline dialysate glucose concentration ratio were similar, D/Pcr in group sLP was lower, and D/D0glu was higher than those in the other two groups (P<0.05 at 12th month. D/D0glu increased (P<0.05, and D/Pcr tended to decrease, (P=0.071 in group sLP. Conclusions. Low-protein diet with keto acids may benefit PD patients by maintaining peritoneum at a lower transport rate.

  8. Amino acid transporter expansions associated with the evolution of obligate endosymbiosis in sap-feeding insects (Hemiptera: sternorrhyncha).

    Science.gov (United States)

    Dahan, Romain A; Duncan, Rebecca P; Wilson, Alex C C; Dávalos, Liliana M

    2015-03-25

    Mutualistic obligate endosymbioses shape the evolution of endosymbiont genomes, but their impact on host genomes remains unclear. Insects of the sub-order Sternorrhyncha (Hemiptera) depend on bacterial endosymbionts for essential amino acids present at low abundances in their phloem-based diet. This obligate dependency has been proposed to explain why multiple amino acid transporter genes are maintained in the genomes of the insect hosts. We implemented phylogenetic comparative methods to test whether amino acid transporters have proliferated in sternorrhynchan genomes at rates grater than expected by chance. By applying a series of methods to reconcile gene and species trees, inferring the size of gene families in ancestral lineages, and simulating the null process of birth and death in multi-gene families, we uncovered a 10-fold increase in duplication rate in the AAAP family of amino acid transporters within Sternorrhyncha. This gene family expansion was unmatched in other closely related clades lacking endosymbionts that provide essential amino acids. Our findings support the influence of obligate endosymbioses on host genome evolution by both inferring significant expansions of gene families involved in symbiotic interactions, and discovering increases in the rate of duplication associated with multiple emergences of obligate symbiosis in Sternorrhyncha.

  9. The role of L-type amino acid transporters in the uptake of glyphosate across mammalian epithelial tissues.

    Science.gov (United States)

    Xu, Jiaqiang; Li, Gao; Wang, Zhuoyi; Si, Luqin; He, Sijie; Cai, Jialing; Huang, Jiangeng; Donovan, Maureen D

    2016-02-01

    Glyphosate is one of the most commonly used herbicides worldwide due to its broad spectrum of activity and reported low toxicity to humans. Glyphosate has an amino acid-like structure that is highly polar and shows low bioavailability following oral ingestion and low systemic toxicity following intravenous exposures. Spray applications of glyphosate in agricultural or residential settings can result in topical or inhalation exposures to the herbicide. Limited systemic exposure to glyphosate occurs following skin contact, and pulmonary exposure has also been reported to be low. The results of nasal inhalation exposures, however, have not been evaluated. To investigate the mechanisms of glyphosate absorption across epithelial tissues, the permeation of glyphosate across Caco-2 cells, a gastrointestinal epithelium model, was compared with permeation across nasal respiratory and olfactory tissues excised from cows. Saturable glyphosate uptake was seen in all three tissues, indicating the activity of epithelial transporters. The uptake was shown to be ATP and Na(+) independent, and glyphosate permeability could be significantly reduced by the inclusion of competitive amino acids or specific LAT1/LAT2 transporter inhibitors. The pattern of inhibition of glyphosate permeability across Caco-2 and nasal mucosal tissues suggests that LAT1/2 play major roles in the transport of this amino-acid-like herbicide. Enhanced uptake into the epithelial cells at barrier mucosae, including the respiratory and gastrointestinal tracts, may result in more significant local and systemic effects than predicted from glyphosate's passive permeability, and enhanced uptake by the olfactory mucosa may result in further CNS disposition, potentially increasing the risk for brain-related toxicities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Effect of light-load resistance exercise on postprandial amino acid transporter expression in elderly men

    DEFF Research Database (Denmark)

    Agergaard, Jakob; Bülow, Jacob; Jensen, Jacob K

    2017-01-01

    An impaired amino acid sensing is associated with age-related loss of skeletal muscle mass. We tested whether light-load resistance exercise (LL-RE) affects postprandial amino acid transporter (AAT) expression in aging skeletal muscle. Untrained, healthy men (age: +65 years) were subjected to 13 h...

  11. Structural variations in wheat HKT1;5 underpin differences in Na+ transport capacity

    KAUST Repository

    Xu, Bo

    2017-11-27

    An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na+) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na+-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (Km) for the Na+ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D471/a gap and D474/G473 that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na+ exclusion in wheat that leads to an improved salinity tolerance in the field.

  12. Structural variations in wheat HKT1;5 underpin differences in Na+ transport capacity

    KAUST Repository

    Xu, Bo; Waters, Shane; Byrt, Caitlin S.; Plett, Darren; Tyerman, Stephen D.; Tester, Mark A.; Munns, Rana; Hrmova, Maria; Gilliham, Mathew

    2017-01-01

    An important trait associated with the salt tolerance of wheat is the exclusion of sodium ions (Na+) from the shoot. We have previously shown that the sodium transporters TmHKT1;5-A and TaHKT1;5-D, from Triticum monoccocum (Tm) and Triticum aestivum (Ta), are encoded by genes underlying the major shoot Na+-exclusion loci Nax1 and Kna1, respectively. Here, using heterologous expression, we show that the affinity (Km) for the Na+ transport of TmHKT1;5-A, at 2.66 mM, is higher than that of TaHKT1;5-D at 7.50 mM. Through 3D structural modelling, we identify residues D471/a gap and D474/G473 that contribute to this property. We identify four additional mutations in amino acid residues that inhibit the transport activity of TmHKT1;5-A, which are predicted to be the result of an occlusion of the pore. We propose that the underlying transport properties of TmHKT1;5-A and TaHKT1;5-D contribute to their unique ability to improve Na+ exclusion in wheat that leads to an improved salinity tolerance in the field.

  13. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures

    DEFF Research Database (Denmark)

    Carvill, Gemma L; McMahon, Jacinta M; Schneider, Amy

    2015-01-01

    GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutatio...

  14. Intracellular ascorbic acid inhibits transport of glucose by neurons, but not by astrocytes.

    Science.gov (United States)

    Castro, Maite A; Pozo, Miguel; Cortés, Christian; García, María de Los Angeles; Concha, Ilona I; Nualart, Francisco

    2007-08-01

    It has been demonstrated that glutamatergic activity induces ascorbic acid (AA) depletion in astrocytes. Additionally, different data indicate that AA may inhibit glucose accumulation in primary cultures of rat hippocampal neurons. Thus, our hypothesis postulates that AA released from the astrocytes during glutamatergic synaptic activity may inhibit glucose uptake by neurons. We observed that cultured neurons express the sodium-vitamin C cotransporter 2 and the facilitative glucose transporters (GLUT) 1 and 3, however, in hippocampal brain slices GLUT3 was the main transporter detected. Functional activity of GLUTs was confirmed by means of kinetic analysis using 2-deoxy-d-glucose. Therefore, we showed that AA, once accumulated inside the cell, inhibits glucose transport in both cortical and hippocampal neurons in culture. Additionally, we showed that astrocytes are not affected by AA. Using hippocampal slices, we observed that upon blockade of monocarboxylate utilization by alpha-cyano-4-hydroxycinnamate and after glucose deprivation, glucose could rescue neuronal response to electrical stimulation only if AA uptake is prevented. Finally, using a transwell system of separated neuronal and astrocytic cultures, we observed that glutamate can reduce glucose transport in neurons only in presence of AA-loaded astrocytes, suggesting the essential role of astrocyte-released AA in this effect.

  15. Transcellular transport of radioiodinated 3-iodo-α-methyl-L-tyrosine across monolayers of kidney epithelial cell line LLC-PK1

    International Nuclear Information System (INIS)

    Shikano, Naoto; Nakajima, Syuichi; Kubota, Nobuo; Ishikawa, Nobuyoshi; Kawai, Keiichi; Kubodera, Akiko; Saji, Hideo

    2004-01-01

    3-[ 123 I]iodo-α-methyl-L-tyrosine ([ 123 I]IMT) is an imaging agent for amino acid transport. In order to obtain fundamental data related to tumor imaging with [ 123 I]IMT and renal physiological accumulation of [ 123 I]IMT, we investigated the transport characteristics of [ 125 I]IMT in porcine kidney epithelial cell line LLC-PK 1 using cell monolayers grown on microporous membrane filters. LLC-PK 1 monolayers were created on a collagen-coated microporous (3 μm) membrane (4.7 cm 2 ). To examine transcellular transport (secretion and reabsorption) and accumulation, the monolayers were incubated for up to 90 min at 37 deg C with 18.5 kBq [ 125 I]IMT in Dulbecco's phosphate-buffered saline (pH 7.4) as an uptake solution. After incubation, transcellular transport was assessed by quantifying the radioactivity of the solutions on each side of the monolayer. For the accumulation experiment, the cells were solubilized in NaOH solution, and the radioactivity was quantified. For the inhibition experiment, the inhibitor was added at a final concentration of 1 mM. For the pH dependence experiment, the pH of the apical-side uptake solution was varied from pH 5 to pH 8. Transport of [ 14 C]Tyr was examined for comparison. Bi-directional transcellular transport of [ 125 I]IMT was observed, corresponding to secretion and reabsorption in proximal tubule. Accumulation of [ 125 I]IMT from the basolateral side (1.62±0.15%) and the apical side (2.62±0.35%) was observed at 90 min. 2-Amino-bicyclo[2,2,1]heptane-2-carboxylic acid (a specific inhibitor of system L), L -Tyr (mother compound of [ 125 I]IMT) and 2-aminoisobutyric acid (an inhibitor of system L and A) inhibited both directional transport (p 125 I]IMT from both sides (p 125 I]IMT transport is system L, rather than Na + -dependent transport, in both apical and basolateral membrane. [ 125 I]IMT was transported by the system that transported L-Tyr, but the observed pH dependence of transport suggests that different

  16. Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate.

    Science.gov (United States)

    Liu, Jingjing; Xie, Zhipeng; Shin, Hyun-Dong; Li, Jianghua; Du, Guocheng; Chen, Jian; Liu, Long

    2017-07-10

    Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1gL -1 to 42.3gL -1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5gL -1 . Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5gL -1 to 89.5gL -1 . Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2gL -1 . The final engineered A. oryzae strain produced 165gL -1 L-malate with a productivity of 1.38gL -1 h -1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/anion transporter, TaALMT1

    Science.gov (United States)

    TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

  18. Air-to-vegetation transport of /sup 131/I as hypoiodous acid (HOI)

    Energy Technology Data Exchange (ETDEWEB)

    Voilleque, P G [Science Applications, Inc., Idaho Falls, ID (USA); Keller, J H [Exxon Nuclear Idaho Co., Inc., Idaho Falls, ID (USA)

    1981-01-01

    A significant fraction of the /sup 131/I in ventilation air in both BWRs and PWRs is present as hypoiodous acid (HOI). While HOI has been observed in the atmosphere its transport through the critical pathway has not been studied in detail. Of particular importance and interest is the deposition velocity used to characterize air-to-vegetation transport. This note describes the measurement of air-to-vegetation transport of HOI in a laboratory environmental chamber. The deposition velocity for HOI is compared with those for elemental I/sub 2/, methyl iodide and iodine associated with airborne particulates to show the relative importance of HOI in transport of /sup 131/I through the air-grass-cow-milk food chain. The data can be used to estimate relative contributions of the four /sup 131/I species to doses via the critical pathway.

  19. Brain Transport Profiles of Ginsenoside Rb1 by Glucose Transporter 1: In Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Yu-Zhu Wang

    2018-04-01

    Full Text Available Ginsenoside Rb1 (Rb1 has been demonstrated its protection for central nervous system and is apparently highly distributed to the brain. The objective of this study was to characterize Rb1 transport at the blood–brain barrier (BBB using primary cultured rat brain microvascular endothelial cells (rBMEC, an in vitro BBB model. The initial uptake velocity of Rb1 in rBMEC was temperature- and concentration-dependent, and was significantly reduced by phloretin, an inhibitor of GLUT1 transporter, but was independent of metabolic inhibitor. Furthermore, the transport of Rb1 into rBMEC was significantly diminished in the presence of natural substrate α-D-glucose, suggesting a facilitated transport of Rb1 via GLUT1 transporter. The impact of GLUT1 on the distribution of Rb1 between brain and plasma was studied experimentally in rats. Administration of phloretin (5 mg/kg, i.v. to normal rats for consecutive 1 week before Rb1 (10 mg/kg, i.v. at 0.5, 2, and 6 h did not alter Rb1 concentrations in plasma, but resulted in significant decreased brain concentrations of Rb1 compared to in the phloretin-untreated normal rats (489.6 ± 58.3 versus 105.1 ± 15.1 ng/g, 193.8 ± 11.1 versus 84.8 ± 4.1 ng/g, and 114.2 ± 24.0 versus 39.9 ± 4.9 ng/g, respectively. The expression of GLUT1 in the phloretin-treated group by western blotting analysis in vitro and in vivo experiments was significantly decreased, indicating that the decreased transport of Rb1 in brain was well related to the down-regulated function and level of GLUT1. Therefore, our in vitro and in vivo results indicate that the transport of Rb1 at the BBB is at least partly mediated by GLUT1 transporter.

  20. Short-Chain Fatty Acids Enhance the Lipid Accumulation of 3T3-L1 Cells by Modulating the Expression of Enzymes of Fatty Acid Metabolism.

    Science.gov (United States)

    Yu, Haining; Li, Ran; Huang, Haiyong; Yao, Ru; Shen, Shengrong

    2018-01-01

    Short-chain fatty acids (SCFA) such as acetic acid, propionic acid, and butyric acid are produced by fermentation by gut microbiota. In this paper, we investigate the effects of SCFA on 3T3-L1 cells and the underlying molecular mechanisms. The cells were treated with acetic acid, propionic acid, or butyric acid when cells were induced to differentiate into adipocytes. MTT assay was employed to detect the viability of 3T3-L1 cells. Oil Red O staining was used to visualize the lipid content in 3T3-L1 cells. A triglyceride assay kit was used to detect the triacylglycerol content in 3T3-L1 cells. qRT-PCR and Western blot were used to evaluate the expression of metabolic enzymes. MTT results showed that safe concentrations of acetic acid, propionic acid, and butyric acid were less than 6.4, 3.2, and 0.8 mM, respectively. Oil Red O staining and triacylglycerols detection results showed that treatment with acetic acid, propionic acid, and butyric acid accelerated the 3T3-L1 adipocyte differentiation. qRT-PCR and Western blot results showed that the expressions of lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4), fatty acid transporter protein 4 (FATP4), and fatty acid synthase (FAS) were significantly increased by acetic acid, propionic acid, and butyric acid treatment during adipose differentiation (p fatty acid metabolism. © 2018 AOCS.

  1. Insulin, concanavalin A, EGF, IFG-I and vanadate activate de novo phosphatidic acid and diacylglycerol synthesis, C-kinase, and glucose transport in BC3H-1 myocytes

    International Nuclear Information System (INIS)

    Cooper, D.R.; Hernandez, H.; Konda, T.S.; Standaert, M.S.; Pollet, R.J.; Farese, R.V.

    1987-01-01

    The authors have reported that insulin stimulates de novo synthesis of phosphatidic acid (PA) which is metabolized directly to diacylglycerol (DG) in BS3H-1 myocytes; this is accompanied by increases in C-kinase activity in membrane and cytosolic extracts. This pathway may be involved in stimulating glucose transport and other metabolic processes. In this study, the authors have compared the effects of concanavalin A, EGF, IGF-I and sodium orthovanadate to insulin on PA/DG synthesis, C-kinase activity and glucose transport. All were found to be effective in stimulating glucose transport. Additionally, all activators rapidly increased the incorporation of [ 3 H]glycerol into DG and total glycerolipids, although none were as effective as insulin, which increased [ 3 H]DG 400% in 1 minute. Increased incorporation into phospholipids and triacylglycerols and to a lesser extent monoacylglycerol was also noted. They examined effects of concanavalin A and EGF on C-kinase activity and found that both agonists, like insulin, increase C-kinase activity in cytosolic and/or membrane fractions. Their findings raise the possibility that activation of receptors having associated tyrosine kinase activity may provoke some cellular responses through de novo PA/GD synthesis and C-kinase activation

  2. Effects of γ-Aminobutyric acid transporter 1 inhibition by tiagabine on brain glutamate and γ-Aminobutyric acid metabolism in the anesthetized rat In vivo.

    Science.gov (United States)

    Patel, Anant B; de Graaf, Robin A; Rothman, Douglas L; Behar, Kevin L

    2015-07-01

    γ-Aminobutyric acid (GABA) clearance from the extracellular space after release from neurons involves reuptake into terminals and astrocytes through GABA transporters (GATs). The relative flows through these two pathways for GABA released from neurons remains unclear. This study determines the effect of tiagabine, a selective inhibitor of neuronal GAT-1, on the rates of glutamate (Glu) and GABA metabolism and GABA resynthesis via the GABA-glutamine (Gln) cycle. Halothane-anesthetized rats were administered tiagabine (30 mg/kg, i.p.) and 45 min later received an intravenous infusion of either [1,6-(13)C2]glucose (in vivo) or [2-(13)C]acetate (ex vivo). Nontreated rats served as controls. Metabolites and (13)C enrichments were measured with (1)H-[(13)C]-nuclear magnetic resonance spectroscopy and referenced to their corresponding endpoint values measured in extracts from in situ frozen brain. Metabolic flux estimates of GABAergic and glutamatergic neurons were determined by fitting a metabolic model to the (13)C turnover data measured in vivo during [1,6-(13)C2]glucose infusion. Tiagabine-treated rats were indistinguishable (P > 0.05) from controls in tissue amino acid levels and in (13)C enrichments from [2-(13)C]acetate. Tiagabine reduced average rates of glucose oxidation and neurotransmitter cycling in both glutamatergic neurons (↓18%, CMR(glc(ox)Glu): control, 0.27 ± 0.05 vs. tiagabine, 0.22 ± 0.04 µmol/g/min; ↓11%, V(cyc(Glu-Gln)): control 0.23 ± 0.05 vs. tiagabine 0.21 ± 0.04 µmol/g/min and GABAergic neurons (↓18-25%, CMR(glc(ox)GABA): control 0.09 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min; V(cyc(GABA-Gln)): control 0.08 ± 0.02 vs. tiagabine 0.07 ± 0.03 µmol/g/min), but the changes in glutamatergic and GABAergic fluxes were not significant (P > 0.10). The results suggest that any reduction in GABA metabolism by tiagabine might be an indirect response to reduced glutamatergic drive rather than direct compensatory effects. © 2015 Wiley

  3. Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology.

    Science.gov (United States)

    Skjørringe, Tina; Burkhart, Annette; Johnsen, Kasper Bendix; Moos, Torben

    2015-01-01

    Iron is required in a variety of essential processes in the body. In this review, we focus on iron transport in the brain and the role of the divalent metal transporter 1 (DMT1) vital for iron uptake in most cells. DMT1 locates to cellular membranes and endosomal membranes, where it is a key player in non-transferrin bound iron uptake and transferrin-bound iron uptake, respectively. Four isoforms of DMT1 exist, and their respective characteristics involve a complex cell-specific regulatory machinery all controlling iron transport across these membranes. This complexity reflects the fine balance required in iron homeostasis, as this metal is indispensable in many cell functions but highly toxic when appearing in excess. DMT1 expression in the brain is prominent in neurons. Of serious dispute is the expression of DMT1 in non-neuronal cells. Recent studies imply that DMT1 does exist in endosomes of brain capillary endothelial cells denoting the blood-brain barrier. This supports existing evidence that iron uptake at the BBB occurs by means of transferrin-receptor mediated endocytosis followed by detachment of iron from transferrin inside the acidic compartment of the endosome and DMT1-mediated pumping iron into the cytosol. The subsequent iron transport across the abluminal membrane into the brain likely occurs by ferroportin. The virtual absent expression of transferrin receptors and DMT1 in glial cells, i.e., astrocytes, microglia and oligodendrocytes, suggest that the steady state uptake of iron in glia is much lower than in neurons and/or other mechanisms for iron uptake in these cell types prevail.

  4. Back to Acid Soil Fields: The Citrate Transporter SbMATE Is a Major Asset for Sustainable Grain Yield for Sorghum Cultivated on Acid Soils

    Directory of Open Access Journals (Sweden)

    Geraldo Carvalho Jr

    2016-02-01

    Full Text Available Aluminum (Al toxicity damages plant roots and limits crop production on acid soils, which comprise up to 50% of the world’s arable lands. A major Al tolerance locus on chromosome 3, AltSB, controls aluminum tolerance in sorghum [Sorghum bicolor (L. Moench] via SbMATE, an Al-activated plasma membrane transporter that mediates Al exclusion from sensitive regions in the root apex. As is the case with other known Al tolerance genes, SbMATE was cloned based on studies conducted under controlled environmental conditions, in nutrient solution. Therefore, its impact on grain yield on acid soils remains undetermined. To determine the real world impact of SbMATE, multi-trait quantitative trait loci (QTL mapping in hydroponics, and, in the field, revealed a large-effect QTL colocalized with the Al tolerance locus AltSB, where SbMATE lies, conferring a 0.6 ton ha–1 grain yield increase on acid soils. A second QTL for Al tolerance in hydroponics, where the positive allele was also donated by the Al tolerant parent, SC283, was found on chromosome 9, indicating the presence of distinct Al tolerance genes in the sorghum genome, or genes acting in the SbMATE pathway leading to Al-activated citrate release. There was no yield penalty for AltSB, consistent with the highly localized Al regulated SbMATE expression in the root tip, and Al-dependent transport activity. A female effect of 0.5 ton ha–1 independently demonstrated the effectiveness of AltSB in hybrids. Al tolerance conferred by AltSB is thus an indispensable asset for sorghum production and food security on acid soils, many of which are located in developing countries.

  5. Identification of a disulfide bridge important for transport function of SNAT4 neutral amino acid transporter.

    Directory of Open Access Journals (Sweden)

    Rugmani Padmanabhan Iyer

    Full Text Available SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT and Tris(2-carboxyethylphosphine (TCEP, indicating the possible involvement of disulfide bridge(s. Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H2O2. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.

  6. 14 CFR 206.1 - Emergency transportation.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Emergency transportation. 206.1 Section 206.1 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS... EXEMPTIONS § 206.1 Emergency transportation. Notwithstanding the provisions of section 41101 of the Statute...

  7. Biodistribution and human dosimetry of enantiomer-1 of the synthetic leucine analog anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid

    International Nuclear Information System (INIS)

    Nye, Jonathon A.; Jarkas, Nashwa; Schuster, David M.; Savir-Baruch, Bital; Voll, Ronald J.; Camp, Vernon M.; Goodman, Mark M.

    2011-01-01

    Introduction: The enantiomerically enriched (ee=90%, enantiomer 1) synthetic amino acid (R,S)-anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid (anti-2-[ 18 F]FACPC-1) accumulates in malignant cells by elevated transport through the sodium-independent system-L (leucine preferring) amino acid transporter. The purpose of this study was to evaluate in vivo uptake and single-dose toxicity of anti-2-[ 18 F]FACPC-1 in animals as well as the individual organ and whole-body dose in humans. Methods: A DU145 xenograft rodent model was used to measure anti-2-[ 18 F]FACPC-1 uptake at 15, 30 and 60 min post-injection. Animals were sacrificed and organs harvested to measure the percent injected activity per organ and to calculate residence time. Anti-2-[ 18 F]FACPC-1 toxicity was assessed using a single microdose (37-74 MBq/kg) in nonhuman primates. Their vital signs were monitored for 2 h post-injection for drug-related effects. Human biodistribution studies were collected by sequential whole-body PET/CT scans on six healthy volunteers (three male and three female) for 120 min following a single 247±61 MBq bolus injection of anti-2-[ 18 F]FACPC-1. Estimates of radiation dose from anti-2-[ 18 F]FACPC-1 to the human body were calculated using recommendations of the MIRD committee and MIRDOSE 3.0 software. Results: High anti-2-[ 18 F]FACPC-1 residence time was observed in the pancreas of the rodent model compared to the human data. No abnormal treatment-related observations were made in the nonhuman primate toxicity studies. Human venous blood showed no metabolites of anti-2-[ 18 F]FACPC-1 in the first 60 min post-injection. All volunteers showed initially high uptake in the kidneys followed by a rapid washout phase. The estimated effective dose equivalent was 0.0196 mSv/MBq. Conclusion: Anti-2-[ 18 F]FACPC-1 showed low background uptake in the brain, thoracic and abdominal cavities of humans, suggesting a possible use for detecting malignant tissues in these regions.

