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Sample records for acid transporter pat1

  1. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    PubMed

    Ögmundsdóttir, Margrét H; Heublein, Sabine; Kazi, Shubana; Reynolds, Bruno; Visvalingam, Shivanthy M; Shaw, Michael K; Goberdhan, Deborah C I

    2012-01-01

    Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+)-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  2. Differential expression of proton-assisted amino acid transporters (PAT[1] and PAT[2]) in tissues of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The PATs have been identified as growth-regulatory nutrient sensors in Drosophila and as activators of mammalian target of rapamycin (mTOR) in mammalian cell cultures. These studies suggest that, beyond their classical function as transporters of simple amino acids (AA), the PATs act as tranceptors,...

  3. PAT-1 safety analysis report addendum.

    SciTech Connect

    Weiner, Ruth F.; Schmale, David T.; Kalan, Robert J.; Akin, Lili A.; Miller, David Russell; Knorovsky, Gerald Albert; Yoshimura, Richard Hiroyuki; Lopez, Carlos; Harding, David Cameron; Jones, Perry L.; Morrow, Charles W.

    2010-09-01

    The Plutonium Air Transportable Package, Model PAT-1, is certified under Title 10, Code of Federal Regulations Part 71 by the U.S. Nuclear Regulatory Commission (NRC) per Certificate of Compliance (CoC) USA/0361B(U)F-96 (currently Revision 9). The purpose of this SAR Addendum is to incorporate plutonium (Pu) metal as a new payload for the PAT-1 package. The Pu metal is packed in an inner container (designated the T-Ampoule) that replaces the PC-1 inner container. The documentation and results from analysis contained in this addendum demonstrate that the replacement of the PC-1 and associated packaging material with the T-Ampoule and associated packaging with the addition of the plutonium metal content are not significant with respect to the design, operating characteristics, or safe performance of the containment system and prevention of criticality when the package is subjected to the tests specified in 10 CFR 71.71, 71.73 and 71.74.

  4. PAT-1 safety analysis report addendum author responses to request for additional information.

    SciTech Connect

    Weiner, Ruth F.; Schmale, David T.; Kalan, Robert J.; Akin, Lili A.; Miller, David Russell; Knorovsky, Gerald Albert; Yoshimura, Richard Hiroyuki; Lopez, Carlos; Harding, David Cameron; Jones, Perry L.; Morrow, Charles W.

    2010-09-01

    The Plutonium Air Transportable Package, Model PAT-1, is certified under Title 10, Code of Federal Regulations Part 71 by the U.S. Nuclear Regulatory Commission (NRC) per Certificate of Compliance (CoC) USA/0361B(U)F-96 (currently Revision 9). The National Nuclear Security Administration (NNSA) submitted SAND Report SAND2009-5822 to NRC that documented the incorporation of plutonium (Pu) metal as a new payload for the PAT-1 package. NRC responded with a Request for Additional Information (RAI), identifying information needed in connection with its review of the application. The purpose of this SAND report is to provide the authors responses to each RAI. SAND Report SAND2010-6106 containing the proposed changes to the Addendum is provided separately.

  5. ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

    PubMed Central

    Cipak, Lubos; Hyppa, Randy; Smith, Gerald; Gregan, Juraj

    2012-01-01

    To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1+ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis. PMID:22487684

  6. The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing

    PubMed Central

    Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

    2016-01-01

    ABSTRACT Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation. PMID:26918764

  7. Bile acid transporters

    PubMed Central

    Dawson, Paul A.; Lan, Tian; Rao, Anuradha

    2009-01-01

    In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na+ taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTα-OSTβ. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers. PMID:19498215

  8. Molecular cloning and characterization of oocyte-specific Pat1a in Rana rugosa frogs.

    PubMed

    Nakamura, Yoriko; Iwasaki, Takehiro; Umei, Yosuke; Saotome, Kazuhiro; Nakajima, Yukiko; Kitahara, Shoichi; Uno, Yoshinobu; Matsuda, Yoichi; Oike, Akira; Kodama, Maho; Nakamura, Masahisa

    2015-10-01

    The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a germ cell-specific protein-and Pat1a, Vasa-immunopositive signals were observed in all of the germ cells, whereas Pat1a signals were confined to the growing oocytes (50-200 μm in diameter), and absent from small germ cells (<50 μm in diameter). Forty days after testosterone injection into tadpoles to induce female-to-male sex-reversal, Pat1a-immunoreactive oocytes had disappeared completely from the sex-reversed gonad, but Vasa-positive small germ cells persisted. Thus, Pat1a would be a good marker for identifying the sexual status of the sex-reversing gonad in amphibians. In addition, fluorescence in situ hybridization analysis showed Pat1a to have an autosomal locus, suggesting that Pat1a transcription is probably regulated by a tissue-specific transcription factor in R. rugosa.

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

    SciTech Connect

    Murakami, Taro Yoshinaga, Mariko

    2013-10-04

    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.

  10. Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase

    PubMed Central

    Cipak, Lubos; Polakova, Silvia; Hyppa, Randy W.; Smith, Gerald R.; Gregan, Juraj

    2014-01-01

    Synchronous cultures are often indispensable for studying meiosis. Here, we present an optimized protocol for induction of synchronous meiosis in the fission yeast Schizosaccharomyces pombe. Chemical inactivation of an ATP analog-sensitive form of the Pat1 kinase (pat1-as2) by adding the ATP-analog 1-NM-PP1 in G1-arrested cells allows induction of synchronous meiosis at optimal temperature (25 °C). Importantly, this protocol eliminates detrimental effects of elevated temperature (34 °C) which is required to inactivate the commonly used temperature-sensitive Pat1 kinase mutant (pat1-114). Addition of the mat-Pc gene to a mat1-M strain further improves chromosome segregation and spore viability. Thus, our protocol offers highly synchronous meiosis at optimal temperature with most characteristics similar to those of wild-type meiosis. The synchronization protocol can be completed in 5 days. PMID:24385151

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

    PubMed

    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.

  12. Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T

    PubMed Central

    Kutumbaka, Kirthi K.; Pasmowitz, Joshua; Mategko, James; Reyes, Dindo; Friedrich, Alex; Han, Sukkyun; Martens-Habbena, Willm; Neal-McKinney, Jason; Janagama, Harish K.; Nadala, Cesar

    2015-01-01

    The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1T. PMID:26358606

  13. Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase.

    PubMed

    Cipak, Lubos; Polakova, Silvia; Hyppa, Randy W; Smith, Gerald R; Gregan, Juraj

    2014-01-01

    Synchronous cultures are often indispensable for studying meiosis. Here we present an optimized protocol for induction of synchronous meiosis in the fission yeast Schizosaccharomyces pombe. Chemical inactivation of an ATP analog-sensitive form of the Pat1 kinase (pat1-as2) by adding the ATP analog 1-NM-PP1 in G1-arrested cells allows the induction of synchronous meiosis at optimal temperature (25°C). Importantly, this protocol eliminates detrimental effects of elevated temperature (34°C), which is required to inactivate the commonly used temperature-sensitive Pat1 kinase mutant (pat1-114). The addition of the mat-Pc gene to a mat1-M strain further improves chromosome segregation and spore viability. Thus, our protocol offers highly synchronous meiosis at optimal temperature, with most characteristics similar to those of wild-type meiosis. The synchronization protocol can be completed in 5 d (not including strain production, which may take as long as 2 or 3 months).

  14. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    NASA Astrophysics Data System (ADS)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  15. The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

    PubMed

    Jungfleisch, Jennifer; Chowdhury, Ashis; Alves-Rodrigues, Isabel; Tharun, Sundaresan; Díez, Juana

    2015-08-01

    The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

  16. Human intestine luminal ACE2 and amino acid transporter expression increased by ACE-inhibitors.

    PubMed

    Vuille-dit-Bille, Raphael N; Camargo, Simone M; Emmenegger, Luca; Sasse, Tom; Kummer, Eva; Jando, Julia; Hamie, Qeumars M; Meier, Chantal F; Hunziker, Schirin; Forras-Kaufmann, Zsofia; Kuyumcu, Sena; Fox, Mark; Schwizer, Werner; Fried, Michael; Lindenmeyer, Maja; Götze, Oliver; Verrey, François

    2015-04-01

    Sodium-dependent neutral amino acid transporter B(0)AT1 (SLC6A19) and imino acid (proline) transporter SIT1 (SLC6A20) are expressed at the luminal membrane of small intestine enterocytes and proximal tubule kidney cells where they exert key functions for amino acid (re)absorption as documented by their role in Hartnup disorder and iminoglycinuria, respectively. Expression of B(0)AT1 was shown in rodent intestine to depend on the presence of the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). This enzyme belongs to the renin-angiotensin system and its expression is induced by treatment with ACE-inhibitors (ACEIs) or angiotensin II AT1 receptor blockers (ARBs) in many rodent tissues. We show here in the Xenopus laevis oocyte expression system that human ACE2 also functionally interacts with SIT1. To investigate in human intestine the potential effect of ACEIs or ARBs on ACE2, we analysed intestinal biopsies taken during routine gastroduodenoscopy and ileocolonoscopy from 46 patients of which 9 were under ACEI and 13 ARB treatment. Analysis of transcript expression by real-time PCR and of proteins by immunofluorescence showed a co-localization of SIT1 and B(0)AT1 with ACE2 in the brush-border membrane of human small intestine enterocytes and a distinct axial expression pattern of the tested gene products along the intestine. Patients treated with ACEIs displayed in comparison with untreated controls increased intestinal mRNA levels of ACE2, peptide transporter PEPT1 (SLC15A1) and AA transporters B(0)AT1 and PAT1 (SLC36A1). This study unravels in human intestine the localization and distribution of intestinal transporters involved in amino acid absorption and suggests that ACEIs impact on their expression.

  17. P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics

    PubMed Central

    Hurto, Rebecca L.; Hopper, Anita K.

    2011-01-01

    The nuclear-cytoplasmic distribution of tRNA depends on the balance between tRNA nuclear export/re-export and retrograde tRNA nuclear import in Saccharomyces cerevisiae. The distribution of tRNA is sensitive to nutrient availability as cells deprived of various nutrients exhibit tRNA nuclear accumulation. Starvation induces numerous events that result in translational repression and P-body formation. This study investigated the possible coordination of these responses with tRNA nuclear-cytoplasmic distribution. Dhh1 and Pat1 function in parallel to promote translation repression and P-body formation in response to starvation. Loss of both, Dhh1 and Pat1, results in a failure to repress translation and to induce P-body formation in response to glucose starvation. This study reports that nutrient deprived dhh1 pat1 cells also fail to accumulate tRNA within nuclei. Conversely, inhibition of translation initiation and induction of P-body formation by overproduction of Dhh1 or Pat1 cause tRNA nuclear accumulation in nutrient-replete conditions. Also, loss of the mRNA decapping activator, Lsm1, causes tRNA nuclear accumulation. However, the coordination between P-body formation, translation repression, and tRNA distribution is limited to the early part of the P-body formation/translation repression pathway as loss of mRNA decapping or 5′ to 3′ degradation does not influence tRNA nuclear-cytoplasmic dynamics. The data provide the first link between P-body formation/translation initiation and tRNA nuclear-cytoplasmic dynamics. The current model is that Dhh1 and Pat1 function in parallel to promote starvation-induced tRNA nuclear accumulation. PMID:21398402

  18. Cysteine transport through excitatory amino acid transporter 3 (EAAT3).

    PubMed

    Watts, Spencer D; Torres-Salazar, Delany; Divito, Christopher B; Amara, Susan G

    2014-01-01

    Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest

  19. Ascorbic acid transport into cultured pituitary cells

    SciTech Connect

    Cullen, E.I.; May, V.; Eipper, R.A.

    1986-05-01

    An amidating enzyme designated peptidyl-glycine ..cap alpha..-amidating monooxygenase (PAM) has been studied in a variety of tissues and is dependent on molecular oxygen and stimulated by copper and ascorbic acid. To continue investigating the relationship among cellular ascorbic acid concentrations, amidating ability, and PAM activity, the authors studied ascorbic acid transport in three cell preparations that contain PAM and produce amidated peptides: primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated in 50 ..mu..M (/sup 14/C)ascorbic acid all three cell preparations concentrated ascorbic acid 20- to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes. All three cell preparations displayed saturable ascorbic acid uptake with half-maximal initial rates occurring between 9 and 18 ..mu..M ascorbate. Replacing NaCl in the uptake buffer with choline chloride significantly diminished ascorbate uptake in all three preparations. Ascorbic acid efflux from these cells was slow, displaying half-lives of 7 hours. Unlike systems that transport dehydroascorbic acid, the transport system for ascorbic acid in these cells was not inhibited by glucose. Thus, ascorbate is transported into pituitary cells by a sodium-dependent, active transport system.

  20. SLC27 fatty acid transport proteins.

    PubMed

    Anderson, Courtney M; Stahl, Andreas

    2013-01-01

    The uptake and metabolism of long chain fatty acids (LCFA) are critical to many physiological and cellular processes. Aberrant accumulation or depletion of LCFA underlie the pathology of numerous metabolic diseases. Protein-mediated transport of LCFA has been proposed as the major mode of LCFA uptake and activation. Several proteins have been identified to be involved in LCFA uptake. This review focuses on the SLC27 family of fatty acid transport proteins, also known as FATPs, with an emphasis on the gain- and loss-of-function animal models that elucidate the functions of FATPs in vivo and how these transport proteins play a role in physiological and pathological situations.

  1. Transport and biological activities of bile acids.

    PubMed

    Zwicker, Brittnee L; Agellon, Luis B

    2013-07-01

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

  2. Selective amino acid substitutions convert the creatine transporter to a gamma-aminobutyric acid transporter.

    PubMed

    Dodd, Joanna R; Christie, David L

    2007-05-25

    The creatine transporter (CRT) is a member of a large family of sodium-dependent neurotransmitter and amino acid transporters. The CRT is closely related to the gamma-aminobutyric acid (GABA) transporter, GAT-1, yet GABA is not an effective substrate for the CRT. The high resolution structure of a prokaryotic homologue, LeuT has revealed precise details of the substrate binding site for leucine (Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., and Gouaux, E. (2005) Nature 437, 215-223). We have now designed mutations based on sequence comparisons of the CRT with GABA transporters and the LeuT structural template in an attempt to alter the substrate specificity of the CRT. Combinations of two or three amino acid substitutions at four selected positions resulted in the loss of creatine transport activity and gain of a specific GABA transport function. GABA transport by the "gain of function" mutants was sensitive to nipecotic acid, a competitive inhibitor of GABA transporters. Our results show LeuT to be a good structural model to identify amino acid residues involved in the substrate and inhibitor selectivity of eukaryotic sodium-dependent neurotransmitter and amino acid transporters. However, modification of the binding site alone appears to be insufficient for efficient substrate translocation. Additional residues must mediate the conformational changes required for the diffusion of substrate from the binding site to the cytoplasm.

  3. Intestinal transport and metabolism of bile acids

    PubMed Central

    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. Amino Acid Transport into Cultured Tobacco Cells

    PubMed Central

    Harrington, H. Michael; Henke, Randolph R.

    1981-01-01

    Lysine transport into suspension-cultured Wisconsin-38 tobacco cells was observed. Uptake was linear (up to 90 minutes) with respect to time and amount of tissue only after 4 to 6 hours preincubation in calcium-containing medium. The observed cellular accumulation of lysine was against a concentration gradient and not due to exchange diffusion. Transport was stimulated by low pH and characterized by a biphasic uptake isotherm with two Km values for lysine. System I (Km ≃ 5 × 10−6 molar; Vmax ≃ 180 nanomoles per gram fresh weight per hour) and system II (Km ≃ 10−4 molar; Vmax ≃ 1900 nanomoles per gram fresh weight per hour) were inhibited by N-ethylmaleimide and a variety of respiratory inhibitors. This inhibition was not due to increased efflux. In antagonism experiments, system I was inhibited most effectively by basic amino acids, followed by the sulfur amino acids. System I was only slightly inhibited by the neutral and aromatic amino acids and was not inhibited by the acidic amino acids aspartic and glutamic acids. Transport by system II was inhibited by all of the tested amino acids (including aspartic and glutamic acids) and analogs; however, this system was not inhibited by d-arginine. Neither system was strongly inhibited by d-lysine or the lysine analog S-2-aminoethyl-l-cysteine. Arginine was shown to be a competitive inhibitor of both systems with values for Ki similar to the respective Km values. These studies suggest the presence of at least two amino acid permeases in W-38 tobacco cells. PMID:16661678

  5. Amino acid transport by prosthecae of Asticcacaulis biprosthecum: evidence for a broad-range transport system.

    PubMed

    Tam, E; Pate, J L

    1985-10-01

    Prosthecae purified from cells of Asticcaulis biprosthecum possess active transport systems that transport all 20 amino acids tested. Using ascorbate-reduced phenazine methosulphate in the presence of oxygen, all 20 amino acids are accumulated against a concentration gradient by isolated prosthecae. Results of experiments testing the inhibition of transport of one amino acid by another, and of experiments testing the exchange of exogenous amino acids with those preloaded in prosthecae, along with characteristics of mutants defective in amino acid transport, suggest the presence in prosthecae of three amino acid transport systems. One, the general or G system, transports at least 18 of the 20 amino acids tested. Another system, referred to as the proline or P system, transports seven amino acids (including proline) that are also transported by the G system. The third system transports only glutamate and aspartate, and is referred to as the acidic amino acid transport system or A system.

  6. Modeling Electrical Transport through Nucleic Acids

    NASA Astrophysics Data System (ADS)

    Qi, Jianqing

    Nucleic acids play a vital role in many biological systems and activities. In recent years, engineers and scientists have been interested in studying their electrical properties. The motivation for these studies stems from the following facts: (1) the bases, which form the building blocks of nucleic acids, have unique ionization potentials. Further, nucleic acids are one of the few nanomaterials that can be reproducibly manufactured with a high degree of accuracy (though admittedly their placement at desired locations remains a challenge). As a result, designed strands with specific sequences may offer unique device properties; (2) electrical methods offer potential for sequencing nucleic acids based on a single molecule; (3) electrical methods for disease detection based on the current flowing through nucleic acids are beginning to be demonstrated. While experiments in the above mentioned areas is promising, a deeper understanding of the electrical current flow through the nucleic acids needs to be developed. The modeling of current flowing in these molecules is complex because: (1) they are based on atomic scale contacts between nucleic acids and metal, which cannot be reproducibly built; (2) the conductivity of nucleic acids is easily influenced by the environment, which is constantly changing; and (3) the nucleic acids by themselves are floppy. This thesis focuses on the modeling of electrical transport through nucleic acids that are connected to two metal electrodes at nanoscale. We first develop a decoherent transport model for the double-stranded helix based on the Landauer-Buttiker framework. This model is rationalized by comparison with an experiment that measured the conductance of four different DNA strands. The developed model is then used to study the: (1) potential to make barriers and wells for quantum transport using specifically engineered sequences; (2) change in the electrical properties of a specific DNA strand with and without methylation; (3

  7. Reactive solute transport in acidic streams

    USGS Publications Warehouse

    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. Abscisic Acid Transport in Human Erythrocytes*

    PubMed Central

    Vigliarolo, Tiziana; Guida, Lucrezia; Millo, Enrico; Fresia, Chiara; Turco, Emilia; De Flora, Antonio; Zocchi, Elena

    2015-01-01

    Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [3H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [3H]ABA and [35S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone. PMID:25847240

  9. Amino Acid Transport in Mycobacterium smegmatis

    PubMed Central

    Yabu, Kunihiko

    1970-01-01

    The transport of d-alanine, d-glutamic acid, and d-valine in Mycobacterium smegmatis was compared quantitatively with that of their l-isomers. It appeared that the uptake of d-alanine was mediated by an active process displaying saturation kinetics characteristic of enzyme function, whereas the uptake of d-glutamic acid was accomplished by a passive process showing diffusion kinetics. Both processes were involved in the uptake of l-alanine, l-glutamic acid, d-valine, and l-valine. d-Valine competed with l-valine for entry into the cell through a single active process. d-Alanine and l-alanine also utilized the same active process, but the d-isomer could not enter the cell through the passive process. The passive process exhibited characteristics of diffusion, but was sensitive to sulfhydryl-blocking reagents and showed competition among structurally related amino acids. These last findings suggested that the passive process is a facilitated diffusion. PMID:5437732

  10. Ascorbic acid participates in a general mechanism for concerted glucose transport inhibition and lactate transport stimulation.

    PubMed

    Castro, Maite A; Angulo, Constanza; Brauchi, Sebastián; Nualart, Francisco; Concha, Ilona I

    2008-11-01

    In this paper, we present a novel function for ascorbic acid. Ascorbic acid is an important water-soluble antioxidant and cofactor in various enzyme systems. We have previously demonstrated that an increase in neuronal intracellular ascorbic acid is able to inhibit glucose transport in cortical and hippocampal neurons. Because of the presence of sodium-dependent vitamin C transporters, ascorbic acid is highly concentrated in brain, testis, lung, and adrenal glands. In this work, we explored how ascorbic acid affects glucose and lactate uptake in neuronal and non-neuronal cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression of glucose and ascorbic acid transporters in non-neuronal cells was studied. Like neurons, HEK293 cells expressed GLUT1, GLUT3, and SVCT2. With radioisotope-based methods, only intracellular ascorbic acid, but not extracellular, inhibits 2-deoxyglucose transport in HEK293 cells. As monocarboxylates such as pyruvate and lactate, are important metabolic sources, we analyzed the ascorbic acid effect on lactate transport in cultured neurons and HEK293 cells. Intracellular ascorbic acid was able to stimulate lactate transport in both cell types. Extracellular ascorbic acid did not affect this transport. Our data show that ascorbic acid inhibits glucose transport and stimulates lactate transport in neuronal and non-neuronal cells. Mammalian cells frequently present functional glucose and monocarboxylate transporters, and we describe here a general effect in which ascorbic acid functions like a glucose/monocarboxylate uptake switch in tissues expressing ascorbic acid transporters.

  11. Air transport of plutonium metal: content expansion initiative for the plutonium air transportable (PAT01) packaging

    SciTech Connect

    Caviness, Michael L; Mann, Paul T

    2010-01-01

    The National Nuclear Security Administration (NNSA) has submitted an application to the Nuclear Regulatory Commission (NRC) for the air shipment of plutonium metal within the Plutonium Air Transportable (PAT-1) packaging. The PAT-1 packaging is currently authorized for the air transport of plutonium oxide in solid form only. The INMM presentation will provide a limited overview of the scope of the plutonium metal initiative and provide a status of the NNSA application to the NRC.

  12. Transport of Aromatic Amino Acids by Pseudomonas aeruginosa

    PubMed Central

    Kay, W. W.; Gronlund, Audrey F.

    1971-01-01

    Kinetic studies of the transport of aromatic amino acids by Pseudomonas aeruginosa revealed the existence of two high-affinity transport systems which recognized the three aromatic amino acids. From competition data and studies on the exchange of preformed aromatic amino acid pools, the first transport system was found to be functional with phenylalanine, tyrosine, and tryptophan (in order of decreasing activity), whereas the second system was active with tryptophan, phenylalanine, and tyrosine. The two systems also transported a number of aromatic amino acid analogues but not other amino acids. Mutants defective in each of the two and in both transport systems were isolated and described. When the amino acids were added at low external concentrations to cells growing logarithmically in glucose minimal medium, the tryptophan pool very quickly became saturated. Under identical conditions, phenylalanine and tyrosine each accumulated in the intracellular pool of P. aeruginosa at a concentration which was 10 times greater than that of tryptophan. PMID:4994029

  13. Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

    PubMed

    Côrte-Real, M; Leão, C

    1990-04-01

    DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.

  14. Novel Lactate Transporters from Carboxylic Acid-Producing Rhizopus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungus Rhizopus is frequently used for fermentative production of lactic acid, but little is known about the mechanisms or proteins for transporting this carboxylic acid. Since transport of the lactate anion across the plasma membrane is critical to prevent acidification of the cytoplasm, we ev...

  15. Structural Determinants for Transport Across the Intestinal Bile Acid Transporter Using C-24 Bile Acid Conjugates

    PubMed Central

    Rais, Rana; Acharya, Chayan; MacKerell, Alexander D.; Polli, James E.

    2010-01-01

    The human apical sodium dependent bile acid transporter (hASBT) re-absorbs gram quantities of bile acid daily and is a potential prodrug target to increase oral drug absorption. In the absence of a high resolution hASBT crystal structure, 3D-QSAR modeling may prove beneficial in designing prodrug targets to hASBT. The objective was to derive a conformationally sampled pharmacophore 3D–QSAR (CSP-SAR) model for the uptake of bile acid conjugates by hASBT. A series of bile acid conjugates of glutamyl chenodeoxycholate were evaluated in terms of Km and normalized Vmax(normVmax) using hASBT-MDCK cells. All mono-anionic conjugates were potent substrates. Dianions, cations and zwitterions, which bound with a high affinity, were not substrates. CSP-SAR models were derived using structural and physicochemical descriptors, and evaluated via cross-validation. The best CSP-SAR model for Km included two structural and two physiochemical descriptors, where substrate hydrophobicity enhanced affinity. A best CSP-SAR model for Km/normVmax employed one structural and three physicochemical descriptors, also indicating hydrophobicity enhanced efficiency. Overall, the bile acid C-24 region accommodated a range of substituted anilines, provided a single negative charge was present near C-24. In comparing uptake findings to prior inhibition results, increased hydrophobicity enhanced activity, with dianions and zwitterions hindering activity. PMID:20939504

  16. Ascorbic acid transport and accumulation in human neutrophils

    SciTech Connect

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

    1989-11-15

    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.

  17. Valproic acid induces the glutamate transporter excitatory amino acid transporter-3 in human oligodendroglioma cells.

    PubMed

    Bianchi, M G; Franchi-Gazzola, R; Reia, L; Allegri, M; Uggeri, J; Chiu, M; Sala, R; Bussolati, O

    2012-12-27

    Glutamate transport in early, undifferentiated oligodendrocytic precursors has not been characterized thus far. Here we show that human oligodendroglioma Hs683 cells are not endowed with EAAT-dependent anionic amino acid transport. However, in these cells, but not in U373 human glioblastoma cells, valproic acid (VPA), an inhibitor of histone deacetylases, markedly induces SLC1A1 mRNA, which encodes for the glutamate transporter EAAT3. The effect is detectable after 8h and persists up to 120h of treatment. EAAT3 protein increase becomes detectable after 24h of treatment and reaches its maximum after 72-96h, when it is eightfold more abundant than control. The initial influx of d-aspartate increases in parallel, exhibiting the typical features of an EAAT3-mediated process. SLC1A1 mRNA induction is associated with the increased expression of PDGFRA mRNA (+150%), a marker of early oligodendrocyte precursor cells, while the expression of GFAP, CNP and TUBB3 remains unchanged. Short term experiments have indicated that the VPA effect is shared by trichostatin A, another inhibitor of histone deacetylases. On the contrary, EAAT3 induction is neither prevented by inhibitors of mitogen-activated protein kinases nor triggered by a prolonged incubation with lithium, thus excluding a role for the GSK3β/β-catenin pathway. Thus, the VPA-dependent induction of the glutamate transporter EAAT3 in human oligodendroglioma cells likely occurs through an epigenetic mechanism and may represent an early indicator of commitment to oligodendrocytic differentiation.

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

    PubMed

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

    2014-10-15

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

  19. Arachidonic acid inhibits glycine transport in cultured glial cells.

    PubMed Central

    Zafra, F; Alcantara, R; Gomeza, J; Aragon, C; Gimenez, C

    1990-01-01

    The effects of arachidonic acid on glycine uptake, exchange and efflux in C6 glioma cells were investigated. Arachidonic acid produced a dose-dependent inhibition of high-affinity glycine uptake. This effect was not due to a simple detergent-like action on membranes, as the inhibition of glycine transport was most pronounced with cis-unsaturated long-chain fatty acids, whereas saturated and trans-unsaturated fatty acids had relatively little or no effect. Endogenous unsaturated non-esterified fatty acids may exert a similar inhibitory effect on the transport of glycine. The mechanism for this inhibitory effect has been examined in a plasma membrane vesicle preparation derived from C6 cells, which avoids metabolic or compartmentation interferences. The results suggest that part of the selective inhibition of glycine transport by arachidonic acid could be due to the effects of the arachidonic acid on the lipid domain surrounding the carrier. PMID:2121132

  20. Ligands targeting the excitatory amino acid transporters (EAATs).

    PubMed

    Dunlop, John; Butera, John A

    2006-01-01

    This review provides an overview of ligands for the excitatory amino acid transporters (EAATs), a family of high-affinity glutamate transporters localized to the plasma membrane of neurons and astroglial cells. Ligand development from the perspective of identifying novel and more selective tools for elucidating transporter subtype function, and the potential of transporter ligands in a therapeutic setting are discussed. Acute pharmacological modulation of EAAT activity in the form of linear and conformationally restricted glutamate and aspartate analogs is presented, in addition to recent strategies aimed more toward modulating transporter expression levels, the latter of particular significance to the development of transporter based therapeutics.

  1. Identification of a novel sialic acid transporter in Haemophilus ducreyi.

    PubMed

    Post, Deborah M B; Mungur, Rachna; Gibson, Bradford W; Munson, Robert S

    2005-10-01

    Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.

  2. Ammonia Transporters and Their Role in Acid-Base Balance.

    PubMed

    Weiner, I David; Verlander, Jill W

    2017-04-01

    Acid-base homeostasis is critical to maintenance of normal health. Renal ammonia excretion is the quantitatively predominant component of renal net acid excretion, both under basal conditions and in response to acid-base disturbances. Although titratable acid excretion also contributes to renal net acid excretion, the quantitative contribution of titratable acid excretion is less than that of ammonia under basal conditions and is only a minor component of the adaptive response to acid-base disturbances. In contrast to other urinary solutes, ammonia is produced in the kidney and then is selectively transported either into the urine or the renal vein. The proportion of ammonia that the kidney produces that is excreted in the urine varies dramatically in response to physiological stimuli, and only urinary ammonia excretion contributes to acid-base homeostasis. As a result, selective and regulated renal ammonia transport by renal epithelial cells is central to acid-base homeostasis. Both molecular forms of ammonia, NH3 and NH4(+), are transported by specific proteins, and regulation of these transport processes determines the eventual fate of the ammonia produced. In this review, we discuss these issues, and then discuss in detail the specific proteins involved in renal epithelial cell ammonia transport.

  3. Identification and application of keto acids transporters in Yarrowia lipolytica

    PubMed Central

    Guo, Hongwei; Liu, Peiran; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2015-01-01

    Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L−1, whereas the concentration of pyruvate decreased to 12.3 g·L−1. Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms. PMID:25633653

  4. Identification and application of keto acids transporters in Yarrowia lipolytica.

    PubMed

    Guo, Hongwei; Liu, Peiran; Madzak, Catherine; Du, Guocheng; Zhou, Jingwen; Chen, Jian

    2015-01-30

    Production of organic acids by microorganisms is of great importance for obtaining building-block chemicals from sustainable biomass. Extracellular accumulation of organic acids involved a series of transporters, which play important roles in the accumulation of specific organic acid while lack of systematic demonstration in eukaryotic microorganisms. To circumvent accumulation of by-product, efforts have being orchestrated to carboxylate transport mechanism for potential clue in Yarrowia lipolytica WSH-Z06. Six endogenous putative transporter genes, YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D24607g, YALI0D20108g and YALI0E32901g, were identified. Transport characteristics and substrate specificities were further investigated using a carboxylate-transport-deficient Saccharomyces cerevisiae strain. These transporters were expressed in Y. lipolytica WSH-Z06 to assess their roles in regulating extracellular keto acids accumulation. In a Y. lipolytica T1 line over expressing YALI0B19470g, α-ketoglutarate accumulated to 46.7 g·L(-1), whereas the concentration of pyruvate decreased to 12.3 g·L(-1). Systematic identification of these keto acids transporters would provide clues to further improve the accumulation of specific organic acids with higher efficiency in eukaryotic microorganisms.

  5. Amino acid transporters: roles in amino acid sensing and signalling in animal cells.

    PubMed Central

    Hyde, Russell; Taylor, Peter M; Hundal, Harinder S

    2003-01-01

    Amino acid availability regulates cellular physiology by modulating gene expression and signal transduction pathways. However, although the signalling intermediates between nutrient availability and altered gene expression have become increasingly well documented, how eukaryotic cells sense the presence of either a nutritionally rich or deprived medium is still uncertain. From recent studies it appears that the intracellular amino acid pool size is particularly important in regulating translational effectors, thus, regulated transport of amino acids across the plasma membrane represents a means by which the cellular response to amino acids could be controlled. Furthermore, evidence from studies with transportable amino acid analogues has demonstrated that flux through amino acid transporters may act as an initiator of nutritional signalling. This evidence, coupled with the substrate selectivity and sensitivity to nutrient availability classically associated with amino acid transporters, plus the recent discovery of transporter-associated signalling proteins, demonstrates a potential role for nutrient transporters as initiators of cellular nutrient signalling. Here, we review the evidence supporting the idea that distinct amino acid "receptors" function to detect and transmit certain nutrient stimuli in higher eukaryotes. In particular, we focus on the role that amino acid transporters may play in the sensing of amino acid levels, both directly as initiators of nutrient signalling and indirectly as regulators of external amino acid access to intracellular receptor/signalling mechanisms. PMID:12879880

  6. Carboxylic Acids Plasma Membrane Transporters in Saccharomyces cerevisiae.

    PubMed

    Casal, Margarida; Queirós, Odília; Talaia, Gabriel; Ribas, David; Paiva, Sandra

    2016-01-01

    This chapter covers the functionally characterized plasma membrane carboxylic acids transporters Jen1, Ady2, Fps1 and Pdr12 in the yeast Saccharomyces cerevisiae, addressing also their homologues in other microorganisms, as filamentous fungi and bacteria. Carboxylic acids can either be transported into the cells, to be used as nutrients, or extruded in response to acid stress conditions. The secondary active transporters Jen1 and Ady2 can mediate the uptake of the anionic form of these substrates by a H(+)-symport mechanism. The undissociated form of carboxylic acids is lipid-soluble, crossing the plasma membrane by simple diffusion. Furthermore, acetic acid can also be transported by facilitated diffusion via Fps1 channel. At the cytoplasmic physiological pH, the anionic form of the acid prevails and it can be exported by the Pdr12 pump. This review will highlight the mechanisms involving carboxylic acids transporters, and the way they operate according to the yeast cell response to environmental changes, as carbon source availability, extracellular pH and acid stress conditions.

  7. Transport of aromatic amino acids by Brevibacterium linens.

    PubMed

    Boyaval, P; Moreira, E; Desmazeaud, M J

    1983-09-01

    Whole metabolizing Brevibacterium linens cells were used to study the transport of aromatic amino acids. Kinetic results followed the Michaelis-Menten equation with apparent Km values for phenylalanine, tyrosine, and tryptophan of 24, 3.5, and 1.8 microM. Transport of these amino acids was optimum at pH 7.5 and 25 degrees C for phenylalanine and pH 8.0 and 35 degrees C for tyrosine and tryptophan. Crossed inhibitions were all noncompetitive. The only marked stereospecificity was for the L form of phenylalanine. Transport was almost totally inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Iodoacetate and N-ethylmaleimide were much more inhibitory for tryptophan transport than for transport of the other two aromatic amino acids.

  8. Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

    PubMed Central

    Côrte-Real, M; Leão, C

    1990-01-01

    DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0. PMID:2339872

  9. Nucleic acids encoding metal uptake transporters and their uses

    DOEpatents

    Schroeder, Julian I.; Antosiewicz, Danuta M.; Schachtman, Daniel P.; Clemens, Stephan

    1999-01-01

    The invention provides LCT1 nucleic acids which encode metal ion uptake transporters. The invention also provides methods of modulating heavy metal and alkali metal uptake in plants. The methods involve producing transgenic plants comprising a recombinant expression cassette containing an LCT1 nucleic acid linked to a plant promoter.

  10. Modeling acid transport in chemically amplified resist films

    NASA Astrophysics Data System (ADS)

    Patil, Abhijit A.; Doxastakis, Manolis; Stein, Gila E.

    2014-03-01

    The acid-catalyzed deprotection of glassy poly(4-hydroxystyrene-co-tert butyl acrylate) films was studied with infrared absorbance spectroscopy and stochastic simulations. Experimental data were interpreted with a simple description of subdiffusive acid transport coupled to second-order acid loss. This model predicts key attributes of observed deprotection rates, such as fast reaction at short times, slow reaction at long times, and a non-linear dependence on acid loading. The degree of anomalous character is reduced by increasing the post-exposure bake temperature or adding plasticizing agents to the polymer resin. These findings indicate that the acid mobility and overall deprotection kinetics are coupled to glassy matrix dynamics. Furthermore, the acid diffusion lengths were calculated from the anomalous transport model and compared with nanopattern line widths. The consistent scaling between experiments and simulations suggests that the anomalous diffusion model could be further developed into a predictive lithography tool.

  11. Luminal Heterodimeric Amino Acid Transporter Defective in Cystinuria

    PubMed Central

    Pfeiffer, Rahel; Loffing, Jan; Rossier, Grégoire; Bauch, Christian; Meier, Christian; Eggermann, Thomas; Loffing-Cueni, Dominique; Kühn, Lukas C.; Verrey, François

    1999-01-01

    Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b0,+ type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b0,+AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b0,+ amino acid transporter complex. We demonstrate its b0,+-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b0,+AT protein is the product of the gene defective in non-type I cystinuria. PMID:10588648

  12. Differential diagnosis of (inherited) amino acid metabolism or transport disorders.

    PubMed

    Blom, W; Huijmans, J G

    1992-02-01

    Disorders of amino acid metabolism or transport are most clearly expressed in urine. Nevertheless the interpretation of abnormalities in urinary amino acid excretion remains difficult. An increase or decrease of almost every amino acid in urine can be due to various etiology. To differentiate between primary and secondary aminoacido-pathies systematic laboratory investigation is necessary. Early diagnosis of disorders of amino acid metabolism or transport is very important, because most of them can be treated, leading to the prevention of (further) clinical abnormalities. In those disorders, which cannot be treated, early diagnosis in an index-patient may prevent the birth of other siblings by means of genetic counseling and prenatal diagnosis.Primary aminoacidopathies can be due to genetically determined transport disorders and enzyme deficiencies in amino acid metabolism or degradation. Secondary aminoacidopathies are the result of abnormal or deficient nutrition, intestinal dysfunction, organ pathology or other metabolic diseases like organic acidurias.A survey of amino acid metabolism and transport abnormalities will be given, illustrated with metabolic pathways and characteristic abnormal amino acid chromatograms.

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

    PubMed Central

    Aleksunes, Lauren M.

    2010-01-01

    Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting β polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) α and β] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of regulatory

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

    PubMed

    Klaassen, Curtis D; Aleksunes, Lauren M

    2010-03-01

    Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of

  15. Functional characterization of Caenorhabditis elegans heteromeric amino acid transporters.

    PubMed

    Veljkovic, Emilija; Stasiuk, Susan; Skelly, Patrick J; Shoemaker, Charles B; Verrey, François

    2004-02-27

    Mammalian heteromeric amino acid transporters (HATs) are composed of a multi-transmembrane spanning catalytic protein covalently associated with a type II glycoprotein (e.g. 4F2hc, rBAT) through a disulfide bond. Caenorhabditis elegans has nine genes encoding close homologues of the HAT catalytic proteins. Three of these genes (designated AAT-1 to AAT-3) have a much higher degree of similarity to the mammalian homologues than the other six, including the presence of a cysteine residue at the position known to form a disulfide bridge to the glycoprotein partner in mammalian HATs. C. elegans also has two genes encoding homologues of the heteromeric amino acid transporter type II glycoprotein subunits (designated ATG-1 and ATG-2). Both ATG, and/or AAT-1, -2, -3 proteins were expressed in Xenopus oocytes and tested for amino acid transport function. This screen revealed that AAT-1 and AAT-3 facilitate amino acid transport when expressed together with ATG-2 but not with ATG-1 or the mammalian type II glycoproteins 4F2hc and rBAT. AAT-1 and AAT-3 covalently bind to both C. elegans ATG glycoproteins, but only the pairs with ATG-2 traffic to the oocyte surface. Both of these functional, surface-expressed C. elegans HATs transport most neutral amino acids and display the highest transport rate for l-Ala and l-Ser (apparent K(m) 100 microm range). Similar to their mammalian counterparts, the C. elegans HATs function as (near) obligatory amino acid exchangers. Taken together, this study demonstrates that the heteromeric structure and the amino acid exchange function of HATs have been conserved throughout the evolution of nematodes to mammals.

  16. Transported acid aerosols measured in southern Ontario

    NASA Astrophysics Data System (ADS)

    Keeler, Gerald J.; Spengler, John D.; Koutrakis, Petros; Allen, George A.; Raizenne, Mark; Stern, Bonnie

    During the period 29 June 1986-9 August 1986, a field health study assessing the acute health effects of air pollutants on children was conducted at a summer girls' camp on the northern shore of Lake Erie in SW Ontario. Continuous air pollution measurements of SO 2, O 3, NO x, particulate sulfates, light scattering, and meteorological measurements including temperature, dew point, and wind speed and direction were made. Twelve-hour integrated samples of size fractioned particles were also obtained using dichotomous samplers and Harvard impactors equipped with an ammonia denuder for subsequent hydrogen ion determination. Particulate samples were analyzed for trace elements by X-ray fluorescence and Neutron Activation, and for organic and elemental carbon by a thermal/optical technique. The measured aerosol was periodically very acidic with observed 12-h averaged H + concentrations in the range < 10-560 nmoles m -3. The aerosol H + appeared to represent the net strong acidity after H 2SO 4 reaction with NH 3(g). Average daytime concentrations were higher than night-time for aerosol H +, sulfate, fine mass and ozone. Prolonged episodes of atmospheric acidity, sulfate, and ozone were associated with air masses arriving at the measurement site from the west and from the southwest over Lake Erie. Sulfate concentrations measured at the lakeshore camp were more than twice those measured at inland sites during extreme pollution episodes. The concentration gradient observed with onshore flow was potentially due to enhanced deposition near the lakeshore caused by discontinuities in the meteorological fields in this region.

  17. Specific lysosomal transport of small neutral amino acids

    SciTech Connect

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

    1986-05-01

    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-(/sup 14/C)proline (50 ..mu..M) uptake by fibroblast lysosomes at 37/sup 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-(/sup 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/sup +/ is required for most proline and alanine transport, lysosomal uptake of these amino acids occurs independently of Na/sup +/.

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

    PubMed

    Pratelli, Réjane; Pilot, Guillaume

    2014-10-01

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

  19. Renal Transport of Uric Acid: Evolving Concepts and Uncertainties

    PubMed Central

    Bobulescu, Ion Alexandru; Moe, Orson W.

    2013-01-01

    In addition to its role as a metabolic waste product, uric acid has been proposed to be an important molecule with multiple functions in human physiology and pathophysiology and may be linked to human diseases beyond nephrolithiasis and gout. Uric acid homeostasis is determined by the balance between production, intestinal secretion, and renal excretion. The kidney is an important regulator of circulating uric acid levels, by reabsorbing around 90% of filtered urate, while being responsible for 60–70% of total body uric acid excretion. Defective renal handling of urate is a frequent pathophysiologic factor underpinning hyperuricemia and gout. In spite of tremendous advances over the past decade, the molecular mechanisms of renal urate transport are still incompletely understood. Many transport proteins are candidate participants in urate handling, with URAT1 and GLUT9 being the best characterized to date. Understanding these transporters is increasingly important for the practicing clinician as new research unveils their physiology, importance in drug action, and genetic association with uric acid levels in human populations. The future may see the introduction of new drugs that specifically act on individual renal urate transporters for the treatment of hyperuricemia and gout. PMID:23089270

  20. Neutral amino acid transport across brain microvessel endothelial cell monolayers

    SciTech Connect

    Audus, K.L.; Borchardt, R.T.

    1986-03-01

    Brain microvessel endothelial cells (BMEC) which form the blood-brain barrier (BBB) possess an amino acid carrier specific for large neutral amino acids (LNAA). The carrier is important for facilitating the delivery of nutrient LNAA's and centrally acting drugs that are LNAA's, to the brain. Bovine BMEC's were isolated and grown up to complete monolayers on regenerated cellulose-membranes in primary culture. To study the transendothelial transport of leucine, the monolayers were placed in a side-by-side diffusion cell, and transport across the monolayers followed with (/sup 3/H)-leucine. The transendothelial transport of leucine in this in vitro model was determined to be bidirectional, and time-, temperature-, and concentration-dependent. The transport of leucine was saturable and the apparent K/sub m/ and V/sub max/, 0.18 mM and 6.3 nmol/mg/min, respectively. Other LNAA's, including the centrally acting drugs, ..cap alpha..-methyldopa, L-DOPA, ..cap alpha..-methyl-tyrosine, and baclofen, inhibited leucine transport. The leucine carrier was also found to be stereospecific and not sensitive to inhibitors of active transport. These results are consistent with previous in vitro and in vivo studies. Primary cultures of BMEC's appear to be a potentially important tool for investigating at the cellular level, the transport mechanisms of the BBB.