  8. Facilitated transport of titanium dioxide nanoparticles by humic substances in saturated porous media under acidic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ruichang [Chinese Academy of Sciences, Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science (China); Zhang, Haibo; Tu, Chen; Hu, Xuefeng; Li, Lianzhen [Chinese Academy of Sciences, Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research (China); Luo, Yongming, E-mail: ymluo@yic.ac.cn; Christie, Peter [Chinese Academy of Sciences, Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science (China)

    2015-04-15

    The transport behavior of titanium dioxide nanoparticles (TiO{sub 2} NPs, 30 nm in diameter) was studied in well-defined porous media composed of clean quartz sand over a range of solution chemistry under acidic conditions. Transport of TiO{sub 2} NPs was dramatically enhanced by humic substances (HS) at acidic pH (4.0, 5.0 and 6.0), even at a low HS concentration of 0.5 mg L{sup −1}. Facilitated transport of TiO{sub 2} NPs was likely attributable to the increased stability of TiO{sub 2} NPs and repulsive interaction between TiO{sub 2} NPs and quartz sands due to the adsorbed HS. The mobility of TiO{sub 2} NPs was also increased with increasing pH from 4.0 to 6.0. Although transport of TiO{sub 2} NPs was insensitive to low ionic strength, it was significantly inhibited by high concentrations of NaCl and CaCl{sub 2}. In addition, calculated Derjaguin–Landau–Verwey–Overbeek (DLVO) interaction energy indicated that high energy barriers were responsible for the high mobility of TiO{sub 2} NPs, while the secondary energy minimum could play an important role in the retention of TiO{sub 2} NPs at 100 mmol L{sup −1} NaCl. Straining and gravitational settlement of larger TiO{sub 2} NPs aggregates at 1 mg L{sup −1} HS, pH 5.0, and 2 mmol L{sup −1} CaCl{sub 2} could be responsible for the significant retention even in the presence of high energy barriers. Moreover, more favorable interaction between approaching TiO{sub 2} NPs and TiO{sub 2} NPs that had been already deposited on the collector resulted in a ripening-shape breakthrough curve at 2 mmol L{sup −1} CaCl{sub 2}. Overall, a combination of mechanisms including DLVO-type force, straining, and physical filtration was involved in the retention of TiO{sub 2} NPs over the range of solution chemistry examined in this study.

  9. Flavonoids Are Inhibitors of Human Organic Anion Transporter 1 (OAT1)–Mediated Transport

    Science.gov (United States)

    An, Guohua; Wang, Xiaodong

    2014-01-01

    Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 μM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway. PMID:25002746

  10. Okadaic acid inhibits cell growth and photosynthetic electron transport in the alga Dunaliella tertiolecta

    Energy Technology Data Exchange (ETDEWEB)

    Perreault, Francois; Matias, Marcelo Seleme; Oukarroum, Abdallah [Department of Chemistry, Universite du Quebec a Montreal, 2101, Rue Jeanne Mance, Montreal, QC, Canada H2X 2J6 (Canada); Matias, William Gerson [Department of Chemistry, Universite du Quebec a Montreal, 2101, Rue Jeanne Mance, Montreal, QC, Canada H2X 2J6 (Canada); Laboratorio de Toxicologia Ambiental, LABTOX, Depto. de Engenharia Sanitaria e Ambiental, Universidade Federal de Santa Catarina, Campus Universitario, CEP: 88040-970, Florianopolis, SC (Brazil); Popovic, Radovan, E-mail: popovic.radovan@uqam.ca [Department of Chemistry, Universite du Quebec a Montreal, 2101, Rue Jeanne Mance, Montreal, QC, Canada H2X 2J6 (Canada)

    2012-01-01

    Okadaic acid (OA), which is produced by several dinoflagellate species, is a phycotoxin known to induce a decrease of biomass production in phytoplankton. However, the mechanisms of OA cytotoxicity are still unknown in microalgae. In this study, we exposed the green microalga Dunaliella tertiolecta to OA concentrations of 0.05 to 0.5 {mu}M in order to evaluate its effects on cell division, reactive oxygen species production and photosynthetic electron transport. After 72 h of treatment under continuous illumination, OA concentrations higher than 0.10 {mu}M decreased culture cell density, induced oxidative stress and inhibited photosystem II electron transport capacity. OA effect in D. tertiolecta was strongly light dependent since no oxidative stress was observed when D. tertiolecta was exposed to OA in the dark. In the absence of light, the effect of OA on culture cell density and photosystem II activity was also significantly reduced. Therefore, light appears to have a significant role in the toxicity of OA in microalgae. Our results indicate that the site of OA interaction on photosynthetic electron transport is likely to be at the level of the plastoquinone pool, which can lead to photo-oxidative stress when light absorbed by the light-harvesting complex of photosystem II cannot be dissipated via photochemical pathways. These findings allowed for a better understanding of the mechanisms of OA toxicity in microalgae. - Highlights: Black-Right-Pointing-Pointer Exposition of Dunaliella tertiolecta to okadaic acid in light conditions results in reactive oxygen species formation. Black-Right-Pointing-Pointer Inhibition of photosystem II is dependent on oxidative stress and effects of okadaic acid on the plastoquinone pool. Black-Right-Pointing-Pointer Oxidative stress and inhibition of photosynthesis increase okadaic acid effect on cell density in light conditions. Black-Right-Pointing-Pointer Okadaic acid induces toxicity in algae via both light-dependent and light

  11. Cloning and characterization of a functional human ¿-aminobutyric acid (GABA) transporter, human GAT-2

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Meinild, Anne-Kristine; Jensen, Anders A.

    2007-01-01

    Plasma membrane gamma-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human....... The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT......-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable...

  12. Placental fatty acid transport in maternal obesity.

    Science.gov (United States)

    Cetin, I; Parisi, F; Berti, C; Mandò, C; Desoye, G

    2012-12-01

    Pregestational obesity is a significant risk factor for adverse pregnancy outcomes. Maternal obesity is associated with a specific proinflammatory, endocrine and metabolic phenotype that may lead to higher supply of nutrients to the feto-placental unit and to excessive fetal fat accumulation. In particular, obesity may influence placental fatty acid (FA) transport in several ways, leading to increased diffusion driving force across the placenta, and to altered placental development, size and exchange surface area. Animal models show that maternal obesity is associated with increased expression of specific FA carriers and inflammatory signaling molecules in placental cotyledonary tissue, resulting in enhanced lipid transfer across the placenta, dislipidemia, fat accumulation and possibly altered development in fetuses. Cell culture experiments confirmed that inflammatory molecules, adipokines and FA, all significantly altered in obesity, are important regulators of placental lipid exchange. Expression studies in placentas of obese-diabetic women found a significant increase in FA binding protein-4 expression and in cellular triglyceride content, resulting in increased triglyceride cord blood concentrations. The expression and activity of carriers involved in placental lipid transport are influenced by the endocrine, inflammatory and metabolic milieu of obesity, and further studies are needed to elucidate the strong association between maternal obesity and fetal overgrowth.

  13. Protein Restriction with Amino Acid-Balanced Diets Shrinks Circulating Pool Size of Amino Acid by Decreasing Expression of Specific Transporters in the Small Intestine.

    Directory of Open Access Journals (Sweden)

    Kai Qiu

    Full Text Available Dietary protein restriction is not only beneficial to health and longevity in humans, but also protects against air pollution and minimizes feeding cost in livestock production. However, its impact on amino acid (AA absorption and metabolism is not quite understood. Therefore, the study aimed to explore the effect of protein restriction on nitrogen balance, circulating AA pool size, and AA absorption using a pig model. In Exp.1, 72 gilts weighting 29.9 ± 1.5 kg were allocated to 1 of the 3 diets containing 14, 16, or 18% CP for a 28-d trial. Growth (n = 24, nitrogen balance (n = 6, and the expression of small intestinal AA and peptide transporters (n = 6 were evaluated. In Exp.2, 12 barrows weighting 22.7 ± 1.3 kg were surgically fitted with catheters in the portal and jejunal veins as well as the carotid artery and assigned to a diet containing 14 or 18% CP. A series of blood samples were collected before and after feeding for determining the pool size of circulating AA and AA absorption in the portal vein, respectively. Protein restriction did not sacrifice body weight gain and protein retention, since nitrogen digestibility was increased as dietary protein content reduced. However, the pool size of circulating AA except for lysine and threonine, and most AA flux through the portal vein were reduced in pigs fed the low protein diet. Meanwhile, the expression of peptide transporter 1 (PepT-1 was stimulated, but the expression of the neutral and cationic AA transporter systems was depressed. These results evidenced that protein restriction with essential AA-balanced diets, decreased AA absorption and reduced circulating AA pool size. Increased expression of small intestinal peptide transporter PepT-1 could not compensate for the depressed expression of jejunal AA transporters for AA absorption.

  14. Low pH, Aluminum, and Phosphorus Coordinately Regulate Malate Exudation through GmALMT1 to Improve Soybean Adaptation to Acid Soils1[W][OA

    Science.gov (United States)

    Liang, Cuiyue; Piñeros, Miguel A.; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V.; Liao, Hong

    2013-01-01

    Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function. PMID:23341359

  15. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

    International Nuclear Information System (INIS)

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J.

    2014-01-01

    Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL

  16. Cholesterol efflux from THP-1 macrophages is impaired by the fatty acid component from lipoprotein hydrolysis by lipoprotein lipase

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J., E-mail: rbrown@mun.ca

    2014-09-05

    Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

  17. Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

    Science.gov (United States)

    Zhang, Yuanyuan; Jackson, Jonathan P; St Claire, Robert L; Freeman, Kimberly; Brouwer, Kenneth R; Edwards, Jeffrey E

    2017-08-01

    Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 μmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OST α ) and OST β increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OST α and OST β, by OCA reduced the intracellular concentrations of d 8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases. © 2017 Intercept Pharmaceuticals. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and

  18. Predominant contribution of L-type amino acid transporter to 4-borono-2-18F-fluoro-phenylalanine uptake in human glioblastoma cells

    International Nuclear Information System (INIS)

    Yoshimoto, Mitsuyoshi; Kurihara, Hiroaki; Honda, Natsuki; Kawai, Keiichi; Ohe, Kazuyo; Fujii, Hirofumi; Itami, Jun; Arai, Yasuaki

    2013-01-01

    Introduction: 4-Borono-2- 18 F-fluoro-phenylalanine ( 18 F-FBPA) has been used to anticipate the therapeutic effects of boron neutron capture therapy (BNCT) with 4-borono-L-phenylalanine (BPA). Similarly, L-[methyl- 11 C]-methionine ( 11 C-MET), the most popular amino acid PET tracer, is a possible candidate for this purpose. We investigated the transport mechanism of 18 F-FBPA and compared it with that of 14 C-MET in human glioblastoma cell lines. Methods: Uptake of 18 F-FBPA and 14 C-MET was examined in A172, T98G, and U-87MG cells using 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (a system L-specific substrate), 2-(methylamino)-isobutyric acid (a system A-specific substrate), and BPA. Gene expression was analyzed by quantitative real time polymerase chain reaction. Results: System L was mainly involved in the uptake of 18 F-FBPA (74.5%–81.1% of total uptake) and 14 C-MET (48.3%–59.4%). System A and ASC also contributed to the uptake of 14 C-MET. Inhibition experiments revealed that BPA significantly decreased the uptake of 18 F-FBPA, whereas 31%–42% of total 14 C-MET uptake was transported by BPA non-sensitive transporters. In addition, 18 F-FBPA uptake correlated with LAT1 and total LAT expressions. Conclusion: This study demonstrated that 18 F-FBPA was predominantly transported by system L in human glioblastoma cells compared to 14 C-MET. Although further studies are needed to elucidate the correlation between 18 F-FBPA uptake and BPA content in tumor tissues, 18 F-FBPA is suitable for the selection of patients who benefit from BNCT with BPA

  19. Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics.

    Science.gov (United States)

    Walker, Aisha L; Lancaster, Cynthia S; Finkelstein, David; Ware, Russell E; Sparreboom, Alex

    2013-12-15

    Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assessing the role of OATP1B transporters in modulating hydroxyurea PK. Using wild-type and Oatp1b knockout (Oatp1b(-/-)) mice, hydroxyurea PK was analyzed in vivo by measuring [(14)C]hydroxyurea distribution in plasma, kidney, liver, urine, or the exhaled (14)CO2 metabolite. Plasma levels were significantly reduced by 20% in Oatp1b(-/-) mice compared with wild-type (area under the curve of 38.64 or 48.45 μg·h(-1)·ml(-1), respectively) after oral administration, whereas no difference was observed between groups following intravenous administration. Accumulation in the kidney was significantly decreased by twofold in Oatp1b(-/-) mice (356.9 vs. 748.1 pmol/g), which correlated with a significant decrease in urinary excretion. Hydroxyurea accumulation in the liver was also decreased (136.6 vs. 107.3 pmol/g in wild-type or Oatp1b(-/-) mice, respectively) correlating with a decrease in exhaled (14)CO2. These findings illustrate that deficiency of Oatp1b transporters alters the absorption, distribution, and elimination of hydroxyurea thus providing the first in vivo evidence that cell membrane transporters may play a significant role in modulating hydroxyurea PK. Future studies to investigate other transporters and their role in hydroxyurea disposition are warranted for understanding the sources of variation in hydroxyurea's PK.

  20. DISORDERS OF CARNITINE TRANSPORT AND THE CARNITINE CYCLE

    OpenAIRE

    Longo, Nicola; di San Filippo, Cristina Amat; Pasquali, Marzia

    2006-01-01

    Carnitine plays an essential role in the transfer of long-chain fatty acids across the inner mitochondrial membrane. This transfer requires enzymes and transporters that accumulate carnitine within the cell (OCTN2 carnitine transporter), conjugate it with long chain fatty acids (carnitine palmitoyl transferase 1, CPT1), transfer the acylcarnitine across the inner plasma membrane (carnitine-acylcarnitine translocase, CACT), and conjugate the fatty acid back to Coenzyme A for subsequent beta ox...

  1. Stereospecific transport of Tyr-MIF-1 across the blood-brain barrier by peptide transport system-1

    Energy Technology Data Exchange (ETDEWEB)

    Banks, W.A.; Kastin, A.J.; Michals, E.A.; Barrera, C.M. (Veterans Affairs Medical Center, New Orleans, LA (USA))

    1990-10-01

    Previous studies have suggested that peptide transport system-1 (PTS-1), the saturable system that transports Tyr-MIF-1, the enkephalins, and related peptides out of the central nervous system (CNS), exhibits stereospecificity. In the present studies, we showed that {sup 125}I-L-Tyr-MIF-1, but not {sup 131}I-D-Tyr-MIF-1, was cleared from the CNS more rapidly than could be accounted for by nonspecific mechanisms. Such clearance was inhibited by a 1.0 nmol dose of L-Tyr-MIF-1, but not by D-Tyr-MIF-1. Neither L- nor D-Tyr-MIF-1 altered the much lower clearance of I-D-Tyr-MIF-1 from the brain. Radioactivity recovered from the vascular space after the injection of {sup 125}I-Tyr-MIF-1 into the lateral ventricle of the brain eluted by HPLC primarily as intact peptide, demonstrating that most of the Tyr-MIF-1 was not degraded during transport. By contrast, the nonsaturable unidirectional influx of Tyr-MIF-1 into the CNS did not distinguish between the isomers. These studies confirm and extend the observations that Tyr-MIF-1 is transported out of the CNS by a saturable, stereospecific transport system as an intact peptide while the influx into the CNS is by a nonsaturable mechanism that does not distinguish between the isomers.

  2. Amino acid transport system - A substrate predicts the therapeutic effects of particle radiotherapy.

    Directory of Open Access Journals (Sweden)

    Tomoya Uehara

    Full Text Available L-[methyl-11C]Methionine (11C-Met is useful for estimating the therapeutic efficacy of particle radiotherapy at early stages of the treatment. Given the short half-life of 11C, the development of longer-lived 18F- and 123I-labeled probes that afford diagnostic information similar to 11C-Met, are being sought. Tumor uptake of 11C-Met is involved in many cellular functions such as amino acid transport System-L, protein synthesis, and transmethylation. Among these processes, since the energy-dependent intracellular functions involved with 11C-Met are more reflective of the radiotherapeutic effects, we evaluated the activity of the amino acid transport System-A as an another energy-dependent cellular function in order to estimate radiotherapeutic effects. In this study, using a carbon-ion beam as the radiation source, the activity of System-A was evaluated by a specific System-A substrate, alpha-[1-14C]-methyl-aminoisobutyric acid (14C-MeAIB. Cellular growth and the accumulation of 14C-MeAIB or 14C-Met were evaluated over time in vitro in cultured human salivary gland (HSG tumor cells (3-Gy or in vivo in murine xenografts of HSG tumors (6- or 25-Gy before and after irradiation with the carbon-ion beam. Post 3-Gy irradiation, in vitro accumulation of 14C-Met and 14C-MeAIB decreased over a 5-day period. In xenografts of HSG tumors in mice, tumor re-growth was observed in vivo on day-10 after a 6-Gy irradiation dose, but no re-growth was detected after the 25-Gy irradiation dose. Consistent with the growth results, the in vivo tumor accumulation of 14C-MeAIB did not decrease after the 6-Gy irradiation dose, whereas a significant decrease was observed after the 25-Gy irradiation dose. These results indicate that the activity of energy dependent System-A transporter may reflect the therapeutic efficacy of carbon-ion radiotherapy and suggests that longer half-life radionuclide-labeled probes for System-A may also provide widely available probes to

  3. The transport of carboxylic acids and important role of the Jen1p transporter during the development of yeast colonies

    Czech Academy of Sciences Publication Activity Database

    Paivo, S.; Strachotová, Dita; Kučerová, Helena; Hlaváček, Otakar; Mota, A.; Casal, M.; Palková, Z.; Váchová, Libuše

    2013-01-01

    Roč. 454, SEP 2013 (2013), s. 551-558 ISSN 0264-6021 R&D Projects: GA ČR GA204/08/0718 Institutional support: RVO:61388971 Keywords : ammonia production * Jen1p protein * monocarboxylic acid import Subject RIV: CE - Biochemistry Impact factor: 4.779, year: 2013

  4. Influence of rye flour enzymatic biotransformation on the antioxidant capacity and transepithelial transport of phenolic acids.

    Science.gov (United States)

    de Lima, Fabíola Aliaga; Martins, Isabela Mateus; Faria, Ana; Calhau, Conceição; Azevedo, Joana; Fernandes, Iva; Mateus, Nuno; Macedo, Gabriela Alves

    2018-03-01

    Phenolic acids have been reported to play a role on the antioxidant activity and other important biological activities. However, as most polyphenolics in food products are either bound to cellular matrices or present as free polymeric forms, the way they are absorbed has not been totally clear until now. Hydrolytic enzymes may act to increase functionalities in polyphenolic-rich foods, enhancing the bioaccessibility of phenolic compounds and minerals from whole grains. The aim of this study was to evaluate the action of tannin acyl hydrolase (tannase) on the total phenols, phenolic acid profile, antioxidant capacity and in vitro bioaccessibility of phenolic acids found in whole rye flour (RF). Besides increasing total phenols and the antioxidant capacity, tannase treatment increased the amounts of ferulic, sinapic and vanillic acids identified in RF, evidencing a new type of feruloyl esterase catalytic action of tannase. Vanillic and sinapic acids in tannase-treated whole rye flour (RFT) were higher than RF after in vitro gastrointestinal digestion, and higher amounts of transported vanillic acid through the Caco-2 monolayer were detected in RFT. However, the bioaccessibility and the transport efficiency of RF phenolic acids were higher than RFT. Underutilized crops like rye and rye-derived products may be an important source of phenolic acids. The tannase biotransformation, even influencing the total phenolics and antioxidant capacity of RF, did not increase the bioaccessibility of phenolic acids under the experimental conditions of this study.

  5. Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

    Science.gov (United States)

    Ramesh, Sunita A; Kamran, Muhammad; Sullivan, Wendy; Chirkova, Larissa; Okamoto, Mamoru; Degryse, Fien; McLaughlin, Michael; Gilliham, Matthew; Tyerman, Stephen D

    2018-05-01

    Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABA A receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA] i ) in both wheat ( Triticum aestivum ) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA] i because TaALMT1 facilitates GABA efflux but GABA does not complex Al 3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14 C-GABA uptake into TaALMT1 -expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1 F213C ) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA] i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status. © 2018 American Society of Plant Biologists. All rights reserved.

  6. NpPDR1, a pleiotropic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense.

    Science.gov (United States)

    Stukkens, Yvan; Bultreys, Alain; Grec, Sébastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

    2005-09-01

    Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family.

  7. SGLT2 inhibitor lowers serum uric acid through alteration of uric acid transport activity in renal tubule by increased glycosuria

    Science.gov (United States)

    Chino, Yukihiro; Samukawa, Yoshishige; Sakai, Soichi; Nakai, Yasuhiro; Yamaguchi, Jun-ichi; Nakanishi, Takeo; Tamai, Ikumi

    2014-01-01

    Sodium glucose cotransporter 2 (SGLT2) inhibitors have been reported to lower the serum uric acid (SUA) level. To elucidate the mechanism responsible for this reduction, SUA and the urinary excretion rate of uric acid (UEUA) were analysed after the oral administration of luseogliflozin, a SGLT2 inhibitor, to healthy subjects. After dosing, SUA decreased, and a negative correlation was observed between the SUA level and the UEUA, suggesting that SUA decreased as a result of the increase in the UEUA. The increase in UEUA was correlated with an increase in urinary d-glucose excretion, but not with the plasma luseogliflozin concentration. Additionally, in vitro transport experiments showed that luseogliflozin had no direct effect on the transporters involved in renal UA reabsorption. To explain that the increase in UEUA is likely due to glycosuria, the study focused on the facilitative glucose transporter 9 isoform 2 (GLUT9ΔN, SLC2A9b), which is expressed at the apical membrane of the kidney tubular cells and transports both UA and d-glucose. It was observed that the efflux of [14C]UA in Xenopus oocytes expressing the GLUT9 isoform 2 was trans-stimulated by 10 mm d-glucose, a high concentration of glucose that existed under SGLT2 inhibition. On the other hand, the uptake of [14C]UA by oocytes was cis-inhibited by 100 mm d-glucose, a concentration assumed to exist in collecting ducts. In conclusion, it was demonstrated that the UEUA could potentially be increased by luseogliflozin-induced glycosuria, with alterations of UA transport activity because of urinary glucose. PMID:25044127

  8. Polymer-immobilized liquid membrane transport of palladium (II) from nitric acid media using some thia extractants as novel receptors

    International Nuclear Information System (INIS)

    Shukla, J.P.

    1996-01-01

    Carrier-facilitated co-transport of Pd (II) from dilute acidic nitrate solutions was examined across a polymer-immobilized liquid membrane (PILM) deploying S 6 -pentano-36 (S 6 -P-36), bis-(2-ethylhexyl) sulfoxide (BESO) and bis (2, 4, 4 trimethyl pentyl) monothio phosphinic acid (Cyanex 302) as the novel receptors. The study carried out to distinguish the driving force between H + and NO 3 - ion for the cation transport across PILM, indicated that NO 3 - ion not the H + ion seems to be the driving force for Pd (II) transport under the present conditions for both BESO-PILM and S 6 -P-36-PILM systems. Recovery of palladium from acidic process effluents generated in Purex reprocessing of spent fuels was successfully achieved. 39 refs., 8 figs., 7 tabs

  9. Hydrogen sulfide: role in ion channel and transporter modulation in the eye

    Directory of Open Access Journals (Sweden)

    Ya Fatou eNjie-Mbye

    2012-07-01

    Full Text Available Hydrogen sulfide (H2S, a colorless gas with a characteristic smell of rotten eggs, has been portrayed for decades as a toxic environmental pollutant. Since evidence of its basal production in mammalian tissues a decade ago, H2S has attracted substantial interest as a potential inorganic gaseous mediator with biological importance in cellular functions. Current research suggests that, next to its counterparts nitric oxide and carbon monoxide, H2S is an important multifunctional signaling molecule with pivotal regulatory roles in various physiological and pathophysiological processes as diverse as learning and memory, modulation of synaptic activities, cell survival, inflammation and maintenance of vascular tone in the central nervous and cardiovascular systems. In contrast, there are few reports of a regulatory role of H2S in the eye. Accumulating reports on the pharmacological role of H2S in ocular tissues indicate the existence of a functional trans-sulfuration pathway and a potential physiological role for H2S as a gaseous neuromodulator in the eye. Thus, understanding the role of H2S in vision-related processes is imperative to our expanding knowledge of this molecule as a gaseous mediator in ocular tissues. This review aims to provide a comprehensive and current understanding of the potential role of H2S as a signaling molecule in the eye. This objective is achieved by discussing the involvement of H2S in the regulation of (1 ion channels such as calcium (L-type, T-type and intracellular stores, potassium (KATP and small conductance channels and chloride channels, (2 glutamate transporters such as EAAT1/GLAST and the L-cystine/glutamate antiporter. The role of H2S as an important mediator in cellular functions and physiological processes that are triggered by its interaction with ion channels/transporters in the eye will also be discussed.

  10. Transport and fate of Herbaspirillum chlorophenolicum FA1 in saturated porous media

    Science.gov (United States)

    Li, X.; Xu, H.; Wu, J.

    2016-12-01

    For the bioremediation of contaminated groundwater, sufficient dispersal of functional microorganisms is one of the most important factors that determine the remediation efficiency. There are extensive studies on the transport of microbes in porous media, while most of them focus on pathogenic bacteria and little attention has been given toward functional bacteria that being used in bioremediation process. Therefore, accurate knowledge of the mechanisms that govern the transport and distribution of such bacteria in groundwater is needed to develop efficient treatment techniques. Herbaspirillum chlorophenolicum FA1, a pure bacterial strain capable of absorbing heavy metals and degrading polycyclic aromatic hydrocarbons (PAHs), was selected as the representative functional bacterium in this study. A series of batch and column experiments were conducted to investigate the transport and deposition behavior of strain FA1 in saturated porous media. The effects of physical (grain size), chemical (ionic strength, humic acid), and biological factors (living/dead cells) were studied in detail. In addition, numerical simulations of breakthrough curve (BTC) data were also performed for information gathering. Results of this study could advance our understanding of functional bacteria transport and help to develop successful bioremediation strategies. This work was financially supported by the National Natural Science Foundation of China -Xinjiang Project (U1503282), the National Natural Science Foundation of China (41030746, 41102148), and the Natural Science Foundation of Jiangsu Province (BK20151385). Keywords: Herbaspirillum chlorophenolicum FA1, bacteria, porous media, transport, modeling

  11. Theophylline–gentisic acid (1/1

    Directory of Open Access Journals (Sweden)

    Srinivasulu Aitipamula

    2009-09-01

    Full Text Available In the title 1:1 cocrystal, C7H8N4O2·C7H6O4, the anti-asthmatic drug theophylline (systematic name: 1,3-dimethyl-7H-purine-2,6-dione and a non-steroidal anti-inflammatory drug, gentisic acid (systematic name: 2,5-dihydroxybenzoic acid crystallize together, forming two-dimensional hydrogen-bonded sheets involving N—H...O and O—H...N hydrogen bonds. The overall crystal packing features π–π stacking interactions [centroid–centroid distance = 3.348 (1 Å]. The cocrystal described herein belongs to the class of pharmaceutical cocrystals involving two active pharmaceutical ingredients which has been relatively unexplored to date.

  12. Impact of FDA-Approved Drugs on the Prostaglandin Transporter OATP2A1/SLCO2A1.

    Science.gov (United States)

    Kamo, Shunsuke; Nakanishi, Takeo; Aotani, Rika; Nakamura, Yoshinobu; Gose, Tomoka; Tamai, Ikumi

    2017-09-01

    To understand interaction of drugs with the prostaglandin transporter OATP2A1/SLCO2A1 that regulates disposition of prostaglandins, we explored the impact of 636 drugs in an FDA-approved drug library on 6-carboxyfluorescein (6-CF) uptake by OATP2A1-expressing HEK293 cells (HEK/2A1). Fifty-one and 10 drugs were found to inhibit and enhance 6-CF uptake by more than 50%, respectively. Effect of the 51 drugs on 6-CF uptake was positively correlated with that on PGE 2 uptake (r = 0.64, p < 0.001). Among those, 5 drugs not structurally related to prostaglandins, suramin, pranlukast, zafirlukast, olmesartan medoxomil, and losartan potassium, exhibited more than 90% PGE 2 uptake inhibition. Inhibitory affinity of suramin to OATP2A1 was the highest (IC 50,2A1 of 0.17 μM), and its IC 50 values to MRP4-mediated PGE 2 transport (IC 50,MRP4 ) and PGE 2 synthesis in human U-937 cells treated with phorbol 12-myristate 13-acetate (IC 50,Syn ) were 73.6 and 336.7 times higher than IC 50,2A1 , respectively. Moreover, structure-activity relationship study in 29 nonsteroidal anti-inflammatory drugs contained in the library displayed inhibitory activities of anthranilic acid derivatives, but enhancing effects of propionic acid derivatives. These results demonstrate that suramin is a potent selective inhibitor of OATP2A1, providing a comprehensive information about drugs in clinical use that interact with OATP2A1. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  13. Ethane-1,1,2-trisphosphonic acid hemihydrate.

    Science.gov (United States)

    Delain-Bioton, Lise; Lohier, Jean François; Villemin, Didier; Sopková-de Oliveira Santos, Jana; Hix, Gary; Jaffrès, Paul Alain

    2008-02-01

    Ethane-1,1,2-trisphosphonic acid crystallizes as a hemihydrate, C(2)H(9)O(9)P(3).0.5H(2)O, in which the water O atom lies on an inversion centre in the space group P2(1)/c. The acid component, which contains a short but noncentred O-H...O hydrogen bond, adopts a gauche conformation. The acid components are linked by an extensive series of O-H...O hydrogen bonds to form layers, which are linked into pairs by the water molecules.

  14. Improved efficiency of NiOx-based p-i-n perovskite solar cells by using PTEG-1 as electron transport layer

    Science.gov (United States)

    Groeneveld, Bart G. H. M.; Najafi, Mehrdad; Steensma, Bauke; Adjokatse, Sampson; Fang, Hong-Hua; Jahani, Fatemeh; Qiu, Li; ten Brink, Gert H.; Hummelen, Jan C.; Loi, Maria Antonietta

    2017-07-01

    We present efficient p-i-n type perovskite solar cells using NiOx as the hole transport layer and a fulleropyrrolidine with a triethylene glycol monoethyl ether side chain (PTEG-1) as electron transport layer. This electron transport layer leads to higher power conversion efficiencies compared to perovskite solar cells with PCBM (phenyl-C61-butyric acid methyl ester). The improved performance of PTEG-1 devices is attributed to the reduced trap-assisted recombination and improved charge extraction in these solar cells, as determined by light intensity dependence and photoluminescence measurements. Through optimization of the hole and electron transport layers, the power conversion efficiency of the NiOx/perovskite/PTEG-1 solar cells was increased up to 16.1%.

  15. Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling.

    Science.gov (United States)

    Papagianni, Maria

    2007-01-01

    Citric acid is regarded as a metabolite of energy metabolism, of which the concentration will rise to appreciable amounts only under conditions of substantive metabolic imbalances. Citric acid fermentation conditions were established during the 1930s and 1940s, when the effects of various medium components were evaluated. The biochemical mechanism by which Aspergillus niger accumulates citric acid has continued to attract interest even though its commercial production by fermentation has been established for decades. Although extensive basic biochemical research has been carried out with A. niger, the understanding of the events relevant for citric acid accumulation is not completely understood. This review is focused on citric acid fermentation by A. niger. Emphasis is given to aspects of fermentation biochemistry, membrane transport in A. niger and modeling of the production process.

  16. The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids*

    Science.gov (United States)

    Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

    2014-01-01

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation. PMID:24652292

  17. The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

    Science.gov (United States)

    Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

    2014-05-09

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

  18. Amino Acid Metabolism and Transport Mechanisms as Potential Antifungal Targets

    Directory of Open Access Journals (Sweden)

    Matthew W. McCarthy

    2018-03-01

    Full Text Available Discovering new drugs for treatment of invasive fungal infections is an enduring challenge. There are only three major classes of antifungal agents, and no new class has been introduced into clinical practice in more than a decade. However, recent advances in our understanding of the fungal life cycle, functional genomics, proteomics, and gene mapping have enabled the identification of new drug targets to treat these potentially deadly infections. In this paper, we examine amino acid transport mechanisms and metabolism as potential drug targets to treat invasive fungal infections, including pathogenic yeasts, such as species of Candida and Cryptococcus, as well as molds, such as Aspergillus fumigatus. We also explore the mechanisms by which amino acids may be exploited to identify novel drug targets and review potential hurdles to bringing this approach into clinical practice.

  19. Humic acid transport in saturated porous media: influence of flow velocity and influent concentration.

    Science.gov (United States)

    Wei, Xiaorong; Shao, Mingan; Du, Lina; Horton, Robert

    2014-12-01

    Understanding the transport of humic acids (HAs) in porous media can provide important and practical evidence needed for accurate prediction of organic/inorganic contaminant transport in different environmental media and interfaces. A series of column transport experiments was conducted to evaluate the transport of HA in different porous media at different flow velocities and influent HA concentrations. Low flow velocity and influent concentration were found to favor the adsorption and deposition of HA onto sand grains packed into columns and to give higher equilibrium distribution coefficients and deposition rate coefficients, which resulted in an increased fraction of HA being retained in columns. Consequently, retardation factors were increased and the transport of HA through the columns was delayed. These results suggest that the transport of HA in porous media is primarily controlled by the attachment of HA to the solid matrix. Accordingly, this attachment should be considered in studies of HA behavior in porous media. Copyright © 2014. Published by Elsevier B.V.

  20. Zn2+ modulation of neurotransmitter transporters

    DEFF Research Database (Denmark)

    Nørgaard-Nielsen, K.; Gether, U.

    2006-01-01

    of neurotransmitter transporters have been identified based on sequence homology: (1) the neurotransmitter sodium symporter family (NSS), which includes the Na+/C1(-)-dependent transporters for dopamine, norepinephrine, and serotonin; and (2) the dicarboxylate/amino acid cation symporter family (DAACS), which...

  1. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486.

    Science.gov (United States)

    Kalandadze, Avtandil; Wu, Ying; Robinson, Michael B

    2002-11-29

    Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an

  2. Biodistribution and human dosimetry of enantiomer-1 of the synthetic leucine analog anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid

    Energy Technology Data Exchange (ETDEWEB)

    Nye, Jonathon A., E-mail: jnye@emory.edu; Jarkas, Nashwa; Schuster, David M.; Savir-Baruch, Bital; Voll, Ronald J.; Camp, Vernon M.; Goodman, Mark M.

    2011-10-15

    Introduction: The enantiomerically enriched (ee=90%, enantiomer 1) synthetic amino acid (R,S)-anti-1-amino-2-fluorocyclopentyl-1-carboxylic acid (anti-2-[{sup 18}F]FACPC-1) accumulates in malignant cells by elevated transport through the sodium-independent system-L (leucine preferring) amino acid transporter. The purpose of this study was to evaluate in vivo uptake and single-dose toxicity of anti-2-[{sup 18}F]FACPC-1 in animals as well as the individual organ and whole-body dose in humans. Methods: A DU145 xenograft rodent model was used to measure anti-2-[{sup 18}F]FACPC-1 uptake at 15, 30 and 60 min post-injection. Animals were sacrificed and organs harvested to measure the percent injected activity per organ and to calculate residence time. Anti-2-[{sup 18}F]FACPC-1 toxicity was assessed using a single microdose (37-74 MBq/kg) in nonhuman primates. Their vital signs were monitored for 2 h post-injection for drug-related effects. Human biodistribution studies were collected by sequential whole-body PET/CT scans on six healthy volunteers (three male and three female) for 120 min following a single 247{+-}61 MBq bolus injection of anti-2-[{sup 18}F]FACPC-1. Estimates of radiation dose from anti-2-[{sup 18}F]FACPC-1 to the human body were calculated using recommendations of the MIRD committee and MIRDOSE 3.0 software. Results: High anti-2-[{sup 18}F]FACPC-1 residence time was observed in the pancreas of the rodent model compared to the human data. No abnormal treatment-related observations were made in the nonhuman primate toxicity studies. Human venous blood showed no metabolites of anti-2-[{sup 18}F]FACPC-1 in the first 60 min post-injection. All volunteers showed initially high uptake in the kidneys followed by a rapid washout phase. The estimated effective dose equivalent was 0.0196 mSv/MBq. Conclusion: Anti-2-[{sup 18}F]FACPC-1 showed low background uptake in the brain, thoracic and abdominal cavities of humans, suggesting a possible use for detecting

  3. Differences in monocarboxylic acid transporter type 1 expression in rumen epithelium of newborn calves due to age and milk or milk replacer feeding.

    Science.gov (United States)

    Flaga, J; Górka, P; Zabielski, R; Kowalski, Z M

    2015-06-01

    The aim of the present study was to investigate whether besides age and solid feed intake, monocarboxylic acid transporter type 1 (MCT1) expression in the rumen epithelium of calves is affected by liquid feed type [whole milk (WM) or milk replacer (MR)]. Thirty bull calves at the mean age of 5 days were randomly allocated to five experimental groups (six calves/group). Six calves were slaughtered immediately after allocation to the trial (5 days of life), eighteen calves were fed MR and slaughtered at week intervals (on 12, 19, 26 days of life respectively), and six calves were fed WM and slaughtered at the 26 days of life. MCT1 protein abundance and the MCT1 mRNA level were investigated in the dorsal and ventral sack of the rumen. Solid feed intake and short-chain fatty acids (SCFA) concentration in the rumen fluid increased linearly with calves' age. The amount of the MCT1 protein and mRNA in the dorsal sac of rumen as well as the amount of MCT1 protein in the cranial ventral sac of rumen also increased linearly with calves' age. Calves fed WM had greater solid feed intake in the last week of the study as compared to calves fed MR, but SCFA concentration in the rumen fluid was not different. MCT1 mRNA expression in the cranial dorsal sac of rumen and protein MCT1 expression in both dorsal and ventral cranial sack of the rumen were higher in calves fed WM as compared to calves fed MR. This study confirmed age-dependent changes of MCT1 expression in the rumen epithelium of newborn calves and showed that its expression might be affected by liquid feed type. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  4. Involvement of the carboxyl-terminal region of the yeast peroxisomal half ABC transporter Pxa2p in its interaction with Pxa1p and in transporter function.

    Directory of Open Access Journals (Sweden)

    Cheng-Yi Chuang

    Full Text Available The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette [ABC] transporter. This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p.Using a yeast two-hybrid assay, we found that the carboxyl-terminal region (CT of Pxa2p, but not of Pxa1p, is required for their interaction. Further analysis indicated that the central part of the CT (designated CT2 of Pxa2p was indispensable for its interaction with the carboxyl terminally truncated Pxa1_NBD. An interaction between the CT of Pxa2p and Pxa1_NBD was not detected, but could be identified in the presence of Pxa2_NBD-CT1. A single mutation of two conserved residues (aligned with X-ALD-associated mutations at the same positions in ALDP in the CT2 of the Pxa2_NBD-CT protein impaired its interaction with Pxa1_NBD or Pxa1_NBD-CT, resulting in a mutant protein that exhibited a proteinase K digestion profile different from that of the wild-type protein. Functional analysis of these mutant proteins on oleate plates indicated that they were defective in transporter function.The CT of Pxa2p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies, helping to establish the pathological mechanism for CT-related X

  5. Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development.

    Science.gov (United States)

    Wong, Bernice H; Chan, Jia Pei; Cazenave-Gassiot, Amaury; Poh, Rebecca W; Foo, Juat Chin; Galam, Dwight L A; Ghosh, Sujoy; Nguyen, Long N; Barathi, Veluchamy A; Yeo, Sia W; Luu, Chi D; Wenk, Markus R; Silver, David L

    2016-05-13

    Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Low-Protein Diet Supplemented with Keto Acids Is Associated with Suppression of Small-Solute Peritoneal Transport Rate in Peritoneal Dialysis Patients

    OpenAIRE

    Jiang, Na; Qian, Jiaqi; Lin, Aiwu; Fang, Wei; Zhang, Weiming; Cao, Liou; Wang, Qin; Ni, Zhaohui; Yao, Qiang

    2011-01-01

    Objective. We investigate whether low-protein diet would show benefits in suppressing peritoneal transport rate in peritoneal dialysis (PD) patients. Methods. This is a supplemented analysis of our previously published trial, which randomized 60 PD patients to receive low- (LP: dietary protein intake of 0.6–0.8 g/kg/d), keto-acid-supplemented low- (sLP: 0.6–0.8 g/kg/d with 0.12 g/kg/d of keto acids), or high- (HP: 1.0–1.2 g/kg/d) protein diet and lasted for one year. In this study, the variat...

  7. Stable isotope tracer reveals that viviparous snakes transport amino acids to offspring during gestation.

    Science.gov (United States)

    Van Dyke, James U; Beaupre, Steven J

    2012-03-01

    Viviparity and placentation have evolved from oviparity over 100 times in squamate reptiles (lizards and snakes). The independent origins of placentation have resulted in a variety of placental morphologies in different taxa, ranging from simple apposition of fetal and maternal tissues to endotheliochorial implantation that is homoplasious with mammalian placentation. Because the eggs of oviparous squamates transport gases and water from the environment and calcium from the eggshell, the placentae of viviparous squamates are thought to have initially evolved to accomplish these functions from within the maternal oviduct. Species with complex placentae have also been shown to rely substantially, or even primarily, on placental transport of organic nutrients for embryonic nutrition. However, it is unclear whether species with only simple placentae are also capable of transporting organic nutrients to offspring. Among viviparous squamates, all of the snakes that have been studied thus far have been shown to have simple placentae. However, most studies of snake placentation are limited to a single lineage, the North American Natricinae. We tested the abilities of four species of viviparous snakes - Agkistrodon contortrix (Viperidae), Boa constrictor (Boidae), Nerodia sipedon (Colubridae: Natricinae) and Thamnophis sirtalis (Colubridae: Natricinae) - to transport diet-derived amino acids to offspring during gestation. We fed [(15)N]leucine to pregnant snakes, and compared offspring (15)N content with that of unlabeled controls. Labeled females allocated significantly more (15)N to offspring than did controls, but (15)N allocation did not differ among species. Our results indicate that viviparous snakes are capable of transporting diet-derived amino acids to their offspring during gestation, possibly via placentation.

  8. On the Evolution of Specificity in Members of the Yeast Amino Acid Transporter Family as Parts of Specific Metabolic Pathways

    Directory of Open Access Journals (Sweden)

    Christos Gournas

    2018-05-01

    Full Text Available In the recent years, molecular modeling and substrate docking, coupled with biochemical and genetic analyses have identified the substrate-binding residues of several amino acid transporters of the yeast amino acid transporter (YAT family. These consist of (a residues conserved across YATs that interact with the invariable part of amino acid substrates and (b variable residues that interact with the side chain of the amino acid substrate and thus define specificity. Secondary structure sequence alignments showed that the positions of these residues are conserved across YATs and could thus be used to predict the specificity of YATs. Here, we discuss the potential of combining molecular modeling and structural alignments with intra-species phylogenetic comparisons of transporters, in order to predict the function of uncharacterized members of the family. We additionally define some orphan branches which include transporters with potentially novel, and to be characterized specificities. In addition, we discuss the particular case of the highly specific l-proline transporter, PrnB, of Aspergillus nidulans, whose gene is part of a cluster of genes required for the utilization of proline as a carbon and/or nitrogen source. This clustering correlates with transcriptional regulation of these genes, potentially leading to the efficient coordination of the uptake of externally provided l-Pro via PrnB and its enzymatic degradation in the cell.

  9. Assaying the proton transport and regulation of UCP1 using solid supported membranes.

    Science.gov (United States)

    Blesneac, Iulia; Ravaud, Stéphanie; Machillot, Paul; Zoonens, Manuela; Masscheylen, Sandrine; Miroux, Bruno; Vivaudou, Michel; Pebay-Peyroula, Eva

    2012-08-01

    The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.

  10. Cellular fatty acid transport in heart and skeletal muscle as facilitated by proteins

    NARCIS (Netherlands)

    Luiken, J. J.; Schaap, F. G.; van Nieuwenhoven, F. A.; van der Vusse, G. J.; Bonen, A.; Glatz, J. F.

    1999-01-01

    Despite the importance of long-chain fatty acids (FA) as fuels for heart and skeletal muscles, the mechanism of their cellular uptake has not yet been clarified. There is dispute as to whether FA are taken up by the muscle cells via passive diffusion and/or carrier-mediated transport. Kinetic

  11. HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein

    Directory of Open Access Journals (Sweden)

    Cara Andrea

    2007-03-01

    Full Text Available Abstract Background The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN (IN inhibitors, IINs has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA derivatives active on the HIV-1 IN strand transfer (ST step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR reversing ability. Results The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells. Conclusion To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.

  12. {sup 18}F-FBPA as a tumor-specific probe of L-type amino acid transporter 1 (LAT1): a comparison study with {sup 18}F-FDG and {sup 11}C-Methionine PET

    Energy Technology Data Exchange (ETDEWEB)

    Watabe, Tadashi [Osaka University Graduate School of Medicine, Department of Nuclear Medicine and Tracer Kinetics, Osaka (Japan); Osaka University Graduate School of Medicine, PET Molecular Imaging Center, Osaka (Japan); Ikeda, Hayato; Aoki, Masanao [Osaka University Graduate School of Medicine, Department of Nuclear Medicine and Tracer Kinetics, Osaka (Japan); Nagamori, Shushi; Wiriyasermkul, Pattama; Tanaka, Yoko; Hagiwara, Kohei; Kanai, Yoshikatsu [Osaka University Graduate School of Medicine, Department of Bio-system Pharmacology, Osaka (Japan); Naka, Sadahiro [Osaka University, Osaka University Graduate School of Medicine, Osaka University Hospital, Osaka (Japan); Kanai, Yasukazu [Osaka University Graduate School of Medicine, PET Molecular Imaging Center, Osaka (Japan); Osaka University Graduate School of Medicine, Department of Molecular Imaging in Medicine, Osaka (Japan); Shimosegawa, Eku [Osaka University Graduate School of Medicine, Department of Nuclear Medicine and Tracer Kinetics, Osaka (Japan); Osaka University Graduate School of Medicine, PET Molecular Imaging Center, Osaka (Japan); Osaka University, Osaka University Graduate School of Medicine, Osaka University Hospital, Osaka (Japan); Hatazawa, Jun [Osaka University Graduate School of Medicine, Department of Nuclear Medicine and Tracer Kinetics, Osaka (Japan); Osaka University Graduate School of Medicine, PET Molecular Imaging Center, Osaka (Japan); Osaka University, Immunology Frontier Research Center, Osaka (Japan)

    2017-02-15

    The purpose of this study was to evaluate the usefulness of L-4-borono-2-{sup 18}F-fluoro-phenylalanine ({sup 18}F-FBPA) as a tumor-specific probe, in comparison to {sup 18}F-FDG and {sup 11}C-methionine (Met), focusing on its transport selectivity by L-type amino acid transporter 1 (LAT1), which is highly upregulated in cancers. Cellular analyses of FBPA were performed to evaluate the transportability and K{sub m} value. PET studies were performed in rat xenograft models of C6 glioma (n = 12) and in rat models of turpentine oil-induced subcutaneous inflammation (n = 9). The kinetic parameters and uptake values on static PET images were compared using the one-tissue compartment model (K{sub 1}, k{sub 2}) and maximum standardized uptake value (SUVmax). The cellular analyses showed that FBPA had a lower affinity to a normal cell-type transporter LAT2 and induced less efflux through LAT2 among FBPA, Met, and BPA, while the efflux through LAT1 induced by FBPA was similar among the three compounds. The K{sub m} value of {sup 18}F-FBPA for LAT1 (196.8 ± 11.4 μM) was dramatically lower than that for LAT2 (2813.8 ± 574.5 μM), suggesting the higher selectivity of {sup 18}F-FBPA for LAT1. K{sub 1} and k{sub 2} values were significantly smaller in {sup 18}F-FBPA PET (K{sub 1} = 0.04 ± 0.01 ml/ccm/min and k{sub 2} = 0.07 ± 0.01 /min) as compared to {sup 11}C-Met PET (0.22 ± 0.09 and 0.52 ± 0.10, respectively) in inflammatory lesions. Static PET analysis based on the SUVmax showed significantly higher accumulation of {sup 18}F-FDG in the tumor and inflammatory lesions (7.2 ± 2.1 and 4.6 ± 0.63, respectively) as compared to both {sup 18}F-FBPA (3.2 ± 0.40 and 1.9 ± 0.19) and {sup 11}C-Met (3.4 ± 0.43 and 1.6 ± 0.11). No significant difference was observed between {sup 18}F-FBPA and {sup 11}C-Met in the static PET images. This study shows the utility of {sup 18}F-FBPA as a tumor-specific probe of LAT1 with low accumulation in the inflammatory lesions. (orig.)

  13. Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

    KAUST Repository

    Aouida, Mustapha

    2013-06-03

    Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

  14. Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

    KAUST Repository

    Aouida, Mustapha; Texeira, Marta Rubio; Thevelein, Johan M.; Poulin, Richard; Ramotar, Dindial

    2013-01-01

    Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.

  15. Survey of 1 1/2D transport codes

    International Nuclear Information System (INIS)

    Grad, H.

    1978-10-01

    A survey is given of a family of classical transport codes, recently termed ''1 1/2D'', which efficiently and accurately follow the evolution of plasma configurations on a long time scale, following coupled changes in plasma shape and topology with transport (but not wave motion). Codes have been constructed and operated (since 1974) which include various combinations of finite beta, general plasma cross-section and aspect, various topologies (Doublet, tearing, reversed-field mirror) including time dependent transitions in topology resulting from external coil variation and plasma transport, with models including (classical) tensor resistivity and heat flow as well as the adiabatic limiting case

  16. SEPARATION OF Fe (III, Cr(III, Cu(II, Ni(II, Co(II, AND Pb(II METAL IONS USING POLY(EUGENYL OXYACETIC ACID AS AN ION CARRIER BY A LIQUID MEMBRANE TRANSPORT METHOD

    Directory of Open Access Journals (Sweden)

    La Harimu

    2010-06-01

    Full Text Available Fe (III, Cr(III, Cu(II, Ni(II, Co(II, and Pb(II  metal ions had been separated using poly(eugenyl oxyacetic acid as an ion carrier by bulk liquid membrane transport method. The effect of pH, polyeugenyl oxyacetic acid ion carrier concentration, nitric acid concentration in the stripping solution, transport time, and metal concentration were optimized. The result showed that the optimum condition for transport of metal ions was at pH 4 for ion Fe(III and at pH 5 for Cr(III, Cu(II, Ni(II, Co(II, and Pb(II ions. The carrier volumes were optimum with concentration of 1 x 10-3 M at 7.5 mL for Cr(III, Cu (II,  Ni(II, Co(II ions and at 8.5 mL for Fe(III and Pb(II ions. The concentration of HNO3 in stripping phase was optimum at 2 M for Fe(III and Cu(II ions, 1 M for Cr(III, Ni(II and Co(II ions, and 0.5 M for Pb(II ion. The optimum transport times were 36 h for Fe(III and Co(II ions, and 48 h for Cr(III, Cu (II, Ni(II, and Pb(II ions. The concentration of metal ions accurately transported were 2.5 x 10-4 M for Fe(III and Cr(III ions, and 1 M for Cu (II, Ni(II, Co(II, and Pb(II ions. Compared to other metal ions the transport of Fe(III was the highest with selectivity order of Fe(III > Cr(III > Pb(II > Cu(II > Ni(II > Co(II. At optimum condition, Fe(III ion was transported through the membrane at 46.46%.   Keywords: poly(eugenyl oxyacetic acid, transport, liquid membrane, Fe (III, Cr(III, Cu(II, Ni(II, Co(II, and Pb(II ions

  17. The β-lactam clavulanic acid mediates glutamate transport-sensitive pain relief in a rat model of neuropathic pain

    DEFF Research Database (Denmark)

    Kristensen, P J; Gegelashvili, G; Munro, G

    2017-01-01

    -regulates glutamate transporters both in vitro and in vivo. Crucially, a similar up-regulation of glutamate transporters in human spinal astrocytes by clavulanic acid supports the development of novel β-lactam-based analgesics, devoid of antibacterial activity, for the clinical treatment of chronic pain.......BACKGROUND: Following nerve injury, down-regulation of astroglial glutamate transporters (GluTs) with subsequent extracellular glutamate accumulation is a key factor contributing to hyperexcitability within the spinal dorsal horn. Some β-lactam antibiotics can up-regulate GluTs, one of which......, ceftriaxone, displays analgesic effects in rodent chronic pain models. METHODS: Here, the antinociceptive actions of another β-lactam clavulanic acid, which possesses negligible antibiotic activity, were compared with ceftriaxone in rats with chronic constriction injury (CCI)-induced neuropathic pain...