  1. Acid-base transport in pancreas—new challenges

    PubMed Central

    Novak, Ivana; Haanes, Kristian A.; 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 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 HCO−3 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 signaling, 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. PMID:24391597

  2. Transport of Amino Acids to the Maize Root 1

    PubMed Central

    Oaks, Ann

    1966-01-01

    When 5-mm maize root tips were excised and placed in an inorganic salts solution for 6 hours, there was a loss of alcohol-insoluble nitrogen. The levels of threonine, proline, valine, isoleucine, leucine, tyrosine, phenylalanine, and lysine in the alcohol soluble fraction were severely reduced, whereas those of glutamate, aspartate, ornithine, and alanine were scarcely affected. There was a 4-fold increase in the level of γ-aminobutyrate. Those amino acids whose synthesis appeared to be deficient in excised root tips also showed poor incorporation of acetate carbon. In addition, the results show that asparagine and the amino acids of the neutral and basic fraction were preferentially transported to the root tip region. The results therefore suggest that the synthesis of certain amino acids in the root tip region is restricted, and that this requirement for amino acids in the growing region could regulate the flow of amino acids to the root tip. PMID:16656225

  3. The involvement of L-type amino acid transporters in theanine transport.

    PubMed

    Yamamoto, Sachiko; Kimura, Toru; Tachiki, Takashi; Anzai, Naohiko; Sakurai, Takuya; Ushimaru, Makoto

    2012-01-01

    L-Theanine has favorable physiological effects in terms of human health, but the mechanisms that transport it to its target organs or cells are not completely defined. To identify the major transport mechanisms of L-theanine, we screened for candidate transporters of L-3H-theanine in several mammal cell lines that intrinsically express multiple transporters with various specificities. All of the cells tested, T24, HepG2, COS1, 293A, Neuro2a, and HuH7, absorbed L-3H-theanine. Uptake was significantly inhibited by the addition of L-leucine and by a specific inhibitor of the system L transport system, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). L-3H-Theanine uptake occurred mostly independently of Na+. These results indicate that L-theanine was taken up via a system L like transport system in all of the cells tested. Additionally, in experiments using cells stably expressing two system L isoforms, LAT1 and LAT2, we found that the two isoforms mediated L-theanine transport to similar extents. Taken together, our results indicate that L-theanine is transported mostly via the system L transport pathway and its isoforms.

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

    PubMed

    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

    2015-06-01

    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 [(14)C]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 [(14)C]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.

  5. Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells.

    PubMed

    Mitchell, Ryan W; On, Ngoc H; Del Bigio, Marc R; Miller, Donald W; Hatch, Grant M

    2011-05-01

    The blood-brain barrier (BBB), formed by the brain capillary endothelial cells, provides a protective barrier between the systemic blood and the extracellular environment of the CNS. Passage of fatty acids from the blood to the brain may occur either by diffusion or by proteins that facilitate their transport. Currently several protein families have been implicated in fatty acid transport. The focus of the present study was to identify the fatty acid transport proteins (FATPs) expressed in the brain microvessel endothelial cells and characterize their involvement in fatty acid transport across an in vitro BBB model. The major fatty acid transport proteins expressed in human brain microvessel endothelial cells (HBMEC), mouse capillaries and human grey matter were FATP-1, -4 and fatty acid binding protein 5 and fatty acid translocase/CD36. The passage of various radiolabeled fatty acids across confluent HBMEC monolayers was examined over a 30-min period in the presence of fatty acid free albumin in a 1 : 1 molar ratio. The apical to basolateral permeability of radiolabeled fatty acids was dependent upon both saturation and chain length of the fatty acid. Knockdown of various fatty acid transport proteins using siRNA significantly decreased radiolabeled fatty acid transport across the HBMEC monolayer. Our findings indicate that FATP-1 and FATP-4 are the predominant fatty acid transport proteins expressed in the BBB based on human and mouse expression studies. While transport studies in HBMEC monolayers support their involvement in fatty acid permeability, fatty acid translocase/CD36 also appears to play a prominent role in transport of fatty acids across HBMEC.

  6. Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter.

    PubMed

    Zhen, Hongmin; Nakamura, Koichi; Kitaura, Yasuyuki; Kadota, Yoshihiro; Ishikawa, Takuya; Kondo, Yusuke; Xu, Minjun; Shimomura, Yoshiharu

    2015-01-01

    Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.

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

    NASA Technical Reports Server (NTRS)

    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.

  8. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  9. Inorganic nanoparticles as nucleic acid transporters into eukaryotic cells

    NASA Astrophysics Data System (ADS)

    Amirkhanov, R. N.; Zarytova, V. F.; Zenkova, M. A.

    2017-02-01

    The review is concerned with inorganic nanoparticles (gold, titanium dioxide, silica, iron oxides, calcium phosphate) used as nucleic acid transporters into mammalian cells. Methods for the synthesis of nanoparticles and approaches to surface modification through covalent or noncovalent attachment of low- or high-molecular-weight compounds are considered. The data available from the literature on biological action of nucleic acids delivered into the cells by nanoparticles and on the effect of nanoparticles and their conjugates and complexes on the cell survival are summarized. Pathways of cellular internalization of nanoparticles and the mechanism of their excretion, as well as the ways of release of nucleic acids from their complexes with nanoparticles after the cellular uptake are described. The bibliography includes 161 references.

  10. Neutralizing aspartate 83 modifies substrate translocation of excitatory amino acid transporter 3 (EAAT3) glutamate transporters.

    PubMed

    Hotzy, Jasmin; Machtens, Jan-Philipp; Fahlke, Christoph

    2012-06-08

    Excitatory amino acid transporters (EAATs) terminate glutamatergic synaptic transmission by removing glutamate from the synaptic cleft into neuronal and glial cells. EAATs are not only secondary active glutamate transporters but also function as anion channels. Gating of EAAT anion channels is tightly coupled to transitions within the glutamate uptake cycle, resulting in Na(+)- and glutamate-dependent anion currents. A point mutation neutralizing a conserved aspartic acid within the intracellular loop close to the end of transmembrane domain 2 was recently shown to modify the substrate dependence of EAAT anion currents. To distinguish whether this mutation affects transitions within the uptake cycle or directly modifies the opening/closing of the anion channel, we used voltage clamp fluorometry. Using three different sites for fluorophore attachment, V120C, M205C, and A430C, we observed time-, voltage-, and substrate-dependent alterations of EAAT3 fluorescence intensities. The voltage and substrate dependence of fluorescence intensities can be described by a 15-state model of the transport cycle in which several states are connected to branching anion channel states. D83A-mediated changes of fluorescence intensities, anion currents, and secondary active transport can be explained by exclusive modifications of substrate translocation rates. In contrast, sole modification of anion channel opening and closing is insufficient to account for all experimental data. We conclude that D83A has direct effects on the glutamate transport cycle and that these effects result in changed anion channel function.

  11. Aluminum in acidic surface waters: chemistry, transport, and effects.

    PubMed Central

    Driscoll, C T

    1985-01-01

    Ecologically significant concentrations of Al have been reported in surface waters draining "acid-sensitive" watersheds that are receiving elevated inputs of acidic deposition. It has been hypothesized that mineral acids from atmospheric deposition have remobilized Al previously precipitated within the soil during soil development. This Al is then thought to be transported to adjacent surface waters. Dissolved mononuclear Al occurs as aquo Al, as well as OH-, F-, SO4(2-), and organic complexes. Although past investigations have often ignored non-hydroxide complexes of Al, it appears that organic and F complexes are the predominant forms of Al in dilute (low ionic strength) acidic surface waters. The concentration of inorganic forms of Al increases exponentially with decreases in solution pH. This response is similar to the theoretical pH dependent solubility of Al mineral phases. The concentration of organic forms of Al, however, is strongly correlated with variations in organic carbon concentration of surface waters rather than pH. Elevated concentrations of Al in dilute acidic waters are of interest because: Al is an important pH buffer; Al may influence the cycling of important elements like P, organic carbon, and trace metals; and Al is potentially toxic to aquatic organisms. An understanding of the aqueous speciation of Al is essential for an evaluation of these processes. PMID:3935428

  12. Choline inhibition of amino acid transport in preimplantation mouse blastocysts

    SciTech Connect

    Campione, A.L.; Haghighat, N.; Gorman, J.; Van Winkle, L.J.

    1987-05-01

    Addition of 70 mM choline chloride to Brinster's medium (140 mM Na/sup +/) inhibited uptake of approx. 1 ..mu..M (/sup 3/H)glycine, leucine, lysine and alanine in blastocysts by about 50% each during a five-minute incubation period at 37/sup 0/C, whereas 70 mM LiCl, sodium acetate and NaCl or 140 mM mannitol had no effect. They attribute the apparent linear relationship between Gly transport in blastocysts and the square of the (Na/sup +/), observed when choline was substituted for Na/sup +/ in Brinster's medium, to concomitant, concentration-dependent enhancement and inhibition of transport by Na/sup +/ and choline, respectively. As expected, Gly uptake and the (Na/sup +/) were linearly related up to 116 mM Na/sup +/, when Na/sup +/ was replaced with Li/sup +/. The rates of Na/sup +/-independent Gly and Ala uptake were <5% and <2% of the total, respectively, and similar when either Li/sup +/ or choline replaced Na/sup +/. Therefore, neither Li/sup +/ nor choline appears to substitute for Na/sup +/ in supporting Na/sup +/-dependent transport in blastocysts. Na/sup +/-independent Leu uptake was 20 times faster than Gly or Ala uptake and appeared to be inhibited by choline in blastocysts since it was about 37% slower when choline instead of Li/sup +/ was substituted for Na/sup +/. In contrast to blastocysts, choline had no effect on amino acid transport in cleavage-stage mouse embryos. The unexpected sensitivity of transport to choline in blastocysts underscores the importance of testing the effects of this substance when it is used to replace Na/sup +/ in new transport studies.

  13. Perfluorocarboxylic acid (PFCA) atmospheric formation and transport to the Arctic.

    NASA Astrophysics Data System (ADS)

    Pike-thackray, C.; Selin, N. E.

    2015-12-01

    Perfluorocarboxylic acids (PFCAs) are highly persistent and toxic environmental contaminants that have been found in remote locations such as the Arctic, far from emission sources. These persistent organic pollutants are emitted directly to the atmosphere as well as being produced by the degradation of precursor compounds in the atmosphere, but recent trends towards increasing precursor emissions and decreasing direct emissions raise the importance of production in the atmosphere. Our work aims to improve understanding of the atmospheric degradation of fluorotelomer precursor compounds to form the long-chain PFCAs PFOA (C8) and PFNA (C9).Using the atmospheric chemical transport model GEOS-Chem, which uses assimilated meteorology to simulate the atmospheric transport of trace gas species, we investigate the interaction of the atmospheric formation of PFCAs and the atmospheric transport of their precursor species. Our simulations are a first application of the GEOS-Chem framework to PFCA chemistry. We highlight the importance of the spatial and temporal variability of background atmospheric chemical conditions experienced during transport. We find that yields and formation times of PFOA and PFNA respond differently and strongly to the photochemical conditions of the atmosphere, such as the abundance of NO, HO2, and other photochemical species.

  14. Endothelium as a gatekeeper of fatty acid transport

    PubMed Central

    Mehrotra, Devi; Wu, Jingxia; Papangeli, Irinna; Chun, Hyung J.

    2013-01-01

    The endothelium transcends all clinical disciplines and is key to the function of every organ system. A crucial, but poorly understood role of the endothelium is its ability to control the transport of energy supply according to organ needs. Fatty acids (FAs) in particular represent a key energy source that is utilized by a number of tissues, but whose utilization must be tightly regulated to avoid potentially deleterious consequences of excess accumulation, including insulin resistance. Recent studies have identified key endothelial signaling mechanisms involving vascular endothelial growth factor B, peroxisome proliferator-activated receptor-γ, and the peptide ligand apelin, that are critical to endothelial regulation of FA transport. Here we discuss the mechanisms by which these signaling pathways regulate this key endothelial function. PMID:24315207

  15. Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1-7-Pat1 complex.

    PubMed

    Chowdhury, Ashis; Raju, Kalidindi K; Kalurupalle, Swathi; Tharun, Sundaresan

    2012-05-01

    Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa- or heptameric RNA-binding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 5'-3' mRNA decay pathway. This complex also protects mRNA 3'-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 3'-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 3'-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.

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

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thévenieau, France; Nicaud, Jean-Marc

    2015-09-01

    Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast.

  17. Antibacterial drug treatment increases intestinal bile acid absorption via elevated levels of ileal apical sodium-dependent bile acid transporter but not organic solute transporter α protein.

    PubMed

    Miyata, Masaaki; Hayashi, Kenjiro; Yamakawa, Hiroki; Yamazoe, Yasushi; Yoshinari, Kouichi

    2015-01-01

    Antibacterial drug treatment increases the bile acid pool size and hepatic bile acid concentration through the elevation of hepatic bile acid synthesis. However, the involvement of intestinal bile acid absorption in the increased bile acid pool size remains unclear. To determine whether intestinal bile acid absorption contributes to the increased bile acid pool in mice treated with antibacterial drugs, we evaluated the levels of bile acid transporter proteins and the capacity of intestinal bile acid absorption. Ileal apical sodium-dependent bile acid transporter (ASBT) mRNA and protein levels were significantly increased in ampicillin (ABPC)-treated mice, whereas organic solute transporter α (OSTα) mRNA levels, but not protein levels, significantly decreased in mice. Similar alterations in the expression levels of bile acid transporters were observed in mice treated with bacitracin/neomycin/streptomycin. The capacity for intestinal bile acid absorption was evaluated by an in situ loop method. Increased ileal absorption of taurochenodeoxycholic acid was observed in mice treated with ABPC. These results suggest that intestinal bile acid absorption is elevated in an ASBT-dependent manner in mice treated with antibacterial drugs.

  18. Intracellular dehydroascorbic acid inhibits SVCT2-dependent transport of ascorbic acid in mitochondria.

    PubMed

    Fiorani, Mara; Azzolini, Catia; Guidarelli, Andrea; Cerioni, Liana; Scotti, Maddalena; Cantoni, Orazio

    2015-09-01

    Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA.

  19. Transport of heptafluorostearate across model membranes. Membrane transport of long-chain fatty acid anions I.

    PubMed

    Schmider, W; Fahr, A; Blum, H E; Kurz, G

    2000-05-01

    Heptafluorostearic acid, an isogeometric derivative of stearic acid, has a pK(a) value of about 0.5. To evaluate the suitability of heptafluorostearate as model compound for anions of long-chain fatty acids in membrane transport, monolayer and liposome studies were performed with lipid mixtures containing phospholipids;-cholesterol-heptafluorostearate or stearate (100:40:20 molar ratios). Transfer of heptafluorostearate and stearate from liposomes to bovine serum albumin (BSA) was followed by measuring the intrinsic fluorescence of BSA. The percentage of heptafluorostearate, equivalent to the amount placed in their outer monolayer, transferred from liposomes (120;-130 nm diameter) to BSA was 55.7 +/- 3.7% within 10 min at 25 degrees C and 55 +/- 2% within 5 min at 37 degrees C. Slow transfer of 22.7 +/- 2.5% of heptafluorostearate at 25 degrees C followed with a half-life of 2.3 +/- 0.4 h and of 20 +/- 4% at 37 degrees C with a half-life of 0.9 +/- 0.1 h until the final equilibrium distributions between BSA and liposomes were reached, 79 +/- 6% to 21 +/- 5% at 25 degrees C and 75 +/- 5% to 25 +/- 4% at 37 degrees C. The pseudounimolecular rate constants for flip-flop of heptafluorostearate equal k(FF,25) = 0.24 +/- 0.05 h(-) and k(FF,37) = 0.6 +/- 0.1 h(-), respectively. By comparison, transfer of stearate required only 3 min to reach equilibrium distribution. The difference between heptafluorostearate and stearate may be explained by a rapid flip-flop movement of the un-ionized fatty acids which exist in different concentrations in accordance with their pK(a) values. Half-life of flip-flop of heptafluorostearate makes it suitable to study mediated membrane transport of long-chain fatty acid anions.

  20. Transport of two naphthoic acids and salicylic acid in soil: experimental study and empirical modeling.

    PubMed

    Hanna, K; Lassabatere, L; Bechet, B

    2012-09-15

    In contrast to the parent compounds, the mechanisms responsible for the transport of natural metabolites of polycyclic aromatic hydrocarbons (PAH) in contaminated soils have been scarcely investigated. In this study, the sorption of three aromatic acids (1-naphthoic acid (NA), 1-hydroxy-2-naphthoic acid (HNA) and salicylic acid (SA)) was examined on soil, in a batch equilibrium single-system, with varying pH and acid concentrations. Continuous flow experiments were also carried out under steady-state water flow. The adsorption behavior of naphthoic and benzoic acids was affected by ligand functionality and molecular structure. All modeling options (equilibrium, chemical nonequilibrium, i.e. chemical kinetics, physical nonequilibrium, i.e. surface sites in the immobile water fraction, and both chemical and physical nonequilibrium) were tested in order to describe the breakthrough behavior of organic compounds in homogeneously packed soil columns. Tracer experiments showed a small fractionation of flow into mobile and immobile compartments, and the related hydrodynamic parameters were used for the modeling of reactive transport. In all cases, the isotherm parameters obtained from column tests differed from those derived from the batch experiments. The best accurate modeling was obtained considering nonequilibrium for the three organic compounds. Both chemical and physical nonequilibrium led to appropriate modeling for HNA and NA, while chemical nonequilibrium was the sole option for SA. SA sorption occurs mainly in mobile water and results from the concomitancy of instantaneous and kinetically limited sites. For all organic compounds, retention is contact condition dependent and differs between batch and column experiments. Such results show that preponderant mechanisms are solute dependent and kinetically limited, which has important implications for the fate and transport of carboxylated aromatic compounds in contaminated soils.

  1. Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Rashotte, Aaron M.; Poupart, Julie; Waddell, Candace S.; Muday, Gloria K.; Brown, C. S. (Principal Investigator)

    2003-01-01

    Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.

  2. Increased Rat Placental Fatty Acid, but Decreased Amino Acid and Glucose Transporters Potentially Modify Intrauterine Programming.

    PubMed

    Nüsken, Eva; Gellhaus, Alexandra; Kühnel, Elisabeth; Swoboda, Isabelle; Wohlfarth, Maria; Vohlen, Christina; Schneider, Holm; Dötsch, Jörg; Nüsken, Kai-Dietrich

    2016-07-01

    Regulation of placental nutrient transport significantly affects fetal development and may modify intrauterine growth restriction (IUGR) and fetal programming. We hypothesized that placental nutrient transporters are differentially affected both by utero-placental insufficiency and prenatal surgical stress. Pregnant rats underwent bilateral uterine artery and vein ligation (LIG), sham operation (SOP) or no operation (controls, C) on gestational day E19. Placentas were obtained by caesarean section 4 h (LIG, n=20 placentas; SOP, n=24; C, n=12), 24 h (LIG, n=28; SOP, n=20; C, n=12) and 72 h (LIG, n=20; SOP, n=20; C, n=24) after surgery. Gene and protein expression of placental nutrient transporters for fatty acids (h-FABP, CD36), amino acids (SNAT1, SNAT2) and glucose (GLUT-1, Connexin 26) were examined by qRT-PCR, western blot and immunohistochemistry. Interestingly, the mean protein expression of h-FABP was doubled in placentas of LIG and SOP animals 4, 24 (SOP significant) and 72 h (SOP significant) after surgery. CD36 protein was significantly increased in LIG after 72 h. SNAT1 and SNAT2 protein and gene expressions were significantly reduced in LIG and SOP after 24 h. Further significantly reduced proteins were GLUT-1 in LIG (4 h, 72 h) and SOP (24 h), and Connexin 26 in LIG (72 h). In conclusion, placental nutrient transporters are differentially affected both by reduced blood flow and stress, probably modifying the already disturbed intrauterine milieu and contributing to IUGR and fetal programming. Increased fatty acid transport capacity may affect energy metabolism and could be a compensatory reaction with positive effects on brain development. J. Cell. Biochem. 117: 1594-1603, 2016. © 2015 Wiley Periodicals, Inc.

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

    PubMed Central

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-01-01

    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. PMID:25915115

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

    PubMed

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-04-23

    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.

  5. Impact of Microbial Growth on Subsurface Perfluoroalkyl Acid Transport

    NASA Astrophysics Data System (ADS)

    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.

  6. Few Amino Acid Exchanges Expand the Substrate Spectrum of Monocarboxylate Transporter 10.

    PubMed

    Johannes, Jörg; Braun, Doreen; Kinne, Anita; Rathmann, Daniel; Köhrle, Josef; Schweizer, Ulrich

    2016-07-01

    Monocarboxylate transporters (MCTs) belong to the SLC16 family within the major facilitator superfamily of transmembrane transporters. MCT8 is a thyroid hormone transporter mutated in the Allan-Herndon-Dudley syndrome, a severe psychomotor retardation syndrome. MCT10 is closely related to MCT8 and is known as T-type amino acid transporter. Both transporters mediate T3 transport, but although MCT8 also transports rT3 and T4, these compounds are not efficiently transported by MCT10, which, in contrast, transports aromatic amino acids. Based on the 58% amino acid identity within the transmembrane regions among MCT8 and MCT10, we reasoned that substrate specificity may be primarily determined by a small number of amino acid differences between MCT8 and MCT10 along the substrate translocation channel. Inspecting the homology model of MCT8 and a structure-guided alignment between both proteins, we selected 8 amino acid positions and prepared chimeric MCT10 proteins with selected amino acids changed to the corresponding amino acids in MCT8. The MCT10 mutant harboring 8 amino acid substitutions was stably expressed in Madin-Darby canine kidney 1 cells and found to exhibit T4 transport activity. We then successively reduced the number of amino acid substitutions and eventually identified a minimal set of 2-3 amino acid exchanges which were sufficient to allow T4 transport. The resulting MCT10 chimeras exhibited KM values for T4 similar to MCT8 but transported T4 at a slower rate. The acquisition of T4 transport by MCT10 was associated with complete loss of the capacity to transport Phe, when Tyr184 was mutated to Phe.

  7. Acid-base transport by the renal proximal tubule

    PubMed Central

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

    2015-01-01

    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. PMID:21170887

  8. Intestinal transport of sugars and amino acids in diabetic rats

    PubMed Central

    Olsen, Ward A.; Rosenberg, Irwin H.

    1970-01-01

    The specificity and mechanism of altered intestinal transport of diabetic rats was studied with an everted ring technique. Increased intracellular accumulation of amino acids, as well as galactose and 3-O-methylglucose, was demonstrated in diabetes. The greater accumulation by diabetic intestine could not be attributed to a direct effect of the agent used to induce diabetes or to an alteration in food consumption. Although the changes were related to the severity of diabetes and could be reversed with treatment with insulin, they could not be modified by addition of insulin in vitro. The changes could not be induced in control intestine either with hyperglycemia from glucose infusion or preincubation with glucose in vitro. Although the higher concentration gradients of amino acids, galactose, and 3-O-methylglucose could result from increased energy utilization by diabetic intestine, an alteration of cell membrane function, as well, is suggested by the demonstration with kinetic studies of increased influx with an increase in Vmax. PMID:5409812

  9. The transport of uric acid across mouse small intestine in vitro.

    PubMed Central

    Bronk, J R; Shaw, M I

    1986-01-01

    The in vitro recirculation technique was used to study the uptake and transport of uric acid by the jejunum of mouse small intestine. Three components of the serosal secretions appeared to be endogenously derived nucleic acid derivatives; two of these were identified as uric acid and uracil. There was no detectable metabolism of uric acid by the intestine. Uric acid transported from the lumen appeared in the serosal fluid at a concentration higher than that in the lumen. The final serosal/luminal concentration ratio of about 1.18 for exogenous uric acid was found to be constant over the concentration range studied (0.01-0.1 mM). The presence of exogenous uric acid in the lumen did not affect the production of endogenous uric acid by the intestine and its release into the serosal secretions. Mucosal concentration of exogenous uric acid was below, but the total mucosal concentration (exogenous+endogenous) was above, that in the lumen. There was no evidence for the secretion of endogenous uric acid into the lumen. Oxypurinol significantly decreased the rate of serosal appearance of exogenous uric acid. Allopurinol did not affect the transport of exogenous uric acid from the lumen and there was negligible metabolism of allopurinol to oxypurinol by the tissue. Uracil did not affect the transport of exogenous uric acid from the lumen, or the serosal appearance of endogenous uric acid. Likewise uracil transport was unaffected by luminal uric acid. PMID:3795104

  10. Butyric acid increases transepithelial transport of ferulic acid through upregulation of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4).

    PubMed

    Ziegler, Kerstin; Kerimi, Asimina; Poquet, Laure; Williamson, Gary

    2016-06-01

    Ferulic acid is released by microbial hydrolysis in the colon, where butyric acid, a major by-product of fermentation, constitutes the main energy source for colonic enterocytes. We investigated how varying concentrations of this short chain fatty acid may influence the absorption of the phenolic acid. Chronic treatment of Caco-2 cells with butyric acid resulted in increased mRNA and protein abundance of the monocarboxylate transporters SLC16A1 (MCT1) and SLC16A3 (MCT4), previously proposed to facilitate ferulic acid absorption in addition to passive diffusion. Short term incubation with butyric acid only led to upregulation of MCT4 while both conditions increased transepithelial transport of ferulic acid in the apical to basolateral, but not basolateral to apical, direction. Chronic treatment also elevated intracellular concentrations of ferulic acid, which in turn gave rise to increased concentrations of ferulic acid metabolites. Immunofluorescence staining of cells revealed uniform distribution of MCT1 protein in the cell membrane, whereas MCT4 was only detected in the lateral plasma membrane sections of Caco-2 cells. We therefore propose that MCT1 may be acting as an uptake transporter and MCT4 as an efflux system across the basolateral membrane for ferulic acid, and that this process is stimulated by butyric acid.

  11. Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

    PubMed Central

    Cássio, F; Leáo, C

    1991-01-01

    Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1664712

  12. Cationic amino acid transporters play key roles in the survival and transmission of apicomplexan parasites

    PubMed Central

    Rajendran, Esther; Hapuarachchi, Sanduni V.; Miller, Catherine M.; Fairweather, Stephen J.; Cai, Yeping; Smith, Nicholas C.; Cockburn, Ian A.; Bröer, Stefan; Kirk, Kiaran; van Dooren, Giel G.

    2017-01-01

    Apicomplexans are obligate intracellular parasites that scavenge essential nutrients from their hosts via transporter proteins on their plasma membrane. The identities of the transporters that mediate amino acid uptake into apicomplexans are unknown. Here we demonstrate that members of an apicomplexan-specific protein family—the Novel Putative Transporters (NPTs)—play key roles in the uptake of cationic amino acids. We show that an NPT from Toxoplasma gondii (TgNPT1) is a selective arginine transporter that is essential for parasite survival and virulence. We also demonstrate that a homologue of TgNPT1 from the malaria parasite Plasmodium berghei (PbNPT1), shown previously to be essential for the sexual gametocyte stage of the parasite, is a cationic amino acid transporter. This reveals a role for cationic amino acid scavenging in gametocyte biology. Our study demonstrates a critical role for amino acid transporters in the survival, virulence and life cycle progression of these parasites. PMID:28205520

  13. Intestinal transport of zinc and folic acid: a mutual inhibitory effect

    SciTech Connect

    Ghishan, F.K.; Said, H.M.; Wilson, P.C.; Murrell, J.E.; Greene, H.L.

    1986-02-01

    Recent observations suggest an inverse relationship between folic acid intake and zinc nutriture and indicate an interaction between folic acid and zinc at the intestinal level. To define that interaction, we designed in vivo and in vitro transport studies in which folic acid transport in the presence of zinc, as well as zinc transport in the presence of folic acid was examined. These studies show that zinc transport is significantly decreased when folate is present in the intestinal lumen. Similarly folic acid transport is significantly decreased with the presence of zinc. To determine whether this intestinal inhibition is secondary to zinc and folate-forming complexes, charcoal-binding studies were performed. These studies indicate that zinc and folate from complexes at pH 2.0, but that at pH 6.0, these complexes dissolve. Therefore, our studies suggest that under normal physiological conditions a mutual inhibition between folate and zinc exists at the site of intestinal transport.

  14. Cationic amino acid transporters play key roles in the survival and transmission of apicomplexan parasites.

    PubMed

    Rajendran, Esther; Hapuarachchi, Sanduni V; Miller, Catherine M; Fairweather, Stephen J; Cai, Yeping; Smith, Nicholas C; Cockburn, Ian A; Bröer, Stefan; Kirk, Kiaran; van Dooren, Giel G

    2017-02-16

    Apicomplexans are obligate intracellular parasites that scavenge essential nutrients from their hosts via transporter proteins on their plasma membrane. The identities of the transporters that mediate amino acid uptake into apicomplexans are unknown. Here we demonstrate that members of an apicomplexan-specific protein family-the Novel Putative Transporters (NPTs)-play key roles in the uptake of cationic amino acids. We show that an NPT from Toxoplasma gondii (TgNPT1) is a selective arginine transporter that is essential for parasite survival and virulence. We also demonstrate that a homologue of TgNPT1 from the malaria parasite Plasmodium berghei (PbNPT1), shown previously to be essential for the sexual gametocyte stage of the parasite, is a cationic amino acid transporter. This reveals a role for cationic amino acid scavenging in gametocyte biology. Our study demonstrates a critical role for amino acid transporters in the survival, virulence and life cycle progression of these parasites.

  15. Functional transformations of bile acid transporters induced by high-affinity macromolecules

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Alam, Farzana; Park, Jooho; Lee, Kyung Eun; Jeon, Hyesung; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2014-01-01

    Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery. PMID:24566561

  16. L-aspartic acid transport by cat erythrocytes

    SciTech Connect

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

    1986-03-01

    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 /sup 3/H-L-asp (typically 2..mu..M) was measured in washed RBCs incubated for 60 s at 37/sup 0/C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl/sub 2/, 15 mM MOPS pH 7.4, 5 mM glucose, and /sup 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 0.5-1000..mu..M, 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 ..mu..M and 148.8 +/- 7.2 ..mu..mol 1. cell/sup -1/h/sup -1/ respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4/sup +/M L-asp, 40/sup +/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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26635971

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

    PubMed

    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.

  19. Retinoic acid induces expression of the thyroid hormone transporter, monocarboxylate transporter 8 (Mct8).

    PubMed

    Kogai, Takahiko; Liu, Yan-Yun; Richter, Laura L; Mody, Kaizeen; Kagechika, Hiroyuki; Brent, Gregory A

    2010-08-27

    Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. Mct8 (monocarboxylate transporter 8), expressed predominantly in the brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells toward neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All-trans-RA (tRA) and other RA receptor (RAR) agonists dramatically (>300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1- and 4.3-fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and 5'-rapid amplification of cDNA ends PCR analysis in F9 cells identified 11 transcription start sites and a proximal Sp1 site but no TATA box. tRA significantly enhanced Mct8 promoter activity through a consensus RA-responsive element located 6.6 kilobases upstream of the coding region. A chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid X receptor to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates cross-talk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.

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

    PubMed

    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.

  1. Report membrane transport of lactic acid in the filamentous fungus Rhizopus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fungus Rhizopus is frequently used for fermentative production of lactic acid, but little is known about the mechanisms or proteins for transporting this carboxylic acid. Since transport of the lactate anion across the plasma membrane is critical to prevent acidification of the cytoplasm, we ev...

  2. Properties of an Inducible C4-Dicarboxylic Acid Transport System in Bacillus subtilis

    PubMed Central

    Ghei, Om. K.; Kay, William W.

    1973-01-01

    The transport of the tricarboxylic acid cycle C4-dicarboxylic acids was studied in both the wild-type strain and tricarboxylic acid cycle mutants of Bacillus subtilis. Active transport of malate, fumarate, and succinate was found to be inducible by these dicarboxylic acids or by precursors to them, whereas glucose or closely related metabolites catabolite-repressed their uptake. l-Malate was found to be the best dicarboxylic acid transport inducer in succinic dehydrogenase, fumarase, and malic dehydrogenase mutants. Succinate and fumarate are accumulated over 100-fold in succinic dehydrogenase and fumarase mutants, respectively, whereas mutants lacking malate dehydrogenase were unable to accumulate significant quantities of the C4-dicarboxylic acids. The stereospecificity of this transport system was studied from a comparison of the rates of competitive inhibition of both succinate uptake and efflux in a succinate dehydrogenase mutant by utilizing thirty dicarboxylic acid analogues. The system was specific for the C4-dicarboxylic acids of the tricarboxylic acid cycle, neither citrate nor α-ketoglutarate were effective competitive inhibitors. Of a wide variety of metabolic inhibitors tested, inhibiors of oxidative phosphorylation and of the formation of proton gradients were the most potent inhibitors of transport. From the kinetics of dicarboxylic acid transport (Km approximately 10−4 M for succinate or fumarate in succinic acid dehydrogenase and fumarase mutants) and from the competitive inhibition studies, it was concluded that an inducible dicarboxylic acid transport system mediates the entry of malate, fumarate, or succinate into B. subtilis. Mutants devoid of α-ketoglutarate dehydrogenase were shown to accumulate both α-ketoglutarate and glutamate, and these metabolites subsequently inhibited the transport of all the C4-dicarboxylic acids, suggesting a regulatory role. Images PMID:4633350

  3. Adsorption and transport of polymaleic acid on Callovo-Oxfordian clay stone: batch and transport experiments.

    PubMed

    Durce, Delphine; Landesman, Catherine; Grambow, Bernd; Ribet, Solange; Giffaut, Eric

    2014-08-01

    Dissolved Organic Matter (DOM) can affect the mobility of radionuclides in pore water of clay-rich geological formations, such as those intended to be used for nuclear waste disposal. The present work studies the adsorption and transport properties of a polycarboxylic acid, polymaleic acid (PMA, Mw=1.9kDa), on Callovo-Oxfordian argillite samples (COx). Even though this molecule is rather different from the natural organic matter found in clay rock, the study of its retention properties on both dispersed and intact samples allows assessing to which extent organic acids may undergo sorption under natural conditions (pH7) and what could be the impact on their mobility. PMA sorption and desorption were investigated in dispersed systems. The degree of sorption was measured after 1, 8 and 21days and for a range of PMA initial concentrations from 4.5×10(-7) to 1.4×10(-3)mol.L(-1). The reversibility of the sorption process was estimated by desorption experiments performed after the sorption experiments. At the sorption steady state, the sorption was described by a two-site Langmuir model. A total sorption capacity of COx for PMA was found to be 1.01×10(-2) mol.kg(-1) distributed on two sorption sites, one weak and one strong. The desorption of PMA was incomplete, independently of the duration of the sorption phase. The amount of desorbable PMA even appeared to decrease for sorption phases from 1 to 21days. To describe the apparent desorption hysteresis, two conceptual models were applied. The two-box diffusion model accounted for intraparticle diffusion and more generally for nonequilibrium processes. The two-box first-order non-reversible model accounted for a first-order non-reversible sorption and more generally for kinetically-controlled irreversible sorption processes. The use of the two models revealed that desorption hysteresis was not the result of nonequilibrium processes but was due to irreversible sorption. Irreversible sorption on the strong site was

  4. Adsorption and transport of polymaleic acid on Callovo-Oxfordian clay stone: Batch and transport experiments

    NASA Astrophysics Data System (ADS)

    Durce, Delphine; Landesman, Catherine; Grambow, Bernd; Ribet, Solange; Giffaut, Eric

    2014-08-01

    Dissolved Organic Matter (DOM) can affect the mobility of radionuclides in pore water of clay-rich geological formations, such as those intended to be used for nuclear waste disposal. The present work studies the adsorption and transport properties of a polycarboxylic acid, polymaleic acid (PMA, Mw = 1.9 kDa), on Callovo-Oxfordian argillite samples (COx). Even though this molecule is rather different from the natural organic matter found in clay rock, the study of its retention properties on both dispersed and intact samples allows assessing to which extent organic acids may undergo sorption under natural conditions (pH 7) and what could be the impact on their mobility. PMA sorption and desorption were investigated in dispersed systems. The degree of sorption was measured after 1, 8 and 21 days and for a range of PMA initial concentrations from 4.5 × 10- 7 to 1.4 × 10- 3 mol.L- 1. The reversibility of the sorption process was estimated by desorption experiments performed after the sorption experiments. At the sorption steady state, the sorption was described by a two-site Langmuir model. A total sorption capacity of COx for PMA was found to be 1.01×10- 2 mol.kg- 1 distributed on two sorption sites, one weak and one strong. The desorption of PMA was incomplete, independently of the duration of the sorption phase. The amount of desorbable PMA even appeared to decrease for sorption phases from 1 to 21 days. To describe the apparent desorption hysteresis, two conceptual models were applied. The two-box diffusion model accounted for intraparticle diffusion and more generally for nonequilibrium processes. The two-box first-order non-reversible model accounted for a first-order non-reversible sorption and more generally for kinetically-controlled irreversible sorption processes. The use of the two models revealed that desorption hysteresis was not the result of nonequilibrium processes but was due to irreversible sorption. Irreversible sorption on the strong site was

  5. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-07

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  6. Study of Tranexamic Acid During Air Medical Prehospital Transport Trial (STAAMP trial)

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-13-2-0080 TITLE: Study of Tranexamic Acid During Air Medical Prehospital Transport Trial (STAAMP trial) PRINCIPAL INVESTIGATOR...TITLE AND SUBTITLE 5a. CONTRACT NUMBER Study of Tranexamic Acid During Air Medical Prehospital Transport Trial (STAAMP trial) 5b. GRANT NUMBER W81XWH...IRB approval regarding changes to the protocol language. 15. SUBJECT TERMS Prehospital; Tranexamic acid 16. SECURITY CLASSIFICATION OF: 17. LIMITATION

  7. Mechanism of L-lactic acid transport in L6 skeletal muscle cells.

    PubMed

    Kobayashi, Masaki; Itagaki, Shirou; Hirano, Takeshi; Iseki, Ken

    2004-10-01

    L-lactic acid transport plays an important role in the regulation of L-lactic acid circulation into and out of muscle. To clarify the transport mechanism of L-lactic acid in skeletal muscle, L-lactic acid uptake was investigated using a L6 cell line. mRNAs of monocarboxylate transporter (MCT) 1, 2 and 4 were found to be expressed in L6 cells. The [(14)C] L-lactic acid uptake by L6 cells increased up to pH of 6.0. The [(14)C] L-lactic acid uptake at pH 6.0 was concentration-dependent with a K(m) of 3.7 mM. This process was reduced by alpha-cyano-4-hydroxycinnamate, a typical MCT1, 2 and 4 inhibitor. These results suggest that an MCT participates in the uptake of L-lactic acid by L6 cells. [(14)C] L-lactic acid uptake was markedly inhibited by monocarboxylic acids and monocarboxylate drugs but not by dicarboxylic acids and amino acids. Moreover, benzoic acid, a substrate for MCT1, competitively inhibited this process with K(i) of 1.7 mM. [(14)C] L-lactic acid efflux in L6 cells was inhibited by alpha-cyano-4-hydroxycinnamate but not by benzoic acid. These results suggest that [(14)C] L-lactic acid efflux in L6 cells is mediated by MCT other than MCT1.

  8. [Human peritoneum in vitro: changes in urate transport after administration of pyrazinoic acid].

    PubMed

    Czyzewska, K; Grzegorzewska, A; Stawny, B; Knapowski, J

    1989-01-01

    The article is an analysis of the dynamics of two-direction transportation of uric acid (UA) through the human peritoneum in vitro, and also changes of the dynamics under the influence of pyrazinoic++ acid. The peritoneum was taken from the anterior abdominal wall of patients undergoing planned abdominal surgery. It was found that the transportation of UA both from the vascular to the mesothelial side of the peritoneal membrane and in the opposite direction remained on a stable level for 120 minutes. The introduction of pyrazinoic++ acid decreased the transportation of UA from the vascular to the mesothelial side of the peritoneum on the average by 50 per cent. The transportation in the opposite direction did not change. The results obtained are consistent with results of clinical examinations. One may suppose that pyrazinoic++ acid induces changes in transportation qualities of the peritoneum.

  9. Amino-acid transporters in T-cell activation and differentiation.

    PubMed

    Ren, W; Liu, G; Yin, J; Tan, B; Wu, G; Bazer, F W; Peng, Y; Yin, Y

    2017-03-02

    T-cell-mediated immune responses aim to protect mammals against cancers and infections, and are also involved in the pathogenesis of various inflammatory or autoimmune diseases. Cellular uptake and the utilization of nutrients is closely related to the T-cell fate decision and function. Research in this area has yielded surprising findings in the importance of amino-acid transporters for T-cell development, homeostasis, activation, differentiation and memory. In this review, we present current information on amino-acid transporters, such as LAT1 (l-leucine transporter), ASCT2 (l-glutamine transporter) and GAT-1 (γ-aminobutyric acid transporter-1), which are critically important for mediating peripheral naive T-cell homeostasis, activation and differentiation, especially for Th1 and Th17 cells, and even memory T cells. Mechanically, the influence of amino-acid transporters on T-cell fate decision may largely depend on the mechanistic target of rapamycin complex 1 (mTORC1) signaling. These discoveries remarkably demonstrate the role of amino-acid transporters in T-cell fate determination, and strongly indicate that manipulation of the amino-acid transporter-mTORC1 axis could ameliorate many inflammatory or autoimmune diseases associated with T-cell-based immune responses.

  10. Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2.

    PubMed

    Muñoz-Montesino, Carola; Roa, Francisco J; Peña, Eduardo; González, Mauricio; Sotomayor, Kirsty; Inostroza, Eveling; Muñoz, Carolina A; González, Iván; Maldonado, Mafalda; Soliz, Carlos; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

    2014-05-01

    Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport Km of 0.6mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is

  11. The role of L-type amino acid transporter 1 in human tumors

    PubMed Central

    Zhao, Yu; Wang, Lin; Pan, Jihong

    2015-01-01

    Summary L-type amino acid transporter 1 (LAT1) is an L-type amino acid transporter and transports large neutral amino acids such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, methionine, and histidine. LAT1 was found to be highly expressed especially in human cancer tissues, and up-regulated LAT1 can lead to dysfunction in human tumor cells. These findings suggest that LAT1 plays an important role in human tumors. This review provides an overview of the current understanding of LAT1 expression and its clinical significance and function in tumors. PMID:26668776

  12. Structural basis of the alternating-access mechanism in a bile acid transporter

    NASA Astrophysics Data System (ADS)

    Zhou, Xiaoming; Levin, Elena J.; Pan, Yaping; McCoy, Jason G.; Sharma, Ruchika; Kloss, Brian; Bruni, Renato; Quick, Matthias; Zhou, Ming

    2014-01-01

    Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBTNM) in detergent was reported recently, showing the protein in an inward-open conformation bound to two Na+ and a taurocholic acid. However, the structural changes that bring bile acid and Na+ across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na+ and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved `crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications

  13. Functional domains of the fatty acid transport proteins: studies using protein chimeras.

    PubMed

    DiRusso, Concetta C; Darwis, Dina; Obermeyer, Thomas; Black, Paul N

    2008-03-01

    Fatty acid transport proteins (FATP) function in fatty acid trafficking pathways, several of which have been shown to participate in the transport of exogenous fatty acids into the cell. Members of this protein family also function as acyl CoA synthetases with specificity towards very long chain fatty acids or bile acids. These proteins have two identifying sequence motifs: The ATP/AMP motif, an approximately 100 amino acid segment required for ATP binding and common to members of the adenylate-forming super family of proteins, and the FATP/VLACS motif that consists of approximately 50 amino acid residues and is restricted to members of the FATP family. This latter motif has been implicated in fatty acid transport in the yeast FATP orthologue Fat1p. In the present studies using a yeast strain containing deletions in FAT1 (encoding Fat1p) and FAA1 (encoding the major acyl CoA synthetase (Acsl) Faa1p) as an experimental platform, the phenotypic and functional properties of specific murine FATP1-FATP4 and FATP6-FATP4 protein chimeras were evaluated in order to define elements within these proteins that further distinguish the fatty acid transport and activation functions. As expected from previous work FATP1 and FATP4 were functional in the fatty acid transport pathway, while and FATP6 was not. All three isoforms were able to activate the very long chain fatty acids arachidonate (C(20:4)) and lignocerate (C(24:0)), but with distinguishing activities between saturated and highly unsaturated ligands. A 73 amino acid segment common to FATP1 and FATP4 and between the ATP/AMP and FATP/VLACS motifs was identified by studying the chimeras, which is hypothesized to contribute to the transport function.