  18. Inhibition of beta-amino acid transport by diamide does not involve the brush border membrane surface

    International Nuclear Information System (INIS)

    Chesney, R.W.; Gusowski, N.; Albright, P.

    1985-01-01

    Diamide (dicarboxylic acid bis-(N,N-dimethylamide) has been shown in previous studies to block the uptake of the beta-amino acid taurine at its high affinity transport site in rat renal cortex slices. Diamide may act by increasing the efflux of taurine from the slice. Studies performed in rat slices again indicate enhanced efflux over 8-12 minutes. The time course of reduced glutathione (GSH) depletion from renal cortex is similar, indicating a potential interaction between GSH depletion and inhibition of taurine accumulation. The effect of 9 mM diamide on the Na+ -dependent accumulation of taurine (10 and 250 microM) by brush border membrane vesicles was examined, and the taurine uptake value both initially and at equilibrium was the same in the presence and absence of diamide. Isolation of the brush border surface and subsequent transport studies of taurine are not influenced by diamide. Thus, diamide inhibition of taurine uptake does not involve physiochemical alteration of the membrane surface where active amino acid transport occurs, despite the thiol-oxidizing properties of this agent. Further, these studies suggest that diamide either acts at the basolateral surface, rather than the brush border surface of rat renal cortex or requires the presence of an intact tubule, capable of metabolism, prior to its inhibitory action

  19. Transport of S-cysteine conjugates in LL-PK1 cells and its role in toxicity

    International Nuclear Information System (INIS)

    Schaeffer, V.; Stevens, J.

    1986-01-01

    In order to study its role in S-cysteine conjugate toxicity, the transport of the nephrotoxic cysteine conjugate, S-1,2-dichlorovinyl-L-cysteine (L-DCVC), was investigated in the kidney cell line, LLC-PK1. When incubated with [ 35 S]-DCVC, accumulation of radioactivity within the cells was linear for at least 5 min with 92% of the 35 S present as unmetabolized L-DCVC. Kinetic analysis indicated two saturable uptake systems; Km = 6.8 μM and 1.6 mM; V/sub max/ = .048 and 1.4 nmol/min/mg protein. Both systems were Na + -independent and were inhibited by amino acid transport system L substrates but not substrates of systems A, ASC or organic anion transport. L-DCVC uptake at 5 μM and 500 μM was significantly greater in subconfluent cells than in confluent cultures by 5 and 2.8 fold, respectively. The presence of the non-toxic conjugates S-methyl-(SMC), S-ethyl-(SEC), S-benzyl-L- cysteine (SBC) and the D isomer of DCVC blocked the toxicity and inhibited the transport of L-DCVC in LLC-PK1 cells in the rank order of SBC > SEC congruent to D-DCVC > SMC. The metabolism of L-DCVC, previously shown to be required for cytotoxicity, was only slightly inhibited by these conjugates and did not correlate with their relative protection against toxicity. This study suggests that L-DCVC is transported into these cells by the system L transporter and that protection against toxicity by non-toxic S-cysteine conjugates is due to the inhibition of transport

  20. Hepatic and renal Bcrp transporter expression in mice treated with perfluorooctanoic acid

    International Nuclear Information System (INIS)

    Eldasher, Lobna M.; Wen, Xia; Little, Michael S.; Bircsak, Kristin M.; Yacovino, Lindsay L.; Aleksunes, Lauren M.

    2013-01-01

    Highlights: ► PFOA increased liver weight and Cyp4a14 mRNA and protein expression in mice. ► PFOA increased kidney Cyp4a14 mRNA in mice. ► PFOA increased Bcrp mRNA and protein in livers, but not kidneys, of mice. ► PFOA inhibited activation of human BCRP ATPase activity in vitro. ► PFOA inhibited human BCRP transport in inverted membrane vesicles. - Abstract: The breast cancer resistance protein (Bcrp) is an efflux transporter that participates in the biliary and renal excretion of drugs and environmental chemicals. Recent evidence suggests that pharmacological activation of the peroxisome proliferator activated receptor alpha (PPARα) can up-regulate the hepatic expression of Bcrp. The current study investigated the regulation of hepatic and renal Bcrp mRNA and protein in mice treated with the PPARα agonist perfluorooctanoic acid (PFOA) and the ability of PFOA to alter human BCRP function in vitro. Bcrp mRNA and protein expression were quantified in the livers and kidneys of male C57BL/6 mice treated with vehicle or PFOA (1 or 3 mg/kg/day oral gavage) for 7 days. PFOA treatment increased liver weights as well as the hepatic mRNA and protein expression of the PPARα target gene, cytochrome P450 4a14. Compared to vehicle-treated control mice, PFOA increased hepatic Bcrp mRNA and protein between 1.5- and 3-fold. Immunofluorescent staining confirmed enhanced canalicular Bcrp staining in liver sections from PFOA-treated mice. The kidney expression of cytochrome P450 4a14 mRNA, but not Bcrp, was increased in mice treated with PFOA. Micromolar concentrations of PFOA decreased human BCRP ATPase activity and inhibited BCRP-mediated transport in inverted membrane vesicles. Together, these studies demonstrate that PFOA induces hepatic Bcrp expression in mice and may inhibit human BCRP transporter function at concentrations that exceed levels observed in humans

  1. The effect of Eimeria maxima infection on the expression of amino acid and sugar transporters aminopeptidase, as well as the di- and tri-peptide transporter PepT1, is not solely due to decreased feed intake

    Science.gov (United States)

    Coccidiosis caused by Eimeria in poultry is endemic to poultry operations and results in decreased feed intake, diarrhea, and decreased weight gain. The goal was to determine the effect infection Eimeria maxima on the expression of genes that encode peptide and amino acid transporters (AATs), and al...

  2. A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

    Science.gov (United States)

    Lu, C.; Fedoroff, N.

    2000-01-01

    Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

  3. L-lactate transport in Ehrlich ascites-tumour cells.

    Science.gov (United States)

    Spencer, T L; Lehninger, A L

    1976-01-01

    Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids. PMID:7237

  4. Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport

    Science.gov (United States)

    Day, Pricilla E.; Ntani, Georgia; Crozier, Sarah R.; Mahon, Pam A.; Inskip, Hazel M.; Cooper, Cyrus; Harvey, Nicholas C.; Godfrey, Keith M.; Hanson, Mark A.; Lewis, Rohan M.; Cleal, Jane K.

    2015-01-01

    Introduction Maternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth. Methods RNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR. Results Increased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001). Conclusion A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of

  5. Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport.

    Directory of Open Access Journals (Sweden)

    Pricilla E Day

    Full Text Available Maternal environment and lifestyle factors may modify placental function to match the mother's capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content on a selection of metabolic and amino acid transporter genes and their associations with fetal growth.RNA was extracted from 102 term Southampton Women's Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR.Increased placental LAT2 (p = 0.01, y+LAT2 (p = 0.03, aspartate aminotransferase 2 (p = 0.02 and decreased aspartate aminotransferase 1 (p = 0.04 mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01, ASCT1 (p = 0.03, mitochondrial branched chain aminotransferase (p = 0.02 and glutamine synthetase (p = 0.05 was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05 associated with higher maternal diet quality (prudent dietary pattern pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05 and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01 associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01. Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001.A number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking

  6. mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

    Science.gov (United States)

    Wyant, Gregory A; Abu-Remaileh, Monther; Wolfson, Rachel L; Chen, Walter W; Freinkman, Elizaveta; Danai, Laura V; Vander Heiden, Matthew G; Sabatini, David M

    2017-10-19

    The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: isolation and pharmacological characterization of rOscp1.

    Science.gov (United States)

    Izuno, Hisanori; Kobayashi, Yasuna; Sanada, Yutaka; Nihei, Daisuke; Suzuki, Masako; Kohyama, Noriko; Ohbayashi, Masayuki; Yamamoto, Toshinori

    2007-09-22

    Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a Km value of 15.7+/-1.9 microM, and rOscp1-mediated organic solutes were exhibited in time- and Na+-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239-1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.

  8. The expression and function of fatty acid transport protein-2 and -4 in the murine placenta.

    Directory of Open Access Journals (Sweden)

    Takuya Mishima

    Full Text Available The uptake and trans-placental trafficking of fatty acids from the maternal blood into the fetal circulation are essential for embryonic development, and involve several families of proteins. Fatty acid transport proteins (FATPs uniquely transport fatty acids into cells. We surmised that placental FATPs are germane for fetal growth, and are regulated during hypoxic stress, which is associated with reduced fat supply to the fetus.Using cultured primary term human trophoblasts we found that FATP2, FATP4 and FATP6 were highly expressed in trophoblasts. Hypoxia enhanced the expression of trophoblastic FATP2 and reduced the expression of FATP4, with no change in FATP6. We also found that Fatp2 and Fatp4 are expressed in the mouse amnion and placenta, respectively. Mice deficient in Fatp2 or Fatp4 did not deviate from normal Mendelian distribution, with both embryos and placentas exhibiting normal weight and morphology, triglyceride content, and expression of genes related to fatty acid mobilization.We conclude that even though hypoxia regulates the expression of FATP2 and FATP4 in human trophoblasts, mouse Fatp2 and Fatp4 are not essential for intrauterine fetal growth.

  9. Workshop 1: Geosynthetics in Transportation Geotechnics

    OpenAIRE

    Tatsuoka, Fumio; Zornberg, Jorge; Machado do Vale, José Luís; Neves, José

    2016-01-01

    This book contains the extended abstracts of the Workshop 1 - Geosynthetics in Transportation Geotechnics - of the 3rd International Conference on Transportation Geotechnics, 4-7 September 2016, Guimarães, Portugal. The main goals of the workshop were: state-of-the-art of the use of geosynthetics in transportation geotechnics; theory and research of geosynthetics engineering for transportation engineering; key issues in practice; and perspective.

  10. Lipid Transport through the Fetoplacental Barrier by the Fatty Acid-Binding Proteins in Pregnant Women with Herpes Virus Infection in the third Trimester

    Directory of Open Access Journals (Sweden)

    Michael T. Lucenko, PhD, ScD

    2012-12-01

    Full Text Available In this study, the transport of the long chain polyunsaturated fatty acids (LCPUFAs from the lacunar blood through the syncytiotrophoblast of the placental villi to the fetal cord blood via a saturable protein-mediated mechanism by the heart-type fatty acid-binding proteins (H-FABPs has been examined. Exacerbation of the herpes simplex viruses (HSV-1 in the third trimester of gestation reduces the delivery of the fatty acid-binding proteins to the syncytiotrophoblast. During exacerbation of the HSV-1 infection, the selective transfer of the LCPUFAs across the syncytiotrophoblast basal plasma membrane into the fetal cord blood was observed. The supply of anti-inflammatory ω-3 PUFAs was reduced; however, the inflow of inflammatory arachidonic acid and other ω-6 PUFAs into the fetal blood was increased.

  11. Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1).

    Science.gov (United States)

    Demidenko, Aleksandr; Akberdin, Ilya R; Allemann, Marco; Allen, Eric E; Kalyuzhnaya, Marina G

    2016-01-01

    Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1) . Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE , was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE -knockout mutants and farE -overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE -strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.

  12. ETMB-RBF: discrimination of metal-binding sites in electron transporters based on RBF networks with PSSM profiles and significant amino acid pairs.

    Science.gov (United States)

    Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

    2013-01-01

    Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation-reduction reactions. In these oxidation-reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins.

  13. Butyrate transcriptionally enhances peptide transporter PepT1 expression and activity.

    Directory of Open Access Journals (Sweden)

    Guillaume Dalmasso

    Full Text Available BACKGROUND: PepT1, an intestinal epithelial apical di/tripeptide transporter, is normally expressed in the small intestine and induced in colon during chronic inflammation. This study aimed at investigating PepT1 regulation by butyrate, a short-chain fatty acid produced by commensal bacteria and accumulated inside inflamed colonocyte. RESULTS: We found that butyrate treatment of human intestinal epithelial Caco2-BBE cells increased human PepT1 (hPepT1 promoter activity in a dose- and time-dependent manner, with maximal activity observed in cells treated with 5 mM butyrate for 24 h. Under this condition, hPepT1 promoter activity, mRNA and protein expression levels were increased as assessed by luciferase assay, real-time RT-PCR and Western blot, respectively. hPepT1 transport activity was accordingly increased by approximately 2.5-fold. Butyrate did not alter hPepT1 mRNA half-life indicating that butyrate acts at the transcriptional level. Molecular analyses revealed that Cdx2 is the most important transcription factor for butyrate-induced increase of hPepT1 expression and activity in Caco2-BBE cells. Butyrate-activated Cdx2 binding to hPepT1 promoter was confirmed by gel shift and chromatin immunoprecipitation. Moreover, Caco2-BBE cells overexpressing Cdx2 exhibited greater hPepT1 expression level than wild-type cells. Finally, treatment of mice with 5 mM butyrate added to drinking water for 24 h increased colonic PepT1 mRNA and protein expression levels, as well as enhanced PepT1 transport activity in colonic apical membranes vesicles. CONCLUSIONS: Collectively, our results demonstrate that butyrate increases PepT1 expression and activity in colonic epithelial cells, which provides a new understanding of PepT1 regulation during chronic inflammation.

  14. ZIFL1.1 transporter modulates polar auxin transport by stabilizing membrane abundance of multiple PINs in Arabidopsis root tip

    Science.gov (United States)

    Remy, Estelle; Baster, Pawel; Friml, Jiří; Duque, Paula

    2013-01-01

    Cell-to-cell directional flow of the phytohormone auxin is primarily established by polar localization of the PIN auxin transporters, a process tightly regulated at multiple levels by auxin itself. We recently reported that, in the context of strong auxin flows, activity of the vacuolar ZIFL1.1 transporter is required for fine-tuning of polar auxin transport rates in the Arabidopsis root. In particular, ZIFL1.1 function protects plasma-membrane stability of the PIN2 carrier in epidermal root tip cells under conditions normally triggering PIN2 degradation. Here, we show that ZIFL1.1 activity at the root tip also promotes PIN1 plasma-membrane abundance in central cylinder cells, thus supporting the notion that ZIFL1.1 acts as a general positive modulator of polar auxin transport in roots. PMID:23857365

  15. Central transport and distribution of labelled glutamic and aspartic acids to the cochlear nucleus in cats

    International Nuclear Information System (INIS)

    Kane, E.S.

    1979-01-01

    Tritiated L-glutamic acid or L-aspartic acid was injected unilaterally into the cochleas of adult cats, and 4 h-7 days later the localization of label was studied by light-microscopic autoradiography in sections of the brain stem. Consistent differences in labelling after glutamate and after aspartate suggest differences in their uptake, metabolic conversion and/or transport to the cochlear nucleus by cochlear fibers. The morphological differences shown here agree with the distribution of those two amino acids in the cat cochlear nucleus as shown by microchemical analyses. (author)

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  18. Distribution of indole-3-acetic acid in Petunia hybrida shoot tip cuttings and relationship between auxin transport, carbohydrate metabolism and adventitious root formation.

    OpenAIRE

    Ahkami, Amir H.; Melzer, Michael; Ghaffari, Mohammad R.; Pollmann, Stephan; Ghorbani, Majid; Shahinnia, Fahimeh; Hajirezaei, Mohammad R.; Druege, Uwe

    2013-01-01

    To determine the contribution of polar auxin transport (PAT) to auxin accumulation and to adventitious root (AR) formation in the stem base of Petunia hybrida shoot tip cuttings, the level of indole-3-acetic acid (IAA) was monitored in non-treated cuttings and cuttings treated with the auxin transport blocker naphthylphthalamic acid (NPA) and was complemented with precise anatomical studies. The temporal course of carbohydrates, amino acids and activities of controlling enzymes was also inves...

  19. Facilitated transport of Cr(III) through activated composite membrane containing di-(2-ethylhexyl)phosphoric acid (DEHPA) as carrier agent

    Energy Technology Data Exchange (ETDEWEB)

    Arslan, Gulsin [Department of Chemistry, Selcuk University, 42031, Campus, Konya (Turkey); Tor, Ali, E-mail: ator@selcuk.edu.tr [Department of Environmental Engineering, Selcuk University, 42031 Campus, Konya (Turkey); Cengeloglu, Yunus; Ersoz, Mustafa [Department of Chemistry, Selcuk University, 42031, Campus, Konya (Turkey)

    2009-06-15

    The facilitated transport of chromium(III) through activated composite membrane (ACM) containing di-(2-ethylhexyl) phosphoric acid (DEHPA) was investigated. DEHPA was immobilised by interfacial polymerisation on polysulfone layer which was deposited on non-woven fabric by using spin coater. Then, ACM was characterised by using scanning electron microscopy (SEM), contact angle measurements and atomic force microscopy (AFM). Initially, batch experiments of liquid-liquid distribution of Cr(III) and the extractant (DEHPA) were carried out to determine the appropriate pH of the feed phase and the results showed that maximum extraction of Cr(III) was achieved at a pH of 4. It was also found that Cr(III) and DEHPA reacted in 1/1 molar ratio. The effects of Cr(III) (in feed phase), HCl (in stripping phase) and DEHPA (in ACM) concentrations were investigated. DEHPA concentration varies from 0.1 to 1.0 M and it was determined that the transport of Cr(III) increased with the carrier concentration up to 0.8 M. It was also observed that the transport of Cr(III) through the ACM tended to increase with Cr(III) and HCl concentrations. The stability of ACM was also confirmed with replicate experiments.

  20. Facilitated transport of Cr(III) through activated composite membrane containing di-(2-ethylhexyl)phosphoric acid (DEHPA) as carrier agent

    International Nuclear Information System (INIS)

    Arslan, Gulsin; Tor, Ali; Cengeloglu, Yunus; Ersoz, Mustafa

    2009-01-01

    The facilitated transport of chromium(III) through activated composite membrane (ACM) containing di-(2-ethylhexyl) phosphoric acid (DEHPA) was investigated. DEHPA was immobilised by interfacial polymerisation on polysulfone layer which was deposited on non-woven fabric by using spin coater. Then, ACM was characterised by using scanning electron microscopy (SEM), contact angle measurements and atomic force microscopy (AFM). Initially, batch experiments of liquid-liquid distribution of Cr(III) and the extractant (DEHPA) were carried out to determine the appropriate pH of the feed phase and the results showed that maximum extraction of Cr(III) was achieved at a pH of 4. It was also found that Cr(III) and DEHPA reacted in 1/1 molar ratio. The effects of Cr(III) (in feed phase), HCl (in stripping phase) and DEHPA (in ACM) concentrations were investigated. DEHPA concentration varies from 0.1 to 1.0 M and it was determined that the transport of Cr(III) increased with the carrier concentration up to 0.8 M. It was also observed that the transport of Cr(III) through the ACM tended to increase with Cr(III) and HCl concentrations. The stability of ACM was also confirmed with replicate experiments.

  1. Characterization of the ZAT1p zinc transporter from Arabidopsis thaliana in microbial model organisms and reconstituted proteoliposomes.

    Science.gov (United States)

    Bloss, Tanja; Clemens, Stephan; Nies, Dietrich H

    2002-03-01

    The ZAT1p zinc transporter from Arabidopsis thaliana (L.) Heynh. is a member of the cation diffusion facilitator (CDF) protein family. When heterologously expressed in Escherichia coli, ZAT1p bound zinc in a metal blot. Binding of zinc occurred mainly to the hydrophilic amino acid region from H182 to H232. A ZAT1p/ZAT1p*Delta(M1-I25) protein mixture was purified and reconstituted into proteoliposomes. Uptake of zinc into the proteoliposomes did not require a proton gradient across the liposomal membrane. ZAT1p did not transport cobalt, and transported cadmium at only 1% of the zinc transport rate. ZAT1p functioned as an uptake system for 65Zn2+ in two strains of the Gram-negative bacterium Ralstonia metallidurans, which were different in their content of zinc-efflux systems. The ZAT1 gene did not rescue increased zinc sensitivity of a Delta ZRC1single-mutant strain or of a Delta ZRC1 Delta COT1 double-mutant strain of Saccharomyces cerevisiae, but ZAT1 complemented this phenotype in a Delta SpZRC1 mutant strain of Schizosaccharomyces pombe.

  2. Transcriptional dysregulation of γ-aminobutyric acid transporter in parvalbumin-containing inhibitory neurons in the prefrontal cortex in schizophrenia.

    Science.gov (United States)

    Bitanihirwe, Byron K Y; Woo, Tsung-Ung W

    2014-12-30

    Parvalbumin (PV)-containing neurons are functionally compromised in schizophrenia. Using double in situ hybridization in postmortem human prefrontal cortex, we found that the messenger RNA (mRNA) for the γ-aminobutyric acid (GABA) transporter GAT-1 was undetectable in 22-41% of PV neurons in layers 3-4 in schizophrenia. In the remaining PV neurons with detectable GAT-1 mRNA, transcript expression was decreased by 26% in layer 3. Hence, the dysfunction of PV neurons involves the molecular dysregulation of presynaptic GABA reuptake. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  3. Crystal structure of a bacterial homologue of glucose transporters GLUT1-4.

    Science.gov (United States)

    Sun, Linfeng; Zeng, Xin; Yan, Chuangye; Sun, Xiuyun; Gong, Xinqi; Rao, Yu; Yan, Nieng

    2012-10-18

    Glucose transporters are essential for metabolism of glucose in cells of diverse organisms from microbes to humans, exemplified by the disease-related human proteins GLUT1, 2, 3 and 4. Despite rigorous efforts, the structural information for GLUT1-4 or their homologues remains largely unknown. Here we report three related crystal structures of XylE, an Escherichia coli homologue of GLUT1-4, in complex with d-xylose, d-glucose and 6-bromo-6-deoxy-D-glucose, at resolutions of 2.8, 2.9 and 2.6 Å, respectively. The structure consists of a typical major facilitator superfamily fold of 12 transmembrane segments and a unique intracellular four-helix domain. XylE was captured in an outward-facing, partly occluded conformation. Most of the important amino acids responsible for recognition of D-xylose or d-glucose are invariant in GLUT1-4, suggesting functional and mechanistic conservations. Structure-based modelling of GLUT1-4 allows mapping and interpretation of disease-related mutations. The structural and biochemical information reported here constitutes an important framework for mechanistic understanding of glucose transporters and sugar porters in general.

  4. The renal urate transporter SLC17A1 locus: confirmation of association with gout.

    Science.gov (United States)

    Hollis-Moffatt, Jade E; Phipps-Green, Amanda J; Chapman, Brett; Jones, Gregory T; van Rij, Andre; Gow, Peter J; Harrison, Andrew A; Highton, John; Jones, Peter B; Montgomery, Grant W; Stamp, Lisa K; Dalbeth, Nicola; Merriman, Tony R

    2012-04-27

    Two major gout-causing genes have been identified, the urate transport genes SLC2A9 and ABCG2. Variation within the SLC17A1 locus, which encodes sodium-dependent phosphate transporter 1, a renal transporter of uric acid, has also been associated with serum urate concentration. However, evidence for association with gout is equivocal. We investigated the association of the SLC17A1 locus with gout in New Zealand sample sets. Five variants (rs1165196, rs1183201, rs9358890, rs3799344, rs12664474) were genotyped across a New Zealand sample set totaling 971 cases and 1,742 controls. Cases were ascertained according to American Rheumatism Association criteria. Two population groups were studied: Caucasian and Polynesian. At rs1183201 (SLC17A1), evidence for association with gout was observed in both the Caucasian (odds ratio (OR) = 0.67, P = 3.0 × 10-6) and Polynesian (OR = 0.74, P = 3.0 × 10-3) groups. Meta-analysis confirmed association of rs1183201 with gout at a genome-wide level of significance (OR = 0.70, P = 3.0 × 10-8). Haplotype analysis suggested the presence of a common protective haplotype. We confirm the SLC17A1 locus as the third associated with gout at a genome-wide level of significance.

  5. In silico characterization of boron transporter (BOR1 protein sequences in Poaceae species

    Directory of Open Access Journals (Sweden)

    Ertuğrul Filiz

    2013-01-01

    Full Text Available Boron (B is essential for the plant growth and development, and its primary function is connected with formation of the cell wall. Moreover, boron toxicity is a shared problem in semiarid and arid regions. In this study, boron transporter protein (BOR1 sequences from some Poaceae species (Hordeum vulgare subsp. vulgare, Zea mays, Brachypodium distachyon, Oryza sativa subsp. japonica, Oryza sativa subsp. indica, Sorghum bicolor, Triticum aestivum were evaluated by bioinformatics tools. Physicochemical analyses revealed that most of BOR1 proteins were basic character and had generally aliphatic amino acids. Analysis of the domains showed that transmembrane domains were identified constantly and three motifs were detected with 50 amino acids length. Also, the motif SPNPWEPGSYDHWTVAKDMFNVPPAYIFGAFIPATMVAGLYYFDHSVASQ was found most frequently with 25 repeats. The phylogenetic tree showed divergence into two main clusters. B. distachyon species were clustered separately. Finally, this study contributes to the new BOR1 protein characterization in grasses and create scientific base for in silico analysis in future.

  6. Oleanolic acid alters bile acid metabolism and produces cholestatic liver injury in mice

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jie, E-mail: JLiu@kumc.edu [University of Kansas Medical Center, Kansas City, KS 66160 (United States); Zunyi Medical College, Zunyi 563003 (China); Lu, Yuan-Fu [University of Kansas Medical Center, Kansas City, KS 66160 (United States); Zunyi Medical College, Zunyi 563003 (China); Zhang, Youcai; Wu, Kai Connie [University of Kansas Medical Center, Kansas City, KS 66160 (United States); Fan, Fang [Cytopathology, University of Kansas Medical Center, Kansas City, KS 66160 (United States); Klaassen, Curtis D. [University of Kansas Medical Center, Kansas City, KS 66160 (United States)

    2013-11-01

    Oleanolic acid (OA) is a triterpenoids that exists widely in plants. OA is effective in protecting against hepatotoxicants. Whereas a low dose of OA is hepatoprotective, higher doses and longer-term use of OA produce liver injury. This study characterized OA-induced liver injury in mice. Adult C57BL/6 mice were given OA at doses of 0, 22.5, 45, 90, and 135 mg/kg, s.c., daily for 5 days, and liver injury was observed at doses of 90 mg/kg and above, as evidenced by increases in serum activities of alanine aminotransferase and alkaline phosphatase, increases in serum total bilirubin, as well as by liver histopathology. OA-induced cholestatic liver injury was further evidenced by marked increases of both unconjugated and conjugated bile acids (BAs) in serum. Gene and protein expression analysis suggested that livers of OA-treated mice had adaptive responses to prevent BA accumulation by suppressing BA biosynthetic enzyme genes (Cyp7a1, 8b1, 27a1, and 7b1); lowering BA uptake transporters (Ntcp and Oatp1b2); and increasing a BA efflux transporter (Ostβ). OA increased the expression of Nrf2 and its target gene, Nqo1, but decreased the expression of AhR, CAR and PPARα along with their target genes, Cyp1a2, Cyp2b10 and Cyp4a10. OA had minimal effects on PXR and Cyp3a11. Taken together, the present study characterized OA-induced liver injury, which is associated with altered BA homeostasis, and alerts its toxicity potential. - Highlights: • Oleanolic acid at higher doses and long-term use may produce liver injury. • Oleanolic acid increased serum ALT, ALP, bilirubin and bile acid concentrations. • OA produced feathery degeneration, inflammation and cell death in the liver. • OA altered bile acid homeostasis, affecting bile acid synthesis and transport.

  7. Metabolism of Mevalonic Acid in Vegetative and Induced Plants of Xanthium strumarium 1

    Science.gov (United States)

    Bledsoe, Caroline S.; Ross, Cleon W.

    1978-01-01

    The metabolism of mevalonic acid in Xanthium strumarium L. Chicago plants was studied to determine how mevalonate was metabolized and whether metabolism was related to induction of flowering. Leaves of vegetative, photoperiodically induced, and chemically inhibited cocklebur plants were supplied with [14C]mevalonic acid prior to or during a 16-hour inductive dark period. Vegetative, induced, and Tris(2-diethylaminoethyl)phosphate trihydrochloride-treated plants did not differ significantly in the amount of [14C]mevalonic acid they absorbed, nor in the distribution of radioactivity among the leaf blade (97%), petiole (2.3%), or shoot tip (0.7%). [14C]Mevalonic acid was rapidly metabolized and transported out of the leaves. Possible metabolites of mevalonate were mevalonic acid phosphates and sterols. No detectable 14C was found in gibberellins, carotenoids, or the phytol alcohol of chlorophyll. Chemically inhibited plants accumulated 14C compounds not found in vegetative or induced plants. When ethanol extracts of leaves, petioles, and buds were chromatographed, comparisons of chromatographic patterns did not show significant differences between vegetative and induced treatments. ImagesFig. 1 PMID:16660583

  8. Methylmercury transport across the blood-brain barrier by molecular mimicry

    International Nuclear Information System (INIS)

    Kerper, L.E.; Ballatori, N.; Clarkson, T.W.

    1990-01-01

    The mechanism by which methylmercury (MeHg) crosses the blood-brain barrier is not known. Co-administration of MeHg with L-cysteine by intravenous injection has been shown to accelerate MeHg uptake into brain tissue in rats. Since the complex of MeHg with L-cysteine is structurally similar to L-methionine, a substrate for the L (leucine-preferring) neutral amino acid transport system, this amino acid carrier may be involved in MeHg uptake into brain. To examine this hypothesis, the rapid carotid infusion technique was used in the rat. The concentration-dependence of initial rates of Me 203 Hg uptake into rat brains following injection of Me 203 Hg-L-cysteine complex was non-linear, exhibiting characteristics of saturable transport (K m 250 μM, V max 700 pmol·g -1 ·15 s -1 ). A slower, nonsaturable uptake was seen following MeHg-D-cysteine injection. MeHg-L-cysteine uptake was inhibited by co-injection of L-methionine (K i 200 μM), D-methionine (K i 600 μM), and amino acid analog 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (K i 1.4 mM), but not by amino acid analog α-methylaminoisobutyric acid. Transport of 14 C-L-phenylalanine was inhibited by MeHg-L-cysteine, but not by MeHgCl. The results suggest that MeHg may enter brain capillary endothelial cells as a cysteine complex, via amino acid transport system L

  9. Major involvement of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) in uptake of biotin and pantothenic acid by human brain capillary endothelial cells.

    Science.gov (United States)

    Uchida, Yasuo; Ito, Katsuaki; Ohtsuki, Sumio; Kubo, Yoshiyuki; Suzuki, Takashi; Terasaki, Tetsuya

    2015-07-01

    The purpose of this study was to clarify the expression of Na(+) -dependent multivitamin transporter (SLC5A6/SMVT) and its contribution to the supply of biotin and pantothenic acid to the human brain via the blood-brain barrier. DNA microarray and immunohistochemical analyses confirmed that SLC5A6 is expressed in microvessels of human brain. The absolute expression levels of SLC5A6 protein in isolated human and monkey brain microvessels were 1.19 and 0.597 fmol/μg protein, respectively, as determined by a quantitative targeted absolute proteomics technique. Using an antibody-free method established by Kubo et al. (2015), we found that SLC5A6 was preferentially localized at the luminal membrane of brain capillary endothelium. Knock-down analysis using SLC5A6 siRNA showed that SLC5A6 accounts for 88.7% and 98.6% of total [(3) H]biotin and [(3) H]pantothenic acid uptakes, respectively, by human cerebral microvascular endothelial cell line hCMEC/D3. SLC5A6-mediated transport in hCMEC/D3 was markedly inhibited not only by biotin and pantothenic acid, but also by prostaglandin E2, lipoic acid, docosahexaenoic acid, indomethacin, ketoprofen, diclofenac, ibuprofen, phenylbutazone, and flurbiprofen. This study is the first to confirm expression of SLC5A6 in human brain microvessels and to provide evidence that SLC5A6 is a major contributor to luminal uptake of biotin and pantothenic acid at the human blood-brain barrier. In humans, it was unclear (not concluded) about what transport system at the blood-brain barrier (BBB) is responsible for the brain uptakes of two vitamins, biotin and pantothenic acid, which are necessary for brain proper function. This study clarified for the first time that the solute carrier 5A6/Na(+) -dependent multivitamin transporter SLC5A6/SMVT is responsible for the supplies of biotin and pantothenic acid into brain across the BBB in humans. DHA, docosahexaenoic acid; NSAID, non-steroidal anti-inflammatory drug; PGE2, prostaglandin E2. © 2015

  10. Regorafenib is transported by the organic anion transporter 1B1 and the multidrug resistance protein 2.

    Science.gov (United States)

    Ohya, Hiroki; Shibayama, Yoshihiko; Ogura, Jiro; Narumi, Katsuya; Kobayashi, Masaki; Iseki, Ken

    2015-01-01

    Regorafenib is a small molecule inhibitor of tyrosine kinases, and has been shown to improve the outcomes of patients with advanced colorectal cancer and advanced gastrointestinal stromal tumors. The transport profiles of regorafenib by various transporters were evaluated. HEK293/organic anion transporting polypeptide 1B1 (OATP1B1) cells exhibited increased drug sensitivity to regorafenib. Regorafenib inhibited the uptake of 3H-estrone sulfate by HEK293/OATP1B1 cells in a dose-dependent manner, but did not affect its elimination by P-glycoproteins. The concentration of regorafenib was significantly lower in LLC-PK1/multidrug resistance protein 2 (MRP2) cells than in LLC-PK1 cells treated with the MRP2 inhibitor, MK571. MK571 abolished the inhibitory effects of regorafenib on intracellular accumulation in LLC-PK1/MRP2 cells. The uptake of regorafenib was significantly higher in HEK293/OATP1B1 cells than in OATP1B1-mock cells. Transport kinetics values were estimated to be Km=15.9 µM and Vmax=1.24 nmol/mg/min. No significant difference was observed in regorafenib concentrations between HEK293/OATP1B3 and OATP1B3-mock cells. These results indicated that regorafenib is a substrate for MRP2 and OATP1B1, and also suggest that the substrate preference of regorafenib may implicate the pharmacokinetic profiles of regorafenib.

  11. Regulators of Slc4 bicarbonate transporter activity

    Directory of Open Access Journals (Sweden)

    Ian M. Thornell

    2015-06-01

    Full Text Available 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 HCO3– 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 HCO3– 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 classical 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.

  12. Aryl hydrocarbon receptor-dependent up-regulation of the heterodimeric amino acid transporter LAT1 (SLC7A5)/CD98hc (SLC3A2) by diesel exhaust particle extract in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Le Vee, Marc; Jouan, Elodie; Lecureur, Valérie [Institut de Recherches en Santé, Environnement et Travail (IRSET), UMR INSERM U1085, Faculté de Pharmacie, 2 Avenue du Pr Léon Bernard, 35043 Rennes (France); Fardel, Olivier, E-mail: olivier.fardel@univ-rennes1.fr [Institut de Recherches en Santé, Environnement et Travail (IRSET), UMR INSERM U1085, Faculté de Pharmacie, 2 Avenue du Pr Léon Bernard, 35043 Rennes (France); Pôle Biologie, Centre Hospitalier Universitaire, 2 rue Henri Le Guilloux, 35033 Rennes (France)

    2016-01-01

    The heterodimeric L-type amino acid transporter (LAT) 1/CD98hc is overexpressed in lung cancers with a poor prognosis factor. Factors that contribute to LAT1/CD98hc overexpression in lung cells remain however to be determined, but the implication of atmospheric pollution can be suspected. The present study was therefore designed to analyze the effects of diesel exhaust particle (DEP) extract (DEPe) on LAT1/CD98hc expression in bronchial epithelial BEAS-2B cells. Exposure to DEPe up-regulated LAT1 and CD98hc mRNA levels in a concentration-dependent manner, with DEPe EC{sub 50} values (around 0.2 μg/mL) relevant to environmental situations. DEPe concomitantly induced LAT1/CD98hc protein expression and LAT1-mediated leucine accumulation in BEAS-2B cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway through the use of a chemical AhR antagonist or the siRNA-mediated silencing of AhR expression was next found to prevent DEPe-mediated induction of LAT1/CD98hc, indicating that this regulation depends on AhR, known to be activated by major chemical DEP components like polycyclic aromatic hydrocarbons. DEPe exposure was finally shown to induce mRNA expression and activity of matrix metalloproteinase (MMP)-2 in BEAS-2B cells, in a CD98hc/focal adhesion kinase (FAK)/extracellular regulated kinase (ERK) manner, thus suggesting that DEPe-mediated induction of CD98hc triggers activation of the integrin/FAK/ERK signaling pathway known to be involved in MMP-2 regulation. Taken together, these data demonstrate that exposure to DEPe induces functional overexpression of the amino acid transporter LAT1/CD98hc in lung cells. Such a regulation may participate to pulmonary carcinogenic effects of DEPs, owing to the well-documented contribution of LAT1 and CD98hc to cancer development. - Highlights: • The amino acid transporter LAT1/CD98hc is up-regulated in DEPe-treated lung cells. • The aryl hydrocarbon receptor is involved in DEPe-triggered induction of LAT1/CD98hc.

  13. Expression Profile of Cationic Amino Acid Transporters in Rats with Endotoxin-Induced Uveitis

    Directory of Open Access Journals (Sweden)

    Yung-Ray Hsu

    2016-01-01

    Full Text Available Purpose. The transcellular arginine transportation via cationic amino acid transporter (CAT is the rate-limiting step in nitric oxide (NO synthesis, which is crucial in intraocular inflammation. In this study, CAT isoforms and inducible nitric oxide synthase (iNOS expression was investigated in endotoxin-induced uveitis (EIU. Methods. EIU was induced in Lewis rats by lipopolysaccharide (LPS injection. In the treatment group, the rats were injected intraperitoneally with the proteasome inhibitor bortezomib before EIU induction. After 24 hours, leukocyte quantification, NO measurement of the aqueous humor, and histopathological examination were evaluated. The expression of CAT isoforms and iNOS was determined by reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence staining. Nuclear factor-kappa B (NF-κB binding activity was evaluated by electrophoretic mobility shift assay. The mouse macrophage cell line RAW 264.7 was used to validate the in vivo findings. Results. LPS significantly stimulated iNOS, CAT-2A, and CAT-2B mRNA and protein expression but did not affect CAT-1 in EIU rats and RAW 264.7 cells. Bortezomib attenuated inflammation and inhibited iNOS, CAT-2A, and CAT-2B expression through NF-κB inhibition. Conclusions. CAT-2 and iNOS, but not CAT-1, are specifically involved in EIU. NF-κB is essential in the induction of CAT-2 and iNOS in EIU.

  14. 26 CFR 1.132-9 - Qualified transportation fringes.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Qualified transportation fringes. 1.132-9... Qualified transportation fringes. (a) Table of contents. This section contains a list of the questions and answers in § 1.132-9. (1) General rules. Q-1. What is a qualified transportation fringe? Q-2. What is...

  15. Genetic alterations in fatty acid transport and metabolism genes are associated with metastatic progression and poor prognosis of human cancers.

    Science.gov (United States)

    Nath, Aritro; Chan, Christina

    2016-01-04

    Reprogramming of cellular metabolism is a hallmark feature of cancer cells. While a distinct set of processes drive metastasis when compared to tumorigenesis, it is yet unclear if genetic alterations in metabolic pathways are associated with metastatic progression of human cancers. Here, we analyzed the mutation, copy number variation and gene expression patterns of a literature-derived model of metabolic genes associated with glycolysis (Warburg effect), fatty acid metabolism (lipogenesis, oxidation, lipolysis, esterification) and fatty acid uptake in >9000 primary or metastatic tumor samples from the multi-cancer TCGA datasets. Our association analysis revealed a uniform pattern of Warburg effect mutations influencing prognosis across all tumor types, while copy number alterations in the electron transport chain gene SCO2, fatty acid uptake (CAV1, CD36) and lipogenesis (PPARA, PPARD, MLXIPL) genes were enriched in metastatic tumors. Using gene expression profiles, we established a gene-signature (CAV1, CD36, MLXIPL, CPT1C, CYP2E1) that strongly associated with epithelial-mesenchymal program across multiple cancers. Moreover, stratification of samples based on the copy number or expression profiles of the genes identified in our analysis revealed a significant effect on patient survival rates, thus confirming prominent roles of fatty acid uptake and metabolism in metastatic progression and poor prognosis of human cancers.

  16. Modulatory effect of ascorbic acid on physiological responses of transported ostrich chicks

    Directory of Open Access Journals (Sweden)

    Minka N. Salka

    2012-02-01

    Full Text Available The aims of this study were to determine the modulating role of ascorbic acid (AA on rectal temperature (RT, heterophil to lymphocyte (H to L ratio and aberrant behaviours of ostrich chicks transported by road for 4 h during hot-dry conditions. Twenty ostrich chicks aged 2.5 months, of both sexes and belonging to the Red Neck breed, served as subjects of the study. The chicks were assigned randomly to AA-treated and control groups, consisting of 10 chicks each. The AA-treated group was administered orally with 100 mg/kg body weight of AA dissolved in 5 mL of sterile water 30 min before transportation, whilst the control group was given the equivalent of sterile water only. The thermal load (TL experienced in the vehicle during transportation fluctuated between 31 °C and 89 °C, as calculated from the ambient temperature and relative humidity. Transportation induced hyperthermia, lymphopenia, heterophilia and aberrant behaviours of pecking, wing fluffing and panting, which were ameliorated by AA administration. The relationships between the TL, journey duration and physiological variables of RT, H to L ratio and aberrant behaviours recorded during transportation were significantly and positively correlated in the control group. In AA-treated group the relationships were not significantly correlated. In conclusion, the results showed for the first time that AA ameliorated the adverse effects of stress caused by road transportation on the aberrant behaviours, RT and H to L ratio of ostrich chicks during the hot-dry season.

  17. Direct interaction between EgFABP1, a fatty acid binding protein from Echinococcus granulosus, and phospholipid membranes.

    Directory of Open Access Journals (Sweden)

    Jorge L Porfido

    Full Text Available Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious.We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs.This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.

  18. The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility.

    Science.gov (United States)

    Rapp, Micah; Schein, Jessica; Hunt, Kevin A; Nalam, Vamsi; Mourad, George S; Schultes, Neil P

    2016-03-01

    The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

  19. Humic acid facilitates the transport of ARS-labeled hydroxyapatite nanoparticles in iron oxyhydroxide-coated sand

    Science.gov (United States)

    Wang, Dengjun; Bradford, Scott A.; Harvey, Ronald W.; Gao, Bin; Cang, Long; Zhou, Dongmei

    2012-01-01

    Hydroxyapatite nanoparticles (nHAP) have been widely used to remediate soil and wastewater contaminated with metals and radionuclides. However, our understanding of nHAP transport and fate is limited in natural environments that exhibit significant variability in solid and solution chemistry. The transport and retention kinetics of Alizarin red S (ARS)-labeled nHAP were investigated in water-saturated packed columns that encompassed a range of humic acid concentrations (HA, 0–10 mg L–1), fractional surface coverage of iron oxyhydroxide coatings on sand grains (λ, 0–0.75), and pH (6.0–10.5). HA was found to have a marked effect on the electrokinetic properties of ARS-nHAP, and on the transport and retention of ARS-nHAP in granular media. The transport of ARS-nHAP was found to increase with increasing HA concentration because of enhanced colloidal stability and the reduced aggregate size. When HA = 10 mg L–1, greater ARS-nHAP attachment occurred with increasing λ because of increased electrostatic attraction between negatively charged nanoparticles and positively charged iron oxyhydroxides, although alkaline conditions (pH 8.0 and 10.5) reversed the surface charge of the iron oxyhydroxides and therefore decreased deposition. The retention profiles of ARS-nHAP exhibited a hyperexponential shape for all test conditions, suggesting some unfavorable attachment conditions. Retarded breakthrough curves occurred in sands with iron oxyhydroxide coatings because of time-dependent occupation of favorable deposition sites. Consideration of the above effects is necessary to improve remediation efficiency of nHAP for metals and actinides in soils and subsurface environments.

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

    Directory of Open Access Journals (Sweden)

    Kyohei Higashi

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

  1. 26 CFR 49.4262(a)-1 - Taxable transportation.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 16 2010-04-01 2010-04-01 true Taxable transportation. 49.4262(a)-1 Section 49...) MISCELLANEOUS EXCISE TAXES FACILITIES AND SERVICES EXCISE TAXES Transportation of Persons § 49.4262(a)-1 Taxable transportation. (a) In general. Unless excluded under section 4262(b) (see § 49.4262(b)-1), taxable...

  2. INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

    Science.gov (United States)

    Grzegorzewicz, Anna E.; Pham, Ha; Gundi, Vijay A. K. B.; Scherman, Michael S.; North, Elton J.; Hess, Tamara; Jones, Victoria; Gruppo, Veronica; Born, Sarah E. M.; Korduláková, Jana; Chavadi, Sivagami Sundaram; Morisseau, Christophe; Lenaerts, Anne J.; Lee, Richard E.; McNeil, Michael R.; Jackson, Mary

    2011-01-01

    New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. PMID:22344175

  3. LAPTM4b recruits the LAT1-4F2hc Leu transporter to lysosomes and promotes mTORC1 activation.

    Science.gov (United States)

    Milkereit, Ruth; Persaud, Avinash; Vanoaica, Liviu; Guetg, Adriano; Verrey, Francois; Rotin, Daniela

    2015-05-22

    Mammalian target of rapamycin 1 (mTORC1), a master regulator of cellular growth, is activated downstream of growth factors, energy signalling and intracellular essential amino acids (EAAs) such as Leu. mTORC1 activation occurs at the lysosomal membrane, and involves V-ATPase stimulation by intra-lysosomal EAA (inside-out activation), leading to activation of the Ragulator, RagA/B-GTP and mTORC1 via Rheb-GTP. How Leu enters the lysosomes is unknown. Here we identified the lysosomal protein LAPTM4b as a binding partner for the Leu transporter, LAT1-4F2hc (SLC7A5-SLAC3A2). We show that LAPTM4b recruits LAT1-4F2hc to lysosomes, leading to uptake of Leu into lysosomes, and is required for mTORC1 activation via V-ATPase following EAA or Leu stimulation. These results demonstrate a functional Leu transporter at the lysosome, and help explain the inside-out lysosomal activation of mTORC1 by Leu/EAA.

  4. Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

    Science.gov (United States)

    Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

    2015-02-01

    Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

  5. Recent insights into the biological functions of liver fatty acid binding protein 1

    Science.gov (United States)

    Wang, GuQi; Bonkovsky, Herbert L.; de Lemos, Andrew; Burczynski, Frank J.

    2015-01-01

    Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency). PMID:26443794

  6. In vitro bioacessibility and transport across Caco-2 monolayers of haloacetic acids in drinking water.

    Science.gov (United States)

    Melo, A; Faria, M A; Pinto, E; Mansilha, C; Ferreira, I M P L V O

    2016-10-01

    Water disinfection plays a crucial role in water safety but it is also a matter of concern as the use of disinfectants promotes the formation of disinfection by-products (DBPs). Haloacetic acids (HAAs) are one of the major classes of DBPs since they are frequently found in treated water, are ubiquitous, pervasive and have high water solubility, so a great concern emerged about their formation, occurrence and toxicity. Exposure to HAAs is influenced by consumption patterns and diet of individuals thus their bioavailability is an important parameter to the overall toxicity. In the current study the bioacessibility of the most representative HAAs (chloroacetic acid - MCAA, bromoacetic acid - MBAA, dichloroacetic acid - DCAA, dibromoacetic acid - DBAA, and trichloroacetic acid - TCAA) after simulated in vitro digestion (SIVD) in tap water and transport across Caco-2 monolayers was evaluated. Compounds were monitored in 8 points throughout the digestion phases by an optimized LC-MS/MS methodology. MCAA and MBAA were not bioaccessible after SIVD whereas DCAA, DBAA and TCAA are highly bioaccessible (85 ± 4%, 97 ± 4% and 106 ± 7% respectively). Concerning transport assays, DCAA and DBAA were highly permeable throughout the Caco-2 monolayer (apparent permeability and calculated fraction absorbed of 13.62 × 10(-6) cm/s and 90% for DCAA; and 8.82 × 10(-6) cm/s and 84% for DBAA), whereas TCAA showed no relevant permeability. The present results may contribute to efficient risk analysis studies concerning HAAs oral exposure from tap water taking into account the different biological behaviour of these chemically similar substances. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins.