  14. LAT1 is the transport competent unit of the LAT1/CD98 heterodimeric amino acid transporter.

    PubMed

    Napolitano, Lara; Scalise, Mariafrancesca; Galluccio, Michele; Pochini, Lorena; Albanese, Leticia Maria; Indiveri, Cesare

    2015-10-01

    LAT1 (SLC7A5) and CD98 (SLC3A2) constitute a heterodimeric transmembrane protein complex that catalyzes amino acid transport. Whether one or both subunits are competent for transport is still unclear. The present work aims to solve this question using different experimental strategies. Firstly, LAT1 and CD98 were immuno-detected in protein extracts from SiHa cells. Under oxidizing conditions, i.e., without addition of SH (thiol) reducing agent DTE, both proteins were revealed as a 120kDa major band. Upon DTE treatment separated bands, corresponding to LAT1(35kDa) or CD98(80kDa), were detected. LAT1 function was evaluated in intact cells as BCH sensitive [(3)H]His transport inhibited by hydrophobic amino acids. Antiport of [(3)H]His was measured in proteoliposomes reconstituted with SiHa cell extract in presence of internal His. Transport was increased by DTE. Hydrophobic amino acids were best inhibitors in addition to hydrophilic Tyr, Gln, Asn and Lys. Cys, Tyr and Gln, included in the intraliposomal space, were transported in antiport with external [(3)H]His. Similar experiments were performed in proteoliposomes reconstituted with the recombinant purified hLAT1. Results overlapping those obtained with native protein were achieved. Lower transport of [(3)H]Leu and [(3)H]Gln with respect to [(3)H]His was detected. Kinetic asymmetry was found with external Km for His lower than internal one. No transport was detected in proteoliposomes reconstituted with recombinant hCD98. The experimental data demonstrate that LAT1 is the sole transport competent subunit of the heterodimer. This conclusion has important outcome for following studies on functional characterization and identification of specific inhibitors with potential application in human therapy.

  15. Oleic acid stimulates system A amino acid transport in primary human trophoblast cells mediated by toll-like receptor 4.

    PubMed

    Lager, Susanne; Gaccioli, Francesca; Ramirez, Vanessa I; Jones, Helen N; Jansson, Thomas; Powell, Theresa L

    2013-03-01

    Obese women have an increased risk to deliver large babies. However, the mechanisms underlying fetal overgrowth in these pregnancies are not well understood. Obese pregnant women typically have elevated circulating lipid levels. We tested the hypothesis that fatty acids stimulate placental amino acid transport, mediated via toll-like receptor 4 (TLR4) and mammalian target of rapamycin (mTOR) signaling pathways. Circulating NEFA levels and placental TLR4 expression were assessed in women with varying prepregnancy body mass index (BMI). The effects of oleic acid on system A and system L amino acid transport, and on the activation of the mTOR (4EBP1, S6K1, rpS6), TLR4 (IĸB, JNK, p38 MAPK), and STAT3 signaling pathways were determined in cultured primary human trophoblast cells. Maternal circulating NEFAs (n = 33), but not placental TLR4 mRNA expression (n = 16), correlated positively with BMI (P < 0.05). Oleic acid increased trophoblast JNK and STAT3 phosphorylation (P < 0.05), whereas mTOR activity was unaffected. Furthermore, oleic acid doubled trophoblast system A activity (P < 0.05), without affecting system L activity. siRNA-mediated silencing of TLR4 expression prevented the stimulatory effect of oleic acid on system A activity. Our data suggest that maternal fatty acids can increase placental nutrient transport via TLR4, thereby potentially affecting fetal growth.

  16. Bile acid transport in sister of P-glycoprotein (ABCB11) knockout mice.

    PubMed

    Lam, Ping; Wang, Renxue; Ling, Victor

    2005-09-20

    In vertebrates, bile flow is essential for movement of water and solutes across liver canalicular membranes. In recent years, the molecular motor of canalicular bile acid secretion has been identified as a member of the ATP binding cassette transporter (ABC) superfamily, known as sister of P-glycoprotein (Spgp) or bile salt export pump (Bsep, ABCB11). In humans, mutations in the BSEP gene are associated with a very low level of bile acid secretion and severe cholestasis. However, as reported previously, because the spgp(-)(/)(-) knockout mice do not express severe cholestasis and have substantial bile acid secretion, we investigated the "alternative transport system" that allows these mice to be physiologically relatively normal. We examined the expression levels of several ABC transporters in spgp(-)(/)(-) mice and found that the level of multidrug resistance Mdr1 (P-glycoprotein) was strikingly increased while those of Mdr2, Mrp2, and Mrp3 were increased to only a moderate extent. We hypothesize that an elevated level of Mdr1 in the spgp(-)(/)(-) knockout mice functions as an alternative pathway to transport bile acids and protects hepatocytes from bile acid-induced cholestasis. In support of this hypothesis, we showed that plasma membrane vesicles isolated from a drug resistant cell line expressing high levels of P-glycoprotein were capable of transporting bile acids, albeit with a 5-fold lower affinity compared to Spgp. This finding is the first direct evidence that P-glycoprotein (Mdr1) is capable of transporting bile acids.

  17. Active transport of amino acids by a guanidiniocarbonyl-pyrrole receptor.

    PubMed

    Urban, Christian; Schmuck, Carsten

    2010-08-16

    Herein we report the synthesis and characterization of a transporter 9 for N-acetylated amino acids. Transporter 9 is a conjugate of a guanidiniocarbonyl pyrrole cation, one of the most efficient carboxylate binding motifs reported so far, and a hydrophobic tris(dodecylbenzyl) group, which ensures solubility in organic solvents. In its protonated form, 9 binds N-acetylated amino acid carboxylates in wet organic solvents with association constants in the range of 10(4) M(-1) as estimated by extraction experiments. Aromatic amino acids are preferred due to additional cation-pi-interactions of the amino acid side chain with the guanidiniocarbonyl pyrrole moiety. U-tube experiments established efficient transport across a bulk liquid chloroform phase with fluxes approaching 10(-6) mol m(-2) s(-1). In experiments with single substrates, the release rate of the amino acid from the receptor-substrate complex at the interface with the receiving phase is rate determining. In contrast to this, in competition experiments with several substrates, the thermodynamic affinity to 9 becomes decisive. As 9 can only transport anions in its protonated form and has a pK(a) value of approximately 7, pH-driven active transport of amino acids is also possible. Transport occurs as a symport of the amino acid carboxylate and a proton.

  18. Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes.

    PubMed

    Schulze, W; Frommer, W B; Ward, J M

    1999-03-01

    Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.

  19. Structure and mechanism of a Na+-independent amino acid transporter.

    PubMed

    Shaffer, Paul L; Goehring, April; Shankaranarayanan, Aruna; Gouaux, Eric

    2009-08-21

    Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.

  20. Transport and metabolic effects of alpha-aminoisobutyric acid in Saccharomyces cerevisiae.

    PubMed

    Kim, K W; Roon, R J

    1982-11-24

    alpha-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a Km for alpha-aminoisobutyric acid of 270 microM, a Vmax of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1-4.3. alpha-Aminoisobutyric acid is also transported by a minor system(s) with a Vmax of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The alpha-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. alpha-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH+4. During nitrogen starvation alpha-aminoisobutyric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that alpha-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because alpha-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.

  1. Characteristics of the transport of ascorbic acid into leucocytes

    SciTech Connect

    Raghoebar, M.; Huisman, J.A.M.; van den Berg, W.B.; van Ginneken, C.A.M.

    1987-02-02

    The degree and the mode of association of (/sup 14/C)-ascorbic acid with leucocytes are examined. The degree of association of ascorbic acid with polymorphonuclear leucocytes (1-3 %) is dependent on cell type, extracellular concentration of ascorbic acid, incubation temperature, intactness of the cells and the extracellular pH. All experiments are performed according to strict protocols as these compounds are labile in aqueous solutions. Further it is noticed that in all experiments an outward gradient of leucocyte endogenic ascorbic acid exists. The results suggest that the association process comprises at least one saturable pathway. The activation of polymorphonuclear leucocytes by phorbol myristate acetate increases the accumulation of ascorbic acid threefold. 30 references, 7 figures, 3 tables.

  2. Relationship between acid precipitation and three-dimensional transport associated with synoptic-scale cyclones

    SciTech Connect

    Haagenson, P.L.; Lazrus, A.L.; Kuo, Y.H.; Caldwell, G.A.

    1985-09-01

    Field data collected during APEX (Acid Precipitation Experiment) are used in combination with an isentropic trajectory model to analyze the relationship between acid precipitation and three-dimensional transport associated with cyclonic storms. Data are presented which indicate that high acidity in precipitation is often associated with slow transport speed and elevated SO2 concentrations in the dry air feeding into the precipitating regions. Conversely, low acidity is usually related to rapid transit, descending motion, and transport above the atmospheric boundary layer. The results also show that precipitation in the cold sector of a cyclone (in advance of the surface warm front) is often more acidic than that in other sectors of the storm. Four case studies are included to detail some of these meteorological effects. 19 references.

  3. The ABC transporter ABC40 encodes a phenylacetic acid export system in Penicillium chrysogenum.

    PubMed

    Weber, Stefan S; Kovalchuk, Andriy; Bovenberg, Roel A L; Driessen, Arnold J M

    2012-11-01

    The filamentous fungus Penicillium chrysogenum is used for the industrial production of β-lactam antibiotics. The pathway for β-lactam biosynthesis has been resolved and involves the enzyme phenylacetic acid CoA ligase that is responsible for the CoA activation of the side chain precursor phenylacetic acid (PAA) that is used for the biosynthesis of penicillin G. To identify ABC transporters related to β-lactam biosynthesis, we analyzed the expression of all 48 ABC transporters present in the genome of P. chryso-genum when grown in the presence and absence of PAA. ABC40 is significantly upregulated when cells are grown or exposed to high levels of PAA. Although deletion of this transporter did not affect β-lactam biosynthesis, it resulted in a significant increase in sensitivity to PAA and other weak acids. It is concluded that ABC40 is involved in weak acid detoxification in P. chrysogenum including resistance to phenylacetic acid.

  4. Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4

    PubMed Central

    Poulsen, Miguel V; Vandenberg, Robert J

    2001-01-01

    The effects of niflumic acid on the substrate-gated currents mediated by the glutamate transporter EAAT4 expressed in Xenopus laevis oocytes were examined using radiolabelled substrate flux measurements and two-electrode voltage clamp techniques. Niflumic acid significantly enhanced the substrate-gated currents in EAAT4, without affecting the affinity of the substrates towards EAAT4. At a concentration of 300 μm, niflumic acid caused a 19 ± 5 % reduction in l-[3H]glutamate uptake and no significant effect on the uptake of dl-[3H]aspartate. Thus, enhancement of the substrate-gated currents in EAAT4 does not correlate with the rate of substrate transport and suggests that the niflumic acid-induced currents are not thermodynamically coupled to the transport of substrate. Niflumic acid and arachidonic acid co-applied with substrate to EAAT4-expressing oocytes had similar functional consequences. However, niflumic acid still enhanced the l-glutamate-gated current to the same extent in the presence and absence of a saturating dose of arachidonic acid, which suggests that the sites of action of the two compounds are distinct. The EAAT4-mediated currents for the two substrates, l-glutamate and l-aspartate, were not enhanced equally by addition of the same dose of niflumic acid and the ionic composition of the niflumic acid-induced currents was not the same for the two substrates. Protons carry the l-glutamate-gated niflumic acid-induced current and both protons and chloride ions carry the l-aspartate-gated niflumic acid-induced current. These results show that niflumic acid can be used to probe the functional aspects of EAAT4 and that niflumic acid and other non-steroid anti-inflammatory drugs could be used as the basis for the development of novel modulators of glutamate transporters. PMID:11432999

  5. Niflumic acid modulates uncoupled substrate-gated conductances in the human glutamate transporter EAAT4.

    PubMed

    Poulsen, M V; Vandenberg, R J

    2001-07-01

    1. The effects of niflumic acid on the substrate-gated currents mediated by the glutamate transporter EAAT4 expressed in Xenopus laevis oocytes were examined using radiolabelled substrate flux measurements and two-electrode voltage clamp techniques. 2. Niflumic acid significantly enhanced the substrate-gated currents in EAAT4, without affecting the affinity of the substrates towards EAAT4. At a concentration of 300 microM, niflumic acid caused a 19 +/- 5 % reduction in L-[(3)H]glutamate uptake and no significant effect on the uptake of DL-[(3)H]aspartate. Thus, enhancement of the substrate-gated currents in EAAT4 does not correlate with the rate of substrate transport and suggests that the niflumic acid-induced currents are not thermodynamically coupled to the transport of substrate. 3. Niflumic acid and arachidonic acid co-applied with substrate to EAAT4-expressing oocytes had similar functional consequences. However, niflumic acid still enhanced the L-glutamate-gated current to the same extent in the presence and absence of a saturating dose of arachidonic acid, which suggests that the sites of action of the two compounds are distinct. 4. The EAAT4-mediated currents for the two substrates, L-glutamate and L-aspartate, were not enhanced equally by addition of the same dose of niflumic acid and the ionic composition of the niflumic acid-induced currents was not the same for the two substrates. Protons carry the L-glutamate-gated niflumic acid-induced current and both protons and chloride ions carry the L-aspartate-gated niflumic acid-induced current. 5. These results show that niflumic acid can be used to probe the functional aspects of EAAT4 and that niflumic acid and other non-steroid anti-inflammatory drugs could be used as the basis for the development of novel modulators of glutamate transporters.

  6. EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells.

    PubMed

    Ye, Jin-Ling; Gao, Chun-Qi; Li, Xiang-Guang; Jin, Cheng-Long; Wang, Dan; Shu, Gang; Wang, Wen-Ce; Kong, Xiang-Feng; Yao, Kang; Yan, Hui-Chao; Wang, Xiu-Qi

    2016-06-21

    Excitatory amino acid transporter 3 (EAAT3, encoded by SLC1A1) is an epithelial type high-affinity anionic amino acid transporter, and glutamate is the major oxidative fuel for intestinal epithelial cells. This study investigated the effects of EAAT3 on amino acid transport and cell proliferation through activation of the mammalian target of the rapamycin (mTOR) pathway in porcine jejunal epithelial cells (IPEC-J2). Anionic amino acid and cystine (Cys) transport were increased (P<0.05) by EAAT3 overexpression and decreased (P<0.05) by EAAT3 knockdown rather than other amino acids. MTT and cell counting assays suggested that IPEC-J2 cell proliferation increased (P<0.05) with EAAT3 overexpression. Phosphorylation of mTOR (Ser2448), ribosomal protein S6 kinase-1 (S6K1, Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1, Thr70) was increased by EAAT3 overexpression and decreased by EAAT3 knockdown (P<0.05), as were levels of activating transcription factor 4 (ATF4) and cystine/glutamate antiporter (xCT) (P<0.05). Our results demonstrate for the first time that EAAT3 facilitates anionic amino acid transport and activates the mTOR pathway, promoting Cys transport and IPEC-J2 cell proliferation.

  7. EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells

    PubMed Central

    Jin, Cheng-long; Wang, Dan; Shu, Gang; Wang, Wen-ce; Kong, Xiang-feng; Yao, Kang; Yan, Hui-chao; Wang, Xiu-qi

    2016-01-01

    Excitatory amino acid transporter 3 (EAAT3, encoded by SLC1A1) is an epithelial type high-affinity anionic amino acid transporter, and glutamate is the major oxidative fuel for intestinal epithelial cells. This study investigated the effects of EAAT3 on amino acid transport and cell proliferation through activation of the mammalian target of the rapamycin (mTOR) pathway in porcine jejunal epithelial cells (IPEC-J2). Anionic amino acid and cystine (Cys) transport were increased (P<0.05) by EAAT3 overexpression and decreased (P<0.05) by EAAT3 knockdown rather than other amino acids. MTT and cell counting assays suggested that IPEC-J2 cell proliferation increased (P<0.05) with EAAT3 overexpression. Phosphorylation of mTOR (Ser2448), ribosomal protein S6 kinase-1 (S6K1, Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1, Thr70) was increased by EAAT3 overexpression and decreased by EAAT3 knockdown (P<0.05), as were levels of activating transcription factor 4 (ATF4) and cystine/glutamate antiporter (xCT) (P<0.05). Our results demonstrate for the first time that EAAT3 facilitates anionic amino acid transport and activates the mTOR pathway, promoting Cys transport and IPEC-J2 cell proliferation. PMID:27231847

  8. Fatty acid binding protein facilitates sarcolemmal fatty acid transport but not mitochondrial oxidation in rat and human skeletal muscle

    PubMed Central

    Holloway, Graham P; Lally, Jamie; Nickerson, James G; Alkhateeb, Hakam; Snook, Laelie A; Heigenhauser, George J F; Calles-Escandon, Jorge; Glatz, Jan F C; Luiken, Joost J F P; Spriet, Lawrence L; Bonen, Arend

    2007-01-01

    The transport of long-chain fatty acids (LCFAs) across mitochondrial membranes is regulated by carnitine palmitoyltransferase I (CPTI) activity. However, it appears that additional fatty acid transport proteins, such as fatty acid translocase (FAT)/CD36, influence not only LCFA transport across the plasma membrane, but also LCFA transport into mitochondria. Plasma membrane-associated fatty acid binding protein (FABPpm) is also known to be involved in sacrolemmal LCFA transport, and it is also present on the mitochondria. At this location, it has been identified as mitochondrial aspartate amino transferase (mAspAT), despite being structurally identical to FABPpm. Whether this protein is also involved in mitochondrial LCFA transport and oxidation remains unknown. Therefore, we have examined the ability of FABPpm/mAspAT to alter mitochondrial fatty acid oxidation. Muscle contraction increased (P < 0.05) the mitochondrial FAT/CD36 content in rat (+22%) and human skeletal muscle (+33%). By contrast, muscle contraction did not alter the content of mitochondrial FABPpm/mAspAT protein in either rat or human muscles. Electrotransfecting rat soleus muscles, in vivo, with FABPpm cDNA increased FABPpm protein in whole muscle (+150%; P < 0.05), at the plasma membrane (+117%; P < 0.05) and in mitochondria (+80%; P < 0.05). In these FABPpm-transfected muscles, palmitate transport into giant vesicles was increased by +73% (P < 0.05), and fatty acid oxidation in intact muscle was increased by +18% (P < 0.05). By contrast, despite the marked increase in mitochondrial FABPpm/mAspAT protein content (+80%), the rate of mitochondrial palmitate oxidation was not altered (P > 0.05). However, electrotransfection increased mAspAT activity by +70% (P < 0.05), and the mitochondrial FABPpm/mAspAT protein content was significantly correlated with mAspAT activity (r= 0.75). It is concluded that FABPpm has two distinct functions depending on its subcellular location: (a) it contributes to

  9. Role of organic acids in promoting colloidal transport of mercury from mine tailings

    USGS Publications Warehouse

    Slowey, A.J.; Johnson, S.B.; Rytuba, J.J.; Brown, Gordon E.

    2005-01-01

    A number of factors affect the transport of dissolved and paniculate mercury (Hg) from inoperative Hg mines, including the presence of organic acids in the rooting zone of vegetated mine waste. We examined the role of the two most common organic acids in soils (oxalic and citric acid) on Hg transport from such waste by pumping a mixed organic acid solution (pH 5.7) at 1 mL/min through Hg mine tailings columns. For the two total organic acid concentrations investigated (20 ??M and 1 mM), particle-associated Hg was mobilized, with the onset of paniculate Hg transport occurring later for the lower organic acid concentration. Chemical analyses of column effluent indicate that 98 wt % of Hg mobilized from the column was paniculate. Hg speciation was determined using extended X-ray absorption fine structure spectroscopy and transmission electron microscopy, showing that HgS minerals are dominant in the mobilized particles. Hg adsorbed to colloids is another likely mode of transport due to the abundance of Fe-(oxyhydr)oxides, Fe-sulfides, alunite, and jarosite in the tailings to which Hg(II) adsorbs. Organic acids produced by plants are likely to enhance the transport of colloid-associated Hg from vegetated Hg mine tailings by dissolving cements to enable colloid release. ?? 2005 American Chemical Society.

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

    PubMed

    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.

  11. Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

    PubMed

    Bentur, Ohad S; Schwartz, Doron; Chernichovski, Tamara; Ingbir, Merav; Weinstein, Talia; Chernin, Gil; Schwartz, Idit F

    2015-08-15

    Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.

  12. Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production.

    PubMed

    Chen, Xiulai; Wang, Yuancai; Dong, Xiaoxiang; Hu, Guipeng; Liu, Liming

    2017-02-22

    L-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the L-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for L-malic acid biosynthesis. Second, the L-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the L-malic acid efflux system. Finally, the L-malic acid pathway was optimized by controlling gene expression levels, and the final L-malic acid concentration, yield, and productivity were up to 30.25 g L(-1), 0.30 g g(-1), and 0.32 g L(-1) h(-1) in the resulting strain W4209 with CaCO3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L(-1), 0.31 g g(-1), and 0.32 g L(-1) h(-1), respectively. The metabolic engineering strategy used here will be useful for efficient production of L-malic acid and other chemicals.

  13. Third system for neutral amino acid transport in a marine pseudomonad.

    PubMed Central

    Pearce, S M; Hildebrandt, V A; Lee, T

    1977-01-01

    Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system. PMID:856786

  14. Maternal bile acid transporter deficiency promotes neonatal demise

    PubMed Central

    Zhang, Yuanyuan; Li, Fei; Wang, Yao; Pitre, Aaron; Fang, Zhong-ze; Frank, Matthew W.; Calabrese, Christopher; Krausz, Kristopher W.; Neale, Geoffrey; Frase, Sharon; Vogel, Peter; Rock, Charles O.; Gonzalez, Frank J.; Schuetz, John D.

    2015-01-01

    Intrahepatic cholestasis of pregnancy (ICP) is associated with adverse neonatal survival and is estimated to impact between 0.4 and 5% of pregnancies worldwide. Here we show that maternal cholestasis (due to Abcb11 deficiency) produces neonatal death among all offspring within 24 h of birth due to atelectasis-producing pulmonary hypoxia, which recapitulates the neonatal respiratory distress of human ICP. Neonates of Abcb11-deficient mothers have elevated pulmonary bile acids and altered pulmonary surfactant structure. Maternal absence of Nr1i2 superimposed on Abcb11 deficiency strongly reduces maternal serum bile acid concentrations and increases neonatal survival. We identify pulmonary bile acids as a key factor in the disruption of the structure of pulmonary surfactant in neonates of ICP. These findings have important implications for neonatal respiratory failure, especially when maternal bile acids are elevated during pregnancy, and highlight potential pathways and targets amenable to therapeutic intervention to ameliorate this condition. PMID:26416771

  15. Acid-base transport in pancreatic cancer: molecular mechanisms and clinical potential.

    PubMed

    Kong, Su Chii; Giannuzzo, Andrea; Gianuzzo, Andrea; Novak, Ivana; Pedersen, Stine Falsig

    2014-12-01

    Solid tumors are characterized by a microenvironment that is highly acidic, while intracellular pH (pHi) is normal or even elevated. This is the result of elevated metabolic rates in the highly proliferative cancer cells, in conjunction with often greatly increased rates of net cellular acid extrusion. Studies in various cancers have suggested that while the acid extrusion mechanisms employed are generally the same as those in healthy cells, the specific transporters upregulated vary with the cancer type. The main such transporters include Na(+)/H(+) exchangers, various HCO3(-) transporters, H(+) pumps, and lactate-H(+) cotransporters. The mechanisms leading to their dysregulation in cancer are incompletely understood but include changes in transporter expression levels, trafficking and membrane localization, and posttranslational modifications. In turn, accumulating evidence has revealed that in addition to supporting their elevated metabolic rate, their increased acid efflux capacity endows the cancer cells with increased capacity for invasiveness, proliferation, and chemotherapy resistance. The pancreatic duct exhibits an enormous capacity for acid-base transport, rendering pHi dysregulation a potentially very important topic in pancreatic ductal adenocarcinoma (PDAC). PDAC - accounting for about 90% of all pancreatic cancers - has one of the highest cancer mortality rates known, and new diagnostic and treatment options are highly needed. However, very little is known about whether pH regulation is altered in PDAC and, if so, the possible role of this in cancer development. Here, we review current models for pancreatic acid-base transport and pH homeostasis and summarize current views on acid-base dysregulation in cancer, focusing where possible on the few studies to date in PDAC. Finally, we present new data-mining analyses of acid-base transporter expression changes in PDAC and discuss essential directions for future work.

  16. In-stream sorption of fulvic acid in an acidic stream: A stream-scale transport experiment

    USGS Publications Warehouse

    McKnight, Diane M.; Hornberger, G.M.; Bencala, K.E.; Boyer, E.W.

    2002-01-01

    The variation of concentration and composition of dissolved organic carbon (DOC) in stream waters cannot be explained solely on the basis of soil processes in contributing subcatchments. To investigate in-stream processes that control DOC, we injected DOC-enriched water into a reach of the Snake River (Summit County, Colorado) that has abundant iron oxyhydroxides coating the streambed. The injected water was obtained from the Suwannee River (Georgia), which is highly enriched in fulvic acid. The fulvic acid from this water is the standard reference for aquatic fulvic acid for the International Humic Substances Society and has been well characterized. During the experimental injection, significant removal of sorbable fulvic acid occurred within the first 141 m of stream reach. We coinjected a conservative tracer (lithium chloride) and analyzed the results with the one-dimensional transport with inflow and storage (OTIS) stream solute transport model to quantify the physical transport mechanisms. The downstream transport of fulvic acid as indicated by absorbance was then simulated using OTIS with a first-order kinetic sorption rate constant applied to the sorbable fulvic acid. The "sorbable" fraction of injected fulvic acid was irreversibly sorbed by streambed sediments at rates (kinetic rate constants) of the order of 10-4-10-3 S-1. In the injected Suwannee River water, sorbable and nonsorbable fulvic acid had distinct chemical characteristics identified in 13C-NMR spectra. The 13C-NMR spectra indicate that during the experiment, the sorbable "signal" of greater aromaticity and carboxyl content decreased downstream; that is, these components were preferentially removed. This study illustrates that interactions between the water and the reactive surfaces will modify significantly the concentration and composition of DOC observed in streams with abundant chemically reactive surfaces on the streambed and in the hyporheic zone.

  17. Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells.

    PubMed

    Komaba, Shigeru; Coluccio, Lynne M

    2015-01-01

    Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM.

  18. Identification and functional characterization of uric acid transporter Urat1 (Slc22a12) in rats.

    PubMed

    Sato, Masanobu; Wakayama, Tomohiko; Mamada, Hideaki; Shirasaka, Yoshiyuki; Nakanishi, Takeo; Tamai, Ikumi

    2011-06-01

    Uric acid transporter URAT1 contributes significantly to reabsorption of uric acid in humans to maintain a constant serum uric acid (SUA) level. Since alteration of SUA level is associated with various diseases, it is important to clarify the mechanism of change in SUA. However, although expression of mRNA of an ortholog of URAT1 (rUrat1) in rats has been reported, functional analysis and localization have not been done. Therefore, rat rUrat1 was functionally analyzed using gene expression systems and isolated brush-border membrane vesicles (BBMVs) prepared from rat kidney, and its localization in kidney was examined immunohistochemically. Uric acid transport by rUrat1 was chloride (Cl-) susceptible with a Km of 1773μM. It was inhibited by benzbromarone and trans-stimulated by lactate and pyrazinecarboxylic acid (PZA). Cl- gradient-susceptible uric acid transport by BBMVs showed similar characteristics to those of uric acid transport by rUrat1. Moreover, rUrat1 was localized at the apical membrane in proximal tubular epithelial cells in rat kidney. Accordingly, rUrat1 is considered to be involved in uric acid reabsorption in rats in the same manner as URAT1 in humans. Therefore, rUrat1 may be a useful model to study issues related to the role of human URAT1.

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

    SciTech Connect

    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.

  20. Transport of Corilagin, Gallic Acid, and Ellagic Acid from Fructus Phyllanthi Tannin Fraction in Caco-2 Cell Monolayers

    PubMed Central

    Zhao, Hai-juan; Liang, Wen-Yi; Chen, Wen-Jing; Han, Shu-Xian; Qi, Qi; Cui, Ya-Ping; Li, Shi; Yang, Guang-Hui; Shao, Yan-Yan; Zhu, Dan

    2016-01-01

    Objective. To investigate the absorption property of the representative hydrolyzable tannin, namely corilagin, and its hydrolysates gallic acid (GA) and ellagic acid (EA) from the Fructus Phyllanthi tannin fraction (PTF) in vitro. Methods. Caco-2 cells monolayer model was established. Influences of PTF on Caco-2 cells viability were detected with MTT assay. The transport across monolayers was examined for different time points, concentrations, and secretory directions. The inhibitors of P-glycoprotein (P-gp), multidrug resistance proteins (MRPs), organic anion transporting polypeptide (OATP) and sodium/glucose cotransporter 1 (SGLT1), and tight junction modulators were used to study the transport mechanism. LC-MS method was employed to quantify the absorption concentration. Results. The apparent permeability coefficient (Papp) values of the three compounds were below 1.0 × 10−6 cm/s. The absorption of corilagin and GA were much lower than their efflux, and the uptake of both compounds was increased in the presence of inhibitors of P-gp and MRPs. The absorption of EA was decreased in the company of OATP and SGLT1 inhibitors. Moreover, the transport of corilagin, GA, and EA was enhanced by tight junction modulators. Conclusion. These observations indicated that the three compounds in PTF were transported via passive diffusion combined with protein mediated transport. P-gp and MRPs might get involved in the transport of corilagin and GA. The absorption of EA could be attributed to OATP and SGLT1 protein. PMID:27738446

  1. Maternal micronutrients and omega 3 fatty acids affect placental fatty acid desaturases and transport proteins in Wistar rats.

    PubMed

    Wadhwani, Nisha S; Dangat, Kamini D; Joshi, Asmita A; Joshi, Sadhana R

    2013-03-01

    Adequate supply of LCPUFA from maternal plasma is crucial for fetal normal growth and development. The present study examines the effect of maternal micronutrients (folic acid and vitamin B12) and omega 3 fatty acids on placental mRNA levels of fatty acid desaturases (Δ5 and Δ6) and transport proteins. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B12. Both the vitamin B12 deficient groups were supplemented with omega 3 fatty acid. Maternal vitamin B12 deficiency reduced placental mRNA and protein levels of Δ5 desaturase, mRNA levels of FATP1 and FATP4 (p<0.05 for all) as compared to control while omega 3 fatty acid supplementation normalized the levels. Our data for the first time indicates that altered maternal micronutrients and omega 3 fatty acids play a key role in regulating fatty acid desaturase and transport protein expression in placenta.

  2. Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti

    PubMed Central

    Boudko, Dmitri Y.; Tsujimoto, Hitoshi; Rodriguez, Stacy D.; Meleshkevitch, Ella A.; Price, David P.; Drake, Lisa L.; Hansen, Immo A.

    2015-01-01

    Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+ dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction. PMID:26449545

  3. Inhibition of ileal bile acid transporter: An emerging therapeutic strategy for chronic idiopathic constipation.

    PubMed

    Mosińska, Paula; Fichna, Jakub; Storr, Martin

    2015-06-28

    Chronic idiopathic constipation is a common disorder of the gastrointestinal tract that encompasses a wide profile of symptoms. Current treatment options for chronic idiopathic constipation are of limited value; therefore, a novel strategy is necessary with an increased effectiveness and safety. Recently, the inhibition of the ileal bile acid transporter has become a promising target for constipation-associated diseases. Enhanced delivery of bile acids into the colon achieves an accelerated colonic transit, increased stool frequency, and relief of constipation-related symptoms. This article provides insight into the mechanism of action of ileal bile acid transporter inhibitors and discusses their potential clinical use for pharmacotherapy of constipation in chronic idiopathic constipation.

  4. Light-activated amino acid transport in Halobacterium halobium envelope vesicles

    NASA Technical Reports Server (NTRS)

    Macdonald, R. E.; Lanyi, J. K.

    1977-01-01

    Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na+ gradient. On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids: arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. The solute carrier family 10 (SLC10): beyond bile acid transport

    PubMed Central

    da Silva, Tatiana Claro; Polli, James E.; Swaan, Peter W.

    2012-01-01

    The solute carrier (SLC) family 10 (SLC10) comprises influx transporters of bile acids, steroidal hormones, various drugs, and several other substrates. Because the seminal transporters of this family, namely, sodium/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile acid transporter (ASBT; SLC10A2), were primarily bile acid transporters, the term “sodium bile salt cotransporting family” was used for the SLC10 family. However, this notion became obsolete with the finding of other SLC10 members that do not transport bile acids. For example, the sodium-dependent organic anion transporter (SOAT; SLC10A6) transports primarily sulfated steroids. Moreover, NTCP was shown to also transport steroids and xenobiotics, including HMG-CoA inhibitors (statins). The SLC10 family contains four additional members, namely, P3 (SLC10A3; SLC10A3), P4 (SLC10A4; SLC10A4), P5 (SLC10A5; SLC10A5) and SLC10A7 (SLC10A7), several of which were unknown or considered hypothetical until approximately a decade ago. While their substrate specificity remains undetermined, great progress has been made towards their characterization in recent years. SLC10A4 may participate in vesicular storage or exocytosis of neurotransmitters or mastocyte mediators, whereas SLC10A5 and SLC10A7 may be involved in solute transport and SLC10A3 may have a role as a housekeeping protein. Finally, the newly found role of bile acids in glucose and energy homeostasis, via the TGR5 receptor, sheds new light on the clinical relevance of ASBT and NTCP. The present mini-review provides a brief summary of recent progress on members of the SLC10 family. PMID:23506869

  7. Transport of acetic acid in Zygosaccharomyces bailii: effects of ethanol and their implications on the resistance of the yeast to acidic environments.

    PubMed Central

    Sousa, M J; Miranda, L; Côrte-Real, M; Leão, C

    1996-01-01

    Cells of Zygosaccharomyces bailii ISA 1307 grown in a medium with acetic acid, ethanol, or glycerol as the sole carbon and energy source transported acetic acid by a saturable transport system. This system accepted propionic and formic acids but not lactic, sorbic, and benzoic acids. When the carbon source was glucose or fructose, the cells displayed activity of a mediated transport system specific for acetic acid, apparently not being able to recognize other monocarboxylic acids. In both types of cells, ethanol inhibited the transport of labelled acetic acid. The inhibition was noncompetitive, and the dependence of the maximum transport rate on the ethanol concentration was found to be exponential. These results reinforced the belief that, under the referenced growth conditions, the acid entered the cells mainly through a transporter protein. The simple diffusion of the undissociated acid appeared to contribute, with a relatively low weight, to the overall acid uptake. It was concluded that in Z. bailii, ethanol plays a protective role against the possible negative effects of acetic acid by inhibiting its transport and accumulation. Thus, the intracellular concentration of the acid could be maintained at levels lower than those expected if the acid entered the cells only by simple diffusion. PMID:8795203

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

    PubMed

    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; Rasmussen, B B

    2014-06-01

    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 < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar 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.

  9. Transport of Palmitic Acid Across the Tegument of the Entomophilic Nematode Romanomermis culicivorax

    PubMed Central

    Gordon, Roger; Burford, Ian R.

    1984-01-01

    Romanomermis culicivorax juveniles, dissected out of Aedes aegypti larvae 7 days after infection, were incubated under controlled conditions in isotonic saline containing ¹⁴C-U-palmitic acid to investigate the nature of the transport mechanism(s) used by the nematode for transcuticular uptake of palmitic acid. Net uptake of the isotope by the nematode was of a logarithmic nature with respect to time. Uptake of palmitic acid was accomplished by a combination of diffusion and a mediated process which was substrate saturable and competitively inhibited by myristic and stearic acids. Both 2,4-dinitrophenol and ouabain inhibited uptake of palmitic acid and thus supported the hypothesis that the carrier system is of the active transport variety and is coupled to a Na⁺K⁺ ATPase pump. PMID:19295867

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

    PubMed

    Melnykov, Artem V

    2016-01-01

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

  11. 1/f fluctuations of amino acids regulate water transportation in aquaporin 1

    NASA Astrophysics Data System (ADS)

    Yamamoto, Eiji; Akimoto, Takuma; Hirano, Yoshinori; Yasui, Masato; Yasuoka, Kenji

    2014-02-01

    Aquaporins (AQPs), which transport water molecules across cell membranes, are involved in many physiological processes. Recently, it is reported that the water-water interactions within the channel are broken at the aromatic/arginine selectivity filter (ar/R region), which prevents proton transportation [U. K. Eriksson et al., Science 340, 1346 (2013), 10.1126/science.1234306]. However, the effects of the conformational fluctuations of amino acids on water transportation remain unclear. Using all-atom molecular dynamics simulations, we analyze water transportation and fluctuations of amino acids within AQP1. The amino acids exhibit 1/f fluctuations, indicating possession of long-term memory. Moreover, we find that water molecules crossing the ar/R region obey a non-Poisson process. To investigate the effect of 1/f fluctuations on water transportation, we perform restrained molecular dynamics simulations of AQP1 and simple Langevin stochastic simulations. As a result, we confirm that 1/f fluctuations of amino acids contribute to water transportation in AQP1. These findings appreciably enhance our understanding of AQPs and suggest possibilities for developing biomimetic nanopores.

  12. Coupling of hydrologic transport and chemical reactions in a stream affected by acid mine drainage

    USGS Publications Warehouse

    Kimball, B.A.; Broshears, R.E.; Bencala, K.E.; McKnight, Diane M.

    1994-01-01

    Experiments in St. Kevin Gulch, an acid mine drainage stream, examined the coupling of hydrologic transport to chemical reactions affecting metal concentrations. Injection of LiCl as a conservative tracer was used to determine discharge and residence time along a 1497-m reach. Transport of metals downstream from inflows of acidic, metal-rich water was evaluated based on synoptic samples of metal concentrations and the hydrologic characteristics of the stream. Transport of SO4 and Mn was generally conservative, but in the subreaches most affected by acidic inflows, transport was reactive. Both 0.1-??m filtered and particulate Fe were reactive over most of the stream reach. Filtered Al partitioned to the particulate phase in response to high instream concentrations. Simulations that accounted for the removal of SO4, Mn, Fe, and Al with first-order reactions reproduced the steady-state profiles. The calculated rate constants for net removal used in the simulations embody several processes that occur on a stream-reach scale. The comparison between rates of hydrologie transport and chemical reactions indicates that reactions are only important over short distances in the stream near the acidic inflows, where reactions occur on a comparable time scale with hydrologic transport and thus affect metal concentrations.

  13. ATP-dependent transport of bile acid intermediates across rat liver peroxisomal membranes.

    PubMed

    Une, Mizuho; Iguchi, Yusuke; Sakamoto, Tomoko; Tomita, Takashi; Suzuki, Yasuyuki; Morita, Masashi; Imanaka, Tsuneo

    2003-08-01

    The bile acid intermediate 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid (THCA) is converted to cholic acid exclusively in peroxisomes by the oxidative cleavage of the side chain. To investigate the mechanism by which the biosynthetic intermediates of bile acids are transported into peroxisomes, we incubated THCA or its CoA ester (THC-CoA) with isolated intact rat liver peroxisomes and analyzed their oxidation products, cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid. The oxidation of both THCA and THC-CoA was dependent on incubation time and peroxisomal proteins, and was stimulated by ATP. THC-CoA was efficiently oxidized to cholic acid and 3alpha,7alpha,12alpha-trihydroxy-5beta-cholest-24-enoic acid as compared with THCA, suggesting that THC-CoA is the preferred substrate for transport into peroxisomes. The oxidation of THC-CoA was significantly inhibited by sodium azide, verapamile, and N-ethylmaleimide. Furthermore, the stimulatory effect of ATP on the oxidation was not replaced by GTP or AMP. In addition, the ATP-dependent oxidation of THC-CoA was markedly inhibited by pretreatment of peroxisomes with proteinase K when peroxisomal matrix proteins were not degraded. These results suggest that an ATP-dependent transport system for THC-CoA exists on peroxisomal membranes.

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

    USGS Publications Warehouse

    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.

  15. Humic acid transport in saturated porous media: influence of flow velocity and influent concentration.

    PubMed

    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.

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

    PubMed Central

    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

  17. Cholesterol reduces the effects of dihydroxy bile acids and fatty acids on water and solute transport in the human jejunum.

    PubMed Central

    Broor, S L; Slota, T; Ammon, H V

    1980-01-01

    Jejunal perfusion studies were performed in 16 healthy volunteers to test the hypothesis that intraluminal cholesterol can mitigate the fluid secretion induced by dihydroxy bile acids and fatty acids. Fluid secretion in the presence of 5 mM taurodeoxycholate was somewhat reduced by 4 mM mono-olein which was used for the solubilization of cholesterol. Addition of 0.8 mM cholesterol reduced fluid secretion further (P less than 0.05). Fluid secretion induced by 4 mM oleic acid was changed to net absorption in a linear fashion with increasing cholesterol concentration in the perfusion solutions. 1 mM cholesterol reduced fluid secretion induced by 6 mM oleic acid (P less than 0.005), but had no effect on fluid secretion induced by 6 mM linolenic acid. Glucose absorption was generally affected in a similar manner as water transport. In vitro, 1 mM cholesterol reduced monomer activity of 6 mM oleic acid to 72.3 +/- 0.9% of control and that of linolenic acid to 81.1 +/- 1.7% of control. Although statistically significant (P less than 0.001), the difference in the effects of cholesterol on monomer activities of the two fatty acids was rather small and it is unlikely that changes in monomer concentration of fatty acids and bile acids account for the protective effect of cholesterol. The in vivo observations point to a new physiological role for biliary cholesterol: the modification of the response of the small intestine to the effects of dihydroxy bile acids and fatty acids. PMID:7358850

  18. Expression of the SNAT2 amino acid transporter during the development of rat cerebral cortex.

    PubMed

    Rodríguez, Angelina; Angelina, Rodríguez; Berumen, Laura C; Francisco, Zafra; Giménez, Cecilio; Cecilio, Giménez; García-Alcocer, María Guadalupe; Guadalupe, García-Alcocer María

    2011-11-01

    The sodium-coupled neutral amino acid transporter 2 (SNAT2) is a protein that is expressed ubiquitously in mammalian tissues and that displays Na(+), voltage and pH dependent activity. This transporter mediates the passage of small zwitterionic amino acids across the cell membrane and regulates the cell homeostasis and its volume. We have examined the expression of SNAT2 mRNA and protein during the development of the rat cerebral cortex, from gestation through the postnatal stages to adulthood. Our data reveal that SNAT2 mRNA and protein expression is higher during embryogenesis, while it subsequently diminishes during postnatal development. Moreover, during embryonic period SNAT2 colocalizes with the radial glial cells marker GLAST, while in postnatal period it is mainly detected in neuronal dendrites. These findings suggest a relevant role for amino acid transport through SNAT2 in the developing embryonic brain.

  19. Carrier-mediated placental transport of cimetidine and valproic acid across differentiating JEG-3 cell layers.

    PubMed

    Ikeda, K; Ueda, C; Yamada, K; Nakamura, A; Hatsuda, Y; Kawanishi, S; Nishii, S; Ogawa, M

    2015-07-01

    Human choriocarcinoma has been used as a model to study trophoblast transcellular drug transport in the placenta. Previous models had limitations regarding low molecular weight drug transport through the intracellular gap junction. The purpose of this study was to evaluate placental carrier-mediated transport across a differentiating JEG-3 choriocarcinoma cell (DJEGs) layer model in which the intracellular gap junction was restricted. Cimetidine is the substrate of an efflux transporter, breast cancer resistance protein (BCRP). BCRP highly expressed in the placenta, and its function in the DJEGs model was investigated. In addition, the placental drug transport of another efflux transporter, multidrug resistance-associated proteins (MRPs), and an influx transporter, monocarboxylate transporter (MCT), were examined with various substrates. Cimetidine permeated from the fetal side to the maternal side at significantly high levels and saturated in a dose-dependent manner. The permeability coefficient of a MRP substrate, fluorescein, across the DJEGs model was significantly increased by inhibiting MRP function with probenecid. On the other hand, permeation in the influx direction to the fetal side with a substrate of MCT, valproic acid, had a gentle dose-dependent saturation. These findings suggest that the DJEGs model could be used to evaluate transcellular placental drug transport mediated by major placental transporters.

  20. Co-dependence of genotype and dietary protein intake to affect expression on amino acid/peptide transporters in porcine skeletal muscle.

    PubMed

    Liu, Y; Kong, X; Li, F; Tan, B; Li, Y; Duan, Y; Yin, Y; He, J; Hu, C; Blachier, F; Wu, Guoyao

    2016-01-01

    A total of 96 barrows (48 pure-bred Bama mini-pigs representing fatty genotype, and 48 Landrace pigs representing lean genotype) were randomly assigned to either a low- or adequate-protein treatment diet. The experimental period commenced at 5 weeks of age and extended to the finishing period. After euthanasia, blood and skeletal muscle samples were collected from pigs at the nursery, growing, and finishing phases. Our results indicate that the concentrations of free AAs in the plasma and muscle decreased as the age of the pigs increased. In addition, a strain × growth phase interaction (P < 0.05) was observed for the free AA pool in the plasma and muscle. The low-protein diet upregulated (P < 0.05) the mRNA levels for T1R1/T1R3 involved in glutamate binding, but downregulated (P < 0.05) the mRNA levels for PAT1, PAT2, and ASCT2, which transport neutral AAs into muscles. Bama mini-pigs had higher (P < 0.05) mRNA levels for LAT1, SNAT2, and EAAC1, but a lower (P < 0.05) mRNA level for PepT1, compared with Landrace pigs. Collectively, our findings indicate that adequate provision of dietary protein plays an important role in regulating profiles of free AA pools and expression of key AA/peptide transporters/transceptors in a genotype- and tissue-specific manner.