    Science.gov (United States)

    Tanaka, Shiho; Miyamoto, Kazuhisa; Noda, Hiroaki; Endo, Haruka; Kikuta, Shingo; Sato, Ryoichi

    2016-04-01

    In a previous report, seven Cry1Ab-resistant strains were identified in the silkworm, Bombyx mori; these strains were shown to have a tyrosine insertion at position 234 in extracellular loop 2 of the ABC transporter C2 (BmABCC2). This insertion was confirmed to destroy the receptor function of BmABCC2 and confer the strains resistance against Cry1Ab and Cry1Ac. However, these strains were susceptible to Cry1Aa. In this report, we examined the mechanisms of the loss of receptor function of the transporter by expressing mutations in Sf9 cells. After replacement of one or two of the five amino acid residues in loop 2 of the susceptible BmABCC2 gene [BmABCC2_S] with alanine, cells still showed susceptibility, retaining the receptor function. Five mutants with single amino acid insertions at position 234 in BmABCC2 were also generated, resulting in loop 2 having six amino acids, which corresponds to replacing the tyrosine insertion in the resistant BmABCC2 gene [BmABCC2_R(+(234)Y)] with another amino acid. All five mutants exhibited loss of function against Cry1Ab and Cry1Ac. These results suggest that the amino acid sequence in loop 2 is less important than the loop size (five vs. six amino acids) or loop structure for Cry1Ab and Cry1Ac activity. Several domain-swapped mutant toxins were then generated among Cry1Aa, Cry1Ab, and Cry1Ac, which are composed of three domains. Swapped mutants containing domain II of Cry1Ab or Cry1Ac did not kill Sf9 cells expressing BmABCC2_R(+(234)Y), suggesting that domain II of the Cry toxin is related to the interaction with the receptor function of BmABCC2. This also suggests that different reactions against Bt-toxins in some B. mori strains, that is, Cry1Ab resistance or Cry1Aa susceptibility, are attributable to structural differences in domain II of Cry1A toxins. Copyright © 2016. Published by Elsevier Inc.

  8. Transforming growth factor β signaling upregulates the expression of human GDP-fucose transporter by activating transcription factor Sp1.

    Science.gov (United States)

    Xu, Yu-Xin; Ma, Anna; Liu, Li

    2013-01-01

    GDP-fucose transporter plays a crucial role in fucosylation of glycoproteins by providing activated fucose donor, GDP-fucose, for fucosyltransferases in the lumen of the Golgi apparatus. Fucose-containing glycans are involved in many biological processes, which are essential for growth and development. Mutations in the GDP-fucose transporter gene cause leukocyte adhesion deficiency syndrome II, a disease characterized by slow growth, mental retardation and immunodeficiency. However, no information is available regarding its transcriptional regulation. Here, by using human cells, we show that TGF-β1 specifically induces the GDP-fucose transporter expression, but not other transporters tested such as CMP-sialic acid transporter, suggesting a diversity of regulatory pathways for the expression of these transporters. The regulatory elements that are responsive to the TGF-β1 stimulation are present in the region between bp -330 and -268 in the GDP-fucose transporter promoter. We found that this region contains two identical octamer GC-rich motifs (GGGGCGTG) that were demonstrated to be essential for the transporter expression. We also show that the transcription factor Sp1 specifically binds to the GC-rich motifs in vitro and Sp1 coupled with phospho-Smad2 is associated with the promoter region covering the Sp1-binding motifs in vivo using chromatin immunoprecipitation (ChIP) assays. In addition, we further confirmed that Sp1 is essential for the GDP-fucose transporter expression stimulated by TGF-β1 using a luciferase reporter system. These results highlight the role of TGF-β signaling in regulation of the GDP-fucose transporter expression via activating Sp1. This is the first transcriptional study for any nucleotide sugar transporters that have been identified so far. Notably, TGF-β1 receptor itself is known to be modified by fucosylation. Given the essential role of GDP-fucose transporter in fucosylation, the finding that TGF-β1 stimulates the expression of

  9. 41 CFR 109-40.103-1 - Domestic transportation.

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false Domestic transportation... Regulations System (Continued) DEPARTMENT OF ENERGY PROPERTY MANAGEMENT REGULATIONS AVIATION, TRANSPORTATION, AND MOTOR VEHICLES 40-TRANSPORTATION AND TRAFFIC MANAGEMENT 40.1-General Provision § 109-40.103-1...

  10. Petrophysical and transport parameters evolution during acid percolation through structurally different limestones

    Science.gov (United States)

    Martinez Perez, Laura; Luquot, Linda

    2017-04-01

    Processes affecting geological media often show complex and unpredictable behavior due to the presence of heterogeneities. This remains problematic when facing contaminant transport problems, in the CO2 storage industry or dealing with the mechanisms underneath natural processes where chemical reactions can be observed during the percolation of rock non-equilibrated fluid (e.g. karst formation, seawater intrusion). To understand the mechanisms taking place in a porous medium as a result of this water-rock interaction, we need to know the flow parameters that control them, and how they evolve with time as a result of that concurrence. This is fundamental to ensure realistic predictions of the behavior of natural systems in response of reactive transport processes. We investigate the coupled influence of structural and hydrodynamic heterogeneities in limestone rock samples tracking its variations during chemical reactions. To do so we use laboratory petrophysical techniques such as helium porosimetry, gas permeability, centrifugue, electrical resistivity and sonic waves measurements to obtain the parameters that characterize flow within rock matrix (porosity, permeability, retention curve and pore size distribution, electrical conductivity, formation factor, cementation index and tortuosity) before and after percolation experiments. We built an experimental setup that allows injection of acid brine into core samples under well controlled conditions, monitor changes in hydrodynamic properties and obtain the chemical composition of the injected solution at different stages. 3D rock images were also acquired before and after the experiments using a micro-CT to locate the alteration processes and perform an acurate analysis of the structural changes. Two limestones with distinct textural classification and thus contrasting transport properties have been used in the laboratory experiments: a crinoid limestone and an oolithic limestone. Core samples dimensions were 1 inch

  11. 26 CFR 31.3121(j)-1 - Covered transportation service.

    Science.gov (United States)

    2010-04-01

    ... 26 Internal Revenue 15 2010-04-01 2010-04-01 false Covered transportation service. 31.3121(j)-1... § 31.3121(j)-1 Covered transportation service. (a) Transportation systems acquired in whole or in part... operation of a public transportation system constitutes covered transportation service if any part of the...

  12. Low pH, aluminum, and phosphorus coordinately regulate malate exudation through GmALMT1 to improve soybean adaptation to acid soils.

    Science.gov (United States)

    Liang, Cuiyue; Piñeros, Miguel A; Tian, Jiang; Yao, Zhufang; Sun, Lili; Liu, Jiping; Shaff, Jon; Coluccio, Alison; Kochian, Leon V; Liao, Hong

    2013-03-01

    Low pH, aluminum (Al) toxicity, and low phosphorus (P) often coexist and are heterogeneously distributed in acid soils. To date, the underlying mechanisms of crop adaptation to these multiple factors on acid soils remain poorly understood. In this study, we found that P addition to acid soils could stimulate Al tolerance, especially for the P-efficient genotype HN89. Subsequent hydroponic studies demonstrated that solution pH, Al, and P levels coordinately altered soybean (Glycine max) root growth and malate exudation. Interestingly, HN89 released more malate under conditions mimicking acid soils (low pH, +P, and +Al), suggesting that root malate exudation might be critical for soybean adaptation to both Al toxicity and P deficiency on acid soils. GmALMT1, a soybean malate transporter gene, was cloned from the Al-treated root tips of HN89. Like root malate exudation, GmALMT1 expression was also pH dependent, being suppressed by low pH but enhanced by Al plus P addition in roots of HN89. Quantitative real-time PCR, transient expression of a GmALMT1-yellow fluorescent protein chimera in Arabidopsis protoplasts, and electrophysiological analysis of Xenopus laevis oocytes expressing GmALMT1 demonstrated that GmALMT1 encodes a root cell plasma membrane transporter that mediates malate efflux in an extracellular pH-dependent and Al-independent manner. Overexpression of GmALMT1 in transgenic Arabidopsis, as well as overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, indicated that GmALMT1-mediated root malate efflux does underlie soybean Al tolerance. Taken together, our results suggest that malate exudation is an important component of soybean adaptation to acid soils and is coordinately regulated by three factors, pH, Al, and P, through the regulation of GmALMT1 expression and GmALMT1 function.

  13. The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

    Science.gov (United States)

    Chen, R.; Hilson, P.; Sedbrook, J.; Rosen, E.; Caspar, T.; Masson, P. H.

    1998-01-01

    Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

  14. Fishy Business: Effect of Omega-3 Fatty Acids on Zinc Transporters and Free Zinc Availability in Human Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Damitha De Mel

    2014-08-01

    Full Text Available Omega-3 (ω-3 fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA. The main omega-3 fatty acids in the mammalian body are α-linolenic acid (ALA, docosahexaenoic acid (DHA and eicosapentaenoic acid (EPA. Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain, DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total. DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on. On the other hand, zinc is the most abundant trace metal in the human brain. There are many scientific studies linking zinc, especially excess amounts of free zinc, to cellular death. Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between omega-3 fatty acids, zinc transporter levels and free zinc availability at cellular levels. Many other studies have also suggested a possible omega-3 and zinc effect on neurodegeneration and cellular death. Therefore, in this review, we will examine the effect of omega-3 fatty acids on zinc transporters and the importance of free zinc for human neuronal cells. Moreover, we will evaluate the collective understanding of mechanism(s for the interaction of these elements in neuronal research and their significance for the diagnosis and treatment of neurodegeneration.

  15. Fishy business: effect of omega-3 fatty acids on zinc transporters and free zinc availability in human neuronal cells.

    Science.gov (United States)

    De Mel, Damitha; Suphioglu, Cenk

    2014-08-15

    Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain, DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total. DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on. On the other hand, zinc is the most abundant trace metal in the human brain. There are many scientific studies linking zinc, especially excess amounts of free zinc, to cellular death. Neurodegenerative diseases, such as Alzheimer's disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between omega-3 fatty acids, zinc transporter levels and free zinc availability at cellular levels. Many other studies have also suggested a possible omega-3 and zinc effect on neurodegeneration and cellular death. Therefore, in this review, we will examine the effect of omega-3 fatty acids on zinc transporters and the importance of free zinc for human neuronal cells. Moreover, we will evaluate the collective understanding of mechanism(s) for the interaction of these elements in neuronal research and their significance for the diagnosis and treatment of neurodegeneration.

  16. Aberrant Bone Density in Aging Mice Lacking the Adenosine Transporter ENT1

    Science.gov (United States)

    Hinton, David J.; McGee-Lawrence, Meghan E.; Lee, Moonnoh R.; Kwong, Hoi K.; Westendorf, Jennifer J.; Choi, Doo-Sup

    2014-01-01

    Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1) is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months) compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density. PMID:24586402

  17. Aberrant bone density in aging mice lacking the adenosine transporter ENT1.

    Directory of Open Access Journals (Sweden)

    David J Hinton

    Full Text Available Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1 is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP, an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density.

  18. Validation of transport measurements in skeletal muscle with N-13 amino acids using a rabbit isolated hindlimb model

    International Nuclear Information System (INIS)

    Conlon, K.C.; Bading, J.R.; DiResta, G.R.; Corbally, M.T.; Gelbard, A.S.; Brennan, M.F.

    1989-01-01

    The authors are studying the transport of C-11 and N-13 labeled amino acids in tumor-bearing rabbits to determine the role of amino acid transport in the pathogenesis of muscle wasting in cancer. To validate a new, in vivo, method for measuring transport in skeletal muscle with these compounds, an isolated hindlimb model was developed in rabbits. The limb was perfused with a non-recirculating, normothermic, constant pressure system and a cell-free perfusate. Hemodynamic and metabolic parameters were measured during the first 75 min. of perfusion and found to remain normal and stable. Flow varied directly with perfusion pressure over the normal range of resting flows in the intact rabbit hindlimb. Time-activity curves (TAC's) were recorded from the medial thigh following bolus co-injection of L-[amide N-13] glutamine or N-13 L-glutamate with Tc-99m human serum albumin (HSA) into the femoral artery. Regional plasma flow was determined from the Tc-99m data

  19. β-Klotho as a Negative Regulator of the Peptide Transporters PEPT1 and PEPT2

    Directory of Open Access Journals (Sweden)

    Abeer Abousaab

    2016-12-01

    Full Text Available Background/Aims: β-Klotho, a transmembrane protein expressed in several tissues including the brain and the kidney, is critically important for inhibition of 1,25(OH2D3 formation by FGF23. The extracellular domain of Klotho protein could be cleaved off, thus being released into blood or cerebrospinal fluid. Soluble klotho is a β-glucuronidase participating in the regulation of several ion channels and carriers. The present study explored the effect of β-Klotho protein on the peptide transporters PEPT1 and PEPT2. Methods: cRNA encoding PEPT1 or PEPT2 was injected into Xenopus laevis oocytes and glycine-glycine (2 mM-induced inward current (IGly taken as measure of glycine-glycine transport. Measurements were made without or with prior 24 h treatment with soluble β-Klotho protein (30 ng/ml in the absence and presence of β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL,10 µM. Ussing chamber experiments were employed to determine electrogenic peptide transport across intestinal epithelia of klotho deficient (kl-/- and corresponding wild type (kl+/+ mice. Results: IGly was observed in PEPT1 and in PEPT2 expressing oocytes but not in water injected oocytes. In both, PEPT1 and PEPT2 expressing oocytes IGly was significantly decreased by treatment with soluble β-Klotho protein. As shown for PEPT1, β-klotho protein decreased significantly the maximal transport rate without significantly modifying the affinity of the carrier. The effect of β-Klotho on PEPT1 was reversed by DSAL. Intestinal IGly was significantly larger in kl-/- than in kl+/+ mice. Conclusion: β-Klotho participates in the regulation of the peptide transporters PEPT1 and PEPT2.

  20. Organic anion transporting polypeptide 1B transporters modulate hydroxyurea pharmacokinetics

    OpenAIRE

    Walker, Aisha L.; Lancaster, Cynthia S.; Finkelstein, David; Ware, Russell E.; Sparreboom, Alex

    2013-01-01

    Hydroxyurea is currently the only FDA-approved drug that ameliorates the pathophysiology of sickle cell anemia. Unfortunately, substantial interpatient variability in the pharmacokinetics (PK) of hydroxyurea may result in variation of the drug's efficacy. However, little is known about mechanisms that modulate hydroxyurea PK. Recent in vitro studies identifying hydroxyurea as a substrate for organic anion transporting polypeptide (OATP1B) transporters prompted the current investigation assess...

  1. Mechanisms of acid-base regulation in peritoneal dialysis.

    Science.gov (United States)

    Sow, Amadou; Morelle, Johann; Hautem, Nicolas; Bettoni, Carla; Wagner, Carsten A; Devuyst, Olivier

    2017-11-22

    Peritoneal dialysis (PD) contributes to restore acid-base homeostasis in patients with end-stage renal disease. The transport pathways for buffers and carbon dioxide (CO2) across the peritoneal membrane remain poorly understood. Combining well-established PD protocols, whole-body plethysmography and renal function studies in mice, we investigated molecular mechanisms of acid-base regulation in PD, including the potential role of the water channel aquaporin-1 (AQP1). After instillation in peritoneal cavity, the pH of acidic dialysis solutions increased within minutes to rapidly equilibrate with blood pH, whereas the neutral pH of biocompatible solutions remained constant. Predictions from the three-pore model of peritoneal transport suggested that local production of HCO3- accounts at least in part for the changes in intraperitoneal pH observed with acidic solutions. Carbonic anhydrase (CA) isoforms were evidenced in the peritoneal membrane and their inhibition with acetazolamide significantly decreased local production of HCO3- and delayed changes in intraperitoneal pH. On the contrary, genetic deletion of AQP1 had no effect on peritoneal transport of buffers and diffusion of CO2. Besides intraperitoneal modifications, the use of acidic dialysis solutions enhanced acid excretion both at pulmonary and renal levels. These findings suggest that changes in intraperitoneal pH during PD are mediated by bidirectional buffer transport and by CA-mediated production of HCO3- in the membrane. The use of acidic solutions enhances acid excretion through respiratory and renal responses, which should be considered in patients with renal failure. © The Author 2017. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  2. Natural and azido fatty acids inhibit phosphate transport and activate fatty acid anion uniport mediated by the mitochondrial phosphate carrier

    Czech Academy of Sciences Publication Activity Database

    Engstová, Hana; Žáčková, Markéta; Růžička, Michal; Meinhardt, A.; Hanuš, Jan; Krämer, R.; Ježek, Petr

    2001-01-01

    Roč. 276, č. 7 (2001), s. 4683-4691 ISSN 0021-9258 R&D Projects: GA ČR GA301/95/0620; GA ČR GA301/98/0568; GA MŠk ME 085; GA MŠk ME 389 Grant - others:US(US) Czechoslovak Science and Technology Program 94043 Institutional research plan: CEZ:AV0Z5011922 Keywords : phosphate transport * fatty acids Subject RIV: CE - Biochemistry Impact factor: 7.258, year: 2001

  3. Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport.

    Science.gov (United States)

    Lin, Shan-Hua; Kuo, Hui-Fen; Canivenc, Geneviève; Lin, Choun-Sea; Lepetit, Marc; Hsu, Po-Kai; Tillard, Pascal; Lin, Huey-Ling; Wang, Ya-Yun; Tsai, Chyn-Bey; Gojon, Alain; Tsay, Yi-Fang

    2008-09-01

    Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-beta-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.

  4. Synthesis and evaluation of radioiodinated 6-Iodo-L-DOPA as a cerebral L-amino acid transport marker

    International Nuclear Information System (INIS)

    Kawai, K.; Ohta, H.; Kubodera, A.; Channing, M.A.; Eckelman, W.C.

    1996-01-01

    Regioselective radioiodination of N-trifluoroacetyl 3,4-dimethoxy-6-trifluoroacetoxymercurio-L-phenylalanine ethyl ester 1 under no-carrier-added condition gave 6-[ 125 I]iodo protected L-DOPA with a labeling efficiency of more than 85%, and no-carrier-added 6-[ 125 I]I-L-DOPA was obtained with a radiochemical purity of over 95% after hydrolysis and chromatography. A nonradioactive standard of 6-iodo protected L-DOPA was synthesized by the iododemercuration of 6-mercuric trifluoroacetate protected L-DOPA 1 using I 2 in chloroform. 6-[ 125 I]I-L-DOPA showed high brain accumulation and rapid blood clearance in mice. The rat brain slice studies indicated high affinity of 6-[ 125 I]I-L-DOPA for carrier-mediated and stereoselective active transport systems. The tissue homogenate analysis revealed that most of the accumulated radioactivity was intact 6-[ 125 I]I-L-DOPA. Thus, 6-[ 123 I]I-L-DOPA appears to be a suitable single photon emission computed tomography (SPECT) tracer for the selective measurement of cerebral L-amino acid transport, having no affinity for dopamine metabolism

  5. BoALMT1, an Al-Induced Malate Transporter in Cabbage, Enhances Aluminum Tolerance in Arabidopsis thaliana

    OpenAIRE

    Zhang, Lei; Wu, Xin-Xin; Wang, Jinfang; Qi, Chuandong; Wang, Xiaoyun; Wang, Gongle; Li, Mingyue; Li, Xingsheng; Guo, Yang-Dong

    2018-01-01

    Aluminum (Al) is present in approximately 50% of the arable land worldwide and is regarded as the main limiting factor of crop yield on acidic soil. Al-induced root malate efflux plays an important role in the Al tolerance of plants. Here, the aluminum induced malate transporter BoALMT1 (KF322104) was cloned from cabbage (Brassica oleracea). BoALMT1 showed higher expression in roots than in shoots. The expression of BoALMT1 was specifically induced by Al treatment, but not the trivalent catio...

  6. Caveolar fatty acids and acylation of caveolin-1.

    Directory of Open Access Journals (Sweden)

    Qian Cai

    Full Text Available Caveolae are cholesterol and sphingolipids rich subcellular domains on plasma membrane. Caveolae contain a variety of signaling proteins which provide platforms for signaling transduction. In addition to enriched with cholesterol and sphingolipids, caveolae also contain a variety of fatty acids. It has been well-established that acylation of protein plays a pivotal role in subcellular location including targeting to caveolae. However, the fatty acid compositions of caveolae and the type of acylation of caveolar proteins remain largely unknown. In this study, we investigated the fatty acids in caveolae and caveolin-1 bound fatty acids.Caveolae were isolated from Chinese hamster ovary (CHO cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS. The caveolin-1 bound fatty acids were immunoprecipitated by anti-caveolin-1 IgG and analyzed with GC/MS.In contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids, 0.48 µg palmitic acid, 0.61 µg stearic acid and 0.83 µg oleic acid/caveolae preparation/5 × 10(7 cells. Unexpectedly, GC/MS analysis indicated that caveolin-1 was not acylated by myristic acid; instead, it was acylated by palmitic acid and stearic acid.Caveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1 was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1 may be important for caveolae formation and for maintaining the function of caveolae.

  7. PAT1 (SLC36A1) shows nuclear localization and affect growth of smooth muscle cells from rats

    DEFF Research Database (Denmark)

    Jensen, Anne; Figueiredo-Larsen, Evan Manuel; Holm, René

    2014-01-01

    The proton-coupled amino acid transporter 1 (PAT1) is a transporter of amino acids in small intestinal enterocytes. PAT1 is, however, also capable of regulating cell growth and sensing the availability of amino acids in other cell types. The aim of the present study was to investigate the localiz...

  8. Identification of Important Amino Acids in Gal2p for Improving the L-arabinose Transport and Metabolism in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Chengqiang Wang

    2017-07-01

    Full Text Available Efficient and cost-effective bioethanol production from lignocellulosic materials requires co-fermentation of the main hydrolyzed sugars, including glucose, xylose, and L-arabinose. Saccharomyces cerevisiae is a glucose-fermenting yeast that is traditionally used for ethanol production. Fermentation of L-arabinose is also possible after metabolic engineering. Transport into the cell is the first and rate-limiting step for L-arabinose metabolism. The galactose permease, Gal2p, is a non-specific, endogenous monosaccharide transporter that has been shown to transport L-arabinose. However, Gal2p-mediated transport of L-arabinose occurs at a low efficiency. In this study, homologous modeling and L-arabinose docking were used to predict amino acids in Gal2p that are crucial for L-arabinose transport. Nine amino acid residues in Gal2p were identified and were the focus for site-directed mutagenesis. In the Gal2p transport-deficient chassis cells, the capacity for L-arabinose transport of the different Gal2p mutants was compared by testing growth rates using L-arabinose as the sole carbon source. Almost all the tested mutations affected L-arabinose transport capacity. Among them, F85 is a unique site. The F85S, F85G, F85C, and F85T point mutations significantly increased L-arabinose transport activities, while, the F85E and F85R mutations decreased L-arabinose transport activities compared to the Gal2p-expressing wild-type strain. These results verified F85 as a key residue in L-arabinose transport. The F85S mutation, having the most significant effect, elevated the exponential growth rate by 40%. The F85S mutation also improved xylose transport efficiency and weakened the glucose transport preference. Overall, enhancing the L-arabinose transport capacity further improved the L-arabinose metabolism of engineered S. cerevisiae.

  9. Brain uptake of pipecolic acid, amino acids, amines following intracarotid injection in the mouse

    International Nuclear Information System (INIS)

    Nishio, H.; Giacobini, E.

    1981-01-01

    The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI of D,L-[3H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid, L-proline, and piperidine show a significant inhibitory effect. Amont the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations

  10. A 9-vinyladenine-based molecularly imprinted polymeric membrane for the efficient recognition of plant hormone 1H-indole-3-acetic acid

    International Nuclear Information System (INIS)

    Chen Changbao; Chen Yanjun; Zhou Jie; Wu Chunhui

    2006-01-01

    9-Vinyladenine was synthesized as a novel functional monomer for molecular imprinting techniques and its structure was established with elemental analysis and 1 H NMR spectroscopy. The binding mechanism between this functional monomer 9-vinyladenine and the plant hormone 1 H-indole-3-acetic acid in acetonitrile was studied with UV-vis spectrophotometry. Based on this study, using 1 H-indole-3-acetic acid as a template molecule, a specific 9-vinyladenine-based molecularly imprinted polymeric membrane was prepared. Then, the resultant polymeric membrane morphologies were visualized with scanning electron microscopy, and the membrane permselectivity for 1 H-indole-3-acetic acid, 1 H-indole-3-butyric acid and kinetin was tested with separate experiments and competitive diffusion experiments. These results showed that the imprinted polymeric membrane prepared with 9-vinyladenine exhibited higher transport selectivity for the template molecule 1 H-indole-3-acetic acid than 1 H-indole-3-butyric acid or kinetin. The membrane prepared with 9-vinyladenine also took on higher permselectivity for 1 H-indole-3-acetic acid in comparison with the imprinted membrane made with methacrylic acid. It is predicted that the 9-vinyladenine-based molecularly imprinted membrane may be applicable to the assay of 1 H-indole-3-acetic acid or for the preparation of a molecularly imprinted polymer sensor for the analysis of 1 H-indole-3-acetic acid in plant samples

  11. Effects of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: Mechanism of impaired ascorbate transport in diabetes

    International Nuclear Information System (INIS)

    Fay, M.J.; Bush, M.J.; Verlangieri, A.J.

    1990-01-01

    Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3 H-2-deoxy-D-glucose by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14 C-L-ascorbic acid. The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus

  12. Incorporation of Oxygen into Abscisic Acid and Phaseic Acid from Molecular Oxygen 1

    Science.gov (United States)

    Creelman, Robert A.; Zeevaart, Jan A. D.

    1984-01-01

    Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% 18O2 and 80% N2 indicates that one atom of 18O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase. Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing 18O2 indicates that one atom of 18O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress. PMID:16663564

  13. Effects of 1 alpha,25-Dihydroxyvitamin D-3 on Transporters and Enzymes of the Rat Intestine and Kidney In Vivo

    NARCIS (Netherlands)

    Chow, Edwin C. Y.; Sun, Huadong; Khan, Ansar A.; Groothuis, Geny M. M.; Pang, K. Sandy

    1 alpha,25-Dihydroxyvitamin D-3 (1,25(OH)(2)D-3), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR-rich target organ in which VDR regulation

  14. Influence of dehydrated nanotubed titanic acid on charge transport and luminescent properties of polymer light-emitting diodes with fluorescent dye

    Science.gov (United States)

    Qian, Lei; Bera, Debasis; Jin, Zhen-Sheng; Du, Zu-Liang; Xu, Zheng; Teng, Feng; Liu, Wei

    2007-09-01

    In this paper, we discuss the influence of dehydrated nanotubed titanic acid (DNTA) on charge transport and luminescent properties of polymer light-emitting diodes (PLEDs) doped with fluorescent dye. Photoluminescence results confirm the efficient energy transfer from PVK to 4-(dicyanom-ethylene)-2- t-butyl-6-(1,1,7,7-tetramethyljulolidyl-9-enyl)-4H-pyran (DCJTB) and tris-(8-hydroxtquinoline) aluminum (Alq 3) in a DNTA-doped device. The device showed lower turn-on voltages and higher charge current by doping with DNTA, which also caused a shift in the exciton's recombination region.

  15. Transepithelial transport of aliphatic carboxylic acids studied in Madin Darby canine kidney (MDCK) cell monolayers

    International Nuclear Information System (INIS)

    Cho, M.J.; Adson, A.; Kezdy, F.J.

    1990-01-01

    Transport of 14C-labeled acetic, propionic (PA), butyric, valeric, heptanoic (HA), and octanoic (OA) acids across the Madin Darby canine kidney (MDCK) epithelial cell monolayer grown on a porous polycarbonate membrane was studied in Hanks' balanced salt solution (HBSS) at 37 degrees C in both apical-to-basolateral and basolateral-to-apical directions. At micromolar concentrations of solutes, metabolic decomposition was significant as evidenced by [14C]CO2 production during the OA transport. The apparent permeability (Pe) indicates that as lipophilicity increases, diffusion across the unstirred boundary layer becomes rate limiting. In support of this notion, transport of OA and HA was enhanced by agitation, showed an activation energy of 3.7 kcal/mol for OA, and resulted in identical Pe values for both transport directions. Analysis of Pe changes with varying alkyl chain length resulted in a delta G of -0.68 +/- 0.09 kcal/mol for -CH2-group transfer from an aqueous phase to the MDCK cells. When the intercellular tight junctions were opened by the divalent chelator EGTA in Ca2+/Mg2(+)-free HBSS, transport of the fluid-phase marker Lucifer yellow greatly increased because of paracellular leakage. PA transport also showed a significant increase, but OA transport was independent of EGTA. Although albumin also undergoes paracellular transport in the presence of EGTA and OA binds strongly to albumin, OA transport in EGTA solution was unchanged by albumin. These observations indicate that transmembrane transport is the major mechanism for lipophilic substances. The present study, together with earlier work on the transport of polar substances, shows that the MDCK cell monolayer is an excellent model of the transepithelial transport barrier

  16. New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid.

    Science.gov (United States)

    Májer, Ferenc; Salomon, Johanna J; Sharma, Ruchika; Etzbach, Simona V; Najib, Mohd Nadzri Mohd; Keaveny, Ray; Long, Aideen; Wang, Jun; Ehrhardt, Carsten; Gilmer, John F

    2012-03-01

    Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 μM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 μM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.

    Science.gov (United States)

    Ligaba, Ayalew; Dreyer, Ingo; Margaryan, Armine; Schneider, David J; Kochian, Leon; Piñeros, Miguel

    2013-12-01

    Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  18. Mucolipin 1 positively regulates TLR7 responses in dendritic cells by facilitating RNA transportation to lysosomes.

    Science.gov (United States)

    Li, Xiaobing; Saitoh, Shin-Ichiroh; Shibata, Takuma; Tanimura, Natsuko; Fukui, Ryutaro; Miyake, Kensuke

    2015-02-01

    Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs. © The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Treatment of attention deficit hyperactivity disorder with monoamine amino acid precursors and organic cation transporter assay interpretation

    Directory of Open Access Journals (Sweden)

    Marty Hinz

    2011-01-01

    Full Text Available Marty Hinz1, Alvin Stein2, Robert Neff3, Robert Weinberg4, Thomas Uncini51NeuroResearch Clinics Inc, Cape Coral, FL; 2Stein Orthopedic Associates, Plantation, FL; 3Mental Training Inc, Dallas, TX; 4Department of Kinesiology and Health, Miami University, Oxford, OH; 5Laboratory, Fairview Regional Medical Center-Mesabi, Hibbing, MN, USABackground: This paper documents a retrospective pilot study of a novel approach for treating attention deficit hyperactivity disorder (ADHD with amino acid precursors of serotonin and dopamine in conjunction with urinary monoamine assays subjected to organic cation transporter (OCT functional status determination. The goal of this research was to document the findings and related considerations of a retrospective chart review study designed to identify issues and areas of concern that will define parameters for a prospective controlled study.Methods: This study included 85 patients, aged 4–18 years, who were treated with a novel amino acid precursor protocol. Their clinical course during the first 8–10 weeks of treatment was analyzed retrospectively. The study team consisted of PhD clinical psychologists, individuals compiling clinical data from records, and a statistician. The patients had been treated with a predefined protocol for administering amino acid precursors of serotonin and dopamine, along with OCT assay interpretation as indicated.Results: In total, 67% of participants achieved significant improvement with only amino acid precursors of serotonin and dopamine. In patients who achieved no significant relief of symptoms with only amino acid precursors, OCT assay interpretation was utilized. In this subgroup, 30.3% achieved significant relief following two or three urine assays and dosage changes as recommended by the assay results. The total percentage of patients showing significant improvement was 77%.Conclusion: The efficacy of this novel protocol appears superior to some ADHD prescription drugs

  20. The conserved basic residues and the charged amino acid residues at the α-helix of the zinc finger motif regulate the nuclear transport activity of triple C2H2 zinc finger proteins

    Science.gov (United States)

    Lin, Chih-Ying

    2018-01-01

    Zinc finger (ZF) motifs on proteins are frequently recognized as a structure for DNA binding. Accumulated reports indicate that ZF motifs contain nuclear localization signal (NLS) to facilitate the transport of ZF proteins into nucleus. We investigated the critical factors that facilitate the nuclear transport of triple C2H2 ZF proteins. Three conserved basic residues (hot spots) were identified among the ZF sequences of triple C2H2 ZF proteins that reportedly have NLS function. Additional basic residues can be found on the α-helix of the ZFs. Using the ZF domain (ZFD) of Egr-1 as a template, various mutants were constructed and expressed in cells. The nuclear transport activity of various mutants was estimated by analyzing the proportion of protein localized in the nucleus. Mutation at any hot spot of the Egr-1 ZFs reduced the nuclear transport activity. Changes of the basic residues at the α-helical region of the second ZF (ZF2) of the Egr-1 ZFD abolished the NLS activity. However, this activity can be restored by substituting the acidic residues at the homologous positions of ZF1 or ZF3 with basic residues. The restored activity dropped again when the hot spots at ZF1 or the basic residues in the α-helix of ZF3 were mutated. The variations in nuclear transport activity are linked directly to the binding activity of the ZF proteins with importins. This study was extended to other triple C2H2 ZF proteins. SP1 and KLF families, similar to Egr-1, have charged amino acid residues at the second (α2) and the third (α3) positions of the α-helix. Replacing the amino acids at α2 and α3 with acidic residues reduced the NLS activity of the SP1 and KLF6 ZFD. The reduced activity can be restored by substituting the α3 with histidine at any SP1 and KLF6 ZFD. The results show again the interchangeable role of ZFs and charge residues in the α-helix in regulating the NLS activity of triple C2H2 ZF proteins. PMID:29381770

  1. Cation-Coupled Bicarbonate Transporters

    OpenAIRE

    Aalkjaer, Christian; Boedtkjer, Ebbe; Choi, Inyeong; Lee, Soojung

    2014-01-01

    Cation-coupled HCO3− transport was initially identified in the mid-1970s when pioneering studies showed that acid extrusion from cells is stimulated by CO2/HCO3− and associated with Na+ and Cl− movement. The first Na+-coupled bicarbonate transporter (NCBT) was expression-cloned in the late 1990s. There are currently five mammalian NCBTs in the SLC4-family: the electrogenic Na,HCO3-cotransporters NBCe1 and NBCe2 (SLC4A4 and SLC4A5 gene products); the electroneutral Na,HCO3-cotransporter NBCn1 ...

  2. Folic acid and pantothenic acid protection against valproic acid-induced neural tube defects in CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Dawson, Jennifer E [Department of Pharmacology and Toxicology and School of Environmental Studies, Queen' s University, Kingston, Ontario, K7L 3N6 (Canada); Raymond, Angela M [Department of Pharmacology and Toxicology and School of Environmental Studies, Queen' s University, Kingston, Ontario, K7L 3N6 (Canada); Winn, Louise M [Department of Pharmacology and Toxicology and School of Environmental Studies, Queen' s University, Kingston, Ontario, K7L 3N6 (Canada)

    2006-03-01

    In utero exposure to valproic acid (VPA) during pregnancy is associated with an increased risk of neural tube defects (NTDs). Although the mechanism by which VPA mediates these effects is unknown, VPA-initiated changes in embryonic protein levels have been implicated. The objectives of this study were to investigate the effect of in utero VPA exposure on embryonic protein levels of p53, NF-{kappa}B, Pim-1, c-Myb, Bax, and Bcl-2 in the CD-1 mouse. We also evaluated the protective effects of folic acid and pantothenic acid on VPA-induced NTDs and VPA-induced embryonic protein changes in this model. Pregnant CD-1 mice were administered a teratogenic dose of VPA prior to neural tube closure and embryonic protein levels were analyzed. In our study, VPA (400 mg/kg)-induced NTDs (24%) and VPA-exposed embryos with an NTD showed a 2-fold increase in p53, and 4-fold decreases in NF-{kappa}B, Pim-1, and c-Myb protein levels compared to their phenotypically normal littermates (P < 0.05). Additionally, VPA increased the ratio of embryonic Bax/Bcl-2 protein levels (P < 0.05). Pretreatment of pregnant dams with either folic acid or pantothenic acid prior to VPA significantly protected against VPA-induced NTDs (P < 0.05). Folic acid also reduced VPA-induced alterations in p53, NF-{kappa}B, Pim-1, c-Myb, and Bax/Bcl-2 protein levels, while pantothenic acid prevented VPA-induced alterations in NF-{kappa}B, Pim-1, and c-Myb. We hypothesize that folic acid and pantothenic acid protect CD-1 embryos from VPA-induced NTDs by independent, but not mutually exclusive mechanisms, both of which may be mediated by the prevention of VPA-induced alterations in proteins involved in neurulation.

  3. Effects of Sodium and Amino Acid Substrate Availability upon the Expression and Stability of the SNAT2 (SLC38A2 Amino Acid Transporter

    Directory of Open Access Journals (Sweden)

    Thorsten M. Hoffmann

    2018-02-01

    Full Text Available The SNAT2 (SLC38A2 System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AAs and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis, and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+ availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that while AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (N-methylaminoisobutyric acid and glutamine, but not non-substrates. This stabilization and substrate-induced repression were critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteasome system, because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes

  4. Soybean SAT1 (Symbiotic Ammonium Transporter 1) encodes a bHLH transcription factor involved in nodule growth and NH4+ transport.

    Science.gov (United States)

    Chiasson, David M; Loughlin, Patrick C; Mazurkiewicz, Danielle; Mohammadidehcheshmeh, Manijeh; Fedorova, Elena E; Okamoto, Mamoru; McLean, Elizabeth; Glass, Anthony D M; Smith, Sally E; Bisseling, Ton; Tyerman, Stephen D; Day, David A; Kaiser, Brent N

    2014-04-01

    Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.

  5. Altered sensitivity of system A amino acid transport to ouabain in normal and transformed C3H-10T1/2 cells during the cell cycle

    International Nuclear Information System (INIS)

    Leister, K.J.; Schenerman, M.A.; Racker, E.

    1989-01-01

    Quiescent C3H-10T1/2 mouse fibroblasts that have not undergone any type of stress have a relatively low rate of 2-aminoisobutyrate (Aib) uptake by means of system A, which is primarily energized by the transmembrane Na + chemical gradient potential. System A activity in these cells is not sensitive to ouabain or proton ionophores. In contrast, methylcholanthrene-transformed and cofluent C3H-10T1/2 cells treated with ouabain utilize the membrane potential generated by the Na + , K + -ATPase pump to drive Aib transport by means of system A as shown by the sensitivity of transport activity to ouabain and proton ionophores. Since glucose is present during the assay, the proton ionophores do not affect the availability of ATP, as indicated by the undiminished uptake of 86 Rb + by the Na + , K + -ATPase pump. As cells progress through the G 1 phase of the cell cycle, they show an increased system A activity prior to entry into the S phase, which is also dependent on the electrogenicity of the Na + , K + -ATPase pump. There appears to be in all these cases a qualitative shift in the bioenergetic mechanism for the uptake of Aib as well as a marked quantitative increase in Aib uptake. The high activity after ouabain treatment was sustained in the transformed cells after removal of the ouabain, whereas in the confluent 10T1/2 cells the rate of uptake decayed rapidly, suggesting a difference in the mode of regulation. The authors conclude that transformed cells and normal cells in late G 1 or under stress make use of the membrane potential generated by the Na + , K + -ATPase pump to drive amino acid uptake by means of system A

  6. 14 CFR 401.1 - The Office of Commercial Space Transportation.

    Science.gov (United States)

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false The Office of Commercial Space Transportation. 401.1 Section 401.1 Aeronautics and Space COMMERCIAL SPACE TRANSPORTATION, FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION GENERAL ORGANIZATION AND DEFINITIONS § 401.1 The Office of Commercial Space Transportation. The Office of...

  7. Na+-taurocholate cotransporting polypeptide (NTCP/SLC10A1) ortholog in the marine skate Leucoraja erinacea is not a physiological bile salt transporter.

    Science.gov (United States)

    Yu, Dongke; Zhang, Han; Lionarons, Daniel A; Boyer, James L; Cai, Shi-Ying

    2017-04-01

    The Na + -dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1) is a hepatocyte-specific solute carrier, which plays an important role in maintaining bile salt homeostasis in mammals. The absence of a hepatic Na + -dependent bile salt transport system in marine skate and rainbow trout raises a question regarding the function of the Slc10a1 gene in these species. Here, we have characterized the Slc10a1 gene in the marine skate, Leucoraja erinacea The transcript of skate Slc10a1 (skSlc10a1) encodes 319 amino acids and shares 46% identity to human NTCP (hNTCP) with similar topology to mammalian NTCP. SkSlc10a1 mRNA was mostly confined to the brain and testes with minimal expression in the liver. An FXR-bile salt reporter assay indicated that skSlc10a1 transported taurocholic acid (TCA) and scymnol sulfate, but not as effectively as hNTCP. An [ 3 H]TCA uptake assay revealed that skSlc10a1 functioned as a Na + -dependent transporter, but with low affinity for TCA ( K m = 92.4 µM) and scymnol sulfate ( K i = 31 µM), compared with hNTCP (TCA, K m = 5.4 µM; Scymnol sulfate, K i = 3.5 µM). In contrast, the bile salt concentration in skate plasma was 2 µM, similar to levels seen in mammals. Interestingly, skSlc10a1 demonstrated transport activity for the neurosteroids dehydroepiandrosterone sulfate and estrone-3-sulfate at physiological concentration, similar to hNTCP. Together, our findings indicate that skSlc10a1 is not a physiological bile salt transporter, providing a molecular explanation for the absence of a hepatic Na + -dependent bile salt uptake system in skate. We speculate that Slc10a1 is a neurosteroid transporter in skate that gained its substrate specificity for bile salts later in vertebrate evolution. Copyright © 2017 the American Physiological Society.

  8. Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

    Science.gov (United States)

    Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

    2013-10-01

    Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Plasmodium falciparum chloroquine resistance transporter (PfCRT) isoforms PH1 and PH2 perturb vacuolar physiology.

    Science.gov (United States)

    Callaghan, Paul S; Siriwardana, Amila; Hassett, Matthew R; Roepe, Paul D

    2016-03-31

    Recent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT). The approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter. During analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions. These data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.

  10. Uptake of biotin by Chlamydia Spp. through the use of a bacterial transporter (BioY and a host-cell transporter (SMVT.

    Directory of Open Access Journals (Sweden)

    Derek J Fisher

    Full Text Available Chlamydia spp. are obligate intracellular Gram-negative bacterial pathogens that cause disease in humans and animals. Minor variations in metabolic capacity between species have been causally linked to host and tissue tropisms. Analysis of the highly conserved genomes of Chlamydia spp. reveals divergence in the metabolism of the essential vitamin biotin with genes for either synthesis (bioF_2ADB and/or transport (bioY. Streptavidin blotting confirmed the presence of a single biotinylated protein in Chlamydia. As a first step in unraveling the need for divergent biotin acquisition strategies, we examined BioY (CTL0613 from C. trachomatis 434/Bu which is annotated as an S component of the type II energy coupling-factor transporters (ECF. Type II ECFs are typically composed of a transport specific component (S and a chromosomally unlinked energy module (AT. Intriguingly, Chlamydia lack recognizable AT modules. Using (3H-biotin and recombinant E. coli expressing CTL0613, we demonstrated that biotin was transported with high affinity (a property of Type II ECFs previously shown to require an AT module and capacity (apparent K(m of 3.35 nM and V(max of 55.1 pmol×min(-1×mg(-1. Since Chlamydia reside in a host derived membrane vacuole, termed an inclusion, we also sought a mechanism for transport of biotin from the cell cytoplasm into the inclusion vacuole. Immunofluorescence microscopy revealed that the mammalian sodium multivitamin transporter (SMVT, which transports lipoic acid, biotin, and pantothenic acid into cells, localizes to the inclusion. Since Chlamydia also are auxotrophic for lipoic and pantothenic acids, SMVT may be subverted by Chlamydia to move multiple essential compounds into the inclusion where BioY and another transporter(s would be present to facilitate transport into the bacterium. Collectively, our data validates the first BioY from a pathogenic organism and describes a two-step mechanism by which Chlamydia transport biotin

  11. Uptake of Biotin by Chlamydia Spp. through the Use of a Bacterial Transporter (BioY) and a Host-Cell Transporter (SMVT)

    Science.gov (United States)

    Fisher, Derek J.; Fernández, Reinaldo E.; Adams, Nancy E.; Maurelli, Anthony T.

    2012-01-01

    Chlamydia spp. are obligate intracellular Gram-negative bacterial pathogens that cause disease in humans and animals. Minor variations in metabolic capacity between species have been causally linked to host and tissue tropisms. Analysis of the highly conserved genomes of Chlamydia spp. reveals divergence in the metabolism of the essential vitamin biotin with genes for either synthesis (bioF_2ADB) and/or transport (bioY). Streptavidin blotting confirmed the presence of a single biotinylated protein in Chlamydia. As a first step in unraveling the need for divergent biotin acquisition strategies, we examined BioY (CTL0613) from C. trachomatis 434/Bu which is annotated as an S component of the type II energy coupling-factor transporters (ECF). Type II ECFs are typically composed of a transport specific component (S) and a chromosomally unlinked energy module (AT). Intriguingly, Chlamydia lack recognizable AT modules. Using 3H-biotin and recombinant E. coli expressing CTL0613, we demonstrated that biotin was transported with high affinity (a property of Type II ECFs previously shown to require an AT module) and capacity (apparent K(m) of 3.35 nM and V(max) of 55.1 pmol×min−1×mg−1). Since Chlamydia reside in a host derived membrane vacuole, termed an inclusion, we also sought a mechanism for transport of biotin from the cell cytoplasm into the inclusion vacuole. Immunofluorescence microscopy revealed that the mammalian sodium multivitamin transporter (SMVT), which transports lipoic acid, biotin, and pantothenic acid into cells, localizes to the inclusion. Since Chlamydia also are auxotrophic for lipoic and pantothenic acids, SMVT may be subverted by Chlamydia to move multiple essential compounds into the inclusion where BioY and another transporter(s) would be present to facilitate transport into the bacterium. Collectively, our data validates the first BioY from a pathogenic organism and describes a two-step mechanism by which Chlamydia transport biotin from the

  12. Regional alternative transportation evaluation report - region 1

    Science.gov (United States)

    2011-01-21

    The U.S. Fish and Wildlife Service (FWS) and the U.S. Department of Transportation (DOT) Volpe Center (Volpe Center) conducted a regional alternative transportation evaluation (RATE) in Region 1, which is comprised of Oregon, Idaho, Washington, and H...

  13. Primary and secondary kinetic isotope effects in the acid-catalyzed dehydration of 1,1'-diadamantylmethylcarbinol in aqueous acetic acid

    International Nuclear Information System (INIS)

    Lomas, J.S.

    1981-01-01

    The sulfuric acid catalyzed dehydration of 1,1'-diadamantyl-methylcarbinol in anhydrous acetic acid proceeds exclusively to 1,1'-bis(1-adamantyl)ethylene. The secondary deuterium isotope effect of 1.32 found for this reaction shows that carbonium ion formation from the protonated alcohol is rate determining. In the presence of water, however, capture of the carbonium ion competes with deprotonation, introducing a primary isotope effect. Consequently, the overall KIE rises, reaching 3.18 for 80% aqueous acetic acid. Analysis of the KIE for 80 to 100% aqueous acetic acid is consistent with a simple classical mechanism involving reversible formation of the intermediate carbonium ion. The primary isotope effect upon deprotonation is at the most 2.98, indicative of an asymmetric transition state close to the carbonium ion

  14. Effects of Equine-Assisted Activities and Therapies on the Affective Network of Adolescents with Internet Gaming Disorder.

    Science.gov (United States)

    Kang, Kyoung Doo; Jung, Tae Woon; Park, In Hui; Han, Doug Hyun

    2018-04-26

    Internet gaming disorder (IGD) has been suggested to be a mental health disorder. Attachment and emotional status in IGD patients are important for understanding the etiology and progression of IGD because both parameters are considered to be associated with the affective network. Equine-assisted activities and therapies (EAAT) have been reported to improve emotional status and attachment in subjects. We hypothesized that EAAT would improve attachment in IGD adolescents with insecure attachment issues and increase functional connectivity (FC) within the affective network. Subjects completed a demographic questionnaire, the Korean Experiences in Close Relationships Scale Revised version (K-ECRS), the Child Depression Inventory, Young's Internet Addiction Scale, the Korean Attention-Deficit Hyperactivity Disorder Rating Scale, and resting-state functional magnetic resonance imaging at baseline at the end of EAAT. Fifteen IGD adolescents with insecure attachment issues and 15 healthy comparison adolescents with secure attachment agreed to participate in this study. After 7 days of EAAT, K-ECRS avoidance and anxiety scores improved in all adolescents. K-ECRS avoidance scores of the IGD group showed marked improvement compared with those of the healthy group. In all participants, FC from the left amygdala to the left parahippocampal gyrus, left medial frontal gyrus, and left inferior frontal gyrus, as well as from the right amygdala to the left caudate, right claustrum, and left inferior frontal gyrus increased. In IGD adolescents, FC from the left amygdala to the left frontal orbital gyrus, as well as from the right amygdala to the right corpus callosum also increased. These findings suggested that EAAT improves attachment, which could lead to a decrease in the severity of IGD symptoms in IGD patients with insecure attachment issues. In addition, EAAT increases FC within the affective network, which was associated with attachment not only in healthy adolescents but

  15. Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy

    International Nuclear Information System (INIS)

    Bakhiya, Nadiya; Arlt, Volker M.; Bahn, Andrew; Burckhardt, Gerhard; Phillips, David H.; Glatt, Hansruedi

    2009-01-01

    Aristolochic acid (AA), present in Aristolochia species, is the major causative agent in the development of severe renal failure and urothelial cancers in patients with AA nephropathy. It may also be a cause of Balkan endemic nephropathy. Epithelial cells of the proximal tubule are the primary cellular target of AA. To study whether organic anion transporters (OATs) expressed in proximal tubule cells are involved in uptake of AA, we used human epithelial kidney (HEK293) cells stably expressing human (h) OAT1, OAT3 or OAT4. AA potently inhibited the uptake of characteristic substrates, p-aminohippurate for hOAT1 and estrone sulfate for hOAT3 and hOAT4. Aristolochic acid I (AAI), the more cytotoxic and genotoxic AA congener, exhibited high affinity for hOAT1 (K i = 0.6 μM) as well as hOAT3 (K i = 0.5 μM), and lower affinity for hOAT4 (K i = 20.6 μM). Subsequently, AAI-DNA adduct formation (investigated by 32 P-postlabelling) was used as a measure of AAI uptake. Significantly higher levels of adducts occurred in hOAT-expressing cells than in control cells: this effect was abolished in the presence of the OAT inhibitor probenecid. In Xenopus laevis oocytes hOAT-mediated efflux of p-aminohippurate was trans-stimulated by extracellular AA, providing further molecular evidence for AA translocation by hOATs. Our study indicates that OATs can mediate the uptake of AA into proximal tubule cells and thereby participate in kidney cell damage by this toxin.

  16. Effect of plasma membrane fluidity on serotonin transport by endothelial cells

    International Nuclear Information System (INIS)

    Block, E.R.; Edwards, D.