  1. Further evaluation of the interrelationship between the hepatocellular transport of bile acids and endocytosed proteins.

    PubMed Central

    Herrera, M. C.; el-Mir, M. Y.; Monte, M. J.; Perez-Barriocanal, F.; Marin, J. J.

    1992-01-01

    Experiments on the relationship between the hepatocellular transport of endogenous or exogenously loaded bile acids (sodium taurocholate, TC, 0.5 mumol/min/100 g body wt) and horseradish peroxidase (HRP) or immunoglobulin A (IgA) (0.5 mg/100 g body wt) were carried out on anaesthetized Wistar rats. The time course of HRP excretion into bile (acceleration in the secretory peak), but not the total amount of HRP output, was affected by TC infusion. Administration of HRP was found to have no stimulatory effect on either spontaneous or TC-induced bile flow, bile acid, lecithin or cholesterol output. Spontaneous bile acid output was increased (25 and 67%, respectively) in rats that were treated for 12-h fasting or by oral administration of TC (45 mg/100 g body wt, every 12 h, for 2 days). These manoeuvres did not change the inability of HRP and IgA to increase bile acid output. Exogenous TC load had no stimulatory effect on the hepatocellular transport of endogenous bile acid pool, that was labelled by a combination of fasting and oral administration of 14C-glycocholic acid 12 h before the experiments. Therefore, exogenous bile acid load-induced stimulation of transcytosis had no effect on endogenous bile acid output. Moreover, bile secretion of both endogenous and exogenously loaded bile acids is unaffected by the administration of proteins, irrespective of whether they are endocytosed by a receptor or nonreceptor mediated process. PMID:1571280

  2. Auxin Activity of Substituted Benzoic Acids and Their Effect on Polar Auxin Transport 1

    PubMed Central

    Keitt, George W.; Baker, Robert A.

    1966-01-01

    Six dichloro-, 3 trichloro-, 2 triiodo-, and 3 heterosubstituted benzoic acids (amiben, dinoben, dicamba), and N-1-naphthylphthalamic acid have been tested for effects on growth and on polar auxin transport. Growth activity with and without kinetin was measured by effects on fresh and dry weights of 30-day cultures of fresh tobacco pith. Transport inhibition was measured by following uptake and output of IAA-2-14C through 10 mm bean epicotyl sections. The distribution of callus growth on vascularized tobacco stem segments was also observed. Avena first internode extension assays established the relative activities: dicamba > amiben > dinoben suggested by pith growth results. Growth effects of active compounds were similar with and without kinetin, except that amiben was less active with kinetin, while 2,3,6-trichlorobenzoic acid was more active with kinetin than alone. The weak auxin activity of NPA was confirmed. Transport experiments showed that NPA was the most inhibitory compound tested, followed by TIBA. Other compounds tested were at least 300 times less inhibitory to IAA transport. The best growth promoters were the least inhibitory to transport, and the most effective transport inhibitors were at best poor auxins. It is suggested that the weak auxin and auxin synergistic activity of TIBA (and perhaps 2,3-dichlorobenzoic acid) in extension growth tests arises from its inhibition of transport of endogenous or added auxin out of the sections, rather than from its intrinsic auxin activity. Chemically induced apolar callus growth on vascularized tobacco stem explants can arise from inhibition of native auxin transport, apolar growth stimulation by auxinic action of the test compound, or both. PMID:16656441

  3. Tuning transport selectivity of ionic species by phosphoric acid gradient in positively charged nanochannel membranes.

    PubMed

    Yang, Meng; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Fan, Xin; Liu, Wei; Liu, Xizhen; Liu, Jianbo; Huang, Jin

    2015-02-03

    The transport of ionic species through a nanochannel plays important roles in fundamental research and practical applications of the nanofluidic device. Here, we demonstrated that ionic transport selectivity of a positively charged nanochannel membrane can be tuned under a phosphoric acid gradient. When phosphoric acid solution and analyte solution were connected by the positively charged nanochannel membrane, the faster-moving analyte through the positively charged nanochannel membrane was the positively charged dye (methylviologen, MV(2+)) instead of the negatively charged dye (1,5-naphthalene disulfonate, NDS(2-)). In other words, a reversed ion selectivity of the nanochannel membranes can be found. It can be explained as a result of the combination of diffusion, induced electroosmosis, and induced electrophoresis. In addition, the influencing factors of transport selectivity, including concentration of phosphoric acid, penetration time, and volume of feed solution, were also investigated. The results showed that the transport selectivity can further be tuned by adjusting these factors. As a method of tuning ionic transport selectivity by establishing phosphoric acid gradient, it will be conducive to improving the separation of ionic species.

  4. Reactive iron transport in an acidic mountain stream in Summit County, Colorado: A hydrologic perspective

    USGS Publications Warehouse

    McKnight, Diane M.; Bencala, K.E.

    1989-01-01

    A pH perturbation experiment was conducted in an acidic, metal-enriched, mountain stream to identify relative rates of chemical and hydrologic processes as they influence iron transport. During the experiment the pH was lowered from 4.2 to 3.2 for three hours by injection of sulfuric acid. Amorphous iron oxides are abundant on the streambed, and dissolution and photoreduction reactions resulted in a rapid increase in the dissolved iron concentration. The increase occurred simultaneously with the decrease in pH. Ferrous iron was the major aqueous iron species. The changes in the iron concentration during the experiment indicate that variation exists in the solubility properties of the hydrous iron oxides on the streambed with dissolution of at least two compartments of hydrous iron oxides contributing to the iron pulse. Spatial variations of the hydrologic properties along the stream were quantified by simulating the transport of a coinjected tracer, lithium. A simulation of iron transport, as a conservative solute, indicated that hydrologie transport had a significant role in determining downstream changes in the iron pulse. The rapidity of the changes in iron concentration indicates that a model based on dynamic equilibrium may be adequate for simulating iron transport in acid streams. A major challenge for predictive solute transport models of geochemical processes may be due to substantial spatial and seasonal variations in chemical properties of the reactive hydrous oxides in such streams, and in the physical and hydrologic properties of the stream. ?? 1989.

  5. Amino acid composition analysis of human secondary transport proteins and implications for reliable membrane topology prediction.

    PubMed

    Saidijam, Massoud; Azizpour, Sonia; Patching, Simon G

    2016-07-08

    Secondary transporters in humans are a large group of proteins that transport a wide range of ions, metals, organic and inorganic solutes involved in energy transduction, control of membrane potential and osmotic balance, metabolic processes and in the absorption or efflux of drugs and xenobiotics. They are also emerging as important targets for development of new drugs and as target sites for drug delivery to specific organs or tissues. We have performed amino acid composition (AAC) and phylogenetic analyses and membrane topology predictions for 336 human secondary transport proteins and used the results to confirm protein classification and to look for trends and correlations with structural domains and specific substrates and/or function. Some proteins showed statistically high contents of individual amino acids or of groups of amino acids with similar physicochemical properties. One recurring trend was a correlation between high contents of charged and/or polar residues with misleading results in predictions of membrane topology, which was especially prevalent in Mitochondrial Carrier family proteins. We demonstrate how charged or polar residues located in the middle of transmembrane helices can interfere with their identification by membrane topology tools resulting in missed helices in the prediction. Comparison of AAC in the human proteins with that in 235 secondary transport proteins from Escherichia coli revealed similar overall trends along with differences in average contents for some individual amino acids and groups of similar amino acids that are presumed to result from a greater number of functions and complexity in the higher organism.

  6. Modeling uranium transport in acidic contaminated groundwater with base addition.

    PubMed

    Zhang, Fan; Luo, Wensui; Parker, Jack C; Brooks, Scott C; Watson, David B; Jardine, Philip M; Gu, Baohua

    2011-06-15

    This study investigates reactive transport modeling in a column of uranium(VI)-contaminated sediments with base additions in the circulating influent. The groundwater and sediment exhibit oxic conditions with low pH, high concentrations of NO(3)(-), SO(4)(2-), U and various metal cations. Preliminary batch experiments indicate that additions of strong base induce rapid immobilization of U for this material. In the column experiment that is the focus of the present study, effluent groundwater was titrated with NaOH solution in an inflow reservoir before reinjection to gradually increase the solution pH in the column. An equilibrium hydrolysis, precipitation and ion exchange reaction model developed through simulation of the preliminary batch titration experiments predicted faster reduction of aqueous Al than observed in the column experiment. The model was therefore modified to consider reaction kinetics for the precipitation and dissolution processes which are the major mechanism for Al immobilization. The combined kinetic and equilibrium reaction model adequately described variations in pH, aqueous concentrations of metal cations (Al, Ca, Mg, Sr, Mn, Ni, Co), sulfate and U(VI). The experimental and modeling results indicate that U(VI) can be effectively sequestered with controlled base addition due to sorption by slowly precipitated Al with pH-dependent surface charge. The model may prove useful to predict field-scale U(VI) sequestration and remediation effectiveness.

  7. Alpha-aminoisobutyric acid transport into human glia and glioma cells in culture.

    PubMed

    Ronquist, G; Agren, G; Ponten, J; Westermark, B

    1976-11-01

    The AIB transport into human glia and glioma cells in culture has been studied. Because of the high affinity of AIB to the plastic culture dishes, a special washing technique had to be developed. With this technique, it was possible to perform transport experiments in a single plate containing about one million cells. The cells were viable, intact and adhered to the supporting medium throughout the experiment. The AIB transport into both types of cells was Na+-dependent and showed saturation kinetics when the small component of the transport due to diffusion had been subtracted. The AIB transport capacity of neoplastic glioma cells was 3.6 times higher than that of glia cells. This difference was related to the Vmax-values for the two types of cells. The apparent Km-values were the same. Inhibition experiments with other amino acids support the view that AIB is transported via System A in both glia and glioma cells. Sulfhydryl reagents (ethacrynic acid and NEM) and cytochalasin B clearly inhibited the AIB transport into glia cells whereas the effect on glioma cells was minimal.

  8. Transport and metabolism of fumaric acid in Saccharomyces cerevisiae in aerobic glucose-limited chemostat culture.

    PubMed

    Shah, Mihir V; van Mastrigt, Oscar; Heijnen, Joseph J; van Gulik, Walter M

    2016-04-01

    Currently, research is being focused on the industrial-scale production of fumaric acid and other relevant organic acids from renewable feedstocks via fermentation, preferably at low pH for better product recovery. However, at low pH a large fraction of the extracellular acid is present in the undissociated form, which is lipophilic and can diffuse into the cell. There have been no studies done on the impact of high extracellular concentrations of fumaric acid under aerobic conditions in S. cerevisiae, which is a relevant issue to study for industrial-scale production. In this work we studied the uptake and metabolism of fumaric acid in S. cerevisiae in glucose-limited chemostat cultures at a cultivation pH of 3.0 (pH < pK). Steady states were achieved with different extracellular levels of fumaric acid, obtained by adding different amounts of fumaric acid to the feed medium. The experiments were carried out with the wild-type S. cerevisiae CEN.PK 113-7D and an engineered S. cerevisiae ADIS 244 expressing a heterologous dicarboxylic acid transporter (DCT-02) from Aspergillus niger, to examine whether it would be capable of exporting fumaric acid. We observed that fumaric acid entered the cells most likely via passive diffusion of the undissociated form. Approximately two-thirds of the fumaric acid in the feed was metabolized together with glucose. From metabolic flux analysis, an increased ATP dissipation was observed only at high intracellular concentrations of fumarate, possibly due to the export of fumarate via an ABC transporter. The implications of our results for the industrial-scale production of fumaric acid are discussed.

  9. Regulation of amino acid transport in isolated rat hepatocytes during development

    SciTech Connect

    Leoni, S.; Spagnuolo, S.; Dini, L.; Devirgiliis, L.C.

    1987-01-01

    The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2-amino (1-/sup 14/C)isobutyric acid (AIB) or N-methyl 2-amino (1-/sup 14/C)isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the Na/sup +/ -dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AIB and MeAIB uptake enhancing the V/sub max/, while the K/sub m/ was unchanged. This effect was evident in cells from adult and 18-20-day-old fetuses, while no response was present before the 18th day of fetal life and in the prenatal period. Actinomycin D or cycloheximide abolished this hormone-dependent increase. A decrease in AIB and MeAIB transport after incubation in an amino acid-rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the release from transinhibition of the transport.

  10. Fatty acids as an energy source for the operation of axoplasmic transport.

    PubMed

    Takenaka, Toshifumi; Hiruma, Hiromi; Hori, Hideaki; Hashimoto, Yoko; Ichikawa, Takafumi; Kawakami, Tadashi

    2003-05-16

    Fatty acids are utilized as a cellular energy source. In the present study, we investigated whether fatty acids could affect axoplasmic transport. Cultured mouse superior cervical ganglion neurons were placed in the glucose-containing medium (145 mM NaCl, 5 mM KCl, 1 mM CaCl(2), 1 mM MgCl(2), 5 mM D-glucose, 10 mM Hepes, pH 7.3, 37 degrees C), and axoplasmic transport of particles in neurites was observed under video-enhanced contrast microscopy. A variety of fatty acids (acetate (C2), caproate (C6), caprylate (C8), caprate (C10), 2-decenoate (C10:1), arachidonate (C20:4); 0.1-1 mM) caused a transient increase in the amount of particles transported in both anterograde and retrograde directions. The increasing effects of fatty acids were dose-dependent. A half-maximum effective dose (ED(50)) for acetate was 0.8 mM, which is similar to the reported K(m) value of acetyl-CoA synthetase for acetate. The ED(50) for caprylate was 28 microM, which is near the K(m) value of acyl-CoA synthetase for medium- and long-chain fatty acids. Application of 5 mM malonate, an inhibitor of the citrate cycle, induced a steady-state decrease in axoplasmic transport, indicating that energy derived from the citrate cycle is required for the maintenance of axoplasmic transport. The increasing effect of acetate (1 mM) on axoplasmic transport was completely abolished by pretreatment with malonate (5 mM), suggesting that acetate produces ATP for axoplasmic transport via the citrate cycle. Alternatively, the effect of caprate (1 mM) was retained after treatment with malonate. Thus, fatty acids except acetate produce ATP probably through both the beta-oxidation pathway and the citrate cycle, increasing axoplasmic transport. Since the effect of fatty acids was transient, certain negative feedback mechanisms might be involved. The removal of glucose from the medium resulted in a low steady-state level of axoplasmic transport. Under such condition, the acetate (1 mM)-induced transient increase in

  11. Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid.

    PubMed

    Zhang, Ying; Wade, Mary Margaret; Scorpio, Angelo; Zhang, Hao; Sun, Zhonghe

    2003-11-01

    Pyrazinamide is an important sterilizing drug that shortens tuberculosis (TB) therapy. However, the mechanism of action of pyrazinamide is poorly understood because of its unusual properties. Here we show that pyrazinoic acid, the active moiety of pyrazinamide, disrupted membrane energetics and inhibited membrane transport function in Mycobacterium tuberculosis. The preferential activity of pyrazinamide against old non-replicating bacilli correlated with their low membrane potential and the disruption of membrane potential by pyrazinoic acid and acid pH. Inhibitors of membrane energetics increased the antituberculous activity of pyrazinamide. These findings shed new light on the mode of action of pyrazinamide and may help in the design of new drugs that shorten therapy.

  12. Aromatic amino acid transporter AAT-9 of Caenorhabditis elegans localizes to neurons and muscle cells.

    PubMed

    Veljkovic, Emilija; Bacconi, Andrea; Stetak, Attila; Hajnal, Alex; Stasiuk, Susan; Skelly, Patrick J; Forster, Ian; Shoemaker, Charles B; Verrey, Francois

    2004-11-19

    The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport.

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

    PubMed

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

    2008-01-01

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

  14. The transport of indole-3-acetic Acid in boron- and calcium-deficient sunflower hypocotyl segments.

    PubMed

    Tang, P M; Dela Fuente, R K

    1986-06-01

    Transfer of sunflower (Helianthus annuus L. cv Russian Mammoth) seedlings from complete nutrient solution to solutions deficient in either boron or calcium resulted in a steady decline in the rate of auxin transport, compared to seedlings that remained in the complete solution. In seedlings transferred to solutions deficient in both B and Ca, the decline in auxin transport was greater than seedlings deficient in only one element. The transfer of B- or Ca-deficient seedlings back to the complete solution prevented further decline in auxin transport, but auxin transport did not increase to the same level as seedlings maintained in complete solution. The significant reduction in auxin transport during the early stages of B or Ca deficiency was not related to (a) reduced growth rate of the hypocotyl, (b) increased acropetal movement of auxin, or (c) lack of respiratory substrates in the hypocotyl. In addition, no difference was found in the water-extractable total and ionic Ca in B-deficient and control nondeficient hypocotyls, indicating a direct effect of B on auxin transport, rather than indirectly by affecting Ca absorption. The rate of auxin transport in hypocotyls deficient in either B or Ca, was inversely correlated with K(+) leakage and rate of respiration. The data presented strongly support the view that there are separate sites for B and Ca in the basipetal transport of the plant hormone indoleacetic acid.

  15. Urinary solute transport by ileal segments. I. Effects of nicotinic acid.

    PubMed

    Martínez-Piñeiro, L; Mateos, F; Montero, A; Madero, R; Martínez-Piñeiro, J A

    1993-12-01

    This study was conducted to quantify urinary solute transport by the ileum, using an in vivo human model, and to determine the effect of nicotinic acid on this process. Patients were studied under both basal conditions and niacin therapy. The rates of solute transport were established by analysis of excretion indexes for each solute. Potassium and ammonium were absorbed by the ileum, while phosphorus, sodium and bicarbonate were secreted. The percentage excretion index of sodium and bicarbonate increased by approximately 100 and 600% respectively, causing a significant rise in urinary pH. Although not statistically significant, there was a tendency for chloride to be absorbed and for water to pass into the bowel lumen. Nicotinic acid 3 g/day had no significant effect on urinary solute transport.

  16. Charge transport and structural dynamics in carboxylic-acid-based deep eutectic mixtures.

    PubMed

    Griffin, Philip J; Cosby, Tyler; Holt, Adam P; Benson, Roberto S; Sangoro, Joshua R

    2014-08-07

    Charge transport and structural dynamics in the 1:2 mol ratio mixture of lidocaine and decanoic acid (LID-DA), a model deep eutectic mixture (DEM), have been characterized over a wide temperature range using broad-band dielectric spectroscopy and depolarized dynamic light scattering. Additionally, Fourier transform infrared spectroscopy measurements were performed to assess the degree of proton transfer between the neutral parent molecules. From our detailed analysis of the dielectric spectra, we have determined that this carboxylic-acid-based DEM is approximately 25% ionic at room temperature. Furthermore, we have found that the characteristic diffusion rate of mobile charge carriers is practically identical to the rate of structural relaxation at all measured temperatures, indicating that fast proton transport does not occur in LID-DA. Our results demonstrate that while LID-DA exhibits the thermal characteristics of a DEM, its charge transport properties resemble those of a protic ionic liquid.

  17. Transport of Glyphosate and Aminomethylphosphonic Acid under Two Soil Management Practices in an Italian Vineyard.

    PubMed

    Napoli, Marco; Marta, Anna Dalla; Zanchi, Camillo A; Orlandini, Simone

    2016-09-01

    Worldwide, glyphosate is the most widely used herbicide in controlling the growth of annual and perennial weeds. An increasing number of studies have highlighted the environmental risk resulting from the use of this molecule in aquatic and terrestrial ecosystems. The objective of the study was to determine the transport of glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), through runoff and transported sediment from a vineyard under two different soil management systems: harrowed inter-row (HR) and permanent grass covered inter-row (GR). The study was performed over a period of 4 yr. Glyphosate and AMPA concentrations were found to be higher in runoff and in transported sediment from HR compared with GR, regardless of the amount of runoff and transported sediment. The mean annual percentages of glyphosate loss, via runoff and transported sediment, were about 1.37 and 0.73% for HR and GR, respectively. Aminomethylphosphonic acid represented approximately 30.9 and 40.0% of the total glyphosate losses in GR and HR, respectively. Moreover, results suggested that rains occurring within 4 wk after treatment could cause the transport of glyphosate and AMPA in high concentrations. Soil analyses indicated that glyphosate content was below detection within 1 yr, whereas AMPA remained in the soil profiles along the vine row and in the inter-row. Results indicated that GR can reduce soil and herbicide loss by runoff in vineyard cropping system.

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

    PubMed Central

    1981-01-01

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

  19. Modulating Effect of Ascorbic Acid on Transport-Induced Immunosuppression in Goats

    PubMed Central

    Minka, Ndazo Salka; Ayo, Joseph Olusegun

    2011-01-01

    The effect of 12 h road transportation on some basic blood cells and the modulating role of ascorbic acid were investigated in 40 adult Red Sokoto goats during the hot dry season. The animals were divided into two groups, GI (experimental; n = 20) and GII (control; n = 20). Group 1 was administered with ascorbic acid (AA) per os at a dosage rate of 100 mg/kg body weight, while GII was given 10 mL of sterile water per goat. Forty minutes after the administration and loading, the goats were transported for 12 h. The result obtained in GII goats showed that loading, transportation, high ambient temperature (AT), and relative humidity (RH) encountered during transportation induced lymphopenia, neutrophilia, and eosinopenia, which can cause immunosuppression. In GI goats, the administration of AA prior to loading and transportation ameliorated the adverse effects of loading and transportation stress on neutrophil/lymphocyte ratio and eosinopenia of the goats. PMID:23738106

  20. Transport of Arginine and Aspartic Acid into Isolated Barley Mesophyll Vacuoles 1

    PubMed Central

    Martinoia, Enrico; Thume, Monika; Vogt, Esther; Rentsch, Doris; Dietz, Karl-Josef

    1991-01-01

    The transport of arginine into isolated barley (Hordeum vulgare L.) mesophyll vacuoles was investigated. In the absence of ATP, arginine uptake was saturable with a Km of 0.3 to 0.4 millimolar. Positively charged amino acids inhibited arginine uptake, lysine being most potent with a Ki of 1.2 millimolar. In the presence of free ATP, but not of its Mg-complex, uptake of arginine was drastically enhanced and a linear function of its concentration up to 16 millimolar. The nonhydrolyzable adenylyl imidodiphosphate, but no other nucleotide tested, could substitute for ATP. Therefore, it is suggested that this process does not require energy and does not involve the tonoplast ATPase. The ATP-dependent arginine uptake was strongly inhibited by p-chloromercuriphenylsulfonic acid. Furthermore, hydrophobic amino acids were inhibitory (I50 phenylalanine 1 millimolar). Similar characteristics were observed for the uptake of aspartic acid. However, rates of ATP-stimulated aspartic acid transport were 10-fold lower as compared to arginine transport. Uptake of aspartate in the absence of ATP was negligible. PMID:16668447

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

    SciTech Connect

    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.

  2. Transport of fluorescent bile acids by the isolated perfused rat liver: kinetics, sequestration, and mobilization.

    PubMed

    Holzinger, F; Schteingart, C D; Ton-Nu, H T; Cerrè, C; Steinbach, J H; Yeh, H Z; Hofmann, A F

    1998-08-01

    Hepatocyte transport of six fluorescent bile acids containing nitrobenzoxadiazolyl (NBD) or a fluorescein derivative on the side chain was compared with that of natural bile acids using the single-pass perfused rat liver. Compounds were infused at 40 nmol/g liver min for 15 minutes; hepatic uptake and biliary recovery were measured; fractional extraction, intrinsic basolateral clearance, and sequestration (nonrecovery after 45 minutes of additional perfusion) were calculated. Fluorescent bile acids were efficiently extracted during the first 3 minutes (70%-97%), but net extraction decreased with time mostly because of regurgitation into the perfusate. For cholylglycine and ursodeoxycholylglycine (UDC-glycine), extraction was 94% to 99%, and regurgitation did not occur. Intrinsic hepatic clearance of fluorescent bile acids (2-7 mL/g liver x min) was lower than that of cholylglycine (9.0 +/- 0.6; mean +/- SD) and UDC-glycine (21.4 +/- 0.4). Sequestration at 60 minutes was 8% to 26% for fluorescent bile acids with a cholyl moiety (cholylglycylaminofluorescein [CGamF], cholyllysylfluorescein [C-L-F], cholyl-[N epsilon-NBD]-lysine [C-L-NBD], and cholylaminofluorescein [CamF]), 32% for ursodeoxycholylaminofluorescein (UDCamF), and 88% for ursodeoxycholyl-(N epsilon-NBD)lysine (UDC-L-NBD). Cholylglycine and UDC-glycine had <3% retention. Biliary secretion of sequestered UDCamF, but not of UDC-L-NBD, was induced by adding dibutyryl cyclic adenosine monophosphate (DBcAMP) to the perfusate, possibly by translocation to the canaliculus of pericanalicular vesicles containing fluorescent bile acids. Biliary secretion of UDC-L-NBD, but not of UDCamF, was induced by adding cholyltaurine or UDC-taurine, possibly by inhibition of binding to intracellular constituents or of transport into organelles. It is concluded that fluorescent bile acids are efficiently transported across the basolateral membrane, but in contrast to natural conjugated bile acids, are sequestered in the

  3. Role of sodium ion in transport of folic acid in the small intestine

    SciTech Connect

    Zimmerman, J.; Selhub, J.; Rosenberg, I.H.

    1986-08-01

    The effect of sodium on folate transport across the intestinal luminal membrane was analyzed using two techniques: the influx chamber and isoalted brush-border membrane vesicles. Preincubation of tissue in Na -free medium did not have a consistent effect on folic acid influx provided that Na was present in the test solution. Replacement of Na in the test solution by choline resulted in a significant reduction of folic acid influx. However, when intestinal sheets that had been equilibrated in Na -free solution were exposed to test solutions containing either Na , Li , K , Rb , Cs , Tris , or guanidinium as main cations, folic acid influx was not significantly decreased. Concentration-dependence studies showed that replacement of Na by Rb did not affect the saturable mechanism of folate transport. Rather, a decrease in nonsaturable folic acid uptake accounted for the slightly reduced influx observed in the presence of Rb . Experiments with brush-border membrane vesicles revealed that methotrexate uptake was significantly higher in the presence of external Na than in the presence of K , but was not different from uptake in the presence of K plus valinomycin. These data suggest that 1) the saturable component of folate transport is not Na dependent, and 2) nonsaturable transport of folic acid across the luminal membrane occurs in part through a conductive pathway that involves a negatively charged species of folate and a cation whose membrane permeability affects the rate of folate transport. The importance of Na in this process in vivo derives from the fact that Na is the most permeant cation available at the absorptive site in the small intestine.

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

    PubMed Central

    2011-01-01

    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. PMID:21917168

  5. SGLT2 inhibitor lowers serum uric acid through alteration of uric acid transport activity in renal tubule by increased glycosuria.

    PubMed

    Chino, Yukihiro; Samukawa, Yoshishige; Sakai, Soichi; Nakai, Yasuhiro; Yamaguchi, Jun-ichi; Nakanishi, Takeo; Tamai, Ikumi

    2014-10-01

    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 (UE(UA)) 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 UE(UA), suggesting that SUA decreased as a result of the increase in the UE(UA). The increase in UE(UA) 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 UE(UA) 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 [(14) C]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 [(14) C]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 UE(UA) could potentially be increased by luseogliflozin-induced glycosuria, with alterations of UA transport activity because of urinary glucose.

  6. Transport of some strong incompletely dissociated acids through anion-exchange membrane.

    PubMed

    Palatý, Zdenek; Záková, Alena

    2003-12-01

    Nitric and sulfuric acids belong among strong incompletely dissociated acids, so that in the description of their transport through an ion-exchange membrane, ionic equilibria have to be taken into account. The paper presents the determination of ionic mobilities and diffusivity of nondissociated form of these acids. For that purpose, data on the dialysis experiments with nitric and sulfuric acids in a batch mixed cell with an anion-exchange membrane NEOSEPTA-AFN, which have been completed by those on the membrane conductivity, have been used. The dependencies of the ionic mobilities and the diffusivity of nondissociated form of nitric acid upon the acid concentration in the membrane have been approximated by second degree polynomials. Their coefficients have been determined by numerical integration of the partial differential equation describing the concentration fields of the acids in the membrane and liquid films on both sides of the membrane, followed by an optimizing procedure. The model used is based on the Nernst-Planck electrodiffusion equation. Using all the experimental data obtained at various acid concentrations and rotational speeds of the stirrers, it has been found that ionic mobility is strongly affected by the acid concentration in the membrane and decreases in the series H(3)O(+), SO(2-)(4), NO(-)(3), HSO(-)(4).

  7. Transport of the aromatic amino acids into isolated rat liver cells. Properties of uptake by two distinct systems.

    PubMed Central

    Salter, M; Knowles, R G; Pogson, C I

    1986-01-01

    The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver. PMID:3954748

  8. Transport of indoleacetic acid in intact corn coleoptiles. [Zea mays L

    SciTech Connect

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

    1990-10-01

    We have characterized the transport of ({sup 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.

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

    NASA Astrophysics Data System (ADS)

    Pabalan, R. T.; Chiang, K.-T. K.

    2013-07-01

    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.

  10. Large amino acid transporter 1 (LAT1) prodrugs of valproic acid: new prodrug design ideas for central nervous system delivery.

    PubMed

    Peura, Lauri; Malmioja, Kalle; Laine, Krista; Leppänen, Jukka; Gynther, Mikko; Isotalo, Antti; Rautio, Jarkko

    2011-10-03

    Central nervous system (CNS) drug delivery is a major challenge in drug development because the blood-brain barrier (BBB) efficiently restricts the entry of drug molecules into the CNS at sufficient amounts. The brain uptake of poorly penetrating drugs could be improved by utilizing the transporters at the BBB with a prodrug approach. In this study, we designed four phenylalanine derivatives of valproic acid and studied their ability to utilize a large amino acid transporter 1 (LAT1) in CNS delivery with an aim to show that the meta-substituted phenylalanine prodrugs bind to LAT1 with a higher affinity compared with the affinity of the para-substituted derivatives. All of the prodrugs crossed the BBB carrier mediatedly via LAT1 in in situ rat brain perfusion. For the first time, we introduced a novel meta-substituted phenylalanine analogue promoiety which improved the LAT1 affinity 10-fold and more importantly the rat brain uptake of the prodrug 2-fold compared with those of the para-substituted derivatives. Therefore, we have characterized a new prodrug design idea for CNS drug delivery utilizing a transporter-mediated prodrug approach.

  11. The plasma transport and metabolism of retinoic acid in the rat

    PubMed Central

    Smith, John Edgar; Milch, Peter O.; Muto, Yasutoshi; Goodman, DeWitt S.

    1973-01-01

    The transport of retinoic acid in plasma was examined in vitamin A-deficient rats maintained on small doses of radioactively labelled retinoic acid. After ultracentrifugation of serum adjusted to density 1.21, most of the radioactivity (83%) was associated with the proteins of density greater than 1.21, and not with the serum lipoproteins. Gel filtration of the labelled serum on Sephadex G-200 showed that the radioactive label was associated with protein in the molecular-weight range of serum albumin. On polyacrylamide-gel electrophoresis almost all of the recovered radioactivity migrated with serum albumin. Similar esults were obtained with serum from a normal control rat given a single oral dose of [14C]retinoic acid. These findings indicate that retinoic acid is transported in rat serum bound to serum albumin, and not by retinol-binding protein (the specific transport protein for plasma retinol). Several tissues and the entire remaining carcase of each rat were extracted with ethanol–acetone to determine the tissue distribution of retinoic acid and some of its metabolites. The total recover of radioactive compounds in in the entire body of the rat was about 7–9μg, representing less than 5% or 10% respectively of the total administered label in the two dosage groups studied. The results confirm that retinoic acid is not stored in any tissue. Most of the radioactive material was found in the carcase, rather than in the specific tissues analysed. Two-thirds of the radioactivity in the carcase appeared to represent unchanged retinoic acid. Of the tissues examined, the liver, kidneys and intestine had relatively high concentrations of radioactive compounds, whereas the testes and fat-pads had the lowest concentrations. PMID:4721615

  12. Transport of indoleacetic acid (IAA) and its conjugates in nodal stem segments of Phaseolus vulgaris L

    SciTech Connect

    Tamas, I.A.; Lim, R.

    1987-04-01

    Donor agar blocks containing either (2-acetyl-/sup 14/C) IAA; (2-acetyl-/sup 14/C) indole-3-acetyl-L-aspartate; (2-aceyl-/sup 14/) indole-3-acetyl-L-glycine; or (2-acetyl-/sup 14/C) indole-3-acetyl-L-alanine were placed on either the apical or the basal cut surface of stem segments each bearing an axillary bud in the middle. A receiver block was placed on the end opposite to the donor. After transport, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segments was counted. For all substances, the amount of /sup 14/C transported to the bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of /sup 14/C in the segment declined sharply from apex to base. The opposite was true for acropetal transport. Transport for the three IAA conjugates did not different substantially from each other. The IAA transport inhibitor, naphthylphthalamic acid (NPA), inhibited basipetal /sup 14/C-IAA transport to the base of the stem segment but did not alter substantially the amount of /sup 14/C-IAA recovered from the bud. The results will be discussed in relation to axillary bud growth regulation.

  13. Effect of maternal micronutrients (folic acid, vitamin B12) and omega 3 fatty acids on liver fatty acid desaturases and transport proteins in Wistar rats.

    PubMed

    Wadhwani, Nisha S; Manglekar, Rupali R; Dangat, Kamini D; Kulkarni, Asmita V; Joshi, Sadhana R

    2012-01-01

    A disturbed fatty acid metabolism increases the risk of adult non-communicable diseases. This study examines the effect of maternal micronutrients on the fatty acid composition, desaturase activity, mRNA levels of fatty acid desaturases and transport proteins in the liver. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B(12). The vitamin B(12) deficient groups were supplemented with omega 3 fatty acid. An imbalance of maternal micronutrients reduces liver docosahexaenoic acid, increases Δ5 desaturase activity but decreases mRNA levels, decreases Δ6 desaturase activity but not mRNA levels as compared to control. mRNA level of Δ5 desaturase reverts back to the levels of the control group as a result of omega 3 fatty acid supplementation. Our data for the first time indicates that maternal micronutrients differentially alter the activity and expression of fatty acid desaturases in the liver.

  14. Molecular Switch Controlling the Binding of Anionic Bile Acid Conjugates to Human Apical Sodium-dependent Bile Acid Transporter

    PubMed Central

    Rais, Rana; Acharya, Chayan; Tririya, Gasirat; MacKerell, Alexander D.; Polli, James E.

    2010-01-01

    The human apical sodium-dependent bile acid transporter (hASBT) may serve as a prodrug target for oral drug absorption. Synthetic, biological, NMR and computational approaches identified the structure-activity relationships of mono- and dianionic bile acid conjugates for hASBT binding. Experimental data combined with a conformationally-sampled pharmacophore/QSAR modeling approach (CSP-SAR) predicted that dianionic substituents with intramolecular hydrogen bonding between hydroxyls on the cholane skeleton and the acid group on the conjugate's aromatic ring increased conjugate hydrophobicity and improved binding affinity. Notably, the model predicted the presence of a conformational molecular switch, where shifting the carboxylate substituent on an aromatic ring by a single position controlled binding affinity. Model validation was performed by effectively shifting the spatial location of the carboxylate by inserting a methylene adjacent to the aromatic ring, resulting in the predicted alteration in binding affinity. This work illustrates conformation as a determinant of ligand binding affinity to a biological transporter. PMID:20504026

  15. Formulating gels for decreased mucociliary transport using rheologic properties: polyacrylic acids.

    PubMed

    Shah, Ankur J; Donovan, Maureen D

    2007-04-20

    The purpose of these studies was to identify the rheologic properties of polyacrylic acid gels necessary for optimal reductions in mucociliary clearance. The mucociliary transport of 2 bioadhesive polyacrylic acid polymers, polycarbophil and carbopol, was assessed in vitro by measuring their clearance rates across explants of ciliated bovine tracheal tissue. The viscoelastic properties of polymer gels were measured in the presence of mucus using controlled stress rheometry. Combinations of apparent viscosity (eta) and complex modulus (G*) were found to be the most useful parameters in the identification of polyacrylic acid formulations capable of decreasing mucociliary transport rate (MTR). A narrow range of eta and G* values suitable for reducing mucociliary clearance, while remaining sufficiently fluid for intranasal administration, were identified. The correlations between the rheologic parameters of the polycarbophil gels and their mucociliary transport rates were used to identify other polyacrylic acid gels that also had suitable mucociliary clearance properties, demonstrating that these parameters can be used to direct the optimization of formulations using simple in vitro rheologic testing.

  16. Structural basis for amino acid export by DMT superfamily transporter YddG.

    PubMed

    Tsuchiya, Hirotoshi; Doki, Shintaro; Takemoto, Mizuki; Ikuta, Tatsuya; Higuchi, Takashi; Fukui, Keita; Usuda, Yoshihiro; Tabuchi, Eri; Nagatoishi, Satoru; Tsumoto, Kouhei; Nishizawa, Tomohiro; Ito, Koichi; Dohmae, Naoshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-06-16

    The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.

  17. Cationic amino acid transporters and beta-defensins in dry eye syndrome.

    PubMed

    Jäger, Kristin; Garreis, Fabian; Dunse, Matthias; Paulsen, Friedrich P

    2010-01-01

    Several diseases concomitant with L-arginine deficiency (diabetes, chronic kidney failure, psoriasis) are significantly associated with dry eye syndrome. One important factor that has so far been neglected is the y(+) transporter. In humans, y(+) accounts for nearly 80% of arginine transport, exclusively carrying the cationic amino acids L-arginine, L-lysine and L-ornithine. y(+) is represented by CAT(cationic amino acid transporter) proteins. L-arginine is a precursor of the moisturizer urea, which has been used in the treatment of dry skin diseases. Although urea has also been shown to be part of the tear film, little attention has been paid to it in this role. Moreover, L-arginine and L-lysine are major components contributing to synthesis of the antimicrobially active beta-defensins induced under dry eye conditions. The first results have demonstrated that transport of L-arginine and L-lysine into epithelial cells is limited by the y(+) transporter at the ocular surface.

  18. Clinical significance of coexpression of L-type amino acid transporter 1 (LAT1) and ASC amino acid transporter 2 (ASCT2) in lung adenocarcinoma

    PubMed Central

    Yazawa, Tomohiro; Shimizu, Kimihiro; Kaira, Kyoichi; Nagashima, Toshiteru; Ohtaki, Yoichi; Atsumi, Jun; Obayashi, Kai; Nagamori, Shushi; Kanai, Yoshikatsu; Oyama, Tetsunari; Takeyoshi, Izumi

    2015-01-01

    Background: L-type amino acid transporter 1 (LAT1) and ASC amino acid transporter 2 (ASCT2) have been associated with tumor growth and progression. However, the clinical significance of LAT1 and ASCT2 coexpression in the prognosis of patients with lung adenocarcinoma remains unclear. Methods: In total, 222 patients with surgically resected lung adenocarcinoma were investigated retrospectively. Tumor sections were stained immunohistochemically for LAT1, ASCT2, CD98, phosphorylated mammalian target-of-rapamycin (p-mTOR), and Ki-67, and microvessel density (MVD) was determined by staining for CD34. Epidermal growth factor receptor (EGFR) mutation status was also examined. Results: LAT1 and ASCT2 were positively expressed in 22% and 40% of cases, respectively. Coexpression of LAT1 and ASCT2 was observed in 12% of cases and was associated significantly with disease stage, lymphatic permeation, vascular invasion, CD98, Ki-67, and p-mTOR. Only LAT1 and ASCT2 coexpression indicated a poor prognosis for lung adenocarcinoma. Furthermore, this characteristic was recognized in early-stage patients, especially those who had wild-type, rather than mutated, EGFR. Multivariate analysis confirmed that the coexpression of LAT1 and ASCT2 was an independent factor for predicting poor outcome. Conclusions: LAT1 and ASCT2 coexpression is an independent prognostic factor for patients with lung adenocarcinoma, especially during the early stages, expressing wild-type EGFR. PMID:26279756

  19. Effect of peracetic acid reprocessing on the transport characteristics of polysulfone hemodialyzers.

    PubMed

    Wolff, Susanne H; Zydney, Andrew L

    2005-02-01

    Peracetic acid is used extensively for reprocessing hemodialyzers, despite several indications that reprocessing alters the dialyzer transport characteristics. The objective of this study was to obtain quantitative data for the effects of peracetic acid reprocessing on the clearance and sieving coefficients of urea, vitamin B12, and polydisperse dextrans using Fresenius F80A polysulfone dialyzers. Reprocessing restored the urea and vitamin B12 clearance to close to their original values. However, the reprocessed dialyzers had substantially lower clearance of the larger molecular weight dextrans, which was attributed to reductions in the effective pore size caused by residual plasma proteins within the membrane. Storage in peracetic acid provided some additional removal of residual proteins, although the clearance and sieving coefficients of the larger dextrans remained well below their original values. Peracetic acid caused no degradation of the membrane polymer, in sharp contrast to results obtained with bleach reprocessing.

  20. Nutrient uptake by marine invertebrates: cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus).

    PubMed

    Meyer, Eli; Manahan, Donal T

    2009-08-01

    Transport of amino acids from low concentrations in seawater by marine invertebrates has been extensively studied, but few of the genes involved in this physiological process have been identified. We have characterized three amino acid transporter genes cloned from embryos of the sea urchin Strongylocentrotus purpuratus. These genes show phylogenetic proximity to classical amino acid transport systems, including Gly and B0+, and the inebriated gene (INE). Heterologous expression of these genes in frog oocytes induced a 40-fold increase in alanine transport above endogenous levels, demonstrating that these genes mediate alanine transport. Antibodies specific to one of these genes (Sp-AT1) inhibited alanine transport, confirming the physiological activity of this gene in larvae. Whole-mount antibody staining of larvae revealed expression of Sp-AT1 in the ectodermal tissues associated with amino acid transport, as independently demonstrated by autoradiographic localization of radioactive alanine. Maximum rates of alanine transport increased 6-fold during early development, from embryonic to larval stages. Analysis of gene expression during this developmental period revealed that Sp-AT1 transcript abundance remained nearly constant, while that of another transporter gene (Sp-AT2) increased 11-fold. The functional characterization of these genes establishes a molecular biological basis for amino acid transport by developmental stages of marine invertebrates.

  1. Effect of heat stress on amino acid digestibility and transporters in meat-type chickens.

    PubMed

    Habashy, W S; Milfort, M C; Adomako, K; Attia, Y A; Rekaya, R; Aggrey, S E

    2017-03-02

    The present study was conducted to investigate the effect of heat stress (HS) on performance, digestibility, and molecular transporters of amino acids in broilers. Cobb 500 chicks were raised from hatch till 13 d in floor pens. At d 14, 48 birds were randomly and equally divided between a control group (25°C) and a HS treatment group (35°C). Birds in both treatment classes were individually caged and fed ad libitum on a diet containing 18.7% CP and 3,560 Kcal ME/Kg. Five birds per treatment at one and 12 d post treatment were euthanized and the Pectoralis major (P. major) and ileum were sampled for gene expression analysis. At d 33, ileal contents were collected and used for digestibility analysis. Broilers under HS had reduced growth and feed intake compared to controls. Although the apparent ileal digestibility (AID) was consistently higher for all amino acids in the HS group, it was not significant except for hydroxylysine. The amino acid consumption and retention were significantly lower in the HS group when compared to the control group. Meanwhile, the retention of amino acids per BWG was higher in the HS group when compared to the control group except for hydroxylysine and ornithine. The dynamics of amino acid transporters in the P. major and ileum was influenced by HS. In P. major and ileum tissues at d one, transporters SNAT1, SNAT2, SNAT7, TAT1, and b0,+AT, were down-regulated in the HS group. Meanwhile, LAT4 and B0AT were down-regulated only in the P. major in the treatment group. The amino acid transporters B0AT and SNAT7 at d 12 post HS were down-regulated in the P. major and ileum, but SNAT2 was down-regulated only in the ileum and TAT1 was down-regulated only in the P. major compared with the control group. These changes in amino acid transporters may explain the reduced growth in meat type chickens under heat stress.

  2. Migration-induced variation of fatty acid transporters and cellular metabolic intensity in passerine birds.

    PubMed

    Zhang, Yufeng; King, Marisa O; Harmon, Erin; Eyster, Kathleen; Swanson, David L

    2015-10-01

    Because lipids are the main fuel supporting avian endurance activity, lipid transport and oxidation capacities may increase during migration. We measured enzyme activities, mRNA expression and protein levels in pectoralis and heart for several key steps of lipid transport and catabolism pathways to investigate whether these pathways were upregulated during migration. We used yellow-rumped (Setophaga coronata) and yellow (S. petechia) warblers and warbling vireos (Vireo gilvus) as study species because they all show migration-induced increases in organismal metabolic capacities. For yellow-rumped warblers, β-hydroxyacyl CoA-dehydrogenase (HOAD) activities and fatty acid transporter mRNA and/or protein levels were higher during spring than fall in pectoralis and heart, except that fatty acid translocase (FAT/CD36) protein levels showed the opposite pattern in heart. Lipid transporter protein levels, but not mRNA expression, in pectoralis and heart of warbling vireos were higher either during spring or fall than summer, but this was not true for HOAD activities. For yellow warblers, pectoralis, but not heart, protein levels of lipid transporters were upregulated during migration relative to summer, but this pattern was not evident for mRNA expression or HOAD activity. Finally, muscle and heart citrate synthase and carnitine palmitoyl transferase activities showed little seasonal variation for any species. These data suggest that pectoralis and heart lipid transport and catabolism capacities are often, but not universally, important correlates of elevated organismal metabolic capacity during migration. In contrast, migration-induced variation in cellular metabolic intensity and mitochondrial membrane transport are apparently not common correlates of the migratory phenotype in passerines.

  3. Organic Anion Transporting Polypeptides Contribute to the Disposition of Perfluoroalkyl Acids in Humans and Rats.

    PubMed

    Zhao, Wen; Zitzow, Jeremiah D; Weaver, Yi; Ehresman, David J; Chang, Shu-Ching; Butenhoff, John L; Hagenbuch, Bruno

    2016-12-24

    Perfluoroalkyl sulfonates (PFSAs) such as perfluorohexane sulfonate (PFHxS) and perfluorooctane sulfonate (PFOS) have very long serum elimination half-lives in humans, and preferentially distribute to serum and liver. The enterohepatic circulation of PFHxS and PFOS likely contributes to their extended elimination half-lives. We previously demonstrated that perfluorobutane sulfonate (PFBS), PFHxS, and PFOS are transported into hepatocytes both in a sodium-dependent and a sodium-independent manner. We identified Na(+)/taurocholate cotransporting polypeptide (NTCP) as the responsible sodium-dependent transporter. Furthermore, we demonstrated that the human apical sodium-dependent bile salt transporter (ASBT) contributes to the intestinal reabsorption of PFOS. However, so far no sodium-independent uptake transporters for PFSAs have been identified in human hepatocytes or enterocytes. In addition, perfluoroalkyl carboxylates (PFCAs) with 8 and 9 carbons were shown to preferentially distribute to the liver of rodents; however, no rat or human liver uptake transporters are known to transport these PFCAs. Therefore, we tested whether PFBS, PFHxS, PFOS, and PFCAs with 7-10 carbons are substrates of organic anion transporting polypeptides (OATPs). We used CHO and HEK293 cells to demonstrate that human OATP1B1, OATP1B3, and OATP2B1 can transport PFBS, PFHxS, PFOS, and the 2 PFCAs (C8 and C9). In addition, we show that rat OATP1A1, OATP1A5, OATP1B2, and OATP2B1 transport all 3 PFSAs. In conclusion, our results suggest that besides NTCP and ASBT, OATPs also are capable of contributing to the enterohepatic circulation and extended human serum elimination half-lives of the tested perfluoroalkyl acids.

  4. Genome expansion and differential expression of amino acid transporters at the aphid/Buchnera symbiotic interface.

    PubMed

    Price, Daniel R G; Duncan, Rebecca P; Shigenobu, Shuji; Wilson, Alex C C

    2011-11-01

    In insects, some of the most ecologically important symbioses are nutritional symbioses that provide hosts with novel traits and thereby facilitate exploitation of otherwise inaccessible niches. One such symbiosis is the ancient obligate intracellular symbiosis of aphids with the γ-proteobacteria, Buchnera aphidicola. Although the nutritional basis of the aphid/Buchnera symbiosis is well understood, the processes and structures that mediate the intimate interactions of symbiotic partners remain uncharacterized. Here, using a de novo approach, we characterize the complement of 40 amino acid polyamine organocation (APC) superfamily member amino acid transporters (AATs) encoded in the genome of the pea aphid, Acyrthosiphon pisum. We find that the A. pisum APC superfamily is characterized by extensive gene duplications such that A. pisum has more APC superfamily transporters than other fully sequenced insects, including a ten paralog aphid-specific expansion of the APC transporter slimfast. Detailed expression analysis of 17 transporters selected on the basis of their phylogenetic relationship to five AATs identified in an earlier bacteriocyte expressed sequence tag study distinguished a subset of eight transporters that have been recruited for amino acid transport in bacteriocyte cells at the symbiotic interface. These eight transporters include transporters that are highly expressed and/or highly enriched in bacteriocytes and intriguingly, the four AATs that show bacteriocyte-enriched expression are all members of gene family expansions, whereas three of the four that are highly expressed but not enriched in bacteriocytes retain one-to-one orthology with transporters in other genomes. Finally, analysis of evolutionary rates within the large A. pisum slimfast expansion demonstrated increased rates of molecular evolution coinciding with two major shifts in expression: 1) a loss of gut expression and possibly a gain of bacteriocyte expression and 2) loss of expression

  5. Molecular physiology of the insect K-activated amino acid transporter 1 (KAAT1) and cation-anion activated amino acid transporter/channel 1 (CAATCH1) in the light of the structure of the homologous protein LeuT.

    PubMed

    Castagna, M; Bossi, E; Sacchi, V F

    2009-06-01

    K-activated amino acid transporter 1 (KAAT1) and cation-anion-activated amino acid transporter/channel 1 (CAATCH1) are amino acid cotransporters, belonging to the Na/Cl-dependent neurotransmitter transporter family (also called SLC6/NSS), that have been cloned from Manduca sexta midgut. They have been thoroughly studied by expression in Xenopus laevis oocytes, and structure/function analyses have made it possible to identify the structural determinants of their cation and amino acid selectivity. About 40 mutants of these proteins have been studied by measuring amino acid uptake and current/voltage relationships. The results obtained since the cloning of KAAT1 and CAATCH1 are here discussed in the light of the 3D model of the first crystallized member of the family, the leucine transporter LeuT.

  6. Novel therapeutic approaches targeting L-type amino acid transporters for cancer treatment

    PubMed Central

    Hayashi, Keitaro; Anzai, Naohiko

    2017-01-01

    L-type amino acid transporters (LATs) mainly assist the uptake of neutral amino acids into cells. Four LATs (LAT1, LAT2, LAT3 and LAT4) have so far been identified. LAT1 (SLC7A5) has been attracting much attention in the field of cancer research since it is commonly up-regulated in various cancers. Basic research has made it increasingly clear that LAT1 plays a predominant role in malignancy. The functional significance of LAT1 in cancer and the potential therapeutic application of the features of LAT1 to cancer management are described in this review. PMID:28144396

  7. Placental glucose and amino acid transport in calorie-restricted wild-type and Glut3 null heterozygous mice.

    PubMed

    Ganguly, Amit; Collis, Laura; Devaskar, Sherin U

    2012-08-01

    Calorie restriction (CR) decreased placenta and fetal weights in wild-type (wt) and glucose transporter (Glut) 3 heterozygous null (glut3(+/-)) mice. Because placental nutrient transport is a primary energy determinant of placentofetal growth, we examined key transport systems. Maternal CR reduced intra- and transplacental glucose and leucine transport but enhanced system A amino acid transport in wt mice. These transport perturbations were accompanied by reduced placental Glut3 and leucine amino acid transporter (LAT) family member 2, no change in Glut1 and LAT family member 1, but increased sodium coupled neutral amino acid transporter (SNAT) and SNAT2 expression. We also noted decreased total and active phosphorylated forms of mammalian target of rapamycin, which is the intracellular nutrient sensor, the downstream total P70S6 kinase, and pS6 ribosomal protein with no change in total and phosphorylated 4E-binding protein 1. To determine the role of placental Glut3 in mediating CR-induced placental transport changes, we next investigated the effect of gestational CR in glut3(+/-) mice. In glut3(+/-) mice, a key role of placental Glut3 in mediating transplacental and intraplacental glucose transport was established. In addition, reduced Glut3 results in a compensatory increase of leucine and system A transplacental transport. On the other hand, diminished Glut3-mediated intraplacental glucose transport reduced leucine transport and mammalian target of rapamycin and preserved LAT and enhancing SNAT. CR in glut3(+/-) mice further reduced transplacental glucose transport and enhanced system A amino acid transport, although the increased leucine transport was lost. In addition, increased Glut3 was seen and preserved Glut1, LAT, and SNAT. These placental changes collectively protect survival of wt and glut3(+/-) fetuses against maternal CR-imposed reduction of macromolecular nutrients.

  8. Placental Glucose and Amino Acid Transport in Calorie-Restricted Wild-Type and Glut3 Null Heterozygous Mice

    PubMed Central

    Ganguly, Amit; Collis, Laura

    2012-01-01

    Calorie restriction (CR) decreased placenta and fetal weights in wild-type (wt) and glucose transporter (Glut) 3 heterozygous null (glut3+/−) mice. Because placental nutrient transport is a primary energy determinant of placentofetal growth, we examined key transport systems. Maternal CR reduced intra- and transplacental glucose and leucine transport but enhanced system A amino acid transport in wt mice. These transport perturbations were accompanied by reduced placental Glut3 and leucine amino acid transporter (LAT) family member 2, no change in Glut1 and LAT family member 1, but increased sodium coupled neutral amino acid transporter (SNAT) and SNAT2 expression. We also noted decreased total and active phosphorylated forms of mammalian target of rapamycin, which is the intracellular nutrient sensor, the downstream total P70S6 kinase, and pS6 ribosomal protein with no change in total and phosphorylated 4E-binding protein 1. To determine the role of placental Glut3 in mediating CR-induced placental transport changes, we next investigated the effect of gestational CR in glut3+/− mice. In glut3+/− mice, a key role of placental Glut3 in mediating transplacental and intraplacental glucose transport was established. In addition, reduced Glut3 results in a compensatory increase of leucine and system A transplacental transport. On the other hand, diminished Glut3-mediated intraplacental glucose transport reduced leucine transport and mammalian target of rapamycin and preserved LAT and enhancing SNAT. CR in glut3+/− mice further reduced transplacental glucose transport and enhanced system A amino acid transport, although the increased leucine transport was lost. In addition, increased Glut3 was seen and preserved Glut1, LAT, and SNAT. These placental changes collectively protect survival of wt and glut3+/− fetuses against maternal CR-imposed reduction of macromolecular nutrients. PMID:22700768

  9. Column experiments to investigate transport of colloidal humic acid through porous media during managed aquifer recharge

    NASA Astrophysics Data System (ADS)

    Liu, Dan; Zhou, Jingjing; Zhang, Wenjing; Huan, Ying; Yu, Xipeng; Li, Fulin; Chen, Xuequn

    2016-09-01

    Colloids act as vectors for pollutants in groundwater, thereby creating a series of environmental problems. While managed aquifer recharge plays an important role in protecting groundwater resources and controlling land subsidence, it has a significant effect on the transport of colloids. In this study, particle size and zeta potential of colloidal humic acid (HA) have been measured to determine the effects of different hydrochemistry conditions. Column experiments were conducted to examine the effects on the transport of colloidal HA under varying conditions of pH (5, 7, 9), ionic strength (<0.0005, 0.02, 0.05 M), cation valence (Na+, Ca2+) and flow rate (0.1, 0.2, 0.4 ml/min) through collectors (glass beads) to model the properties and quality of artificial recharge water and changes in the hydrodynamic field. Breakthrough curves showed that the behavior of colloidal HA being transported varied depending on the conditions. Colloid transport was strongly influenced by hydrochemical and hydrodynamic conditions. With decreasing pH or increasing ionic strength, a decrease in the peak effluent concentration of colloidal HA and increase in deposition could be clearly seen. Comparison of different cation valence tests indicated that changes in transport and deposition were more pronounced with divalent Ca2+ than with monovalent Na+. Changes in hydrodynamic field (flow rate) also had an impact on transportation of colloidal HA. The results of this study highlight the need for further research in this area.

  10. Theory of ion transport with fast acid-base equilibrations in bioelectrochemical systems.

    PubMed

    Dykstra, J E; Biesheuvel, P M; Bruning, H; Ter Heijne, A

    2014-07-01

    Bioelectrochemical systems recover valuable components and energy in the form of hydrogen or electricity from aqueous organic streams. We derive a one-dimensional steady-state model for ion transport in a bioelectrochemical system, with the ions subject to diffusional and electrical forces. Since most of the ionic species can undergo acid-base reactions, ion transport is combined in our model with infinitely fast ion acid-base equilibrations. The model describes the current-induced ammonia evaporation and recovery at the cathode side of a bioelectrochemical system that runs on an organic stream containing ammonium ions. We identify that the rate of ammonia evaporation depends not only on the current but also on the flow rate of gas in the cathode chamber, the diffusion of ammonia from the cathode back into the anode chamber, through the ion exchange membrane placed in between, and the membrane charge density.

  11. Theory of ion transport with fast acid-base equilibrations in bioelectrochemical systems

    NASA Astrophysics Data System (ADS)

    Dykstra, J. E.; Biesheuvel, P. M.; Bruning, H.; Ter Heijne, A.

    2014-07-01

    Bioelectrochemical systems recover valuable components and energy in the form of hydrogen or electricity from aqueous organic streams. We derive a one-dimensional steady-state model for ion transport in a bioelectrochemical system, with the ions subject to diffusional and electrical forces. Since most of the ionic species can undergo acid-base reactions, ion transport is combined in our model with infinitely fast ion acid-base equilibrations. The model describes the current-induced ammonia evaporation and recovery at the cathode side of a bioelectrochemical system that runs on an organic stream containing ammonium ions. We identify that the rate of ammonia evaporation depends not only on the current but also on the flow rate of gas in the cathode chamber, the diffusion of ammonia from the cathode back into the anode chamber, through the ion exchange membrane placed in between, and the membrane charge density.

  12. Evidence for transport intermediates in aromatic amino acid synthesis of non-green tissues

    SciTech Connect

    Leuschner, C.; Schultz, G. )

    1990-05-01

    Quinate (QA) is the predominant pre-aromatic compound formed at high rates in leaves of many plants at the early vegetation stage and transported through the phloem. The transfer of 3-dehydroquinate, 3-dehydroshikimate and (SkA) across the plastidial membranes has been evidenced. The question was whether the rate of QA uptake is comparable to that of the 3 SkA-pathway intermediates. To demonstrate this, /U-{sup 14}C/QA and /U-{sup 14}C/SkA were applied to Brassica rapa roots. Both compounds were uptaken at considerable rates and incorporated into aromatic amino acids (Phe + Tyr + Trp formation, in nmol/g fresh wt x h: applying 145 {mu}mol QA: 21.2; applying 156 {mu}mol Ska: 31.8). Thus, QA is a possible candidate for transport into non-green tissues for aromatic amino acid synthesis.

  13. Fatty Acid-Binding Protein in Small Intestine IDENTIFICATION, ISOLATION, AND EVIDENCE FOR ITS ROLE IN CELLULAR FATTY ACID TRANSPORT

    PubMed Central

    Ockner, Robert K.; Manning, Joan A.

    1974-01-01

    A soluble fatty acid-binding protein (FABP), mol wt ∼ 12,000 is present in intestinal mucosa and other tissues that utilize fatty acids, including liver, myocardium, adipose, and kidney. This protein binds long chain fatty acids both in vivo and in vitro. FABP was isolated from rat intestine by gel filtration and isoelectric focusing. It showed a reaction of complete immunochemical identity with proteins in the 12,000 mol wt fatty acid-binding fractions of liver, myocardium, and adipose tissue supernates. (The presence of immunochemically nonidentical 12,000 mol wt FABP in these tissues is not excluded.) By quantitative radial immunodiffusion, supernatant FABP concentration in mucosa from proximal and middle thirds of jejuno-ileum significantly exceeded that in distal third, duodenum, and liver, expressed as micrograms per milligram soluble protein, micrograms per gram DNA, and micrograms per gram tissue. FABP concentration in villi was approximately three times greater than in crypts. Small quantities of FABP were present in washed nuclei-cell membrane, mitochondrial and microsomal fractions. However, the amount of FABP solubilized per milligram membrane protein was similar for all particulate fractions, and total membrane-associated FABP was only about 16% of supernatant FABP. Intestinal FABP concentration was significantly greater in animals maintained on high fat diets than on low fat; saturated and unsaturated fat diets did not differ greatly in this regard. The preponderance of FABP in villi from proximal and middle intestine, its ability to bind fatty acids in vivo as well as in vitro, and its response to changes in dietary fat intake support the concept that this protein participates in cellular fatty acid transport during fat absorption. Identical or closely related 12,000 mol wt proteins may serve similar functions in other tissues. Images PMID:4211161

  14. The effects of the carboxyl-terminus amino acids of the Shiga toxin B-subunit on retrograde transport.

    PubMed

    Liu, Dan; Fan, Yuying; Li, Jie; Gao, Xiaoge; Hao, Miao; Xue, Huiting; Tai, Guihua

    2012-07-01

    The Shiga toxin B-subunit (STxB), from the enteric pathogen, Shigella dysenteriae, is responsible for the attachment of its receptor, globotriaosylceramide (Gb3), and navigates the retrograde pathway from the plasma membrane to the endoplasmic reticulum (ER). In this study, in order to demonstrate the role of carboxyl-terminus (C-terminus/al) amino acids of the B-fragment on the retrograde transport speed and the retrograde transport pathway, STxB was modified by site-directed mutagenesis and by the addition of an amino acid tail. The results showed that when the C-terminal amino acid, arginine [Arg (R)], was mutated to serine [Ser (S)], the speed of the B-fragment transportation into the ER at 37 ˚C was slower. When an acidic amino acid tail 'glutamine (Glu)-Ser' (ES) was added to the C-terminal amino acid 'R', the B-fragment transporting speed slowed down and remained in the Golgi apparatus. Further experiments showed that the effects induced by mutations of the amino acid tail resulted in STxB-EEEES ≥-EEES>-EES>-ES, demonstrating that the retardation effect on the tail was increased and the length of the acidic amino acid was augmented. The effect was possibly produced by an acidic amino acid tail, not only by the amino acid 'E'. The significant inhibitory effect on the speed of B-fragment retrograde transport was observed only when the mutations of the acidic amino acid tail were linked near to the C-terminus. These results may provide important insights for the study of transport mechanisms and for the development of STxB serial proteins as vectors for drug delivery.

  15. Absorption and lymphatic transport of exogenous and endogenous arachidonic and linoleic acid in the rat

    SciTech Connect

    Nilsson, A.; Landin, B.; Jensen, E.; Akesson, B.

    1987-06-01

    (/sup 3/H)Arachidonic (20:4) and (/sup 14/C)linoleic acid (18:2) were fed to thoracic duct-cannulated rats in test meals of either tracers alone, cream, Intralipid, pure arachidonic acid, or pure linoleic acid. Less (/sup 3/H)20:4 than (/sup 14/C)18:2 was recovered in chyle during the first 5 h. After cream feeding, the proportion of radioactivity found in phospholipids was high and increased during the first 3 h. After the meal 61 +/- 6% of the /sup 3/H and 57 +/- 10% of the /sup 14/C was in phosphatidylcholine, and 11 +/- 3% of the /sup 3/H and 3.0 +/- 4% of the /sup 14/C was in phosphatidylethanolamine. Changing the fat vehicle to Intralipid or pure 18:2 decreased the proportion of label in the phospholipds and increased the /sup 3/H and /sup 14/C radioactivity in the triacylglycerol fraction, the distribution of /sup 14/C radioactivity in the triacylglycerol fraction, the distribution of /sup 14/C being influenced more than that of /sup 3/H. After feeding the tracers in 200 ..mu..l of pure 20:4, >90% of both isotopes was in triacylglycerol. During fasting, triacylglycerol transported 56% (0.7 ..mu..mol/h), phosphatidylethanolamine transported 10% (0.1 ..mu..mol/h) of the 20:4 mass. After cream or Intralipid feeding, the output of 20:4-containing phosphatidylcholine and phosphatidylethanolamine increased 2.1- to 2.8-fold, whereas the transport of 20:4 with triacylglycerol remained constant. Phospholipids thus became the predominant transport form for 20:4. After feeding 200 ..mu..l of 20:4, the intestine produced, however, 20:4-rich triacylglycerols that transported 80% of the chyle 20:4.

  16. Improved Experimental Techniques for Analyzing Nucleic Acid Transport Through Protein Nanopores in Planar Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Costa, Justin A.

    The translocation of nucleic acid polymers across cell membranes is a fundamental requirement for complex life and has greatly contributed to genomic molecular evolution. The diversity of pathways that have evolved to transport DNA and RNA across membranes include protein receptors, active and passive transporters, endocytic and pinocytic processes, and various types of nucleic acid conducting channels known as nanopores. We have developed a series of experimental techniques, collectively known as "Wicking", that greatly improves the biophysical analysis of nucleic acid transport through protein nanopores in planar lipid bilayers. We have verified the Wicking method using numerous types of classical ion channels including the well-studied chloride selective channel, CLIC1. We used the Wicking technique to reconstitute α-hemolysin and found that DNA translocation events of types A and B could be routinely observed using this method. Furthermore, measurable differences were observed in the duration of blockade events as DNA length and composition was varied, consistent with previous reports. Finally, we tested the ability of the Wicking technology to reconstitute the dsRNA transporter Sid-1. Exposure to dsRNAs of increasing length and complexity showed measurable differences in the current transitions suggesting that the charge carrier was dsRNA. However, the translocation events occurred so infrequently that a meaningful electrophysiological analysis was not possible. Alterations in the lipid composition of the bilayer had a minor effect on the frequency of translocation events but not to such a degree as to permit rigorous statistical analysis. We conclude that in many instances the Wicking method is a significant improvement to the lipid bilayer technique, but is not an optimal method for analyzing transport through Sid-1. Further refinements to the Wicking method might have future applications in high throughput DNA sequencing, DNA computation, and

  17. [Hepatocellular transport of bile acids and organic anions in infection and SIRS--evidence for different mechanisms for regulating membrane transport proteins].

    PubMed

    Bolder, U; Thasler, W E; Hofmann, A F; Jauch, K W

    1998-01-01

    The alteration of proinflammatory mediators during sepsis and SIRS results in a large variety of adaptive changes of metabolic and physiologic variables. This study investigated the alterations of hepatocellular transport in a rat sepsis model (LPS i.p.) as well as in a model inducing SIRS by sterile abscess formation (turpentine i.m.). Two bile acids (Cholyltaurine and Chemodeoxycholyltaurine) and one organic anion (Sulfolithocholyltaurine) were used as marker substrates to investigate the time course of hepatocellular transport function. Experiments were performed in isolated perfused rat livers and plasma membrane vesicles. During sepsis, both, the transport of bile acids and that of the organic anion was markedly reduced. In contrast no alteration of transport was detected during SIRS. However, biliary secretion of glutathione (+90%) and bile acid independent bile flow (%) were increased. mRNA levels of bile acid and organic anion transport proteins were reduced. The lowest values were noted 12 h after injection of LPS or turpentine. Almost unchanged kinetic parameters during SIRS pointed to a normal population of transporters with regard to quantity and substrate affinity. Therefore it seems that transcriptional regulation plays an important role for the expression of transport proteins during sepsis, whereas posttranscriptional regulation may be of importance during SIRS. The clinical phenomenon of septic cholestasis including jaundice implies endotoxemia and differenciates against SIRS.

  18. Klotho sensitivity of the neuronal excitatory amino acid transporters EAAT3 and EAAT4.

    PubMed

    Almilaji, Ahmad; Munoz, Carlos; Pakladok, Tatsiana; Alesutan, Ioana; Feger, Martina; Föller, Michael; Lang, Undine E; Shumilina, Ekaterina; Lang, Florian

    2013-01-01

    Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)2D3 formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (Iglu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased Iglu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.

  19. Interaction of α-Lipoic Acid with the Human Na+/Multivitamin Transporter (hSMVT).

    PubMed

    Zehnpfennig, Britta; Wiriyasermkul, Pattama; Carlson, David A; Quick, Matthias

    2015-06-26

    The human Na(+)/multivitamin transporter (hSMVT) has been suggested to transport α-lipoic acid (LA), a potent antioxidant and anti-inflammatory agent used in therapeutic applications, e.g. in the treatment of diabetic neuropathy and Alzheimer disease. However, the molecular basis of the cellular delivery of LA and in particular the stereospecificity of the transport process are not well understood. Here, we expressed recombinant hSMVT in Pichia pastoris and used affinity chromatography to purify the detergent-solubilized protein followed by reconstitution of hSMVT in lipid bilayers. Using a combined approach encompassing radiolabeled LA transport and equilibrium binding studies in conjunction with the stabilized R-(+)- and S-(-)-enantiomers and the R,S-(+/-) racemic mixture of LA or lipoamide, we identified the biologically active form of LA, R-LA, to be the physiological substrate of hSMVT. Interaction of R-LA with hSMVT is strictly dependent on Na(+). Under equilibrium conditions, hSMVT can simultaneously bind ~2 molecules of R-LA in a biphasic binding isotherm with dissociation constants (Kd) of 0.9 and 7.4 μm. Transport of R-LA in the oocyte and reconstituted system is exclusively dependent on Na(+) and exhibits an affinity of ~3 μm. Measuring transport with known amounts of protein in proteoliposomes containing hSMVT in outside-out orientation yielded a catalytic turnover number (kcat) of about 1 s(-1), a value that is well in agreement with other Na(+)-coupled transporters. Our data suggest that hSMVT-mediated transport is highly specific for R-LA at our tested concentration range, a finding with wide ramifications for the use of LA in therapeutic applications.

  20. Transport of. cap alpha. -aminoisobutyric acid by Streptococcus pyogenes and its derived L-form

    SciTech Connect

    Reizer, J.; Panos, C.

    1982-01-01

    We studied the uptake of ..cap alpha..-aminoisobutyric acid (AIB) in Streptococcus pyogenes and its physiologically isotonic L-form. S. pyogenes cells starved for glucose or treated with carbonyl cyanide-m-chlorophenyl hydrazone accumulated limited amounts of AIB. A high apparent K/sub m/ value characterized the glucose-independent transport of AIB. The rate and extent of AIB accumulation significantly increased in the presence of glucose. Two saturable transport components with distinct apparent K/sub m/values characterized glycolysis-coupled transport of AIB. A biphasic Lineweaver-Burk plot was also obtained for L-alanine transport by glycolyzing S. pyogenes cells. AIB seems to share a common transport system(s) with glycine, L- and D-anine, L-serine, and L-valine. This was shown by the competitive exchange efflux of accumulated AIB. About 30% of the AIB uptake was not inhibited by a saturating amount of L-valine, indicating the existence of more than one system for AIB transport, p-Chloromercuribenzoate markedly inhibited the accumulation of AIB by both glycolyzing and glucose-starved cells. In contrast, carbonyl cyanide-m-chlorophenyl hydrazone affected only metabolism-dependent uptake of AIB, which was also sensitive to dinitrophenol, N-ethylmaleimide, iodoacetate, fluoride (NaF), arsenate, and N,N'-dicyclohexylcarbodiimide. These results are interpreted according to the chemiosmotic theory of Mitchell, whereby a proton motive force constitutes the driving force for AIB accumulation. AIB was not accumulated by the L-form. However, a temporary accumulation of AIB by a counterflow mechanism and a saturable system with a low apparent affinity were demonstrated for AIB transport by this organism. We suggest that a deficiency in the coupling of energy to AIB transport is responsible for the apparent lack of active AIB accumulation by the L-form.

  1. Stimulation of the amino acid transporter SLC6A19 by JAK2

    SciTech Connect

    Bhavsar, Shefalee K.; Hosseinzadeh, Zohreh; Merches, Katja; Gu, Shuchen; Broeer, Stefan; Lang, Florian

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer The amino acid transporter SLC6A19 is upregulated by Janus kinase-2 JAK2. Black-Right-Pointing-Pointer The {sup V617F}JAK2 mutant, causing myeloproliferative disease, is more effective. Black-Right-Pointing-Pointer JAK2 inhibitor AG490 reverses stimulation of SLC6A19 by {sup V617F}JAK2. Black-Right-Pointing-Pointer JAK2 enhances SLC6A19 protein insertion into the cell membrane. Black-Right-Pointing-Pointer SLC6A19 may contribute to amino acid uptake into {sup V617F}JAK2 expressing tumor cells. -- Abstract: JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation {sup V617F}JAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na{sup +} coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, {sup V617F}JAK2 or inactive {sup K882E}JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I{sub le}), which was significantly increased following coexpression of JAK2 or {sup V617F}JAK2, but not by coexpression of {sup K882E}JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 {mu}M) resulted in a gradual decline of I{sub le}. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I{sub le} following inhibition of carrier insertion by brefeldin A (5 {mu}M) was similar

  2. Studies on the renal transport of trimethylpentanoic acid metabolites of 2,2,4-trimethylpentane in rat renal cortical slices.

    PubMed

    Lock, E A; Strasser, J; Bus, J S; Charbonneau, M

    1993-01-01

    2,2,4-Trimethylpentane (TMP), a nephrotoxic component of unleaded gasoline in male but not female rats, undergoes oxidative metabolism to yield 2,2,4- and 2,4,4-trimethylpentanol, pentanoic acid and 5-hydroxypentanoic acid. We have examined the effect of three of these pentanoic acid metabolites on the renal transport of the organic anion p-aminohippurate (PAH) and the organic cation tetraethylammonium (TEA) in renal cortical slices from male Fischer 344 rats. 2,4,4-Trimethylpentanoic acid, the major urinary metabolite in rats, produced a selective decrease in the accumulation of PAH without affecting TEA accumulation. Kinetic analysis showed that 2,4,4-trimethylpentanoic acid was a competitive inhibitor of the organic anion transport system, with a Ki of 4 mM. 2,4,4-Trimethyl-5-hydroxypentanoic acid also showed selective inhibition of PAH transport, while 2,2,4-trimethylpentanoic acid was less selective and reduced both PAH and TEA transport. Additional studies with radiolabeled 2,4,4-trimethylpentanoic acid showed that there was a time- and concentration-dependent accumulation of radioactivity into slices of renal cortex. However, experiments conducted at 4 degrees C and studies with metabolic inhibitors, or with an inhibitor of organic anion transport, indicated that little of the accumulated material was entering the cell. We conclude from these studies that the pentanoic acid metabolites formed from 2,2,4-trimethylpentane are not actively transported by the renal organic anion transport system. In summary, in vitro the pentanoic acid metabolites appear to bind to renal cortical tissue and thereby reduce the transport of PAH.

  3. cAMP increases mitochondrial cholesterol transport through the induction of arachidonic acid release inside this organelle in Leydig cells.

    PubMed

    Castillo, Ana Fernanda; Cornejo Maciel, Fabiana; Castilla, Rocío; Duarte, Alejandra; Maloberti, Paula; Paz, Cristina; Podestá, Ernesto J

    2006-11-01

    We have investigated the direct effect of arachidonic acid on cholesterol transport in intact cells or isolated mitochondria from steroidogenic cells and the effect of cyclic-AMP on the specific release of this fatty acid inside the mitochondria. We show for the first time that cyclic-AMP can regulate the release of arachidonic acid in a specialized compartment of MA-10 Leydig cells, e.g. the mitochondria, and that the fatty acid induces cholesterol transport through a mechanism different from the classical pathway. Arachidonic acid and arachidonoyl-CoA can stimulate cholesterol transport in isolated mitochondria from nonstimulated cells. The effect of arachidonoyl-CoA is inhibited by the reduction in the expression or in the activity of a mitochondrial thioesterase that uses arachidonoyl-CoA as a substrate to release arachidonic acid. cAMP-induced arachidonic acid accumulation into the mitochondria is also reduced when the mitochondrial thioesterase activity or expression is blocked. This new feature in the regulation of cholesterol transport by arachidonic acid and the release of arachidonic acid in specialized compartment of the cells could offer novel means for understanding the regulation of steroid synthesis but also would be important in other situations such as neuropathological disorders or oncology disorders, where cholesterol transport plays an important role.

  4. Effects of ethanol and acetic acid on the transport of malic acid and glucose in the yeast Schizosaccharomyces pombe: implications in wine deacidification.

    PubMed

    Sousa, M J; Mota, M; Leão, C

    1995-02-15

    Ethanol and acetic acid, at concentrations which may occur during wine-making, inhibited the transport of L-malic acid in Schizosaccharomyces pombe. The inhibition was non-competitive, the decrease of the maximum initial velocity following exponential kinetics. Glucose transport was not significantly affected either by ethanol (up to 13%, w/v) or by acetic acid (up to 1.5%, w/v). The uptake of labelled acetic acid followed simple diffusion kinetics, indicating that a carrier was not involved in its transport. Therefore, the undissociated acid appears to be the only form that enters the cells and is probably responsible for the toxic effects. Accordingly, deacidification by Ss. pombe during wine fermentation should take place before, rather than after, the main alcoholic fermentation by Saccharomyces cerevisiae.

  5. Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

    PubMed Central

    Schlisselberg, Doreen; Mazarib, Eldar; Inbar, Ehud; Rentsch, Doris; Myler, Peter J.; Zilberstein, Dan

    2015-01-01

    Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters. PMID:26549185

  6. Peptide modules for overcoming barriers of nucleic acids transport to cells.

    PubMed

    Egorova, Anna A; Kiselev, Anton V

    2016-01-01

    Absence of safe and efficient methods of nucleic acids delivery is one of the major issues which limits the development of human gene therapy. Highly efficient viral vectors raise questions due to safety reasons. Among non-viral vectors peptide-based carriers can be considered as good candidates for the development of "artificial viruses"--multifunctional polyplexes that mimic viruses. Suggested strategy to obtain multifunctionality is to combine several peptide modules into one modular carrier. Different kinds of peptide modules are needed for successful overcoming barriers of nucleic acids transport into the cells. Design of such modules and establishment of structure-function relationships are issues of importance to researchers working in the field of nucleic acids delivery.

  7. The evolution of Jen3 proteins and their role in dicarboxylic acid transport in Yarrowia

    PubMed Central

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Michely, Stéphanie; Thevenieau, France; Neuvéglise, Cécile; Nicaud, Jean-Marc

    2015-01-01

    Jen proteins in yeast are involved in the uptake of mono/dicarboxylic acids. The Jen1 subfamily transports lactate and pyruvate, while the Jen2 subfamily transports fumarate, malate, and succinate. Yarrowia lipolytica has six JEN genes: YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D20108g, YALI0D24607g, and YALI0E32901g. Through phylogenetic analyses, we found that these genes represent a new subfamily, Jen3 and that these three Jen subfamilies derivate from three putative ancestral genes. Reverse transcription-PCR. revealed that only four YLJEN genes are expressed and they are upregulated in the presence of lactate, pyruvate, fumarate, malate, and/or succinate, suggesting that they are able to transport these substrates. Analysis of deletion mutant strains revealed that Jen3 subfamily proteins transport fumarate, malate, and succinate. We found evidence that YALI0C15488 encodes the main transporter because its deletion was sufficient to strongly reduce or suppress growth in media containing fumarate, malate, or succinate. It appears that the other YLJEN genes play a minor role, with the exception of YALI0E32901g, which is important for malate uptake. However, the overexpression of each YLJEN gene in the sextuple-deletion mutant strain ΔYLjen1-6 revealed that all six genes are functional and have evolved to transport different substrates with varying degrees of efficacy. In addition, we found that YALI0E32901p transported succinate more efficiently in the presence of lactate or fumarate. PMID:25515252

  8. Substrate Selectivity of YgfU, a Uric Acid Transporter from Escherichia coli*

    PubMed Central

    Papakostas, Konstantinos; Frillingos, Stathis

    2012-01-01

    The ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family includes more than 2,000 members, but only 15 have been characterized experimentally. Escherichia coli has 10 members, of which the uracil permease UraA and the xanthine permeases XanQ and XanP are functionally known. Of the remaining members, YgfU is closely related in sequence and genomic locus with XanQ. We analyzed YgfU and showed that it is a proton-gradient dependent, low-affinity (Km 0.5 mm), and high-capacity transporter for uric acid. It also shows a low capacity for transport of xanthine at 37 °C but not at 25 °C. Based on the set of positions delineated as important from our previous Cys-scanning analysis of permease XanQ, we subjected YgfU to rationally designed site-directed mutagenesis. The results show that the conserved His-37 (TM1), Glu-270 (TM8), Asp-298 (TM9), and Gln-318 and Asn-319 (TM10) are functionally irreplaceable, and Thr-100 (TM3) is essential for the uric acid selectivity because its replacement with Ala allows efficient uptake of xanthine. The key role of these residues is corroborated by the conservation pattern and homology modeling on the recently described x-ray structure of permease UraA. In addition, site-specific replacements at TM8 (S271A, M274D, V282S) impair expression in the membrane, and V320N (TM10) inactivates the permease, whereas R327G (TM10) or S426N (TM14) reduces the affinity for uric acid (4-fold increased Km). Our study shows that comprehensive analysis of structure-function relationships in a newly characterized transporter can be accomplished with relatively few site-directed replacements, based on the knowledge available from Cys-scanning mutagenesis of a prototypic homolog. PMID:22437829

  9. SNAT2 amino acid transporter is regulated by amino acids of the SLC6 gamma-aminobutyric acid transporter subfamily in neocortical neurons and may play no role in delivering glutamine for glutamatergic transmission.

    PubMed

    Grewal, Sukhjeevan; Defamie, Norah; Zhang, Xiong; De Gois, Stéphanie; Shawki, Ali; Mackenzie, Bryan; Chen, Chu; Varoqui, Hélène; Erickson, Jeffrey D

    2009-04-24

    System A transporters SNAT1 and SNAT2 mediate uptake of neutral alpha-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, gamma-aminobutyric acid, and beta-alanine (substrates of the SLC6 gamma-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10x) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.

  10. Stable isotope tracer reveals that viviparous snakes transport amino acids to offspring during gestation.

    PubMed

    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.

  11. Assessment of Amino Acid/Drug Transporters for Renal Transport of [18F]Fluciclovine (anti-[18F]FACBC) in Vitro

    PubMed Central

    Ono, Masahiro; Baden, Atsumi; Okudaira, Hiroyuki; Kobayashi, Masato; Kawai, Keiichi; Oka, Shuntaro; Yoshimura, Hirokatsu

    2016-01-01

    [18F]Fluciclovine (trans-1-amino-3-[18F]fluorocyclobutanecarboxylic acid; anti-[18F]FACBC), a positron emission tomography tracer used for the diagnosis of recurrent prostate cancer, is transported via amino acid transporters (AATs) with high affinity (Km: 97–230 μM). However, the mechanism underlying urinary excretion is unknown. In this study, we investigated the involvement of AATs and drug transporters in renal [18F]fluciclovine reuptake. [14C]Fluciclovine (trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid) was used because of its long half-life. The involvement of AATs in [14C]fluciclovine transport was measured by apical-to-basal transport using an LLC-PK1 monolayer as model for renal proximal tubules. The contribution of drug transporters herein was assessed using vesicles/cells expressing the drug transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 4 (MRP4), organic anion transporter 1 (OAT1), organic anion transporter 3 (OAT3) , organic cation transporter 2 (OCT2), organic anion transporting polypeptide 1B1 (OATP1B1), and organic anion transporting polypeptide 1B3 (OATP1B3). The apical-to-basal transport of [14C]fluciclovine was attenuated by l-threonine, the substrate for system alanine-serine-cysteine (ASC) AATs. [14C]Fluciclovine uptake by drug transporter-expressing vesicles/cells was not significantly different from that of control vesicles/cells. Fluciclovine inhibited P-gp, MRP4, OAT1, OCT2, and OATP1B1 (IC50 > 2.95 mM). Therefore, system ASC AATs may be partly involved in the renal reuptake of [18F]fluciclovine. Further, given that [18F]fluciclovine is recognized as an inhibitor with millimolar affinity for the tested drug transporters, slow urinary excretion of [18F]fluciclovine may be mediated by system ASC AATs, but not by drug transporters. PMID:27754421

  12. Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2.

    PubMed

    Weerachayaphorn, Jittima; Pajor, Ana M

    2008-04-01

    Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1.

  13. The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

    SciTech Connect

    Desforges, M.; Greenwood, S.L.; Glazier, J.D.; Westwood, M.; Sibley, C.P.

    2010-07-16

    Research highlights: {yields} mRNA levels for SNAT1 are higher than other system A subtype mRNAs in primary human cytotrophoblast. {yields} SNAT1 knockdown in cytotrophoblast cells significantly reduces system A activity. {yields} SNAT1 is a key contributor to system A-mediated amino acid transport in human placenta. -- Abstract: System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66 h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis-Menten constants for uptake of the system A-specific substrate, {sup 14}C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of {sup 14}C-MeAIB uptake revealed two distinct transport systems; system 1: K{sub m} = 0.38 {+-} 0.12 mM, V{sub max} = 27.8 {+-} 9.0 pmol/mg protein/20 min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: K{sub m} = 45.4 {+-} 25.0 mM, V{sub max} = 1190 {+-} 291 pmol/mg protein/20 min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific si

  14. Interactions Between Fatty Acid Transport Proteins, Genes That Encode for Them, and Exercise: A Systematic Review.

    PubMed

    Jayewardene, Avindra F; Mavros, Yorgi; Reeves, Anneliese; Hancock, Dale P; Gwinn, Tom; Rooney, Kieron B

    2016-08-01

    Long-chain fatty acid (LCFA) movement into skeletal muscle involves a highly mediated process in which lipid rafts are utilized in the cellular membrane, involving numerous putative plasma membrane-associated LCFA transport proteins. The process of LCFA uptake and oxidation is of particular metabolic significance both at rest and during light to moderate exercise. A comprehensive systematic search of electronic databases was conducted to investigate whether exercise alters protein and/or gene expression of putative LCFA transport proteins. There were 31 studies meeting all eligibility criteria, of these 13 utilized an acute exercise protocol and 18 examined chronic exercise adaptations. Seventeen involved a study design incorporating an exercise stimulus, while the remaining 14 incorporated a combined exercise and diet stimulus. Divergent data relating to acute exercise, as well as prolonged exercise training (≥3 weeks), on protein content (PC) response was identified for proteins CD36, FABPpm and CAV1. Messenger ribonucleic acid (mRNA) data did not always correspond to functional PC, supporting previous suggestions of a disconnect due to potentially limiting factors post gene expression. The large array of study designs, cohorts, and primary dependent variables within the studies included in the present review elucidate the complexity of the interaction between exercise and LCFA transport proteins. Summary of the results in the present review validate the need for further targeted investigation within this topic, and provide an important information base for such research. J. Cell. Physiol. 231: 1671-1687, 2016. © 2015 Wiley Periodicals, Inc.

  15. Influence of Perfluorooctanoic Acid on the Transport and Deposition Behaviors of Bacteria in Quartz Sand.

    PubMed

    Wu, Dan; Tong, Meiping; Kim, Hyunjung

    2016-03-01

    The significance of perfluorooctanoic acid (PFOA) on the transport and deposition behaviors of bacteria (Gram-negative Escherichia coli and Gram-positive Bacillus subtilis) in quartz sand is examined in both NaCl and CaCl2 solutions at pH 5.6 by comparing both breakthrough curves and retained profiles with PFOA in solutions versus those without PFOA. All test conditions are found to be highly unfavorable for cell deposition regardless of the presence of PFOA; however, 7%-46% cell deposition is observed depending on the conditions. The cell deposition may be attributed to micro- or nanoscale roughness and/or to chemical heterogeneity of the sand surface. The results show that, under all examined conditions, PFOA in suspensions increases cell transport and decreases cell deposition in porous media regardless of cell type, presence or absence of extracellular polymeric substances, ionic strength, and ion valence. We find that the additional repulsion between bacteria and quartz sand caused by both acid-base interaction and steric repulsion as well as the competition for deposition sites on quartz sand surfaces by PFOA are responsible for the enhanced transport and decreased deposition of bacteria with PFOA in solutions.

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

    PubMed

    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.