    1987-01-01

    To evaluate the effect of plasma membrane fluidity of lung endothelial cells on serotonin transport, porcine pulmonary artery endothelial cells were incubated for 3 h with either 0.1 mM cholesterol hemisuccinate, 0.1 mM cis-vaccenic acid, or vehicle (control), after which plasma membrane fluidity and serotinin transport were measured. Fluorescence spectroscopy was used to measure fluidity in the plasma membrane. Serotonin uptake was calculated from the disappearance of [ 14 C]-serotonin from the culture medium. Cholesterol decreased fluidity in the subpolar head group and central and midacyl side-chain regions of the plasma membrane and decreased serotonin transport, whereas cis-vaccenic acid increased fluidity in the central and midacyl side-chain regions of the plasma membrane and also increased serotonin transport. Cis-vaccenic acid had no effect of fluidity in the subpolar head group region of the plasma membrane. These results provide evidence that the physical state of the central and midacyl chains within the pulmonary artery endothelial cell plasma membrane lipid bilayer modulates transmembrane transport of serotonin by these cells

  17. l-Arginine Uptake by Cationic Amino Acid Transporter Promotes Intra-Macrophage Survival of Leishmania donovani by Enhancing Arginase-Mediated Polyamine Synthesis

    Directory of Open Access Journals (Sweden)

    Abhishek Mandal

    2017-07-01

    Full Text Available The survival of intracellular protozoan parasite, Leishmania donovani, the causative agent of Indian visceral leishmaniasis (VL, depends on the activation status of macrophages. l-Arginine, a semi-essential amino acid plays a crucial regulatory role for activation of macrophages. However, the role of l-arginine transport in VL still remains elusive. In this study, we demonstrated that intra-macrophage survival of L. donovani depends on the availability of extracellular l-arginine. Infection of THP-1-derived macrophage/human monocyte-derived macrophage (hMDM with Leishmania, resulted in upregulation of l-arginine transport. While investigating the involvement of the transporters, we observed that Leishmania survival was greatly impaired when the transporters were blocked either using inhibitor or siRNA-mediated downregulation. CAT-2 was found to be the main isoform associated with l-arginine transport in L. donovani-infected macrophages. l-arginine availability and its transport regulated the host arginase in Leishmania infection. Arginase and inducible nitric oxide synthase (iNOS expression were reciprocally regulated when assayed using specific inhibitors and siRNA-mediated downregulation. Interestingly, induction of iNOS expression and nitric oxide production were observed in case of inhibition of arginase in infected macrophages. Furthermore, inhibition of l-arginine transport as well as arginase resulted in decreased polyamine production, limiting parasite survival inside macrophages. l-arginine availability and transport regulated Th1/Th2 cytokine levels in case of Leishmania infection. Upregulation of l-arginine transport, induction of host arginase, and enhanced polyamine production were correlated with increased level of IL-10 and decreased level of IL-12 and TNF-α in L. donovani-infected macrophages. Our findings provide clear evidence for targeting the metabolism of l-arginine and l-arginine-metabolizing enzymes as an important

  18. Central transport and distribution of labelled glutamic and aspartic acids to the cochlear nucleus in cats. An autoradiographic study

    Energy Technology Data Exchange (ETDEWEB)

    Kane, E S [University of Massachusetts Medical School, Worcester, MA (USA). Dept. of Anatomy

    1979-01-01

    Tritiated L-glutamic acid or L-aspartic acid was injected unilaterally into the cochleas of adult cats, and 4 h-7 days later the localization of label was studied by light-microscopic autoradiography in sections of the brain stem. Consistent differences in labelling after glutamate and after aspartate suggest differences in their uptake, metabolic conversion and/or transport to the cochlear nucleus by cochlear fibers. The morphological differences shown here agree with the distribution of those two amino acids in the cat cochlear nucleus as shown by microchemical analyses.

  19. Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

    Science.gov (United States)

    Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

    2016-08-05

    A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Connecting Source with Sink: The Role of Arabidopsis AAP8 in Phloem Loading of Amino Acids1[OPEN

    Science.gov (United States)

    Santiago, James P.; Tegeder, Mechthild

    2016-01-01

    Allocation of large amounts of nitrogen to developing organs occurs in the phloem and is essential for plant growth and seed development. In Arabidopsis (Arabidopsis thaliana) and many other plant species, amino acids represent the dominant nitrogen transport forms in the phloem, and they are mainly synthesized in photosynthetically active source leaves. Following their synthesis, a broad spectrum of the amino nitrogen is actively loaded into the phloem of leaf minor veins and transported within the phloem sap to sinks such as developing leaves, fruits, or seeds. Controlled regulation of the source-to-sink transport of amino acids has long been postulated; however, the molecular mechanism of amino acid phloem loading was still unknown. In this study, Arabidopsis AMINO ACID PERMEASE8 (AAP8) was shown to be expressed in the source leaf phloem and localized to the plasma membrane, suggesting its function in phloem loading. This was further supported by transport studies with aap8 mutants fed with radiolabeled amino acids and by leaf exudate analyses. In addition, biochemical and molecular analyses revealed alterations in leaf nitrogen pools and metabolism dependent on the developmental stage of the mutants. Decreased amino acid phloem loading and partitioning to sinks led to decreased silique and seed numbers, but seed protein levels were unchanged, demonstrating the importance of AAP8 function for sink development rather than seed quality. Overall, these results show that AAP8 plays an important role in source-to-sink partitioning of nitrogen and that its function affects source leaf physiology and seed yield. PMID:27016446

  1. Proton transport properties of tin phosphate, chromotropic acid ...

    Indian Academy of Sciences (India)

    The functionalized materials of tin (IV) phosphate (SnP) like chromotropic acid anchored tin ... elemental analysis (ICP–AES), thermal analysis, X-ray analysis and FTIR spectroscopy. .... nal level below 1 V, interfaced to a minicomputer for data.

  2. Serum biochemical activities and muscular soreness in transported goats administered with ascorbic acid during the hot-dry season

    Directory of Open Access Journals (Sweden)

    Ndazo S Minka

    2010-12-01

    Full Text Available The effects of handling, loading and 12 h of road transportation during the hot-dry season on muscular metabolism of 20 experimental goats administered orally with 100 mg/kg body weight of ascorbic acid (AA dissolved in 10 ml of sterile water, and other 20 control goats given equivalent of sterile water 40 min prior to transportation were investigated. The result obtained post-transportation showed that handling, loading and transportation were stressful to the goats, especially the control goats and resulted into muscular damage and the development of delayed-onset-muscular-soreness (DOMS, which may lead to dark-firm-dry (DFD syndrome meat with undesirable effects on its quality. In the experimental goats administered AA such transportation effects were minimal or completely abolished. The result demonstrated that AA reduced the incidence of DOMS and muscular damage in transported goats, therefore it may be used to improve the welfare and quality of meat obtained from goats subjected to long period of road transportation under adverse climatic conditions.

  3. Effects of Mutations and Ligands on the Thermostability of the l-Arginine/Agmatine Antiporter AdiC and Deduced Insights into Ligand-Binding of Human l-Type Amino Acid Transporters

    Directory of Open Access Journals (Sweden)

    Hüseyin Ilgü

    2018-03-01

    Full Text Available The l-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (Tms of the purified AdiC variants in the absence and presence of the selected ligands l-arginine (Arg, agmatine, l-arginine methyl ester, and l-arginine amide. The resulting Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member l-type amino acid transporter-1 (LAT1; SLC7A5, an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for l-amino acid substrates to LATs from the SLC7 family.

  4. Protective effect of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) on irradiation-induced inhibition of intestinal transport function

    International Nuclear Information System (INIS)

    Chen, T.S.; Ando, M.

    1983-01-01

    The purpose of this study was to investigate the protective effect of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) on whole-body irradiation-induced inhibition of intestinal transport function. The jejunal transport of fluid and sugars was studied in male Swiss-Webster mice before and 3 days after whole-body irradiation (1000 rads). The rates of glucose and water transport were decreased by 86 and 70%, respectively, in irradiated animals. However, the rate of transport of 3-O-methyl-D-glucose (3MG) was not affected. In mice receiving WR-2721 (500 mg/kg, ip) 15 to 30 min prior to whole-body irradiation, net water flux was unaffected and the rate of D-glucose transport was decreased only 8%. WR-2721 administered alone (500 mg/kg, ip) had no effect on either D-glucose transport or net water flux across the jejunal mucosa. The results suggest that WR-2721 protects against irradiation-induced inhibition of some intestinal transport functions

  5. Evaluating remedial alternatives for an acid mine drainage stream: Application of a reactive transport model

    Science.gov (United States)

    Runkel, R.L.; Kimball, B.A.

    2002-01-01

    A reactive transport model based on one-dimensional transport and equilibrium chemistry is applied to synoptic data from an acid mine drainage stream. Model inputs include streamflow estimates based on tracer dilution, inflow chemistry based on synoptic sampling, and equilibrium constants describing acid/base, complexation, precipitation/dissolution, and sorption reactions. The dominant features of observed spatial profiles in pH and metal concentration are reproduced along the 3.5-km study reach by simulating the precipitation of Fe(III) and Al solid phases and the sorption of Cu, As, and Pb onto freshly precipitated iron-(III) oxides. Given this quantitative description of existing conditions, additional simulations are conducted to estimate the streamwater quality that could result from two hypothetical remediation plans. Both remediation plans involve the addition of CaCO3 to raise the pH of a small, acidic inflow from ???2.4 to ???7.0. This pH increase results in a reduced metal load that is routed downstream by the reactive transport model, thereby providing an estimate of post-remediation water quality. The first remediation plan assumes a closed system wherein inflow Fe(II) is not oxidized by the treatment system; under the second remediation plan, an open system is assumed, and Fe(II) is oxidized within the treatment system. Both plans increase instream pH and substantially reduce total and dissolved concentrations of Al, As, Cu, and Fe(II+III) at the terminus of the study reach. Dissolved Pb concentrations are reduced by ???18% under the first remediation plan due to sorption onto iron-(III) oxides within the treatment system and stream channel. In contrast, iron(III) oxides are limiting under the second remediation plan, and removal of dissolved Pb occurs primarily within the treatment system. This limitation results in an increase in dissolved Pb concentrations over existing conditions as additional downstream sources of Pb are not attenuated by

  6. Ascorbic acid transported by sodium-dependent vitamin C transporter 2 stimulates steroidogenesis in human choriocarcinoma cells.

    Science.gov (United States)

    Wu, Ximei; Iguchi, Takuma; Itoh, Norio; Okamoto, Kousuke; Takagi, Tatsuya; Tanaka, Keiichi; Nakanishi, Tsuyoshi

    2008-01-01

    Reduced vitamin C [ascorbic acid (AA)], which is taken up into cells by sodium-dependent vitamin C transporter (SVCT) 1 and 2, is believed to be important for hormone synthesis, but its role in generating placental steroids needed to maintain pregnancy and fetal development is not clear. To determine the steroidogenic effect of AA and the role of SVCT2 in AA-induced steroidogenesis, we tested the effects of AA treatment and SVCT2 knockdown on steroidogenesis in human choriocarcinoma cell lines. AA treatment of JEG-3, BeWo, and JAR cells for 48-h dose dependently increased progesterone and estradiol levels. In JEG-3 cells, AA increased the mRNA expression of P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase, key enzymes for steroidogenesis. Stable knockdown of SVCT2 in JEG-3 cells by retrovirally mediated RNA interference decreased the maximal velocity of AA uptake by approximately 50%, but apparent affinity values were not affected. SVCT2 knockdown in JEG-3 cells significantly suppressed the AA-induced mRNA expression of placental P450 cholesterol side-chain cleavage enzyme, 3beta-hydroxysteroid dehydrogenase type 1, and aromatase. This suppression of the AA-induced mRNA expression of steroidogenic enzymes subsequently decreased progesterone and estradiol production. In addition, inhibition of MAPK kinase-ERK signaling, which is a major pathway for AA-regulated gene expression, failed to affect AA-induced steroidogenesis. Our observations indicate that SVCT2-mediated AA uptake into cells is necessary for AA-induced steroidogenesis in human choriocarcinoma cell, but MAPK kinase-ERK signaling is not involved in AA-induced steroidogenesis.

  7. Potassium co-transport and antiport during the uptake of sucrose and glutamic acid from the xylem vessels

    NARCIS (Netherlands)

    Bel, A.J.E. van; Erven, A.J. van

    Perfusion experiments with excised internodes of tomato (Lycopersicon esculentum cv Moneymaker) showed that the uptake of glutamic acid and sucrose from the xylem vessels is accompanied with coupled proton co-transport and potassium antiport at low pH (<5.5). At high pH (5.5) both proton and

  8. Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

    Science.gov (United States)

    Aduri, Nanda G; Ernst, Heidi A; Prabhala, Bala K; Bhatt, Shweta; Boesen, Thomas; Gajhede, Michael; Mirza, Osman

    2018-01-08

    The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. OCT2 and MATE1 Provide Bi-directional Agmatine Transport

    Science.gov (United States)

    Winter, Tate N.; Elmquist, William F.; Fairbanks, Carolyn A.

    2015-01-01

    Agmatine is a biogenic amine (l-arginine metabolite) of potential relevance to several central nervous system (CNS) conditions. The identities of transporters underlying agmatine and polyamine disposition in mammalian systems are not well defined. The SLC-family organic cation transporters (OCT) OCT1 and OCT2 and multidrug and toxin extrusion transporter-1 (MATE1) are transport systems that may be of importance for the cellular disposition of agmatine and putrescine. We investigated the transport of [3H]-agmatine and [3H]-putrescine in human embryonic kidney (HEK293) cells stably-transfected with hOCT1-, hOCT2-, and hMATE1. Agmatine transport by hOCT1 and hOCT2 was concentration-dependent, whereas only hOCT2 demonstrated pH-dependent transport. hOCT2 exhibited a greater affinity for agmatine (Km = 1.84 ± 0.38 mM) than did hOCT1 (Km = 18.73 ± 4.86 mM). Putrescine accumulation was pH- and concentration-dependent in hOCT2-HEK cells (Km = 11.29 ± 4.26 mM) but not hOCT1-HEK cells. Agmatine accumulation, in contrast to putrescine, was significantly enhanced by hMATE1 over-expression, and was saturable (Km = 240 ± 31 μM; Vmax = 192 ± 10 pmol/min/mg protein). Intracellular agmatine was also trans-stimulated (effluxed) from hMATE1-HEK cells in the presence of an inward proton-gradient. The hMATE1-mediated transport of agmatine was inhibited by polyamines, the prototypical substrates MPP+ and paraquat, as well as guanidine and arcaine, but not l-arginine. These results suggest that agmatine disposition may be influenced by hOCT2 and hMATE1, two transporters critical in the renal elimination of xenobiotic compounds. PMID:21128598

  10. Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.

    Science.gov (United States)

    Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny

    2007-06-01

    Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.

  11. Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Q.; Chen, S.Y.; Deng, L.D.; Feng, L.P.; Huang, L.Z.; Yu, R.R. [Department of Pharmacy, Guilin Medical University, Guilin (China)

    2013-11-18

    Excessive oxidative stress in pancreatic β cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides.

  12. Interactive Effects of Jasmonic Acid, Salicylic Acid, and Gibberellin on Induction of Trichomes in Arabidopsis1

    Science.gov (United States)

    Traw, M. Brian; Bergelson, Joy

    2003-01-01

    Leaf trichomes protect plants from attack by insect herbivores and are often induced following damage. Hormonal regulation of this plant induction response has not been previously studied. In a series of experiments, we addressed the effects of artificial damage, jasmonic acid, salicylic acid, and gibberellin on induction of trichomes in Arabidopsis. Artificial damage and jasmonic acid caused significant increases in trichome production of leaves. The jar1-1 mutant exhibited normal trichome induction following treatment with jasmonic acid, suggesting that adenylation of jasmonic acid is not necessary. Salicylic acid had a negative effect on trichome production and consistently reduced the effect of jasmonic acid, suggesting negative cross-talk between the jasmonate and salicylate-dependent defense pathways. Interestingly, the effect of salicylic acid persisted in the nim1-1 mutant, suggesting that the Npr1/Nim1 gene is not downstream of salicylic acid in the negative regulation of trichome production. Last, we found that gibberellin and jasmonic acid had a synergistic effect on the induction of trichomes, suggesting important interactions between these two compounds. PMID:14551332

  13. ESKIMO1 disruption in Arabidopsis alters vascular tissue and impairs water transport.

    Directory of Open Access Journals (Sweden)

    Valérie Lefebvre

    Full Text Available Water economy in agricultural practices is an issue that is being addressed through studies aimed at understanding both plant water-use efficiency (WUE, i.e. biomass produced per water consumed, and responses to water shortage. In the model species Arabidopsis thaliana, the ESKIMO1 (ESK1 gene has been described as involved in freezing, cold and salt tolerance as well as in water economy: esk1 mutants have very low evapo-transpiration rates and high water-use efficiency. In order to establish ESK1 function, detailed characterization of esk1 mutants has been carried out. The stress hormone ABA (abscisic acid was present at high levels in esk1 compared to wild type, nevertheless, the weak water loss of esk1 was independent of stomata closure through ABA biosynthesis, as combining mutant in this pathway with esk1 led to additive phenotypes. Measurement of root hydraulic conductivity suggests that the esk1 vegetative apparatus suffers water deficit due to a defect in water transport. ESK1 promoter-driven reporter gene expression was observed in xylem and fibers, the vascular tissue responsible for the transport of water and mineral nutrients from the soil to the shoots, via the roots. Moreover, in cross sections of hypocotyls, roots and stems, esk1 xylem vessels were collapsed. Finally, using Fourier-Transform Infrared (FTIR spectroscopy, severe chemical modifications of xylem cell wall composition were highlighted in the esk1 mutants. Taken together our findings show that ESK1 is necessary for the production of functional xylem vessels, through its implication in the laying down of secondary cell wall components.

  14. Amino acid derived 1,4-dialkyl substituted imidazolones

    DEFF Research Database (Denmark)

    Diness, Frederik; Meldal, Morten Peter

    2010-01-01

    A general method for synthesis of 1,4-substituted imidazolones from amino acids on solid support or in solution has been developed. Amino acid derived 3-Boc-(1,3)-oxazinane (Box) protected amino aldehyde building blocks were coupled through urea bonds to the amino terminal of dipeptides or amino...... acids. Upon acidic release, the aldehyde instantaneously formed the cyclic N-carbamyliminium ion, which rearranged to the corresponding imidazolone. Under strongly acidic conditions the imidazolones acted as nuclophiles in the Pictet-Spengler reaction....

  15. Potential role of sea spray generation in the atmospheric transport of perfluorocarboxylic acids.

    Science.gov (United States)

    Webster, Eva; Ellis, David A

    2010-08-01

    The observed environmental concentrations of perfluorooctanoic acid (PFOA) and its conjugate base (PFO) in remote regions such as the Arctic have been primarily ascribed to the atmospheric transport and degradation of fluorotelomer alcohols (FTOHs) and to direct PFO transport in ocean currents. These mechanisms are each capable of only partially explaining observations. Transport within marine aerosols has been proposed and may explain transport over short distances but will contribute little over longer distances. However, PFO(A) has been shown to have a very short half-life in aqueous aerosols and thus sea spray was proposed as a mechanism for the generation of PFOA in the gas phase from PFO in a water body. Using the observed PFO concentrations in oceans of the Northern Hemisphere and estimated spray generation rates, this mechanism is shown to have the potential for contributing large amounts of PFOA to the atmosphere and may therefore contribute significantly to the concentrations observed in remote locations. Specifically, the rate of PFOA release into the gas phase from oceans in the Northern Hemisphere is calculated to be potentially comparable to global stack emissions to the atmosphere. The subsequent potential for atmospheric degradation of PFOA and its global warming potential are considered. Observed isomeric ratios and predicted atmospheric concentrations due to FTOH degradation are used to elucidate the likely relative importance of transport pathways. It is concluded that gas phase PFOA released from oceans may help to explain observed concentrations in remote regions. The model calculations performed in the present study strongly suggest that oceanic aerosol and gas phase field monitoring is of vital importance to obtain a complete understanding of the global dissemination of PFCAs. Copyright 2010 SETAC

  16. p27Kip1 Modulates Axonal Transport by Regulating α-Tubulin Acetyltransferase 1 Stability

    Directory of Open Access Journals (Sweden)

    Giovanni Morelli

    2018-05-01

    Full Text Available Summary: The protein p27Kip1 plays roles that extend beyond cell-cycle regulation during cerebral cortex development, such as the regulation of neuronal migration and neurite branching via signaling pathways that converge on the actin and microtubule cytoskeletons. Microtubule-dependent transport is essential for the maturation of neurons and the establishment of neuronal connectivity though synapse formation and maintenance. Here, we show that p27Kip1 controls the transport of vesicles and organelles along the axon of mice cortical projection neurons in vitro. Moreover, suppression of the p27Kip1 ortholog, dacapo, in Drosophila melanogaster disrupts axonal transport in vivo, leading to the reduction of locomotor activity in third instar larvae and adult flies. At the molecular level, p27Kip1 stabilizes the α-tubulin acetyltransferase 1, thereby promoting the acetylation of microtubules, a post-translational modification required for proper axonal transport. : Morelli et al. report that p27Kip1/Dacapo modulates the acetylation of microtubules in axons via stabilization of ATAT1, the main α-tubulin acetyltransferase. Its conditional loss leads to the reduction of bidirectional axonal transport of vesicles and mitochondria in vitro in mice and in vivo in Drosophila. Keywords: p27Kip1, dacapo, acetylation, axonal transport, ATAT1, alpha-tubulin, HDAC6, Drosophila, mouse, cerebral cortex

  17. Studies on radiolysis of amino acids, 1

    International Nuclear Information System (INIS)

    Oku, Tadatake

    1977-01-01

    In order to elucidate the radiolysis of amino acid, peptide, protein and enzyme, the radiolytic mechanisms of neutral amino acids (glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-serine, and L-threonine) and acidic amino acids (L-aspartic acid, L-glutamic acid and DL-amino-n-adipic acid) were studied in the presence of air or in the atmosphere nitrogen. An aqueous solution of 1 mM. of each amino acid was sealed in a glass ampoule under air or nitrogen. Irradiation of amino acid solutions was carried out with γ-rays of 60 Co at doses of 4.4-2,640x10 3 rads. The amino acids and the radiolytic products formed were determined by ion-exchange chromatography. From the results of determining amino acids and the radiolytic products formed and their G-values, the radiolytic mechanisms of the amino acids were discussed. (auth.)

  18. Modification on ursodeoxycholic acid (UDCA) scaffold. discovery of bile acid derivatives as selective agonists of cell-surface G-protein coupled bile acid receptor 1 (GP-BAR1).

    Science.gov (United States)

    Sepe, Valentina; Renga, Barbara; Festa, Carmen; D'Amore, Claudio; Masullo, Dario; Cipriani, Sabrina; Di Leva, Francesco Saverio; Monti, Maria Chiara; Novellino, Ettore; Limongelli, Vittorio; Zampella, Angela; Fiorucci, Stefano

    2014-09-25

    Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

  19. Accidents with sulfuric acid

    Directory of Open Access Journals (Sweden)

    Rajković Miloš B.

    2006-01-01

    Full Text Available Sulfuric acid is an important industrial and strategic raw material, the production of which is developing on all continents, in many factories in the world and with an annual production of over 160 million tons. On the other hand, the production, transport and usage are very dangerous and demand measures of precaution because the consequences could be catastrophic, and not only at the local level where the accident would happen. Accidents that have been publicly recorded during the last eighteen years (from 1988 till the beginning of 2006 are analyzed in this paper. It is very alarming data that, according to all the recorded accidents, over 1.6 million tons of sulfuric acid were exuded. Although water transport is the safest (only 16.38% of the total amount of accidents in that way 98.88% of the total amount of sulfuric acid was exuded into the environment. Human factor was the common factor in all the accidents, whether there was enough control of the production process, of reservoirs or transportation tanks or the transport was done by inadequate (old tanks, or the accidents arose from human factor (inadequate speed, lock of caution etc. The fact is that huge energy, sacrifice and courage were involved in the recovery from accidents where rescue teams and fire brigades showed great courage to prevent real environmental catastrophes and very often they lost their lives during the events. So, the phrase that sulfuric acid is a real "environmental bomb" has become clearer.

  20. Monocarboxylate Transporters 1 and 4 Are Associated with CD147 in Cervical Carcinoma

    Directory of Open Access Journals (Sweden)

    Céline Pinheiro

    2009-01-01

    Full Text Available Due to the highly glycolytic metabolism of solid tumours, there is an increased acid production, however, cells are able to maintain physiological pH through plasma membrane efflux of the accumulating protons. Acid efflux through MCTs (monocarboxylate transporters constitutes one of the most important mechanisms involved in tumour intracellular pH maintenance. Still, the molecular mechanisms underlying the regulation of these proteins are not fully understood. We aimed to evaluate the association between CD147 (MCT1 and MCT4 chaperone and MCT expression in cervical cancer lesions and the clinico-pathological significance of CD147 expression, alone and in combination with MCTs. The series included 83 biopsy samples of precursor lesions and surgical specimens of 126 invasive carcinomas. Analysis of CD147 expression was performed by immunohistochemistry. CD147 expression was higher in squamous and adenocarcinoma tissues than in the non-neoplastic counterparts and, importantly, both MCT1 and MCT4 were more frequently expressed in CD147 positive cases. Additionally, co-expression of CD147 with MCT1 was associated with lymph-node and/or distant metastases in adenocarcinomas. Our results show a close association between CD147 and MCT1 and MCT4 expressions in human cervical cancer and provided evidence for a prognostic value of CD147 and MCT1 co-expression.