  17. A branched chain amino acid metabolite drives vascular transport of fat and causes insulin resistance

    PubMed Central

    Jang, Cholsoon; Oh, Sungwhan F; Wada, Shogo; Rowe, Glenn C; Liu, Laura; Chan, Mun Chun; Rhee, James; Hoshino, Atsushi; Kim, Boa; Ibrahim, Ayon; Baca, Luisa G; Kim, Esl; Ghosh, Chandra C; Parikh, Samir M; Jiang, Aihua; Chu, Qingwei; Forman, Daniel E.; Lecker, Stewart H.; Krishnaiah, Saikumari; Rabinowitz, Joshua D; Weljie, Aalim M; Baur, Joseph A; Kasper, Dennis L; Arany, Zoltan

    2016-01-01

    Epidemiological and experimental data implicate branched chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms underlying this link remain unclear.1–3 Insulin resistance in skeletal muscle stems from excess accumulation of lipid species4, a process that requires blood-borne lipids to first traverse the blood vessel wall. Little is known, however, of how this trans-endothelial transport occurs or is regulated. Here, we leverage PGC-1α, a transcriptional coactivator that regulates broad programs of FA consumption, to identify 3-hydroxy-isobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a novel paracrine regulator of trans-endothelial fatty acids (FA) transport. 3-HIB is secreted from muscle cells, activates endothelial FA transport, stimulates muscle FA uptake in vivo, and promotes muscle lipid accumulation and insulin resistance in animals. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the promotion of endothelial FA uptake. 3-HIB levels are elevated in muscle from db/db mice and from subjects with diabetes. These data thus unveil a novel mechanism that regulates trans-endothelial flux of FAs, revealing 3-HIB as a new bioactive signaling metabolite that links the regulation of FA flux to BCAA catabolism and provides a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes. PMID:26950361

  18. Fatty Acid Transporter CD36 Mediates Hypothalamic Effect of Fatty Acids on Food Intake in Rats

    PubMed Central

    Moullé, Valentine S.; Le Foll, Christelle; Philippe, Erwann; Kassis, Nadim; Rouch, Claude; Marsollier, Nicolas; Bui, Linh-Chi; Guissard, Christophe; Dairou, Julien; Lorsignol, Anne; Pénicaud, Luc; Levin, Barry E.; Cruciani-Guglielmacci, Céline; Magnan, Christophe

    2013-01-01

    Variations in plasma fatty acid (FA) concentrations are detected by FA sensing neurons in specific brain areas such as the hypothalamus. These neurons play a physiological role in the control of food intake and the regulation of hepatic glucose production. Le Foll et al. previously showed in vitro that at least 50% of the FA sensing in ventromedial hypothalamic (VMH) neurons is attributable to the interaction of long chain FA with FA translocase/CD36 (CD36). The present work assessed whether in vivo effects of hypothalamic FA sensing might be partly mediated by CD36 or intracellular events such as acylCoA synthesis or β-oxidation. To that end, a catheter was implanted in the carotid artery toward the brain in male Wistar rats. After 1 wk recovery, animals were food-deprived for 5 h, then 10 min infusions of triglyceride emulsion, Intralipid +/− heparin (IL, ILH, respectively) or saline/heparin (SH) were carried out and food intake was assessed over the next 5 h. Experimental groups included: 1) Rats previously injected in ventromedian nucleus (VMN) with shRNA against CD36 or scrambled RNA; 2) Etomoxir (CPT1 inhibitor) or saline co-infused with ILH/SH; and 3) Triacsin C (acylCoA synthase inhibitor) or saline co-infused with ILH/SH. ILH significantly lowered food intake during refeeding compared to SH (p<0.001). Five hours after refeeding, etomoxir did not affect this inhibitory effect of ILH on food intake while VMN CD36 depletion totally prevented it. Triacsin C also prevented ILH effects on food intake. In conclusion, the effect of FA to inhibit food intake is dependent on VMN CD36 and acylCoA synthesis but does not required FA oxidation. PMID:24040150

  19. Amino acid contents and transport of fixed N in nodules of Leucaena leucocephala variety K-8

    SciTech Connect

    DuBois, J.D.

    1987-04-01

    Seedlings of Leucaena leucocephala var. K-8 were grown with a N-free fertilizer or fertilizer containing /sup 15/N-depleted (NH/sub 4/)/sub 2/SO/sub 4/ (0.01 atom /sup 15/N; 10 ppm). The nodules of 5 month old trees grown on N-free media were used for /sup 15/N-enriched treatment and as controls. Nodules from plants grown on /sup 15/N-depleted media were also used. Nodules were extracted with 0.5% aqueous toluene and aliquots were analyzed with a Beckman 120B Amino Acid Analyzer. Samples were separated into free ammonium, Asp-N, Glu-N, Asn and Gln amide- and amino-N, and remaining amino acids. Fractions were then analyzed for /sup 15/N content. Asn (27.3 umol/gfw) represented 56% of the total free amino acid pool in the nodules. Asn (amide-N and amino-N) also represented approximately 77% of the total N fixed during the one hour /sup 15/N-enriched N/sub 2/ and the /sup 15/N-depleted treatments. Based on these findings and the fact that the ureide fraction is barely detectable in the nodules (0.25 ..mu..mol/gfw), the authors considers L. leucocephala an amide transporter of fixed N. Additional information will be presented on the amino acid contents of tissues, as well as a time course of amino acid content from seed through nodulation.

  20. Transport of citrate across renal brush border membrane: effects of dietary acid and alkali loading

    SciTech Connect

    Jenkins, A.D.; Dousa, T.P.; Smith, L.H.

    1985-10-01

    Dietary acid or alkali loading was given to rats by providing 150 mM NH4Cl or 150 mM NaHCO3 in place of drinking water for 6 days; control animals received 150 mM NaCl. After 6 days, the citrate clearance was 0.04 +/- 0.01 ml/min (mean +/- SE) in the acid-loaded group, 0.9 +/- 0.1 ml/min in the control group, and 2.5 +/- 0.2 ml/min in the alkali-loaded group. At the end of the experiment, the rats were killed, and the Na gradient-dependent citrate uptake was measured in brush border membrane (BBM) vesicles prepared from each group. At 0.3 min, the ( UC)citrate uptake was 198 +/- 8 pmol/mg protein (mean +/- SE) in the acid-loaded group, 94 +/- 16 pmol/mg protein in the control group, and 94 +/- 13 pmol/mg protein in the alkali-loaded group. The rate of Na -independent (NaCl in medium replaced by KCl) ( UC)-citrate uptake by BBM vesicles was the same for acid-loaded, control, and alkali-loaded animals. Thus, the increased capacity of the proximal tubular BBM to transport citrate from the tubular lumen into the cell interior may be an important factor that contributes to decreased urinary citrate in the presence of metabolic acidosis induced by chronic dietary acid loading.

  1. Circumvention of defective neutral amino acid transport in Hartnup disease using tryptophan ethyl ester.

    PubMed Central

    Jonas, A J; Butler, I J

    1989-01-01

    Tryptophan ethyl ester, a lipid-soluble tryptophan derivative, was used to bypass defective gastrointestinal neutral amino acid transport in a child with Hartnup disease. The child's baseline tryptophan concentrations in serum (20 +/- 6 microM) and cerebrospinal fluid (1.0 +/- 0.2 microM) were persistently less than 50% of normal values. Cerebrospinal fluid 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite, was also less than 50% of normal (21 +/- 2 ng/ml). Serum tryptophan concentrations increased only modestly and briefly after an oral challenge with 200 mg/kg of oral L-tryptophan, reflecting the absorptive defect. An oral challenge with 200 mg/kg of tryptophan ethyl ester resulted in a prompt increase in serum tryptophan to a peak of 555 microM. Sustained treatment with 20 mg/kg q6h resulted in normalization of serum (66 +/- 15 microM) and cerebrospinal fluid tryptophan concentrations (mean = 2.3 microM). Cerebrospinal fluid 5-HIAA increased to more normal concentrations (mean = 33 ng/ml). No toxicity was observed over an 8-mo period of treatment, chronic diarrhea resolved, and body weight, which had remained unchanged for 7 mo before ester therapy, increased by approximately 26%. We concluded that tryptophan ethyl ester is effective at circumventing defective gastrointestinal neutral amino acid transport and may be useful in the treatment of Hartnup disease. PMID:2472426

  2. Enhanced charge transport in highly conducting PEDOT-PSS films after acid treatment

    NASA Astrophysics Data System (ADS)

    Shiva, V. Akshaya; Bhatia, Ravi; Menon, Reghu

    The high electrical conductivity, good stability, high strength, flexibility and good transparency of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS), make it useful for many applications including polymeric anodes for organic photovoltaics, light-emitting diodes, flexible electrodes, supercapacitors, electrochromic devices, field-effect transistors and antistatic-coatings. However, the electrical conductivity of PEDOT-PSS has to be increased significantly for replacement of indium tin oxide (ITO) as the transparent electrode in optoelectronic devices. The as prepared (pristine) PEDOT-PSS film prepared from the PEDOT-PSS aqueous solution usually has conductivity below 1Scm-1, remarkably lower than ITO. Significant conductivity enhancement has been observed on transparent and conductive PEDOT-PSS films after a treatment with inorganic acids. Our study investigates the charge transport in pristine and H2SO4, HNO3, HCl treated PEDOT-PSS films. We have treated the films with various concentrations of acids to probe the effect of the acid treatment on the conduction mechanism. The study includes the measurement of dc and electric field dependent conductivity of films in the temperature range of 4.2K-300K. We have also performed magneto-resistance measurements in the range of 0-5T. An enhancement by a factor of~103 has been observed in the room temperature conductivity. The detailed magneto-transport studies explain the various mechanisms for the conductivity enhancement observed.

  3. Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi.

    PubMed

    Reigada, Chantal; Valera-Vera, Edward A; Sayé, Melisa; Errasti, Andrea E; Avila, Carla C; Miranda, Mariana R; Pereira, Claudio A

    2017-03-01

    Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6-10.3 μM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 μM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of

  4. Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi

    PubMed Central

    Reigada, Chantal; Valera-Vera, Edward A.; Sayé, Melisa; Errasti, Andrea E.; Avila, Carla C.; Miranda, Mariana R.; Pereira, Claudio A.

    2017-01-01

    Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6–10.3 μM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 μM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of

  5. Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles 1

    PubMed Central

    Parker, Karen E.; Briggs, Winslow R.

    1990-01-01

    We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature. PMID:16667914

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Transport and cycling of iron and hydrogen peroxide in a freshwater stream: Influence of organic acids

    USGS Publications Warehouse

    Scott, D.T.; Runkel, R.L.; McKnight, Diane M.; Voelker, B.M.; Kimball, B.A.; Carraway, E.R.

    2003-01-01

    An in-stream injection of two dissolved organic acids (phthalic and aspartic acids) was performed in an acidic mountain stream to assess the effects of organic acids on Fe photoreduction and H2O2 cycling. Results indicate that the fate of Fe is dependent on a net balance of oxidative and reductive processes, which can vary over a distance of several meters due to changes in incident light and other factors. Solution phase photoreduction rates were high in sunlit reaches and were enhanced by the organic acid addition but were also limited by the amount of ferric iron present in the water column. Fe oxide photoreduction from the streambed and colloids within the water column resulted in an increase in the diurnal load of total filterable Fe within the experimental reach, which also responded to increases in light and organic acids. Our results also suggest that Fe(II) oxidation increased in response to the organic acids, with the result of offsetting the increase in Fe(II) from photoreductive processes. Fe(II) was rapidly oxidized to Fe(III) after sunset and during the day within a well-shaded reach, presumably through microbial oxidation. H2O 2, a product of dissolved organic matter photolysis, increased downstream to maximum concentrations of 0.25 ??M midday. Kinetic calculations show that the buildup of H2O2 is controlled by reaction with Fe(III), but this has only a small effect on Fe(II) because of the small formation rates of H2O2 compared to those of Fe(II). The results demonstrate the importance of incorporating the effects of light and dissolved organic carbon into Fe reactive transport models to further our understanding of the fate of Fe in streams and lakes.

  9. Reduced amino acid transport in skeletal muscle caused by a circulating factor during endotoxemia.

    PubMed Central

    Warner, B W; Hasselgren, P O; James, J H; Hummel, R P; Rigel, D F; Fischer, J E

    1990-01-01

    The present study was designed to determine whether reduced amino acid uptake in skeletal muscle during endotoxemia is due to associated hypotension or is caused by a factor present in plasma. Three series of experiments were performed. In the first series of experiments, mean arterial pressure (MAP), heart rate, and amino acid uptake in incubated soleus muscles were measured after intravenous injection of endotoxin (1 mg/kg) in male Sprague-Dawley rats (40 to 60 g). Amino acid transport was measured by determining intracellular uptake of [3H]-alpha-amino-isobutyric acid (AIB) during 2 hours of incubation. In the second series of experiments, hypotension was induced by bleeding and muscle amino acid uptake was measured. In the third series of experiments, whole plasma or a low molecular weight fraction (less than 10,000 d) of plasma from endotoxin-injected rats was added in vitro to incubated muscles and amino acid uptake was determined. One hour after injection of endotoxin, MAP was reduced from 80 +/- 2 mmHg to 54 +/- 4 mmHg (p less than 0.05). AIB uptake was reduced by 20% (p less than 0.05) 2 hours after endotoxin injection. When MAP was maintained at 50 mmHg for 1 hour by bleeding, no changes in muscle AIB uptake were noted. When plasma obtained from rats 2 hours after endotoxin injection was added to incubated soleus muscles, AIB uptake was reduced by 22%. This effect was duplicated by a fraction of endotoxic plasma containing substances with a molecular weight less than 10,000 d. The present results suggest that reduced muscle amino acid uptake during endotoxemia is not due to associated hypotension, but may be caused by a circulating factor(s) with a molecular weight less than 10,000 d. PMID:2178567

  10. The anti-tumor drug 2-hydroxyoleic acid (Minerval) stimulates signaling and retrograde transport

    PubMed Central

    Torgersen, Maria L.; Klokk, Tove Irene; Kavaliauskiene, Simona; Klose, Christian; Simons, Kai; Skotland, Tore; Sandvig, Kirsten

    2016-01-01

    2-hydroxyoleic acid (OHOA, Minerval®) is an example of a substance used for membrane lipid therapy, where the cellular membranes rather than specific proteins constitute the therapeutical target. OHOA is thought to mediate its anti-tumor effect by affecting the biophysical properties of membranes, which leads to altered recruitment and activation of amphitropic proteins, altered cellular signaling, and eventual cell death. Little is known about the initial signaling events upon treatment with OHOA, and whether the altered membrane properties would have any impact on the dynamic intracellular transport system. In the present study we demonstrate that treatment with OHOA led to a rapid release of intracellular calcium and activation of multiple signaling pathways in HeLa cells, including the PI3K-AKT1-MTOR pathway and several MAP kinases, in a process independent of the EGFR. By lipidomics we confirmed that OHOA was incorporated into several lipid classes. Concomitantly, OHOA potently increased retrograde transport of the plant toxin ricin from endosomes to the Golgi and further to the endoplasmic reticulum. The OHOA-stimulated ricin transport seemed to require several amphitropic proteins, including Src, phospholipase C, protein kinase C, and also Ca2+/calmodulin. Interestingly, OHOA induced a slight increase in endosomal localization of the retromer component VPS35. Thus, our data show that addition of a lipid known to alter membrane properties not only affects signaling, but also intracellular transport. PMID:27894086

  11. Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

    PubMed Central

    Estrella, M R; Brusseau, M L; Maier, R S; Pepper, I L; Wierenga, P J; Miller, R M

    1993-01-01

    The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments. PMID:8285717

  12. A functional splice variant of the human Golgi CMP-sialic acid transporter.

    PubMed

    Salinas-Marín, Roberta; Mollicone, Rosella; Martínez-Duncker, Iván

    2016-12-01

    The human Golgi Cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Sia) transporter SLC35A1, a member of the nucleotide sugar transporter family, translocates CMP-Sia from the cytosol into the Golgi lumen where sialyltransferases use it as donor substrate for the synthesis of sialoglycoconjugates. In 2005, we reported a novel Congenital Disorder of Glycosylation (CDG) termed CDG-IIf or SLC35A1-CDG, characterized by macrothrombocytopenia, neutropenia and complete lack of the sialyl-Le(x) antigen (NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-R) on polymorphonuclear cells. This disease was caused by the presence of inactive SLC35A1 alleles. It was also found that the SLC35A1 generates additional isoforms through alternative splicing. In this work, we demonstrate that one of the reported isoforms, the del177 with exon 6 skipping, is able to maintain sialylation in HepG2 cells submitted to wt knockdown and restore sialylation to normal levels in the Chinese Hamester Ovary (CHO) cell line Lec2 mutant deficient in CMP-Sia transport. The characteristics of the alternatively spliced protein are discussed as well as therapeutic implications of this finding in CDGs caused by mutations in nucleotide sugar transporters (NSTs).

  13. gamma-aminobutyric acid transporter-mediated current from bipolar cells in tiger salamander retinal slices.

    PubMed

    Yang, C Y

    1998-09-01

    About 10% of bipolar cells in salamander retina synthesize and take up gamma-aminobutyric acid (GABA), and may use GABA as a neurotransmitter. As GABA uptake is electrogenic, bipolar cells expressing GABA transporters (GATs) should give transport current (IGAT) to extracellular GABA. Using whole-cell patch recording, 28 bipolar cells responded to 30-200 microM GABA puffed to the axon terminals with a picrotoxin (PTX)-sensitive chloride current (ICI) only. Another three bipolar cells had, in addition to ICI, a PTX-resistant, sodium-dependent current that was completely and reversibly blocked by NO-711, an IGAT inhibitor, indicating that this component was an IGAT. This finding provides further support for a subset of GABAergic bipolar cells in the salamander retina.

  14. Effects of morpholine and boric acid implementation on secondary chemistry and corrosion product transport

    SciTech Connect

    Siegwarth, D.P.; Pearl, W.L.; Sawochka, S.G.; Clouse, M.E.

    1992-07-01

    This project expanded the database on the effects of morpholine use as a pH control additive in the secondary water system of pressurized water reactors. Tests carried out at Alabama Power`s Joseph M. Farley Units 1 and 2, Northern States Power`s Prairie Island Unit 2, and Commonwealth Edison`s Byron Unit 1 demonstrated that the use of morpholine reduced corrosion product transport in PWR secondary systems by up to a factor of 2. Further reductions were achieved by eliminating copper alloy tubing and increasing secondary cycles pH. Steam generator boric acid treatment in plants with all ferrous secondary cycles operated with morpholine was shown to have a minimal effect on iron transport.

  15. Effects of morpholine and boric acid implementation on secondary chemistry and corrosion product transport

    SciTech Connect

    Siegwarth, D.P.; Pearl, W.L.; Sawochka, S.G.; Clouse, M.E. )

    1992-07-01

    This project expanded the database on the effects of morpholine use as a pH control additive in the secondary water system of pressurized water reactors. Tests carried out at Alabama Power's Joseph M. Farley Units 1 and 2, Northern States Power's Prairie Island Unit 2, and Commonwealth Edison's Byron Unit 1 demonstrated that the use of morpholine reduced corrosion product transport in PWR secondary systems by up to a factor of 2. Further reductions were achieved by eliminating copper alloy tubing and increasing secondary cycles pH. Steam generator boric acid treatment in plants with all ferrous secondary cycles operated with morpholine was shown to have a minimal effect on iron transport.

  16. TRANSPORT

    EPA Science Inventory

    Presentation outline: transport principles, effective solubility; gasoline composition; and field examples (plume diving).
    Presentation conclusions: MTBE transport follows from - phyiscal and chemical properties and hydrology. Field examples show: MTBE plumes > benzene plu...

  17. Light-Activated Amino Acid Transport Systems in Halobacterium halobium Envelope Vesicles: Role of Chemical and Electrical Gradients

    NASA Technical Reports Server (NTRS)

    MacDonald, Russell E.; Greene, Richard V.; Lanyi, Janos K.

    1977-01-01

    The accumulation of 20 commonly occurring L-amino acids by cell envelope vesicles of Halobacterium halobium, in response to light-induced membrane potential and an artificially created sodium gradient, has been studied. Nineteen of these amino acids are actively accumulated under either or both of these conditions. Glutamate is unique in that its uptake is driven only by a chemical gradient for sodium. Amino acid concentrations at half-maximal uptake rates (Km) and maximal transport rates (V(sub max) have been determined for the uptake of all 19 amino acids. The transport systems have been partially characterized with respect to groups of amino acids transported by common carriers, cation effects, and relative response to the electrical and chemical components of the sodium gradient, the driving forces for uptake. The data presented clearly show that the carrier systems, which are responsible for uptake of individual amino acids, are as variable in their properties as those found in other organisms, i. e., some are highly specific for individual amino acids, some transport several amino acids competitively, some are activated by a chemical gradient of sodium only, and some function also in the complete absence of such a gradient. For all amino acids, Na(+) and K(+) are both required for maximal rate of uptake. The carriers for L-leucine and L-histidine are symmetrical in that these amino acids are transported in both directions across the vesicle membrane. It is suggested that coupling of substrate transport to metabolic energy via transient ionic gradients may be a general phenomenon in procaryotes.

  18. Design and Evaluation of a Novel Trifluorinated Imaging Agent for Assessment of Bile Acid Transport Using Fluorine Magnetic Resonance Imaging

    PubMed Central

    Vivian, Diana; Cheng, Kunrong; Khurana, Sandeep; Xu, Su; Dawson, Paul A.; Raufman, Jean-Pierre; Polli, James E.

    2014-01-01

    Previously, we developed a trifluorinated bile acid, CA-lys-TFA, with the objective of noninvasively assessing bile acid transport in vivo using 19F magnetic resonance imaging (MRI). CA-lys-TFA was successfully imaged in the mouse gallbladder, but was susceptible to deconjugation in vitro by choloylglycine hydrolase (CGH), a bacterial bile acid deconjugating enzyme found in the terminal ileum and colon. The objective of the present study was to develop a novel trifluorinated bile acid resistant to deconjugation by CGH. CA-sar-TFMA was designed, synthesized, and tested for in vitro transport properties, stability, imaging properties, and its ability to differentially accumulate in the gallbladders of normal mice, compared with mice with known impaired bile acid transport (deficient in the apical sodium-dependent bile acid transporter, ASBT). CA-sar-TFMA was a potent inhibitor and substrate of ASBT and the Na+/taurocholate cotransporting polypeptide. Stability was favorable in all conditions tested, including the presence of CGH. CA-sar-TFMA was successfully imaged and accumulated at 16.1-fold higher concentrations in gallbladders from wild-type mice compared with those from Asbt-deficient mice. Our results support the potential of using MRI with CA-sar-TFMA as a noninvasive method to assess bile acid transport in vivo. PMID:25196788

  19. Hypomorphic variants of cationic amino acid transporter 3 in males with autism spectrum disorders.

    PubMed

    Nava, Caroline; Rupp, Johanna; Boissel, Jean-Paul; Mignot, Cyril; Rastetter, Agnès; Amiet, Claire; Jacquette, Aurélia; Dupuits, Céline; Bouteiller, Delphine; Keren, Boris; Ruberg, Merle; Faudet, Anne; Doummar, Diane; Philippe, Anne; Périsse, Didier; Laurent, Claudine; Lebrun, Nicolas; Guillemot, Vincent; Chelly, Jamel; Cohen, David; Héron, Delphine; Brice, Alexis; Closs, Ellen I; Depienne, Christel

    2015-12-01

    Cationic amino acid transporters (CATs) mediate the entry of L-type cationic amino acids (arginine, ornithine and lysine) into the cells including neurons. CAT-3, encoded by the SLC7A3 gene on chromosome X, is one of the three CATs present in the human genome, with selective expression in brain. SLC7A3 is highly intolerant to variation in humans, as attested by the low frequency of deleterious variants in available databases, but the impact on variants in this gene in humans remains undefined. In this study, we identified a missense variant in SLC7A3, encoding the CAT-3 cationic amino acid transporter, on chromosome X by exome sequencing in two brothers with autism spectrum disorder (ASD). We then sequenced the SLC7A3 coding sequence in 148 male patients with ASD and identified three additional rare missense variants in unrelated patients. Functional analyses of the mutant transporters showed that two of the four identified variants cause severe or moderate loss of CAT-3 function due to altered protein stability or abnormal trafficking to the plasma membrane. The patient with the most deleterious SLC7A3 variant had high-functioning autism and epilepsy, and also carries a de novo 16p11.2 duplication possibly contributing to his phenotype. This study shows that rare hypomorphic variants of SLC7A3 exist in male individuals and suggest that SLC7A3 variants possibly contribute to the etiology of ASD in male subjects in association with other genetic factors.

  20. Evaluating remedial alternatives for an acid mine drainage stream: Application of a reactive transport model

    USGS Publications Warehouse

    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

  1. Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats.

    PubMed

    Ikeda, Saori; Tachikawa, Masanori; Akanuma, Shin-ichi; Fujinawa, Jun; Hosoya, Ken-ichi

    2012-08-01

    Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.

  2. Charge transport through dicarboxylic-acid-terminated alkanes bound to graphene-gold nanogap electrodes

    NASA Astrophysics Data System (ADS)

    Liu, Longlong; Zhang, Qian; Tao, Shuhui; Zhao, Cezhou; Almutib, Eman; Al-Galiby, Qusiy; Bailey, Steven W. D.; Grace, Iain; Lambert, Colin J.; Du, Jun; Yang, Li

    2016-07-01

    Graphene-based electrodes are attractive for single-molecule electronics due to their high stability and conductivity and reduced screening compared with metals. In this paper, we use the STM-based matrix isolation I(s) method to measure the performance of graphene in single-molecule junctions with one graphene electrode and one gold electrode. By measuring the length dependence of the electrical conductance of dicarboxylic-acid-terminated alkanes, we find that the transport is consistent with phase-coherent tunneling, but with an attenuation factor of βN = 0.69 per methyl unit, which is lower than the value measured for Au-molecule-Au junctions. Comparison with density-functional-theory calculations of electron transport through graphene-molecule-Au junctions and Au-molecule-Au junctions reveals that this difference is due to the difference in Fermi energies of the two types of junction, relative to the frontier orbitals of the molecules. For most molecules, their electrical conductance in graphene-molecule-Au junctions is higher than that in Au-molecule-Au junctions, which suggests that graphene offers superior electrode performance, when utilizing carboxylic acid anchor groups.Graphene-based electrodes are attractive for single-molecule electronics due to their high stability and conductivity and reduced screening compared with metals. In this paper, we use the STM-based matrix isolation I(s) method to measure the performance of graphene in single-molecule junctions with one graphene electrode and one gold electrode. By measuring the length dependence of the electrical conductance of dicarboxylic-acid-terminated alkanes, we find that the transport is consistent with phase-coherent tunneling, but with an attenuation factor of βN = 0.69 per methyl unit, which is lower than the value measured for Au-molecule-Au junctions. Comparison with density-functional-theory calculations of electron transport through graphene-molecule-Au junctions and Au

  3. Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1

    PubMed Central

    Geier, Ethan G.; Schlessinger, Avner; Fan, Hao; Gable, Jonathan E.; Irwin, John J.; Sali, Andrej; Giacomini, Kathleen M.

    2013-01-01

    The Large-neutral Amino Acid Transporter 1 (LAT-1)—a sodium-independent exchanger of amino acids, thyroid hormones, and prescription drugs—is highly expressed in the blood–brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood–brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism. PMID:23509259

  4. Rapid chemoenzymatic route to glutamate transporter inhibitor l-TFB-TBOA and related amino acids.

    PubMed

    Fu, Haigen; Younes, Sabry H H; Saifuddin, Mohammad; Tepper, Pieter G; Zhang, Jielin; Keller, Erik; Heeres, André; Szymanski, Wiktor; Poelarends, Gerrit J

    2017-03-21

    The complex amino acid (l-threo)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate (l-TFB-TBOA) and its derivatives are privileged compounds for studying the roles of excitatory amino acid transporters (EAATs) in regulation of glutamatergic neurotransmission, animal behavior, and in the pathogenesis of neurological diseases. The wide-spread use of l-TFB-TBOA stems from its high potency of EAAT inhibition and the lack of off-target binding to glutamate receptors. However, one of the main challenges in the evaluation of l-TFB-TBOA and its derivatives is the laborious synthesis of these compounds in stereoisomerically pure form. Here, we report an efficient and step-economic chemoenzymatic route that gives access to enantio- and diastereopure l-TFB-TBOA and its derivatives at multigram scale.

  5. Transport mechanism for L-lactic acid in human myocytes using human prototypic embryonal rhabdomyosarcoma cell line (RD cells).

    PubMed

    Kobayashi, Masaki; Fujita, Itaru; Itagaki, Shirou; Hirano, Takeshi; Iseki, Ken

    2005-07-01

    Monocarboxylate transporter (MCT), which cotransport L-lactic acid and protons across cell membranes, are important for regulation of muscle pH. However, it has not been demonstrated in detail whether MCT isoform contribute to the transport of L-lactic acid in skeletal muscle. The aim of this study was to characterize L-lactic acid transport using an human rhabdomyosarcoma (RD) cell line as a model of human skeletal muscle. mRNAs of MCT 1, 2 and 4 were found to be expressed in RD cells. The [14C] L-lactic acid uptake was concentration-dependent with a Km of 1.19 mM. This Km value was comparable to its Km values for MCT1 or MCT2. MCT1 mRNA was found to be present markedly greater than that MCT2. Therefore, MCT1 most probably acts on L-lactic acid uptake at RD cells. [14C] L-Lactic acid efflux in RD cells was inhibited by alpha-cyano-4-hydroxycinnamate (CHC) but not by butyric acid, a substrate of MCT1. Accordingly, MCT2 or MCT4 is responsible for L-lactic acid efflux by RD cells. MCT4 mRNA was found to be present significantly greater than that MCT2. We conclude that MCT1 is responsible for L-lactic acid uptake and L-lactic acid efflux is mediated by MCT4 in RD cells.

  6. Protein kinase C restricts transport of carnitine by amino acid transporter ATB(0,+) apically localized in the blood-brain barrier.

    PubMed

    Michalec, Katarzyna; Mysiorek, Caroline; Kuntz, Mélanie; Bérézowski, Vincent; Szczepankiewicz, Andrzej A; Wilczyński, Grzegorz M; Cecchelli, Roméo; Nałęcz, Katarzyna A

    2014-07-15

    Carnitine (3-hydroxy-4-trimethylammoniobutyrate) is necessary for transfer of fatty acids through the inner mitochondrial membrane. Carnitine, not synthesized in the brain, is delivered there through the strongly polarized blood-brain barrier (BBB). Expression and presence of two carnitine transporters - organic cation/carnitine transporter (OCTN2) and amino acid transporter B(0,+) (ATB(0,+)) have been demonstrated previously in an in vitro model of the BBB. Due to potential protein kinase C (PKC) phosphorylation sites within ATB(0,+) sequence, the present study verified effects of this kinase on transporter function and localization in the BBB. ATB(0,+) can be regulated by estrogen receptor α and up-regulated in vitro, therefore its presence in vivo was verified with the transmission electron microscopy. The analyses of brain slices demonstrated ATB(0,+) luminal localization in brain capillaries, confirmed by biotinylation experiments in an in vitro model of the BBB. Brain capillary endothelial cells were shown to control carnitine gradient. ATB(0,+) was phosphorylated by PKC, what correlated with inhibition of carnitine transport. PKC activation did not change the amount of ATB(0,+) present in the apical membrane of brain endothelial cells, but resulted in transporter exclusion from raft microdomains. ATB(0,+) inactivation by a lateral movement in plasma membrane after transporter phosphorylation has been postulated.

  7. Effect of cyclic fatty acid monomers on fat absorption and transport depends on their positioning within the ingested triacylglycerols.

    PubMed

    Martin, J C; Caselli, C; Broquet, S; Juanéda, P; Nour, M; Sébédio, J L; Bernard, A

    1997-08-01

    We investigated the intestinal digestion of cyclic fatty acid monomers (CFAM) isolated from heated linseed oil and their effects upon fatty acid lymphatic transport and lipoprotein profile in lymph. These cyclic fatty acid monomers were acylated in specific positions in the glycerol backbone of triacylglycerols (sn-(1/3) position for the 1C oil, sn-2 position for the 2C oil and together in the sn-1,2, and 3 positions for the 3C oil) and administered intragastrically to lymph-canulated rats. Their lumenal digestibility was also assessed in vitro using a pancreatic lipase assay. The lipase activity was 1.9 to 6.6 less towards the triacylglycerols acylated with cyclic fatty acids compared to control. The lowest activity was with the 2C oil. In the hydrolytic products, the cyclic fatty acid contents were similar between the experimental groups. When absorbed as 2-monoacyl-sn-glycerol (2C oil), cyclic fatty acid monomers were better and unselectively recovered into the lymph than when absorbed as free fatty acids (1C oil). In that latter situation, the bulkier cyclic fatty acids (C6 and cis membered-ring CFAM) were transported into the lymph to a lesser extent. The appearance of the lymphatic chylomicrons was delayed in rats fed the 1C oil. Cyclic fatty acid monomers from the 2C oil only increased the lymphatic transport of saturated fatty acids (80%). Cyclic fatty acids from the 3C oil (absorbed as 2-monoacyl-sn-glycerol and free fatty acid) usually elicited intermediary effects. We conclude that the effects of cyclic fatty acid monomers upon the intestinal metabolism are greatly influenced by their positioning within the triacylglycerol and that the structure of the cyclic fatty acids influences their lymphatic recovery only when they are absorbed as free fatty acid.

  8. Reactive transport controls on sandy acid sulfate soils and impacts on shallow groundwater quality

    NASA Astrophysics Data System (ADS)

    Salmon, S. Ursula; Rate, Andrew W.; Rengel, Zed; Appleyard, Steven; Prommer, Henning; Hinz, Christoph

    2014-06-01

    Disturbance or drainage of potential acid sulfate soils (PASS) can result in the release of acidity and degradation of infrastructure, water resources, and the environment. Soil processes affecting shallow groundwater quality have been investigated using a numerical code that integrates (bio)geochemical processes with water, solute, and gas transport. The patterns of severe and persistent acidification (pH < 4) in the sandy, carbonate-depleted podzols of a coastal plain could be reproduced without calibration, based on oxidation of microcrystalline pyrite after groundwater level decrease and/or residual groundwater acidity, due to slow vertical solute transport rates. The rate of acidification was limited by gas phase diffusion of oxygen and hence was sensitive to soil water retention properties and in some cases also to oxygen consumption by organic matter mineralization. Despite diffusion limitation, the rate of oxidation in sandy soils was rapid once pyrite-bearing horizons were exposed, even to a depth of 7.5 m. Groundwater level movement was thus identified as an important control on acidification, as well as the initial pyrite content. Increase in the rate of Fe(II) oxidation lead to slightly lower pH and greater accumulation of Fe(III) phases, but had little effect on the overall amount of pyrite oxidized. Aluminosilicate (kaolinite) dissolution had a small pH-buffering effect but lead to the release of Al and associated acidity. Simulated dewatering scenarios highlighted the potential of the model for risk assessment of (bio)geochemical impacts on soil and groundwater over a range of temporal and spatial scales.

  9. Characterization and Regulation of the Amino Acid Transporter SNAT2 in the Small Intestine of Piglets

    PubMed Central

    Tan, Bie; Wang, Jing; Kong, Xiangfeng; Guan, Guiping; Li, Fengna; Yin, Yulong

    2015-01-01

    The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine. PMID:26107628

  10. The mRNA expression of amino acid transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1 in the intestine and liver of post-hatch broiler chicks

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Amino acid transporter (AAT) proteins are responsible for the movement of amino acids (AA) in and out of cells. Aminopeptidase (APN) cleaves AAs from the N terminus of polypeptides making them available for transport, while PepT1 is a di- and tri- peptide transporter. In the intestine, these prote...

  11. Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3′-Diiodothyronine across the Plasma Membrane

    PubMed Central

    Kinne, Anita; Wittner, Melanie; Wirth, Eva K.; Hinz, Katrin M.; Schülein, Ralf; Köhrle, Josef; Krause, Gerd

    2015-01-01

    Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g. LAT2) and a heavy chain (CD98), which is essential for their cell surface expression and functionality. The specificity of Lat2 for thyroid hormones and their metabolites and its role in their transport was not fully clear. This fact motivated us to establish a cell system to elucidate the uptake of thyroid hormones and their metabolites by mouse Lat2. The coinjection of cRNA coding for Lat2 and CD98 into Xenopus laevis oocytes resulted in a markedly increased level of 3,3′-diiodo-L-thyronine (3,3′-T2) and to some extent also enhanced T3 transport. To gain insight into properties of thyroid hormones and their metabolites transported by Lat2, we inhibited 3,3′-T2 uptake by various iodothyronine derivatives. T1 and T2 derivatives as well as 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid strongly competed with 3,3′-T2 uptake. In addition, we performed T2 uptake measurements with the thyroid hormone-specific transporter MCT8. For both Lat2 and MCT8, Km values in a low micromolar range were calculated. We demonstrated that oocytes are a suitable system for thyroid hormone transport studies mediated by Lat2. Our data indicates that Lat2 compared to other thyroid hormone transporters prefers 3,3′-T2 as the substrate. Thus, Lat2 might contribute to the availability of thyroid hormone by importing and/or exporting 3,3′-T2, which is generated either by T3 inactivation or by rapid deiodinase 1-mediated rT3 degradation. PMID:26601072

  12. Geochemical and biological controls on trace metal transport in an acid mine impacted watershed.

    PubMed

    Butler, Thomas W

    2006-06-01

    Water samples collected in an acid mine impacted watershed indicated that the concentrations of dissolved trace metals were diurnally influenced by mineral saturation, which is controlled primarily by pH and water temperature. Measurements taken suggested that these variations only occur at sample locations immediately downstream from the confluence of acidic and alkaline waters. It is at these locations where initial mineral precipitation occurred and where subtle changes in solubility were most affected, increasing trace metal removal when both the rate of photosynthesis (influencing pH in headwaters) and water temperature were at a maximum. The role of iron photoreduction (increased midday production of ferrous iron) on overall Cu, Mn, and Zn transport was also evaluated, but found to be inconclusive. Iron photoreduction may however influence adsorption and/or coprecipitation of trace metals through associated changes in oxidation state, solubility, and mineralogy of various iron colloids, which are produced upon the neutralization of acidic, metal enriched water. Furthermore, measured values of copper and zinc were compared to relative USEPA chronic criterion for exposure to continuous concentration (CCC) of metals by the calculation of a "toxicity unit" (TU). It was found that average values of both copper and zinc only exceeded the CCC (TU>1) in the acid mine-impacted Leona Creek. In general, zinc toxicity decreased while copper toxicity increased downstream of the confluence of the mine impacted Leona Creek and background Lion Creek (sampled at Lake Aliso), indicating a significant source of zinc in upstream, non mine-impacted samples.

  13. Anterograde transport of horseradish peroxidase in the nigrostriatal pathway after neostriatal kainic acid lesions.

    PubMed

    Walker, P D; McAllister, J P

    1986-08-01

    We used the anterograde transport of HRP to analyze the nigrostriatal pathway after intrastriatal injections of kainic acid. A total volume of 1 microliter kainic acid (3 nM) was injected unilaterally into the neostriatum of adult rats. After 5, 10, or 35 days, HRP was injected into the ipsilateral substantia nigra. Sections stained for Nissl substance revealed that kainic acid damaged as much as three-quarters of the neostriatum. Lesion sites were characterized by gliosis and the absence of neurons. Alternate sections processed for HRP histochemistry and analyzed with bright- and dark-field microscopy revealed labeled axons and terminals in the lesion site. These findings were consistent in all three time periods. Much of the labeling was similar to that seen in neostriatal of control animals. However, the normal homogeneous pattern of the nigrostriatal terminal field was disrupted in all experimental groups, illustrated by changes in some labeling characteristics in the lesion site. These findings provide morphologic evidence for the preservation of much of the nigrostriatal pathway but indicate that some axons and their terminals may be altered after kainic acid injection.

  14. Suppression of asymmetric acid efflux and gravitropism in maize roots treated with auxin transport inhibitors of sodium orthovanadate

    NASA Technical Reports Server (NTRS)

    Mulkey, T. J.; Evans, M. L.

    1982-01-01

    In gravitropically stimulated roots of maize (Zea mays L., hybrid WF9 x 38MS), there is more acid efflux on the rapidly growing upper side than on the slowly growing lower side. In light of the Cholodny/Went hypothesis of gravitropism which states that gravitropic curvature results from lateral redistribution of auxin, the effects of auxin transport inhibitors on the development of acid efflux asymmetry and curvature in gravistimulated roots were examined. All the transport inhibitors tested prevented both gravitropism and the development of asymmetric acid efflux in gravistimulated roots. The results indicate that auxin redistribution may cause the asymmetry of acid efflux, a finding consistent with the Cholodny/Went hypothesis of gravitropism. As further evidence that auxin-induced acid efflux asymmetry may mediate gravitropic curvature, sodium orthovanadate, an inhibitor of auxin-induced H+ efflux was found to prevent both gravitropism and the development of asymmetric acid efflux in gravistimulated roots.

  15. Unusual effects of monocarboxylic acids on the structure and on the transport and mechanical properties of chitosan films.

    PubMed

    Chen, Fei; Gällstedt, Mikael; Olsson, Richard T; Gedde, Ulf W; Hedenqvist, Mikael S

    2015-11-05

    The purpose of this study was to study the transport of monocarboxylic acids in chitosan films, since this is important for understanding and predicting the drying kinetics of chitosan from aqueous solutions. Despite the wealth of data on chitosan films prepared from aqueous monocarboxylic acid solutions, this transport has not been reported. Chitosan films were exposed to formic, acetic, propionic and butyric acid vapours, it was found that the rate of uptake decreased with increasing molecular size. The equilibration time was unexpectedly long, especially for propionic and butyric acid, nine months. A clear two-stage uptake curve was observed for propionic acid. Evidently, the rate of uptake was determined by acid-induced changes in the material. X-ray diffraction and infrared spectroscopy indicated that the structure of the chitosan acetate and buffered chitosan films changed during exposure to acid and during the subsequent drying. The dried films previously exposed to the acid showed less crystalline features than the original material and a novel repeating structure possibly involving acid molecules. The molar mass of the chitosan decreased on exposure to acid but tensile tests revealed that the films were always ductile. The films exposed to acid vapour (propionic and butyric acid) for the longest period of time were insoluble in the size-exclusion chromatography eluent, and they were also the most ductile/extensible of all samples studied.

  16. Identification of a Novel N-Acetylmuramic Acid Transporter in Tannerella forsythia.

    PubMed

    Ruscitto, Angela; Hottmann, Isabel; Stafford, Graham P; Schäffer, Christina; Mayer, Christoph; Sharma, Ashu

    2016-11-15

    Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) that form the disaccharide repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia coli MurQ etherase involved in the conversion of MurNAc-6-phosphate (MurNAc-6-P) to GlcNAc-6-P. The genes encoding these components were identified on a three-gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans, but not individually, rescued the inability of an E. coli mutant deficient in the phosphotransferase (PTS) system-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria.

  17. The hepatic bile acid transporters Ntcp and Mrp2 are downregulated in experimental necrotizing enterocolitis.

    PubMed

    Cherrington, Nathan J; Estrada, Teresa E; Frisk, Harrison A; Canet, Mark J; Hardwick, Rhiannon N; Dvorak, Bohuslav; Lux, Katie; Halpern, Melissa D

    2013-01-01

    Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed.

  18. Role of hepatic transporters in prevention of bile acid toxicity after partial hepatectomy in mice

    PubMed Central

    Csanaky, Iván L.; Aleksunes, Lauren M.; Tanaka, Yuji; Klaassen, Curtis D.

    2009-01-01

    The enterohepatic recirculation of bile acids (BAs) is important in several physiological processes. Although there has been considerable research on liver regeneration after two-thirds partial hepatectomy (PHx), little is known about how the liver protects itself against BA toxicity during regeneration. In this study, various BAs in plasma and liver, the composition of micelle-forming bile constituents, as well as gene expression of the main hepatobiliary transporters were quantified in sham-operated and PHx mice 24 and 48 h after surgery. PHx did not influence the hepatic concentrations of taurine-conjugated BAs (T-BA) but increased the concentration of glycine-conjugated (G-BA) and unconjugated BAs. Total BA excretion (μg·min−1·g liver wt−1) increased 2.4-fold and was accompanied by a 55% increase in bile flow after PHx. The plasma concentrations of T-BAs (402-fold), G-BAs (17-fold), and unconjugated BAs (500-fold) increased. The mRNA and protein levels of the BA uptake transporter Ntcp were unchanged after PHx, whereas the canalicular Bsep protein increased twofold at 48 h. The basolateral efflux transporter Mrp3 was induced at the mRNA (2.6-fold) and protein (3.1-fold) levels after PHx, which may contribute to elevated plasma BA and bilirubin levels. Biliary phospholipid excretion was nearly doubled in PHx mice, most likely owing to increased mRNA expression of the phospholipid transporter, Mdr2. In conclusion, the remnant liver after PHx excretes 2.5-fold more BAs and three times more phospholipids per gram liver than the sham-operated mouse liver. Upregulation of phospholipid transport may be important in protecting the biliary tract from BA toxicity during PHx. PMID:19497955

  19. A novel glutamate transport system in poly(γ-glutamic acid)-producing strain Bacillus subtilis CGMCC 0833.

    PubMed

    Wu, Qun; Xu, Hong; Zhang, Dan; Ouyang, Pingkai

    2011-08-01

    Bacillus subtilis CGMCC 0833 is a poly(γ-glutamic acid) (γ-PGA)-producing strain. It has the capacity to tolerate high concentration of extracellular glutamate and to utilize glutamate actively. Such a high uptake capacity was owing to an active transport system for glutamate. Therefore, a specific transport system for L-glutamate has been observed in this strain. It was a novel transport process in which glutamate was symported with at least two protons, and an inward-directed sodium gradient had no stimulatory effect on it. K(m) and V(m) for glutamate transport were estimated to be 67 μM and 152 nmol⁻¹ min⁻¹ mg⁻¹ of protein, respectively. The transport system showed structural specificity and stereospecificity and was strongly dependent on extracellular pH. Moreover, it could be stimulated by Mg²⁺, NH₄⁺, and Ca²⁺. In addition, the glutamate transporter in this strain was studied at the molecular level. As there was no important mutation of the transporter protein, it appeared that the differences of glutamate transporter properties between this strain and other B. subtilis strains were not due to the differences of the amino acid sequence and the structure of transporter protein. This is the first extensive report on the properties of glutamate transport system in γ-PGA-producing strain.

  20. Osteoblast protects osteoclast devoid of sodium-dependent vitamin C transporters from oxidative cytotoxicity of ascorbic acid.

    PubMed

    Takarada, Takeshi; Hinoi, Eiichi; Kambe, Yuki; Sahara, Koichi; Kurokawa, Shintaro; Takahata, Yoshifumi; Yoneda, Yukio

    2007-12-01

    The view that ascorbic acid indirectly benefits osteoclastogenesis through expression of receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) by osteoblasts is prevailing. In this study, we have examined the direct effect of ascorbic acid on osteoclastogenesis in cultured mouse osteoclasts differentiated from bone marrow precursors. The absence of alkaline phosphatase and osteoblastic marker genes validated the usefulness of isolation procedures. Sustained exposure to ascorbic acid, but not to dehydroascorbic acid, significantly reduced the number of multinucleated cells positive to tartrate-resistant acid phosphatase (TRAP) staining. In cultured osteoclasts, mRNA expression was seen for glucose transporter-1 involved in membrane transport of dehydroascorbic acid, but not for sodium-dependent vitamin C transporters-1 and -2 that are both responsible for the transport of ascorbic acid. The inhibition by ascorbic acid was completely prevented by catalase, while ascorbic acid or hydrogen peroxide drastically increased the number of cells stained with propidium iodide and the generation of reactive oxygen species, in addition to inducing mitochondrial membrane depolarization in cultured osteoclasts. In pre-osteoclastic cell line RAW264.7 cells, ascorbic acid similarly inhibited the formation of TRAP-positive multinucleated cells, with a significant decrease in RANKL-induced NF-kappaB transactivation. Moreover, co-culture with osteoblastic MC3T3-E1 cells significantly prevented the ascorbic acid-induced decrease in the number of TRAP-positive multinucleated cells in RAW264.7 cells. These results suggest that ascorbic acid may play a dual repulsive role in osteoclastogenesis toward bone remodeling through the direct cytotoxicity mediated by oxidative stress to osteoclasts, in addition to the indirect trophism mediated by RANKL from osteoblasts.

  1. Proton transport in triflic acid pentahydrate studied via ab initio path integral molecular dynamics.

    PubMed

    Hayes, Robin L; Paddison, Stephen J; Tuckerman, Mark E

    2011-06-16

    Trifluoromethanesulfonic acid hydrates provide a well-defined system to study proton dissociation and transport in perfluorosulfonic acid membranes, typically used as the electrolyte in hydrogen fuel cells, in the limit of minimal water. The triflic acid pentahydrate crystal (CF(3)SO(3)H·5H(2)O) is sufficiently aqueous that it contains an extended three-dimensional water network. Despite it being extended, however, long-range proton transport along the network is structurally unfavorable and would require considerable rearrangement. Nevertheless, the triflic acid pentahydrate crystal system can provide a clear picture of the preferred locations of local protonic defects in the water network, which provides insights about related structures in the disordered, low-hydration environment of perfluorosulfonic acid membranes. Ab initio molecular dynamics simulations reveal that the proton defect is most likely to transfer to the closest water that has the expected presolvation and only contains water in its first solvation shell. Unlike the tetrahydrate of triflic acid (CF(3)SO(3)H·4H(2)O), there is no evidence of the proton preferentially transferring to a water molecule bridging two of the sulfonate groups. However, this could be an artifact of the crystal structure since the only such water molecule is separated from the proton by long O-O distances. Hydrogen bonding criteria, using the two-dimensional potential of mean force, are extracted. Radial distribution functions, free energy profiles, radii of gyration, and the root-mean-square displacement computed from ab initio path integral molecular dynamics simulations reveal that quantum effects do significantly extend the size of the protonic defect and increase the frequency of proton transfer events by nearly 15%. The calculated IR spectra confirm that the dominant protonic defect mostly exists as an Eigen cation but contains some Zundel ion characteristics. Chain lengths and ring sizes determined from the

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

    PubMed Central

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

    2016-01-01

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

  3. Homology Modeling of Human γ-Butyric Acid Transporters and the Binding of Pro-Drugs 5-Aminolevulinic Acid and Methyl Aminolevulinic Acid Used in Photodynamic Therapy

    PubMed Central

    Baglo, Yan; Gabrielsen, Mari; Sylte, Ingebrigt; Gederaas, Odrun A.

    2013-01-01

    Photodynamic therapy (PDT) is a safe and effective method currently used in the treatment of skin cancer. In ALA-based PDT, 5-aminolevulinic acid (ALA), or ALA esters, are used as pro-drugs to induce the formation of the potent photosensitizer protoporphyrin IX (PpIX). Activation of PpIX by light causes the formation of reactive oxygen species (ROS) and toxic responses. Studies have indicated that ALA and its methyl ester (MAL) are taken up into the cells via γ-butyric acid (GABA) transporters (GATs). Uptake via GATs into peripheral sensory nerve endings may also account for one of the few adverse side effects of ALA-based PDT, namely pain. In the present study, homology models of the four human GAT subtypes were constructed using three x-ray crystal structures of the homologous leucine transporter (LeuT) as templates. Binding of the native substrate GABA and the possible substrates ALA and MAL was investigated by molecular docking of the ligands into the central putative substrate binding sites in the outward-occluded GAT models. Electrostatic potentials (ESPs) of the putative substrate translocation pathway of each subtype were calculated using the outward-open and inward-open homology models. Our results suggested that ALA is a substrate of all four GATs and that MAL is a substrate of GAT-2, GAT-3 and BGT-1. The ESP calculations indicated that differences likely exist in the entry pathway of the transporters (i.e. in outward-open conformations). Such differences may be exploited for development of inhibitors that selectively target specific GAT subtypes and the homology models may hence provide tools for design of therapeutic inhibitors that can be used to reduce ALA-induced pain. PMID:23762315

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

    PubMed

    Bjørn-Yoshimoto, Walden E; Underhill, Suzanne M

    2016-09-01

    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 localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity. It is also the main neuronal cysteine uptake system acting as the rate-limiting factor for the synthesis of glutathione, a potent antioxidant, in EAAT3 expressing neurons, while on GABAergic neurons, it is important in supplying glutamate as a precursor for GABA synthesis. Several diseases implicate EAAT3, and modulation of this transporter could prove a useful therapeutic approach. Regulation of EAAT3 could be targeted at several points for functional modulation, including the level of transcription, trafficking and direct pharmacological modulation, and indeed, compounds and experimental treatments have been identified that regulate EAAT3 function at different stages, which together with observations of EAAT3 regulation in patients is giving us insight into the endogenous function of this transporter, as well as the consequences of altered function. This review summarizes work done on elucidating the role and regulation of EAAT3.

  5. Investigating Mass Transport Limitations on Xylan Hydrolysis During Dilute Acid Pretreatment of Poplar

    SciTech Connect

    Mittal, Ashutosh; Pilath, Heid M.; Parent, Yves; Chatterjee, Siddharth G.; Donohoe, Bryon S.; Yarbrough, John M.; Himmel, Michael E.; Nimlos, Mark R.; Johnson, David K.

    2014-04-28

    Mass transport limitations could be an impediment to achieving high sugar yields during biomass pretreatment and thus be a critical factor in the economics of biofuels production. The objective of this work was to study the mass transfer restrictions imposed by the structure of biomass on the hydrolysis of xylan during dilute acid pretreatment of biomass. Mass transfer effects were studied by pretreating poplar wood at particle sizes ranging from 10 micrometers to 10 mm. This work showed a significant reduction in the rate of xylan hydrolysis in poplar when compared to the intrinsic rate of hydrolysis for isolated xylan that is possible in the absence of mass transfer. In poplar samples we observed no significant difference in the rates of xylan hydrolysis over more than two orders of magnitude in particle size. It appears that no additional mass transport restrictions are introduced by increasing particle size from 10 micrometers to 10 mm. This work suggests that the rates of xylan hydrolysis in biomass particles are limited primarily by the diffusion of hydrolysis products out of plant cell walls. A mathematical description is presented to describe the kinetics of xylan hydrolysis that includes transport of the hydrolysis products through biomass into the bulk solution. The modeling results show that the effective diffusion coefficient of the hydrolysis products in the cell wall is several orders of magnitude smaller than typical values in other applications signifying the role of plant cell walls in offering resistance to diffusion of the hydrolysis products.

  6. Fate and transport of glyphosate and aminomethylphosphonic acid in surface waters of agricultural basins

    USGS Publications Warehouse

    Coupe, R.H.; Kalkhoff, S.J.; Capel, P.D.; Gregoire, C.

    2012-01-01

    Background: Glyphosate [N-(phosphonomethyl)glycine] is a herbicide used widely throughout the world in the production of many crops and is heavily used on soybeans, corn and cotton. Glyphosate is used in almost all agricultural areas of the United States, and the agricultural use of glyphosate has increased from less than 10 000 Mg in 1992 to more than 80 000 Mg in 2007. The greatest intensity of glyphosate use is in the midwestern United States, where applications are predominantly to genetically modified corn and soybeans. In spite of the increase in usage across the United States, the characterization of the transport of glyphosate and its degradate aminomethylphosphonic acid (AMPA) on a watershed scale is lacking. Results: Glyphosate and AMPA were frequently detected in the surface waters of four agricultural basins. The frequency and magnitude of detections varied across basins, and the load, as a percentage of use, ranged from 0.009 to 0.86% and could be related to three general characteristics: source strength, rainfall runoff and flow route. Conclusions: Glyphosate use in a watershed results in some occurrence in surface water; however, the watersheds most at risk for the offsite transport of glyphosate are those with high application rates, rainfall that results in overland runoff and a flow route that does not include transport through the soil. ?? 2011 Society of Chemical Industry.

  7. Numerical simulations of ethacrynic acid transport from precorneal region to trabecular meshwork.

    PubMed

    Lin, Cheng-Wen; Yuan, Fan

    2010-03-01

    Topical application of drugs for treatment of intraocular diseases is often limited by inadequate transport and induced toxicity in corneal tissues. To improve the drug delivery, a mathematical model was developed to numerically simulate the transport process of ethacrynic acid (ECA), a potential drug for glaucoma treatment, in the anterior segment of a typical human eye. The model considered diffusion of ECA in all tissues and the aqueous humor (AH) as well as convection of ECA in the AH. The simulation results showed that ECA concentration in the eye depended on the rate of AH production, the half-life of ECA in the precorneal tear film, and the transport parameters in the model. In addition, the main pathway for ECA clearance from the eye was the trabecular meshwork (TM) and the rate of clearance was approximately proportional to the AH production rate. The model predicted that the most effective approach to improving topical drug delivery was to prolong its half-life in the precorneal tear film. These simulation results and model prediction, which could be verified experimentally, might be useful for improving delivery of ECA and other therapeutic agents to the TM as well as other tissues in the anterior segment of the eye.

  8. Amino acid transport in schistosomes: Characterization of the permeaseheavy chain SPRM1hc.

    PubMed

    Krautz-Peterson, Greice; Camargo, Simone; Huggel, Katja; Verrey, François; Shoemaker, Charles B; Skelly, Patrick J

    2007-07-27

    Schistosomes are human parasitic flatworms that constitute an important public health problem globally. Adult parasites live in the bloodstream where they import nutrients such as amino acids across their body surface (the tegument). One amino acid transporter, Schistosome Permease 1 light chain, SPRM1lc, a member of the glycoprotein-associated family of transporters (gpaAT), has been characterized in schistosomes. Only a single member of the SLC3 family of glycoproteins that associate with gpaATs is found following extensive searching of the genomes of Schistosoma mansoni and S. japonicum. In this report, we characterize this schistosome permease heavy chain (SPRM1hc) gene and protein. The 72-kDa gene product is predicted to possess a single transmembrane domain, a (betaalpha)(8) (TIM barrel) conformation and a catalytic triad. Xenopus oocytes functionally expressing SPRM1hc with SPRM1lc import phenylalanine, arginine, lysine, alanine, glutamine, histidine, tryptophan, and leucine. Biochemical characterization demonstrates that in Xenopus extracts and in schistosome extracts SPRM1hc is associated into a high molecular weight complex with SPRM1lc that is disrupted by reducing agents. Quantitative real-time PCR and Western analysis demonstrate that SPRM1hc is expressed in each schistosome life stage examined (eggs, cercariae, schistosomula, adult males and females). SPRM1hc is widely distributed throughout adult male and female worms as determined by immunolocalization. Consistent with the hypothesis that SPRM1hc functions to facilitate nutrient uptake from host blood, immunogold electron microscopy confirms that the protein is distributed on the host-interactive tegumental membranes. We propose that surface-exposed, host-interactive, nutrient-transporting proteins like the SPRM1 heterodimer are promising vaccine candidates.

  9. Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii.

    PubMed

    Sousa, M J; Rodrigues, F; Côrte-Real, M; Leão, C

    1998-03-01

    Zygosaccharomyces bailii ISA 1307 displays biphasic growth in a medium containing a mixture of glucose (0.5%, w/v) and acetic acid (0.5%, w/v), pH 5.0 and 3.0. In cells harvested during the first growth phase, no activity of a mediated acetic acid transport system was found. Incubation of these cells in phosphate buffer with cycloheximide for 1 h restored activity of an acetic acid carrier which behaved as the one present in glucose-grown cells. These results indicated that the acetic acid carrier is probably present in cells from the first growth phase of the mixed medium but its activity was affected by the presence of acetic acid in the culture medium. In glucose-grown cells, after incubation in phosphate buffer with glucose and acetic acid, the activity of the acetic acid carrier decreased significantly with increased acid concentration in the incubation buffer. At acid concentrations above 16.7 mM, no significant carrier activity was detectable. Furthermore, the intracellular acid concentration increased with the extracellular one and was inversely correlated with the activity of the acetic acid carrier, suggesting the involvement of a feedback inhibition mechanism in the regulation of the carrier. During biphasic growth, the first phase corresponded to a simultaneous consumption of glucose and acetic acid, and the second to the utilization of the remaining acid. The enzyme acetyl-CoA synthetase was active in both growth phases, even in the presence of glucose. Activity of isocitrate lyase and phosphoenolpyruvate carboxykinase was found only in acetic-acid-grown cells. Thus it appears that both membrane transport and acetyl-CoA synthetase and their regulation are important for Z. bailii to metabolize acetic acid in the presence of glucose. This fact correlates with the high resistance of this yeast to environments with mixtures of sugars and acetic acid such as those often present during wine fermentation.

  10. Concentration-dependent mode of interaction of angiotensin II receptor blockers with uric acid transporter.

    PubMed

    Iwanaga, Takashi; Sato, Masanobu; Maeda, Tomoji; Ogihara, Toshio; Tamai, Ikumi

    2007-01-01

    Serum uric acid (SUA) is currently recognized as a risk factor for cardiovascular disease. It has been reported that an angiotensin II receptor blocker (ARB), losartan, decreases SUA level, whereas other ARBs, such as candesartan, have no lowering effect. Because the renal uric acid transporter (URAT1) is an important factor controlling the SUA level, we examined the involvement of URAT1 in those differential effects of various ARBs on SUA level at clinically relevant concentrations. This study was done by using URAT1-expressing Xenopus oocytes. Losartan, pratosartan, and telmisartan exhibited cis-inhibitory effects on the uptake of uric acid by URAT1, whereas at higher concentrations, only telmisartan did, and these ARBs reduced the uptake in competitive inhibition kinetics. On the other hand, candesartan, EXP3174 [2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yI)methyl]imidazole-5-carboxylic acid] (a major metabolite of losartan), olmesartan, and valsartan were not inhibitory. Preloading of those ARBs in the oocytes enhanced the URAT1-mediated uric acid uptake, showing a trans-stimulatory effect. The present study is a first demonstration of the differential effects of ARBs on URAT1 that some ARBs are both cis-inhibitory and trans-stimulatory, depending on concentration, whereas others exhibit either a trans-stimulatory or cis-inhibitory effect alone, which could explain the clinically observed differential effects of ARBs on SUA level. Furthermore, it was found that such differential effects of ARBs on URAT1 could be predicted from the partial chemical structures of ARBs, which will be useful information for the appropriate use and development of ARBs without an increase of SUA.

  11. Amino Acid Residues in the Putative Transmembrane Domain 11 of Human Organic Anion Transporting Polypeptide 1B1 Dictate Transporter Substrate Binding, Stability, and Trafficking.

    PubMed

    Hong, Weifang; Wu, Zhixuan; Fang, Zihui; Huang, Jiujiu; Huang, Hong; Hong, Mei

    2015-12-07

    Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are membrane proteins that mediate the sodium-independent transport of a wide range of endogenous and exogenous compounds. Due to their broad substrate specificity, wide tissue distribution, and involvement in drug-drug interactions, OATPs have been considered as key players in drug absorption, distribution, and excretion. Transmembrane domains (TMs) are crucial structural features involved in proper functions of many transporters. According to computer-based modeling and previous studies of our laboratory and others, TM11 of OATP1B1 may face the substrate interaction pocket and thus play an important role in the transport function of the protein. Alanine-scanning of the transmembrane domain identified seven critical amino acid residues within the region. Further analysis revealed that alanine substitution of these residues resulted in reduced protein stability, which led to significantly decreased protein expression on the plasma membrane. In addition, all mutants exhibited an altered Km for ES uptake (either high affinity or low affinity component, or both), though Km for taurocholate transport only changed in R580A, G584A, and F591A. These results suggested that critical residues in TM11 not only affect protein stability of the transporter, but its interaction with substrates as well. The identification of seven essential residues out of 21 TM amino acids highlighted the importance of this transmembrane domain in the proper function of OATP1B1.

  12. Fluid transport by the cornea endothelium is dependent on buffering lactic acid efflux.

    PubMed

    Li, Shimin; Kim, Edward; Bonanno, Joseph A

    2016-07-01

    Maintenance of corneal hydration is dependent on the active transport properties of the corneal endothelium. We tested the hypothesis that lactic acid efflux, facilitated by buffering, is a component of the endothelial fluid pump. Rabbit corneas were perfused with bicarbonate-rich (BR) or bicarbonate-free (BF) Ringer of varying buffering power, while corneal thickness was measured. Perfusate was collected and analyzed for lactate efflux. In BF with no added HEPES, the maximal corneal swelling rate was 30.0 ± 4.1 μm/h compared with 5.2 ± 0.9 μm/h in BR. Corneal swelling decreased directly with [HEPES], such that with 60 mM HEPES corneas swelled at 7.5 ± 1.6 μm/h. Perfusate [lactate] increased directly with [HEPES]. Similarly, reducing the [HCO3 (-)] increased corneal swelling and decreased lactate efflux. Corneal swelling was inversely related to Ringer buffering power (β), whereas lactate efflux was directly related to β. Ouabain (100 μM) produced maximal swelling and reduction in lactate efflux, whereas carbonic anhydrase inhibition and an monocarboxylic acid transporter 1 inhibitor produced intermediate swelling and decreases in lactate efflux. Conversely, 10 μM adenosine reduced the swelling rate to 4.2 ± 0.8 μm/h and increased lactate efflux by 25%. We found a strong inverse relation between corneal swelling and lactate efflux (r = 0.98, P < 0.0001). Introducing lactate in the Ringer transiently increased corneal thickness, reaching a steady state (0 ± 0.6 μm/h) within 90 min. We conclude that corneal endothelial function does not have an absolute requirement for bicarbonate; rather it requires a perfusing solution with high buffering power. This facilitates lactic acid efflux, which is directly linked to water efflux, indicating that lactate flux is a component of the corneal endothelial pump.

  13. Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

    PubMed Central

    GUO, CHENYING; STELLA, SALVATORE L.; HIRANO, ARLENE A.; BRECHA, NICHOLAS C.

    2009-01-01

    Plasmalemmal and vesicular γ-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. PMID:18975268

  14. Transport in Porous Media of Poly(Acrylic Acid) Coated Ferrihydrite Nanoparticles

    NASA Astrophysics Data System (ADS)

    Jaffe, P. R.; Xiang, A.; Koel, B. E.

    2012-12-01

    Augmentation of soils with iron to enhance biological processes such as uranium reduction via iron reducing bacteria, e.g., Geobacter sp., might be achieved via the injection of iron nanoparticles into the subsurface. The challenge is to make these nanoparticles transportable in the subsurface while not affecting the iron bioavailability. Poorly crystallized 2-line ferrihydrite iron oxide nanoparticles were synthesized and coated with different amounts of poly(acrylic acid) polymers (Na-PAA6K or Na-PAA140K). Analyses were then performed on these particles, including sorption/desorption of the polymer onto the iron nanoparticles, particle size, zeta potential, transport in sand and soil columns, and bioavailabity of the Fe(III) in the absence and presence of the coating to iron reducing organisms. Results showed that at pH values of environmental relevance, the zeta potential of the particles varied from about 3 mV (pH=8.2) for the non-coated particles to about -30 mV for the particles coated with the polymers to their highest sorption capacity. The coated particle diameter was shown to be in the range of 200 nm. Column transport experiments showed that for the highest polymer coating the nanoparticle breakthrough was virtually identical to that of bromide, while significant filtration was observed for particles with an intermediate coating, and complete particle removal via filtration was observed for the non-coated particles. These results held for sand as well as for soil, which had been previously characterized, from a field site at Rifle, CO. Bioavailability experiments showed no difference in the iron reduction rate between the untreated and treated nanoparticles. These results show that it is possible to manufacture iron nanoparticles to enhance biological iron reduction, and that the transport properties of these treated particles is tunable so that a desired retention in the porous medium can be achieved.

  15. Regulation of dipeptide transport in Saccharomyces cerevisiae by micromolar amino acid concentrations

    SciTech Connect

    Island, M.D.; Naider, F.; Becker, J.M.

    1987-05-01

    Prototrophic Saccharomyces cerevisiae X2180, when grown on unsupplemented minimal medium, displayed little sensitivity to ethionine- and m-fluorophenylalanine-containing toxic dipeptides. The authors examined the influence of the 20 naturally occurring amino acids on sensitivity to toxic dipeptides. A number of these amino acids, at concentrations as low as 1 ..mu..M (leucine and tryptophan), produced large increases in sensitivity to leucyl-ethionine, alanyl-ethionine, and leucyl-m-fluorophenylalanine. Sensitivity to ethionine and m-fluorophenylalanine remained high under either set of conditions. The addition of 0.15 mM tryptophan to a growing culture resulted in the induction of dipeptide transport, as indicated by a 25-fold increase in the initial rate of L-leucyl-L(/sup 3/H)leucine accumulation. This increase, which was prevented by the addition of cycloheximide, began within 30 min and peaked approximately 240 min after a shift to medium containing tryptophan. Comparable increases in peptidase activity were not apparent in crude cell extracts form tryptophan-induced cultures. The authors concluded that S. cerevisiae possesses a specific mechanism for the induction of dipeptidetransport that can respond to very low concentrations of amino acids.

  16. Mechanism of proton transport in ionic-liquid-doped perfluorosulfonic acid membranes.

    PubMed

    Kumar, Milan; Venkatnathan, Arun

    2013-11-21

    Ionic-liquid-doped perfluorosulfonic acid membranes (PFSA) are promising electrolytes for intermediate/high-temperature fuel cell applications. In the present study, we examine proton-transport pathways in a triethylammonium-triflate (TEATF) ionic liquid (IL)-doped Nafion membrane using quantum chemistry calculations. The IL-doped membrane matrix contains triflic acid (TFA), triflate anions (TFA(-)), triethylamine (TEA), and triethylammonium cations (TEAH(+)). Results show that proton abstraction from the sulfonic acid end groups in the membrane by TFA(-) facilitates TEAH(+) interaction with the side-chains. In the IL-doped PFSA membrane matrix, proton transfer from TFA to TEA and TFA to TFA(-) occurs. However, proton transfer from a tertiary amine cation (TEAH(+)) to a tertiary amine (TEA) does not occur without an interaction with an anion (TFA(-)). An anion interaction with the amine increases its basicity, and as a consequence, it takes a proton from a cation either instantly (if the cation is freely moving) or with a small activation energy barrier of 2.62 kcal/mol (if the cation is interacting with another anion). The quantum chemistry calculations predict that anions are responsible for proton-exchange between cations and neutral molecules of a tertiary amine. Results from this study can assist the experimental choice of IL to provide enhanced proton conduction in PFSA membrane environments.

  17. The Fate and Transport of Glyphosate and its Degradation Product, Aminomethylphosphonic Acid (AMPA), in Water

    NASA Astrophysics Data System (ADS)

    Scribner, E.; Meyer, M. T.

    2006-05-01

    Since 2001, the U.S. Geological Survey (USGS) has investigated the fate and transport of glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), in surface water, and more recently in tile-drain flow, soil, and wet deposition. According to U.S. Environmental Protection Agency sources, glyphosate is among the world's most widely used herbicides. In 2004, glyphosate usage estimates indicated that between 103 and 113 million pounds were applied annually to crops in the United States. The use of glyphosate over a wide geographic area suggests that this herbicide might be a potential concern for air, water, and soil quality as well as measured in high concentrations in streams; therefore, it is important to monitor its fate and transport in ground-water/surface-water systems. National, regional, and field-scale studies conducted by the USGS National Water-Quality Assessment and Toxic Substance Hydrology Programs have studied the fate and transport of glyphosate in overland flow, tile- drain flow, surface water, soil, and wet-deposition samples. The samples were analyzed for glyphosate and AMPA by using derivatization and online solid-phase extraction with liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS methods developed by the USGS Organic Geochemistry Research Laboratory in Lawrence, Kansas. During spring, summer, and fall 2002 runoff periods in 50 Midwestern streams, glyphosate was detected at or above the 0.10 micrograms per liter detection limit in 35, 41, and 31 percent of samples, respectively. AMPA was detected in 53, 82, and 75 percent of samples, respectively. Results of 128 samples from a field study showed that glyphosate was transported as a narrow high- concentration pulse during the first period of runoff after application and that the concentration of glyphosate in runoff was greater than the concentration of AMPA. In tile-drain flow, glyphosate and AMPA were transported in a broad low-concentration pulse during these same

  18. Amino acid transport system - A substrate predicts the therapeutic effects of particle radiotherapy

    PubMed Central

    Watanabe, Mariko; Suzuki, Hiroyuki; Furusawa, Yoshiya; Arano, Yasushi

    2017-01-01

    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 evaluate the effects

  19. Facilitated transport of titanium dioxide nanoparticles by humic substances in saturated porous media under acidic conditions

    NASA Astrophysics Data System (ADS)

    Zhang, Ruichang; Zhang, Haibo; Tu, Chen; Hu, Xuefeng; Li, Lianzhen; Luo, Yongming; Christie, Peter

    2015-04-01

    The transport behavior of titanium dioxide nanoparticles (TiO2 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 TiO2 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-1. Facilitated transport of TiO2 NPs was likely attributable to the increased stability of TiO2 NPs and repulsive interaction between TiO2 NPs and quartz sands due to the adsorbed HS. The mobility of TiO2 NPs was also increased with increasing pH from 4.0 to 6.0. Although transport of TiO2 NPs was insensitive to low ionic strength, it was significantly inhibited by high concentrations of NaCl and CaCl2. In addition, calculated Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy indicated that high energy barriers were responsible for the high mobility of TiO2 NPs, while the secondary energy minimum could play an important role in the retention of TiO2 NPs at 100 mmol L-1 NaCl. Straining and gravitational settlement of larger TiO2 NPs aggregates at 1 mg L-1 HS, pH 5.0, and 2 mmol L-1 CaCl2 could be responsible for the significant retention even in the presence of high energy barriers. Moreover, more favorable interaction between approaching TiO2 NPs and TiO2 NPs that had been already deposited on the collector resulted in a ripening-shape breakthrough curve at 2 mmol L-1 CaCl2. Overall, a combination of mechanisms including DLVO-type force, straining, and physical filtration was involved in the retention of TiO2 NPs over the range of solution chemistry examined in this study.

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

    PubMed

    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 < 0.05) between 0.0001 and 0.1 µM folic acid. Above 0.1 µM, the slope of the regression line was not significantly different from zero (P < 0.137). Folic acid uptake in the jejunum showed a maximum rate of transport at pH 6.0, but was lowest at pH 7.5. The presence of 5-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.

  1. Analysis of acidity production during enhanced reductive dechlorination using a simplified reactive transport model

    NASA Astrophysics Data System (ADS)

    Brovelli, A.; Barry, D. A.; Robinson, C.; Gerhard, J. I.

    2012-07-01

    Build-up of fermentation products and hydrochloric acid at a contaminated site undergoing enhanced reductive dechlorination can result in groundwater acidification. Sub-optimal pH conditions can inhibit microbial activity and lead to reduced dechlorination rates. The extent of acidification likely to occur is site-specific and depends primarily on the extent of fermentation and dechlorination, the geochemical composition of soil and groundwater, and the pH-sensitivity of the active microbial populations. Here, the key chemical and physical mechanisms that control the extent of groundwater acidification in a contaminated site were examined, and the extent to which the remediation efficiency was affected by variations in groundwater pH was evaluated using a simplified process-based reactive-transport model. This model was applied successfully to a well-documented field site and was then employed in a sensitivity analysis to identify the processes likely to significantly influence acidity production and subsequent microbial inhibition. The accumulation of organic acids produced from the fermentation of the injected substrate was the main cause of the pH change. The concentration of dissolved sulphates controlled substrate utilisation efficiency because sulphate-reducing biomass competed with halo-respiring biomass for the fermentation products. It was shown further that increased groundwater velocity increases dilution and reduces the accumulation of acidic products. As a consequence, the flow rate corresponding to the highest remediation efficiency depends on the fermentation and dechlorination rates. The model enables investigation and forecasting of the extent and areal distribution of pH change, providing a means to optimise the application of reductive dechlorination for site remediation.

  2. Determination of thermodynamic and transport parameters of naphthenic acids and organic process chemicals in oil sand tailings pond water.

    PubMed

    Wang, Xiaomeng; Robinson, Lisa; Wen, Qing; Kasperski, Kim L

    2013-07-01

    Oil sand tailings pond water contains naphthenic acids and process chemicals (e.g., alkyl sulphates, quaternary ammonium compounds, and alkylphenol ethoxylates). These chemicals are toxic and can seep through the foundation of the tailings pond to the subsurface, potentially affecting the quality of groundwater. As a result, it is important to measure the thermodynamic and transport parameters of these chemicals in order to study the transport behavior of contaminants through the foundation as well as underground. In this study, batch adsorption studies and column experiments were performed. It was found that the transport parameters of these chemicals are related to their molecular structures and other properties. The computer program (CXTFIT) was used to further evaluate the transport process in the column experiments. The results from this study show that the transport of naphthenic acids in a glass column is an equilibrium process while the transport of process chemicals seems to be a non-equilibrium process. At the end of this paper we present a real-world case study in which the transport of the contaminants through the foundation of an external tailings pond is calculated using the lab-measured data. The results show that long-term groundwater monitoring of contaminant transport at the oil sand mining site may be necessary to avoid chemicals from reaching any nearby receptors.

  3. Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.

    PubMed Central

    Albritton, L M; Kim, J W; Tseng, L; Cunningham, J M

    1993-01-01

    Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses. PMID:8445722

  4. A glial amino-acid transporter controls synapse strength and courtship in Drosophila.

    PubMed

    Grosjean, Yael; Grillet, Micheline; Augustin, Hrvoje; Ferveur, Jean-François; Featherstone, David E

    2008-01-01

    Mate choice is an evolutionarily critical decision that requires the detection of multiple sex-specific signals followed by central integration of these signals to direct appropriate behavior. The mechanisms controlling mate choice remain poorly understood. Here, we show that the glial amino-acid transporter genderblind controls whether Drosophila melanogaster males will attempt to mate with other males. Genderblind (gb) mutant males showed no alteration in heterosexual courtship or copulation, but were attracted to normally unappealing male species-specific chemosensory cues. As a result, genderblind mutant males courted and attempted to copulate with other Drosophila males. This homosexual behavior could be induced within hours using inducible RNAi, suggesting that genderblind controls nervous system function rather than its development. Consistent with this, and indicating that glial genderblind regulates ambient extracellular glutamate to suppress glutamatergic synapse strength in vivo, homosexual behavior could be turned on and off by altering glutamatergic transmission pharmacologically and/or genetically.

  5. Fatty Acid Transport Protein-2 inhibitor Grassofermata/CB5 protects cells against lipid accumulation and toxicity

    PubMed Central

    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 (IC50 8–11μM) 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 (IC50 58μM). 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 13C-oleate demonstrating its potential as a therapeutic agent. PMID:26284975

  6. Amino acid ester prodrugs conjugated to the α-carboxylic acid group do not display affinity for the L-type amino acid transporter 1 (LAT1).

    PubMed

    Rautio, Jarkko; Kärkkäinen, Jussi; Huttunen, Kristiina M; Gynther, Mikko

    2015-01-23

    L-type amino acid transporter (LAT1) is an intriguing target for carrier-mediated transport of drugs as it is highly expressed in the blood-brain barrier and also in various types of cancer. Several studies have proposed that in order for compounds to act as LAT1 substrates they should possess both negatively charged α-carboxyl and positively charged α-amino groups. However, in some reports, such as in two recent publications describing an isoleucine-quinidine ester prodrug (1), compounds having no free α-carboxyl group have been reported to exhibit high affinity for LAT1 in vitro. In the present study, 1 was synthesized and its affinity for LAT1 was evaluated both with an in situ rat brain perfusion technique and in the human breast cancer cell line MCF-7 in vitro. 1 showed no affinity for LAT1 in either model nor did it show any affinity for LAT2 in an in vitro study. Our results confirm the earlier reported requirements for LAT1 substrates. Thus drugs or prodrugs with substituted α-carboxyl group cannot bind to LAT with high affinity.

  7. Vesicular γ-Aminobutyric Acid Transporter Expression in Amacrine and Horizontal Cells

    PubMed Central

    Cueva, Juan G.; Haverkamp, Silke; Reimer, Richard J.; Edwards, Robert; Wässle, Heinz; Brecha, Nicholas C.

    2010-01-01

    The vesicular γ-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preem-bedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release. PMID:11920703

  8. Expression and purification of a functional uric acid-xanthine transporter (UapA).

    PubMed

    Leung, James; Karachaliou, Mayia; Alves, Claudia; Diallinas, George; Byrne, Bernadette

    2010-07-01

    The Nucleobase-Ascorbate Transporters (NATs) family includes carriers with fundamental functions in uptake of key cellular metabolites, such as uric acid or vitamin C. The best studied example of a NAT transporter is the uric acid-xanthine permease (UapA) from the model ascomycete Aspergillus nidulans. Detailed genetic and biochemical analyses have revealed much about the mechanism of action of this protein; however, the difficulties associated with handling eukaryotic membrane proteins have limited efforts to elucidate the precise structure-function relationships of UapA by structural analysis. In this manuscript, we describe the heterologous overexpression of functional UapA as a fusion with GFP in different strains of Saccharomyces cerevisiae. The UapA-GFP construct expressed to 2.3 mg/L in a pep4Delta deletion strain lacking a key vacuolar endopeptidase and 3.8 mg/L in an npi1-1 mutant strain with defective Rsp5 ubiquitin ligase activity. Epifluorescence microscopy revealed that the UapA-GFP was predominately localized to the plasma membrane in both strains, although a higher intensity of fluorescence was observed for the npi1-1 mutant strain plasma membrane. In agreement with these observations, the npi1-1 mutant strain demonstrated a approximately 5-fold increase in uptake of [(3)H]-xanthine compared to the pep4Delta deletion strain. Despite yielding the best results for functional expression, in-gel fluorescence of the UapA-GFP expressed in the npi1-1 mutant strain revealed that the protein was subject to significant proteolytic degradation. Large scale expression of the protein using the pep4Delta deletion strain followed by purification produced mg quantities of pure, monodispersed protein suitable for further structural and functional studies. In addition, this work has generated a yeast cell based system for performing reverse genetics and other targeted approaches, in order to further understand the mechanism of action of this important model protein.

  9. Effect of acid stress on sodium transport by isolated skins and on osmotic permeability of intact frogs

    SciTech Connect

    Fromm, P.O.

    1981-08-01

    The experiments reported here were designed to determine the effects of increased external hydrogen ion concentrations on the ion transport capability of isolated frog skins measured as short-circuit current and to determine the nature of the interaction of hydrogen ions to sodium transport. Results from a study of the effects of acid exposure on the osmotic permeability of intact frogs are also reported.

  10. Ursolic Acid Inhibits Na+/K+-ATPase Activity and Prevents TNF-α-Induced Gene Expression by Blocking Amino Acid Transport and Cellular Protein Synthesis

    PubMed Central

    Yokomichi, Tomonobu; Morimoto, Kyoko; Oshima, Nana; Yamada, Yuriko; Fu, Liwei; Taketani, Shigeru; Ando, Masayoshi; Kataoka, Takao

    2011-01-01

    Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, induce the expression of a wide variety of genes, including intercellular adhesion molecule-1 (ICAM-1). Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid) was identified to inhibit the cell-surface ICAM-1 expression induced by pro-inflammatory cytokines in human lung carcinoma A549 cells. Ursolic acid was found to inhibit the TNF-α-induced ICAM-1 protein expression almost completely, whereas the TNF-α-induced ICAM-1 mRNA expression and NF-κB signaling pathway were decreased only partially by ursolic acid. In line with these findings, ursolic acid prevented cellular protein synthesis as well as amino acid uptake, but did not obviously affect nucleoside uptake and the subsequent DNA/RNA syntheses. This inhibitory profile of ursolic acid was similar to that of the Na+/K+-ATPase inhibitor, ouabain, but not the translation inhibitor, cycloheximide. Consistent with this notion, ursolic acid was found to inhibit the catalytic activity of Na+/K+-ATPase. Thus, our present study reveals a novel molecular mechanism in which ursolic acid inhibits Na+/K+-ATPase activity and prevents the TNF-α-induced gene expression by blocking amino acid transport and cellular protein synthesis. PMID:24970122

  11. Amino acid transporter LAT3 is required for podocyte development and function.

    PubMed

    Sekine, Yuji; Nishibori, Yukino; Akimoto, Yoshihiro; Kudo, Akihiko; Ito, Noriko; Fukuhara, Daisuke; Kurayama, Ryota; Higashihara, Eiji; Babu, Ellappan; Kanai, Yoshikatsu; Asanuma, Katsuhiko; Nagata, Michio; Majumdar, Arindam; Tryggvason, Karl; Yan, Kunimasa

    2009-07-01

    LAT3 is a Na+-independent neutral l-amino acid transporter recently isolated from a human hepatocellular carcinoma cell line. Although liver, skeletal muscle, and pancreas are known to express LAT3, the tissue distribution and physiologic function of this transporter are not completely understood. Here, we observed that glomeruli express LAT3. Immunofluorescence, confocal microscopy, and immunoelectron microscopy revealed that LAT3 localizes to the apical plasma membrane of podocyte foot processes. In mice, starvation upregulated glomerular LAT3, phosphorylated AKT1, reconstituted the actin network, and elongated foot processes. In the fetal kidney, we observed intense LAT3 expression at the capillary loops stage of renal development. Finally, zebrafish morphants lacking lat3 function showed collapsed glomeruli with thickened glomerular basement membranes. Permeability studies of the glomerular filtration barrier in these zebrafish morphants demonstrated a disruption of selective glomerular permeability. Our data suggest that LAT3 may play a crucial role in the development and maintenance of podocyte structure and function by regulating protein synthesis and the actin cytoskeleton.

  12. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

    PubMed

    Kim, J W; Closs, E I; Albritton, L M; Cunningham, J M

    1991-08-22

    Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.

  13. Recent Advance in the Relationship between Excitatory Amino Acid Transporters and Parkinson's Disease

    PubMed Central

    Zhang, Yunlong; Tan, Feng; Xu, Pingyi; Qu, Shaogang

    2016-01-01

    Parkinson's disease (PD) is the most common movement disorder disease in the elderly and is characterized by degeneration of dopamine neurons and formation of Lewy bodies. Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS). If glutamate is not removed promptly in the synaptic cleft, it will excessively stimulate the glutamate receptors and induce excitotoxic effects on the CNS. With lack of extracellular enzyme to decompose glutamate, glutamate uptake in the synaptic cleft is mainly achieved by the excitatory amino acid transporters (EAATs, also known as high-affinity glutamate transporters). Current studies have confirmed that decreased expression and function of EAATs appear in PD animal models. Moreover, single unilateral administration of EAATs inhibitor in the substantia nigra mimics several PD features and this is a solid evidence supporting that decreased EAATs contribute to the process of PD. Drugs or treatments promoting the expression and function of EAATs are shown to attenuate dopamine neurons death in the substantia nigra and striatum, ameliorate the behavior disorder, and improve cognitive abilities in PD animal models. EAATs are potential effective drug targets in treatment of PD and thus study of relationship between EAATs and PD has predominant medical significance currently. PMID:26981287

  14. Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3

    PubMed Central

    Li, Wei; Obregón-Henao, Andrés; Wallach, Joshua B.; North, E. Jeffrey; Lee, Richard E.; Gonzalez-Juarrero, Mercedes; Schnappinger, Dirk

    2016-01-01

    In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies. PMID:27297488

  15. Expression Profile of Cationic Amino Acid Transporters in Rats with Endotoxin-Induced Uveitis

    PubMed Central

    Chang, Shu-Wen; Lee, Yi-An; Kao, Tzu-Yun

    2016-01-01

    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. PMID:27413255

  16. The action of flufenamic acid and other nonsteroidal anti-inflammatories on sulfate transport in the isolated perfused rat liver.

    PubMed

    Lopez, C H; Bracht, A; Yamamoto, N S; Ishii-Iwamoto, E L; Sampaio, E; Kelmer-Bracht, A M

    1999-06-01

    The influence of flufenamic acid and other nonsteroidal anti-inflammatories on sulfate transport in the liver was investigated. The experimental system was the isolated perfused rat liver. Perfusion was accomplished in an open, nonrecirculating system. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4), saturated with a mixture of oxygen and carbon dioxide (95:5) by means of a membrane oxygenator and heated to 37 degrees C. Sulfate transport (equilibrium exchange) was measured by employing the multiple-indicator dilution technique with simultaneous injection (impulse input) of [35S]sulfate. [3H]sucrose (indicator for the distribution of the sinusoidal transit times), and [3H]water (indicator for the total aqueous space). Analysis was accomplished by means of a space-distributed variable transit time model. Flufenamic acid and other anti-inflammatories inhibited sulfate transport in the liver. For a concentration of 100 microM, the following decreasing series of potency could be established: flufenamic acid (53.4 +/- 2.9%) > niflumic acid (41.1 +/- 1.4%) > mefenamic acid (35.6 +/- 3.3%) > piroxicam (16.6 +/- 1.9%) > naproxen (13.5 +/- 8.4)%) nimesulide (11.6 +/- 5.8%). Inhibition of sulfate transport by flufenamic acid was clearly concentration dependent; 250 microM flufenamic acid produced more than 95% inhibition. Flufenamic acid in the range between 50 and 250 microM did not affect the mean transit times of tritiated water (t water) and [3H]sucrose (t suc), the same applying to all other anti-inflammatory agents (100 microM) tested in this work. This means that these agents do not affect vascular and cellular spaces, even when present at high concentrations. The ratio of the intra- to extracellular sulfate concentrations ([C]i/[C]e), generally between 0.4 and 0.5 under control conditions, was affected only by 250 microM flufenamic acid and 100 microM niflumic acid. In the first case, this phenomenon is possibly due to the high degree of transport

  17. Transport and Metabolism of 1-Aminocyclopropane-1-carboxylic Acid in Sunflower (Helianthus annuus L.) Seedlings 1

    PubMed Central

    Finlayson, Scott A.; Foster, Kenneth R.; Reid, David M.

    1991-01-01

    Transport and metabolism of [2,3-14C] 1-aminocyclopropane-1-carboxylic acid (ACC) from roots to shoots in 4-day-old sunflower (Helianthus annuus L.) seedlings were studied. [14C]ACC was detected in, and 14C2H4 was evolved from, shoots 0.5 hours after [14C]ACC was supplied to roots. Ethylene emanation from the shoots returned to normal levels after 6 hours. The roots showed a similar pattern, although at 24 hours ethylene emanation was still slightly higher than in those plants that did not receive ACC. [14C]N-malonyl-ACC (MACC) was detected in both tissues at all times sampled. [14C]MACC levels surpassed [14C]ACC levels in the shoot at 2 hours, whereas [14C]MACC levels in the root remained below [14C]ACC levels until 6 hours, after which they were higher. Thin-layer chromatography analysis identified [14C] ACC in 1-hour shoot extracts, and [14C]MACC was identified in root tissues at 1 and 12 hours after treatment. [14C]ACC and [14C] MACC in the xylem sap of treated seedlings were identified by thin-layer chromatography. Xylem transport of [14C]ACC in treated seedlings, and transport of ACC in untreated seedlings, was confirmed by gas chromatography-mass spectrometry. Some evidence for the presence of [14C]MACC in xylem sap in [14C]ACC-treated seedlings is presented. A substantial amount of radioactivity in both ACC and MACC fractions was detected leaking from the roots over 24 hours. A second radiolabeled volatile compound was trapped in a CO2-trapping solution but not in mercuric perchlorate. Levels of this compound were highest after the peak of ACC levels and before peak MACC levels in both tissues, suggesting that an alternate pathway of ACC metabolism was operating in this system. PMID:16668342

  18. Application of slightly acidic electrolyzed water for decontamination of stainless steel surfaces in animal transport vehicles.

    PubMed

    Ni, Li; Zheng, Weichao; Zhang, Qiang; Cao, Wei; Li, Baoming

    2016-10-01

    The effectiveness of slightly acidic electrolyzed water (SAEW) in reducing Escherichia coli, Salmonella typhimurim, Staphylococcus aureus or bacterial mixtures on stainless steel surfaces was evaluated and compared its efficacy with composite phenol solution for reducing total aerobic bacteria in animal transport vehicles. Stainless steel surfaces were inoculated with these strains individually or in a mixture, and sprayed with SAEW, composite phenol, or alkaline electrolyzed water for 0.5, 1, 1.5 and 2min. The bactericidal activity of SAEW increased with increasing available chlorine concentration and spraying duration. The SAEW solution of 50mgl(-1) of available chlorine concentration showed significantly higher effectiveness than composite phenol in reducing the pathogens on stainless steel surfaces (P<0.05). Complete inactivation of pathogens on stainless steel surfaces were observed after treatment with alkaline electrolyzed water followed by SAEW at 50mgl(-1) of available chlorine concentration for 2min or alkaline electrolyzed water treatment followed by SAEW treatment at 90mgl(-1) of available chlorine concentration for 0.5min. The efficacy of SAEW in reducing total aerobic bacteria in animal transport vehicles was also determined. Vehicles in the disinfection booth were sprayed with the same SAEW, alkaline electrolyzed water and composite phenol solutions using the automatic disinfection system. Samples from vehicle surfaces were collected with sterile cotton swabs before and after each treatment. No significant differences in bactericidal efficiency were observed between SAEW and composite phenol for reducing total aerobic bacteria in the vehicles (P>0.05). SAEW was also found to be more effective when used in conjunction with alkaline electrolyzed water. Results suggest that the bactericidal efficiency of SAEW was higher than or equivalent to that of composite phenol and SAEW may be used as effective alternative for reducing microbial contamination of

  19. The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids*

    PubMed Central

    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

  20. The human gene SLC25A29, of solute carrier family 25, encodes a mitochondrial transporter of basic amino acids.

    PubMed

    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.

  1. Purification and identification of the functional sodium- and chloride-coupled gamma-aminobutyric acid transport glycoprotein from rat brain.

    PubMed

    Radian, R; Bendahan, A; Kanner, B I

    1986-11-25

    Using the reconstitution conditions developed recently (Radian, R., and Kanner, B. I. (1985) J. Biol. Chem. 260, 11859-11865) we have now purified the sodium- and chloride-coupled gamma-aminobutyric acid transporter from rat brain to apparent homogeneity. A partially purified transporter preparation was passed over wheat germ agglutinin-Sepharose 6MB and non-bound proteins were washed away. The transport activity, as expressed upon reconstitution of the protein into liposomes, was eluted by a solution containing Triton X-100 and N-acetylglucosamine. The specific transport activity was increased almost 400-fold over that of the crude extract. Taking into account an approximately 2.5-fold inactivation during the lectin column chromatography, the actual purification is about 1000-fold. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the active fractions revealed one band of 80 kDa and small amounts of a band which ran at an apparent molecular mass of 160 kDa. The ratio between the two could be experimentally changed such as, for instance, by lyophilization. Polyclonal antibodies were prepared against the 80-kDa band which also cross-reacted with the 160-kDa band, indicating that the latter apparently represents a dimer form of the first. Using Protein A-Sepharose Cl-4B and the antibody against the 80-kDa band, we were able to quantitatively immunoprecipitate the potential gamma-aminobutyric acid transport activity from a crude transporter preparation. The pure transporter preparation exhibited the same features of the transporter in synaptic plasma membrane vesicles, namely dependence on sodium and chloride, electrogeneity, affinity, and efflux and exchange properties. We conclude that the 80-kDa band represents the gamma-aminobutyric acid transporter.

  2. Sialic acid transporter NanT participates in Tannerella forsythia biofilm formation and survival on epithelial cells.

    PubMed

    Honma, Kiyonobu; Ruscitto, Angela; Frey, Andrew M; Stafford, Graham P; Sharma, Ashu

    2016-05-01

    Tannerella forsythia is a periodontal pathogen implicated in periodontitis. This gram-negative pathogen depends on exogenous peptidoglycan amino sugar N-acetylmuramic acid (NAM) for growth. In the biofilm state the bacterium can utilize sialic acid (Neu5Ac) instead of NAM to sustain its growth. Thus, the sialic acid utilization system of the bacterium plays a critical role in the growth and survival of the organism in the absence of NAM. We sought the function of a T. forsythia gene annotated as nanT coding for an inner-membrane sugar transporter located on a sialic acid utilization genetic cluster. To determine the function of this putative sialic acid transporter, an isogenic nanT-deletion mutant generated by allelic replacement strategy was evaluated for biofilm formation on NAM or Neu5Ac, and survival on KB epithelial cells. Moreover, since T. forsythia forms synergistic biofilms with Fusobacterium nucleatum, co-biofilm formation activity in mixed culture and sialic acid uptake in culture were also assessed. The data showed that the nanT-inactivated mutant of T. forsythia was attenuated in its ability to uptake sialic acid. The mutant formed weaker biofilms compared to the wild-type strain in the presence of sialic acid and as co-biofilms with F. nucleatum. Moreover, compared to the wild-type T. forsythia nanT-inactivated mutant showed reduced survival when incubated on KB epithelial cells. Taken together, the data presented here demonstrate that NanT-mediated sialic transportation is essential for sialic acid utilization during biofilm growth and survival of the organism on epithelial cells and implies sialic acid might be key for its survival both in subgingival biofilms and during infection of human epithelial cells in vivo.

  3. Deletion of the amino acid transporter Slc6a14 suppresses tumour growth in spontaneous mouse models of breast cancer.

    PubMed

    Babu, Ellappan; Bhutia, Yangzom D; Ramachandran, Sabarish; Gnanaprakasam, Jaya P; Prasad, Puttur D; Thangaraju, Muthusamy; Ganapathy, Vadivel

    2015-07-01

    SLC6A14 mediates Na(+)/Cl(-)-coupled concentrative uptake of a broad-spectrum of amino acids. It is expressed at low levels in many tissues but up-regulated in certain cancers. Pharmacological blockade of SLC6A14 causes amino acid starvation in estrogen receptor positive (ER+) breast cancer cells and suppresses their proliferation in vitro and in vivo. In the present study, we interrogated the role of this transporter in breast cancer by deleting Slc6a14 in mice and monitoring the consequences of this deletion in models of spontaneous breast cancer (Polyoma middle T oncogene-transgenic mouse and mouse mammary tumour virus promoter-Neu-transgenic mouse). Slc6a14-knockout mice are viable, fertile and phenotypically normal. The plasma amino acids were similar in wild-type and knockout mice and there were no major compensatory changes in the expression of other amino acid transporter mRNAs. There was also no change in mammary gland development in the knockout mouse. However, when crossed with PyMT-Tg mice or MMTV/Neu (mouse mammary tumour virus promoter-Neu)-Tg mice, the development and progression of breast cancer were markedly decreased on Slc6a14(-/-) background. Analysis of transcriptomes in tumour tissues from wild-type mice and Slc6a14-null mice indicated no compensatory changes in the expression of any other amino acid transporter mRNA. However, the tumours from the null mice showed evidence of amino acid starvation, decreased mTOR signalling and decreased cell proliferation. These studies demonstrate that SLC6A14 is critical for the maintenance of amino acid nutrition and optimal mammalian target of rapamycin (mTOR) signalling in ER+ breast cancer and that the transporter is a potential target for development of a novel class of anti-cancer drugs targeting amino acid nutrition in tumour cells.

  4. Critical amino acid residues involved in the electrogenic sodium-bicarbonate cotransporter kNBC1-mediated transport.

    PubMed

    Abuladze, Natalia; Azimov, Rustam; Newman, Debra; Sassani, Pakan; Liu, Weixin; Tatishchev, Sergei; Pushkin, Alexander; Kurtz, Ira

    2005-06-15

    We have previously reported a topological model of the electrogenic Na(+)-HCO(3)(-) cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na(+)-dependent base flux of the mutants was determined using intracellular pH measurements with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na(+)-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented.

  5. Critical amino acid residues involved in the electrogenic sodium–bicarbonate cotransporter kNBC1-mediated transport

    PubMed Central

    Abuladze, Natalia; Azimov, Rustam; Newman, Debra; Sassani, Pakan; Liu, Weixin; Tatishchev, Sergei; Pushkin, Alexander; Kurtz, Ira

    2005-01-01

    We have previously reported a topological model of the electrogenic Na+–HCO3− cotransporter (NBC1) in which the cotransporter spans the plasma membrane 10 times with N- and C-termini localized intracellularly. An analysis of conserved amino acid residues among members of the SLC4 superfamily in both the transmembrane segments (TMs) and intracellular/extracellular loops (ILs/ELs) provided the basis for the mutagenesis approach taken in the present study to determine amino acids involved in NBC1-mediated ion transport. Using large-scale mutagenesis, acidic and basic amino acids putatively involved in ion transport mediated by the predominant variant of NBC1 expressed in the kidney (kNBC1) were mutated to neutral and/or oppositely charged amino acids. All mutant kNBC1 cotransporters were expressed in HEK-293T cells and the Na+-dependent base flux of the mutants was determined using intracellular pH measurements with 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Critical glutamate, aspartate, lysine, arginine and histidine residues in ILs/ELs and TMs were detected that were essential for kNBC1-mediated Na+-dependent base transport. In addition, critical phenylalanine, serine, tyrosine, threonine and alanine residues in TMs and ILs/ELs were detected. Furthermore, several amino acid residues in ILs/ELs and TMs were shown to be essential for membrane targeting. The data demonstrate asymmetry of distribution of kNBC1 charged amino acids involved in ion recognition in putative outward-facing and inward-facing conformations. A model summarizing key amino acid residues involved in kNBC1-mediated ion transport is presented. PMID:15817634

  6. Isomer-specific effects of conjugated linoleic acid on HDL functionality associated with reverse cholesterol transport.

    PubMed

    Nicod, Nathalie; Parker, Robert S; Giordano, Elena; Maestro, Virginia; Davalos, Alberto; Visioli, Francesco

    2015-02-01

    High-density lipoproteins (HDLs) are atheroprotective because of their role in reverse cholesterol transport. The intestine is involved in this process because it synthesizes HDL, removes cholesterol from plasma and excretes it into the lumen. We investigated the role of selected dietary fatty acids on intestinal cholesterol uptake and HDL functionality. Caco-2 monolayers grown on Transwells were supplemented with either palmitic, palmitoleic, oleic, linoleic, docosahexaenoic, eicosapentaenoic, arachidonic or conjugated linoleic acids (CLAs): c9,t11-CLA; t9,t11-CLA; c10,t12-CLA. Cells synthesized HDL in the basolateral compartment for 24 h in the absence or presence of an antibody to SR-BI (aSR-BI), which inhibits its interaction with HDL. Free cholesterol (FC) accumulated to a greater extent in the presence than in the absence of aSR-BI, indicating net uptake of FC by SR-BI. Uptake's efficiency was significantly decreased when cells were treated with c9,t11-CLA relative to the other fatty acids. These differences were associated with lower HDL functionality, since neither SR-BI protein expression nor expression and alternative splicing of other genes involved lipid metabolism were affected. Only INSIG2 expression was decreased, with no increase of its target genes. Increasing pre-β-HDL synthesis, by inducing ABCA1 and adding APOA1, resulted in reduced uptake of FC by SR-BI after c9,t11-CLA treatment, indicating reduced functionality of pre-β-HDL. Conversely, treatment with c9,t11-CLA resulted in a greater uptake of FC and esterified cholesterol from mature HDL. Therefore, Caco-2 monolayers administered c9,t11-CLA produced a nonfunctional pre-β-HDL but took up cholesterol more efficiently via SR-BI from mature HDL.

  7. Computational models for drug inhibition of the human apical sodium-dependent bile acid transporter.

    PubMed

    Zheng, Xiaowan; Ekins, Sean; Raufman, Jean-Pierre; Polli, James E

    2009-01-01

    The human apical sodium-dependent bile acid transporter (ASBT; SLC10A2) is the primary mechanism for intestinal bile acid reabsorption. In the colon, secondary bile acids increase the risk of cancer. Therefore, drugs that inhibit ASBT have the potential to increase the risk of colon cancer. The objectives of this study were to identify FDA-approved drugs that inhibit ASBT and to derive computational models for ASBT inhibition. Inhibition was evaluated using ASBT-MDCK monolayers and taurocholate as the model substrate. Computational modeling employed a HipHop qualitative approach, a Hypogen quantitative approach, and a modified Laplacian Bayesian modeling method using 2D descriptors. Initially, 30 compounds were screened for ASBT inhibition. A qualitative pharmacophore was developed using the most potent 11 compounds and applied to search a drug database, yielding 58 hits. Additional compounds were tested, and their K(i) values were measured. A 3D-QSAR and a Bayesian model were developed using 38 molecules. The quantitative pharmacophore consisted of one hydrogen bond acceptor, three hydrophobic features, and five excluded volumes. Each model was further validated with two external test sets of 30 and 19 molecules. Validation analysis showed both models exhibited good predictability in determining whether a drug is a potent or nonpotent ASBT inhibitor. The Bayesian model correctly ranked the most active compounds. In summary, using a combined in vitro and computational approach, we found that many FDA-approved drugs from diverse classes, such as the dihydropyridine calcium channel blockers and HMG CoA-reductase inhibitors, are ASBT inhibitors.

  8. Computational Models for Drug Inhibition of the Human Apical Sodium-dependent Bile Acid Transporter

    PubMed Central

    Zheng, Xiaowan; Ekins, Sean; Raufman, Jean-Pierre; Polli, James E.

    2009-01-01

    The human apical sodium-dependent bile acid transporter (ASBT; SLC10A2) is the primary mechanism for intestinal bile acid re-absorption. In the colon, secondary bile acids increase the risk of cancer. Therefore, drugs that inhibit ASBT have the potential to increase the risk of colon cancer. The objectives of this study were to identify FDA-approved drugs that inhibit ASBT and to derive computational models for ASBT inhibition. Inhibition was evaluated using ASBT-MDCK monolayers and taurocholate as the model substrate. Computational modeling employed a HipHop qualitative approach, a Hypogen quantitative approach, as well as a modified Laplacian Bayesian modeling method using 2D descriptors. Initially, 30 compounds were screened for ASBT inhibition. A qualitative pharmacophore was developed using the most potent 11 compounds and applied to search a drug database, yielding 58 hits. Additional compounds were tested and their Ki values were measured. A 3D-QSAR and a Bayesian model were developed using 38 molecules. The quantitative pharmacophore consisted of one hydrogen bond acceptor, three hydrophobic features, and five excluded volumes. Each model was further validated with two external test sets of 30 and 19 molecules. Validation analysis showed both models exhibited good predictability in determining whether a drug is a potent or non-potent ASBT inhibitor. The Bayesian model correctly ranked the most active compounds. In summary, using a combined in vitro and computational approach, we found that many FDA-approved drugs from diverse classes, such as the dihydropyridine calcium channel blockers and HMG CoA-reductase inhibitors, are ASBT inhibitors. PMID:19673539

  9. Salicylic Acid Is an Uncoupler and Inhibitor of Mitochondrial Electron Transport1

    PubMed Central

    Norman, Christel; Howell, Katharine A.; Millar, A. Harvey; Whelan, James M.; Day, David A.

    2004-01-01

    The effect of salicylic acid (SA) on respiration and mitochondrial function was examined in tobacco (Nicotiana tabacum) suspension cell cultures in the range of 0.01 to 5 mm. Cells rapidly accumulated SA up to 10-fold of the externally applied concentrations. At the lower concentrations, SA accumulation was transitory. When applied at 0.1 mm or less, SA stimulated respiration of whole cells and isolated mitochondria in the absence of added ADP, indicating uncoupling of respiration. However, at higher concentrations, respiration was severely inhibited. Measurements of ubiquinone redox poise in isolated mitochondria suggested that SA blocked electron flow from the substrate dehydrogenases to the ubiquinone pool. This inhibition could be at least partially reversed by re-isolating the mitochondria. Two active analogs of SA, benzoic acid and acetyl-SA, had the same effect as SA on isolated tobacco mitochondria, whereas the inactive p-hydroxybenzoic acid was without effect at the same concentration. SA induced an increase in Aox protein levels in cell suspensions, and this was correlated with an increase in Aox1 transcript abundance. However, when applied at 0.1 mm, this induction was transient and disappeared as SA levels in the cells declined. SA at 0.1 mm also increased the expression of other SA-responsive genes, and this induction was dependent on active mitochondria. The results indicate that SA is both an uncoupler and an inhibitor of mitochondrial electron transport and suggest that this underlies the induction of some genes by SA. The possible implications of this for the interpretation of SA action in plants are discussed. PMID:14684840

  10. Proton transport in triflic acid hydrates studied via path integral car-parrinello molecular dynamics.

    PubMed

    Hayes, Robin L; Paddison, Stephen J; Tuckerman, Mark E

    2009-12-31

    The mono-, di-, and tetrahydrates of trifluoromethanesulfonic acid, which contain characteristic H(3)O(+), H(5)O(2)(+), and H(9)O(4)(+) structures, provide model systems for understanding proton transport in materials with high perfluorosulfonic acid density such as perfluorosulfonic acid membranes commonly employed in hydrogen fuel cells. Ab initio molecular dynamics simulations indicate that protons in these solids are predisposed to transfer to the water most strongly bound to sulfonate groups via a Grotthuss-type mechanism, but quickly return to the most solvated defect structure either due to the lack of a nearby species to stabilize the new defect or a preference for the proton to be maximally hydrated. Path integral molecular dynamics of the mono- and dihydrate reveal significant quantum effects that facilitate proton transfer to the "presolvated" water or SO(3)(-) in the first solvation shell and increase the Zundel character of all the defects. These trends are quantified in free energy profiles for each bonding environment. Hydrogen bonding criteria for HOH-OH(2) and HOH-O(3)S are extracted from the two-dimensional potential of mean force. The quantum radial distribution function, radius of gyration, and root-mean-square displacement position correlation function show that the protonic charge is distributed over two or more water molecules. Metastable structural defects with one excess proton shared between two sulfonate groups and another Zundel or Eigen type cation defect are found for the mono- and dihydrate but not for the tetrahydrate crystal. Results for the tetrahydrate native crystal exhibit minor differences at 210 and 250 K. IR spectra are calculated for all native and stable defect structures. Graph theory techniques are used to characterize the chain lengths and ring sizes in the hydrogen bond network. Low conductivities when limited water is present may be attributable to trapping of protons between SO(3)(-) groups and the increased

  11. Prepartum maternal diets supplemented with oilseeds alter the fatty acid profile in bovine neonatal plasma possibly through reduced placental expression of fatty acid transporter protein 4 and fatty acid translocase.

    PubMed

    Salehi, Reza; Ambrose, Divakar J

    2016-12-12

    In the present study, we determined the effects of maternal dietary fat and the type of fat on plasma fatty acids and the expression of placental fatty acid transporter genes. In Experiment 1, Holstein cows in the last 35 days of gestation received diets containing sunflower seed (n=8; high in linoleic acid (LA)), canola seed (n=7; high in oleic acid (OLA)) or no oilseed (n=7; control). Fatty acids were quantified in dam and neonate plasma at calving. In Experiment 2, placental cotyledons were collected (LA: n=4; OLA: n=4; control: n=5) to quantify gene expression. Maternal long-chain polyunsaturated fatty acids, neonatal total n-3 fatty acids and eicosapentaenoic acid (EPA) declined, whereas docosahexaenoic acid (DHA) and total fat tended to decline following fat supplementation prepartum. Feeding of LA versus OLA prepartum tended to increase peroxisome proliferator-activated receptor α (PPARA) expression, whereas peroxisome proliferator-activated receptor δ (PPARD) and peroxisome proliferator-activated receptor γ (PPARG) expression tended to be higher in OLA- than LA-fed cows. Expression of fatty acid transporter protein 4 (FATP4) and fatty acid translocase (FAT/CD36) expression was lower in placental tissue of cows fed fat compared with control cows. Reduced total n-3 fatty acids, EPA and DHA in neonates born of dams fed fat prepartum is likely due to changes in PPARs and reduced expression of placental FATP4 and FAT/CD36.

  12. A single-amino-acid substitution eliminates the stringent carbohydrate requirement for intracellular transport of a viral glycoprotein.

    PubMed

    Pitta, A M; Rose, J K; Machamer, C E

    1989-09-01

    In this report, we have investigated the contribution of primary sequence to the carbohydrate requirement for intracellular transport of two closely related glycoproteins, the G proteins of the San Juan and Orsay strains of vesicular stomatitis virus. We used site-directed mutagenesis of the coding sequence to eliminate the two consensus sites for glycosylation in the Orsay G protein. Whereas the nonglycosylated San Juan G protein required at least one of its two asparagine-linked oligosaccharides for transport to the plasma membrane at 37 degrees C, a fraction of the Orsay G protein was transported without carbohydrate. Of the 10 amino acid differences between these two proteins, residue 172 (tyrosine in San Juan, aspartic acid in Orsay) played the major role in determining the stringency for the carbohydrate requirement. The rates at which the glycosylated and nonglycosylated Orsay G proteins were transported to the cell surface were the same, although a smaller fraction of the nonglycosylated protein was transported. These results suggest that the carbohydrate does not promote intracellular transport directly but influences a polypeptide folding or oligomerization step which is critical for transport.

  13. Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2.

    PubMed

    Pacheco, António; Talaia, Gabriel; Sá-Pessoa, Joana; Bessa, Daniela; Gonçalves, Maria José; Moreira, Roxana; Paiva, Sandra; Casal, Margarida; Queirós, Odília

    2012-05-01

    We aimed to manipulate the metabolism of Saccharomyces cerevisiae to produce lactic acid and search for the potential influence of acid transport across the plasma membrane in this process. Saccharomyces cerevisiae W303-1A is able to use l-lactic acid but its production in our laboratory has not previously been detected. When the l-LDH gene from Lactobacillus casei was expressed in S. cerevisiae W303-1A and in the isogenic mutants jen1∆, ady2∆ and jen1∆ ady2∆, all strains were able to produce lactic acid, but higher titres were achieved in the mutant strains. In strains constitutively expressing both LDH and JEN1 or ADY2, a higher external lactic acid concentration was found when glucose was present in the medium, but when glucose was exhausted, its consumption was more pronounced. These results demonstrate that expression of monocarboxylate permeases influences lactic acid production. Ady2 has been previously characterized as an acetate permease but our results demonstrated its additional role in lactate uptake. Overall, we demonstrate that monocarboxylate transporters Jen1 and Ady2 are modulators of lactic acid production and may well be used to manipulate lactic acid export in yeast cells.

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

    PubMed Central

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

    1987-01-01

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

  15. Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR

    PubMed Central

    Rosario, Fredrick J.; Shehab, Majida Abu; Powell, Theresa L.; Gupta, Madhulika B.; Jansson, Thomas

    2015-01-01

    Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (–72%, P<0.0001) and SNAT-1 (–42%, P<0.05) and SNAT-2 (–31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR. PMID:26374858

  16. Increased ubiquitination and reduced plasma membrane trafficking of placental amino acid transporter SNAT-2 in human IUGR.

    PubMed

    Chen, Yi-Yung; Rosario, Fredrick J; Shehab, Majida Abu; Powell, Theresa L; Gupta, Madhulika B; Jansson, Thomas

    2015-12-01

    Placental amino acid transport is decreased in intrauterine growth restriction (IUGR); however, the underlying mechanisms remain largely unknown. We have shown that mechanistic target of rapamycin (mTOR) signalling regulates system A amino acid transport by modulating the ubiquitination and plasma membrane trafficking of sodium-coupled neutral amino acid transporter 2 (SNAT-2) in cultured primary human trophoblast cells. We hypothesize that IUGR is associated with (1) inhibition of placental mTORC1 and mTORC2 signalling pathways, (2) increased amino acid transporter ubiquitination in placental homogenates and (3) decreased protein expression of SNAT-2 in the syncytiotrophoblast microvillous plasma membrane (MVM). To test this hypothesis, we collected placental tissue and isolated MVM from women with pregnancies complicated by IUGR (n=25) and gestational age-matched women with appropriately grown control infants (n=19, birth weights between the twenty-fifth to seventy-fifth percentiles). The activity of mTORC1 and mTORC2 was decreased whereas the protein expression of the ubiquitin ligase NEDD4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2; +72%, P<0.0001) and the ubiquitination of SNAT-2 (+180%, P<0.05) were increased in homogenates of IUGR placentas. Furthermore, IUGR was associated with decreased system A amino acid transport activity (-72%, P<0.0001) and SNAT-1 (-42%, P<0.05) and SNAT-2 (-31%, P<0.05) protein expression in MVM. In summary, these findings are consistent with the possibility that decreased placental mTOR activity causes down-regulation of placental system A activity by shifting SNAT-2 trafficking towards proteasomal degradation, thereby contributing to decreased fetal amino acid availability and restricted fetal growth in IUGR.

  17. Incorporating Geochemical And Microbial Kinetics In Reactive Transport Models For Generation Of Acid Rock Drainage

    NASA Astrophysics Data System (ADS)

    Andre, B. J.; Rajaram, H.; Silverstein, J.

    2010-12-01

    Acid mine drainage, AMD, results from the oxidation of metal sulfide minerals (e.g. pyrite), producing ferrous iron and sulfuric acid. Acidophilic autotrophic bacteria such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans obtain energy by oxidizing ferrous iron back to ferric iron, using oxygen as the electron acceptor. Most existing models of AMD do not account for microbial kinetics or iron geochemistry rigorously. Instead they assume that oxygen limitation controls pyrite oxidation and thus focus on oxygen transport. These models have been successfully used for simulating conditions where oxygen availability is a limiting factor (e.g. source prevention by capping), but have not been shown to effectively model acid generation and effluent chemistry under a wider range of conditions. The key reactions, oxidation of pyrite and oxidation of ferrous iron, are both slow kinetic processes. Despite being extensively studied for the last thirty years, there is still not a consensus in the literature about the basic mechanisms, limiting factors or rate expressions for microbially enhanced oxidation of metal sulfides. An indirect leaching mechanism (chemical oxidation of pyrite by ferric iron to produce ferrous iron, with regeneration of ferric iron by microbial oxidation of ferrous iron) is used as the foundation of a conceptual model for microbially enhanced oxidation of pyrite. Using literature data, a rate expression for microbial consumption of ferrous iron is developed that accounts for oxygen, ferrous iron and pH limitation. Reaction rate expressions for oxidation of pyrite and chemical oxidation of ferrous iron are selected from the literature. A completely mixed stirred tank reactor (CSTR) model is implemented coupling the kinetic rate expressions, speciation calculations and flow. The model simulates generation of AMD and effluent chemistry that qualitatively agrees with column reactor and single rock experiments. A one dimensional reaction

  18. Fetal hydantoin syndrome: inhibition of placental folic acid transport as a potential mechanism for fetal growth retardation in the rat

    SciTech Connect

    Will, M.; Barnard, J.A.; Said, H.M.; Ghishan, F.K.

    1985-04-01

    Maternal hydantoin ingestion during pregnancy results in a well defined clinical entity termed ''fetal hydantoin syndrome''. The clinical characteristics of this syndrome includes growth retardation, and congenital anomalies. Because folic acid is essential for protein synthesis and growth, and since hydantoin interferes with intestinal transport of folic acid, the authors postulated that part of the fetal hydantoin syndrome may be due to inhibition of placental folic acid by maternal hydantoin. Therefore, they studied in vivo placental folate transport in a well-established model for fetal hydantoin syndrome in the rat. Our results indicate that maternal hydantoin ingestion, significantly decreased fetal weight and placental and fetal uptake of folate compared to controls. To determine whether maternal hydantoin ingestion has a generalized or specific effect on placental function, they examined placental and fetal zinc transport in the same model. Our results indicate that zinc transport is not altered by hydantoin ingestion. They conclude that maternal hydantoin ingestion results in fetal growth retardation which may be due in part to inhibition of placental folate transport.

  19. LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen.

    PubMed Central

    Schwacke, R; Grallath, S; Breitkreuz, K E; Stransky, E; Stransky, H; Frommer, W B; Rentsch, D

    1999-01-01

    During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes. PMID:10072398

  20. Distinct Effects of Humic Acid on Transport and Retention of TiO2 Rutile Nanoparticles in Saturated Sand Columns

    EPA Science Inventory

    The distinct effects of humic acid (HA, 0−10 mg L−1) on the transport of titanium dioxide (rutile) nanoparticles (nTiO2) through saturated sand columns were observed under conditions of environmental relevance (ionic strength 3−200 mM NaCl, pH 5.7 and 9.0). Specifically, the tra...

  1. Distinct Effects of Humic Acid on Transport and Retention of TiO2 Rutile Nanoparticles in Saturated Sand Column

    EPA Science Inventory

    Distinct effects of humic acid (HA, 0 – 10 mg L-1) on the transport of titanium dioxide (rutile) nanoparticles (nTiO2) through saturated sand columns were observed under conditions of environmental relevance (ionic strength 3 – 200 mM NaCl, pH 5.7 and 9.0). Specifical...

  2. Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

    PubMed

    Cohen, S; Tzuri, G; Harel-Beja, R; Itkin, M; Portnoy, V; Sa'ar, U; Lev, S; Yeselson, L; Petrikov, M; Rogachev, I; Aharoni, A; Ophir, R; Tadmor, Y; Lewinsohn, E; Burger, Y; Katzir, N; Schaffer, A A

    2012-07-01

    Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit

  3. The fatty acid transport protein Fat1p is involved in the export of fatty acids from lipid bodies in Yarrowia lipolytica.

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Dulermo, Thierry; Thevenieau, France; Nicaud, Jean-Marc

    2014-09-01

    In order to live, cells need to import different molecules, such as sugars, amino acids or lipids, using transporters. In Saccharomyces cerevisiae, the ScFAT1 gene encodes the long-chain fatty acid transporter; however, the transport of fatty acids (FAs) in the oleaginous yeast Yarrowia lipolytica has not yet been studied. In contrast to what has previously been found for ΔScfat1 strains, ΔYlfat1 yeast was still able to grow on substrates containing short-, medium- or long-chain FAs. We observed a notable difference in cell lipid content between wild-type (WT) and deletion mutant strains after 24 h of culture in minimal oleate medium: in the WT strain, lipids represented 24% of cell dry weight (CDW), while they accounted for 37% of CDW in the ΔYlfat1 strain. This result indicates that YlFat1p is not involved in cell lipid uptake. Moreover, we also observed that fatty acid remobilisation was decreased in the ΔYlfat1 strain and that fluorescence-tagged YlFat1p proteins localised to the interfaces between lipid bodies, which suggests that YlFat1p may play a role in the export of FAs from lipid bodies.

  4. Surfactant-modified fatty acid composition of Citrobacter sp. SA01 and its effect on phenanthrene transmembrane transport.

    PubMed

    Li, Feng; Zhu, Lizhong

    2014-07-01

    The effects of the surfactants, Tween 80 and sodium dodecyl benzene sulfonate (SDBS) on a membrane's fatty acid composition and the transmembrane transport of phenanthrene were investigated. The results indicated that both surfactants could modify the composition of fatty acids of Citrobacter sp. Strain SA01 cells, 50 mg L(-1) of both surfactants changed the composition of the fatty acids the most, increasing the amount of unsaturated fatty acids. The comparison of fatty acid profiles with diphenylhexatriene fluorescence anisotropy, a probe for plasma membrane fluidity, suggested that an increased amount of unsaturated fatty acids corresponded to greater membrane fluidity. In addition, increased unsaturated fatty acids promoted phenanthrene to partition from the extracellular matrix to cell debris, which increased reverse partitioning from the cell debris to the cytochylema. The results of this study were expected in that the addition of a surfactant is a simple and effective method for accelerating the rate-limiting step of transmembrane transport of hydrophobic organic compounds (HOCs) in bioremediation.

  5. Plasma membrane H+ and K+ transporters are involved in the weak-acid preservative response of disparate food spoilage yeasts.

    PubMed

    Macpherson, Neil; Shabala, Lana; Rooney, Henrietta; Jarman, Marcus G; Davies, Julia M

    2005-06-01

    The food spoilage yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae have been proposed to resist weak-acid preservative stress by different means; Z. bailii by limiting influx of preservative combined with its catabolism, S. cerevisiae by active extrusion of the preservative weak-acid anion and H(+). Measurement of H(+) extrusion by exponential-phase Z. bailii cells suggest that, in common with S. cerevisiae, this yeast uses a plasma membrane H(+)-ATPase to expel H(+) when challenged by weak-acid preservative (benzoic acid). Simultaneous measurement of Z. bailii net H(+) and K(+) fluxes showed that net K(+) influx accompanies net H(+) efflux during acute benzoic acid stress. Such ionic coupling is known for S. cerevisiae in short-term preservative stress. Both yeasts significantly accumulated K(+) on long-term exposure to benzoic acid. Analysis of S. cerevisiae K(+) transporter mutants revealed that loss of the high affinity K(+) uptake system Trk1 confers sensitivity to growth in preservative. The results suggest that cation accumulation is an important factor in adaptation to weak-acid preservatives by spoilage yeasts and that Z. bailii and S. cerevisiae share hitherto unsuspected adaptive responses at the level of plasma membrane ion transport.

  6. Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development.

    PubMed

    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.

  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.

    PubMed

    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.

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

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

    Mochizuki, Tomoki; Kawamura, Kimitaka; Aoki, Kazuma; Sugimoto, Nobuo

    2016-11-01

    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 acids.

  10. Fishy Business: Effect of Omega-3 Fatty Acids on Zinc Transporters and Free Zinc Availability in Human Neuronal Cells

    PubMed Central

    De Mel, Damitha; Suphioglu, Cenk

    2014-01-01

    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. PMID:25195602

  11. Uptake of 4-chloro-2-methylphenoxyacetic acid (MCPA) from the apical membrane of Caco-2 cells by the monocarboxylic acid transporter

    SciTech Connect

    Kimura, Osamu; Tsukagoshi, Kensuke; Endo, Tetsuya

    2008-03-15

    The cellular uptake mechanism of 4-chloro-2-methylphenoxyacetic acid (MCPA), a phenoxyacetic acid derivative, was investigated using Caco-2 epithelial cells. The cells were incubated with 50 {mu}M MCPA at pH 6.0 and 37 deg. C, and the uptake of MCPA from the apical membranes was measured. The uptake of MCPA was significantly decreased by incubation at low temperature (4 {sup o}C) and markedly increased by lowering the extracellular pH. Pretreatment with a protonophore, carbonylcyanide-p-(trifluoromethoxy)phenylhydrazone (25 {mu}M), or metabolic inhibitors, 2,4-dinitrophenol (1 mM) and sodium azide (10 mM), significantly decreased the uptake of MCPA by 53%, 45% and 48%, respectively. Coincubation of MCPA with 10 mM L-lactic acid or {alpha}-cyano-4-hydroxycinnamate, which is a substrate or an inhibitor of the monocarboxylic acid transporters (MCTs), significantly decreased the uptake of MCPA by 31% and 20%, respectively, and coincubation with benzoic acid profoundly decreased the uptake by 68%. In contrast, coincubation with succinic acid (a dicarboxylic acid) did not affect the uptake. Kinetic analysis of initial MCPA uptake suggested that MCPA is taken up via a carrier-mediated process [K{sub m} = 1.37 {+-} 0.15 mM, V{sub max} = 115 {+-} 6 nmol (mg protein){sup -1} (3 min){sup -1}]. Lineweaver-Burk plots show that benzoic acid competitively inhibits the uptake of MCPA with a K{sub i} value of 4.68 {+-} 1.76 mM. A trans-stimulation effect on MCPA uptake was found in cells preloaded with benzoic acid. These results suggest that the uptake of MCPA from the apical membrane of Caco-2 cells is mainly mediated by common MCTs along with benzoic acid but also in part by L-lactic acid.

  12. Unsaturated fatty acids and phytosterols regulate cholesterol transporter genes in Caco-2 and HepG2 cell lines.

    PubMed

    Park, Youngki; Carr, Timothy P

    2013-02-01

    Dietary consumption of phytosterols and certain fatty acids has been shown to reduce cholesterol absorption and plasma cholesterol concentrations. However, it has not been fully elucidated whether phytosterols or fatty acids can alter the expression of cholesterol transporters by functioning as signaling molecules. This study tested the hypothesis that various fatty acids and phytosterols commonly found in the food supply can modulate the expression of transporters including Niemann-Pick C1-like 1, low-density lipoprotein receptor, and scavenger receptor class B type I and 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the intestine and liver. Caco-2 cells were used as models of enterocytes, and HepG2 cells were used as a model of hepatocytes. The cells were treated for 18 hours with 100 μmol/L of a fatty acid, or for 24 hours with 10 μmol/L of 25α-hydroxycholesterol, or 100 μmol/L of cholesterol, sitosterol, and stigmasterol to measure expression of genes involved in cholesterol transport using quantitative real-time polymerase chain reaction. Polyunsaturated fatty acids in Caco-2 cells and sterols in HepG2 cells significantly reduced the messenger RNA expression levels of Niemann-Pick C1-like 1, scavenger receptor class B type I, low-density lipoprotein receptor, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Importantly, sitosterol and stigmasterol reduced the messenger RNA levels of genes to a similar extent as cholesterol. The data support the hypothesis that unsaturated fatty acid and phytosterols can act as signaling molecules and alter the expression of genes involved in cholesterol transport and metabolism.

  13. Formation and transport of the sulfonic acid metabolites of alachlor and metolachlor in soil

    USGS Publications Warehouse

    Aga, D.S.; Thurman, E.M.

    2001-01-01

    Alachlor and metolachlor are dechlorinated and transformed into their corresponding ethane sulfonic acid (ESA) metabolites in soil. In a field-disappearance study, it was shown that alachlor ESA was formed at a faster rate and at concentrations 2-4 times higher than metolachlor ESA, conforming with the observed longer disappearance half-life of metolachlor (15.5 d) in the field as compared to alachlor (8 d). Runoff data also showed higher concentrations of alachlor ESA as compared to metolachlor ESA, even though they were applied at the same levels. Data from soil cores showed transport of the ESA compounds in soil to as far down as 75-90 cm below the surface, at concentrations ranging from less than 0.5 ??g/L to about 50 ??g/L. In contrast, no parent herbicide was detected at these depths. This observation correlates with the higher log KOC values for alachlor (3.33) and metolachlor (3.01) relative to their corresponding ESA metabolites, alachlor ESA (2.26), and metolachlor ESA (2.29).

  14. Separate responses of karyopherins to glucose and amino acid availability regulate nucleocytoplasmic transport

    PubMed Central

    Huang, Hsiao-Yun; Hopper, Anita K.

    2014-01-01

    The importin-β family members (karyopherins) mediate the majority of nucleocytoplasmic transport. Msn5 and Los1, members of the importin-β family, function in tRNA nuclear export. tRNAs move bidirectionally between the nucleus and the cytoplasm. Nuclear tRNA accumulation occurs upon amino acid (aa) or glucose deprivation. To understand the mechanisms regulating tRNA subcellular trafficking, we investigated whether Msn5 and Los1 are regulated in response to nutrient availability. We provide evidence that tRNA subcellular trafficking is regulated by distinct aa-sensitive and glucose-sensitive mechanisms. Subcellular distributions of Msn5 and Los1 are altered upon glucose deprivation but not aa deprivation. Redistribution of tRNA exportins from the nucleus to the cytoplasm likely provides one mechanism for tRNA nuclear distribution upon glucose deprivation. We extended our studies to other members of the importin-β family and found that all tested karyopherins invert their subcellular distributions upon glucose deprivation but not aa deprivation. Glucose availability regulates the subcellular distributions of karyopherins likely due to alteration of the RanGTP gradient since glucose deprivation causes redistribution of Ran. Thus nuclear–cytoplasmic distribution of macromolecules is likely generally altered upon glucose deprivation due to collapse of the RanGTP gradient and redistribution of karyopherins between the nucleus and the cytoplasm. PMID:25057022

  15. Caveolin-1 Sensitivity of Excitatory Amino Acid Transporters EAAT1, EAAT2, EAAT3, and EAAT4.

    PubMed

    Abousaab, Abeer; Warsi, Jamshed; Elvira, Bernat; Lang, Florian

    2016-06-01

    Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear L-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The L-glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.

  16. Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor.

    PubMed

    Van Zeebroeck, Griet; Rubio-Texeira, Marta; Schothorst, Joep; Thevelein, Johan M

    2014-07-01

    The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. L-lysine, L-histidine and L-tryptophan are transported by Gap1 but do not trigger signalling. Unlike L-histidine, L-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and D-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, L-Asp-γ-L-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of L-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1(Y395C) by ubiquitination- and endocytosis-deficient Gap1(K9R,K16R). Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes.

  17. Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor

    PubMed Central

    Van Zeebroeck, Griet; Rubio-Texeira, Marta; Schothorst, Joep; Thevelein, Johan M

    2014-01-01

    The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. l-lysine, l-histidine and l-tryptophan are transported by Gap1 but do not trigger signalling. Unlike l-histidine, l-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and d-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, l-Asp-γ-l-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of l-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1Y395C by ubiquitination- and endocytosis-deficient Gap1K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes. PMID:24852066

  18. Impact of humic acid fouling on membrane performance and transport of pharmaceutically active compounds in forward osmosis.

    PubMed

    Xie, Ming; Nghiem, Long D; Price, William E; Elimelech, Menachem

    2013-09-01

    The impact of humic acid fouling on the membrane transport of two pharmaceutically active compounds (PhACs) - namely carbamazepine and sulfamethoxazole - in forward osmosis (FO) was investigated. Deposition of humic acid onto the membrane surface was promoted by the complexation with calcium ions in the feed solution and the increase in ionic strength at the membrane surface due to the reverse transport of NaCl draw solute. The increase in the humic acid deposition on the membrane surface led to a substantial decrease in the membrane salt (NaCl) permeability coefficient but did not result in a significant decrease in the membrane pure water permeability coefficient. As the deposition of humic acid increased, the permeation of carbamazepine and sulfamethoxazole decreased, which correlated well with the decrease in the membrane salt (NaCl) permeability coefficient. It is hypothesized that the hydrated humic acid fouling layer hindered solute diffusion through the membrane pore and enhanced solute rejection by steric hindrance, but not the permeation of water molecules. The membrane water and salt (NaCl) permeability coefficients were fully restored by physical cleaning of the membrane, suggesting that humic acid did not penetrate into the membrane pores.

  19. Assessment of thermal load on transported goats administered with ascorbic acid during the hot-dry conditions

    NASA Astrophysics Data System (ADS)

    Minka, N. S.; Ayo, J. O.

    2012-03-01

    The major factor in the induction of physiological stress during road transportation of livestock is the complex fluctuations of the thermal transport microenvironment, encountered when animals are transported across different ecological zones. Recommended guidelines on optimum "on-board" conditions in which goats should be transported are lacking, and there are no acceptable ranges and limits for the thermal loads to which goats may be subjected during long-distance road transportation in hot-dry conditions. Panting score (PS), rectal temperature (RT), heart rate (HR) and respiratory rate (RR) were employed as reliable stress indices to assess the effects of different thermal loads, measured as temperature humidity index (THI), encountered in the vehicle during 12 h of road transportation of 40 goats, and to suggest the administration of 100 mg/kg body weight of ascorbic acid (AA) as an ameliorating agent. The results obtained showed that the PS, RT, HR and RR rose above normal reference values with increase in the THI and journey duration. The rise in PS value, which is a visual indicator of the severity of thermal load, was the most pronounced. The results suggest that values of THI in the vehicle up to 94.6 constitute no risk, while at of 100 it presents a moderate risk and above 100 may result in severe stress. The relationships between the thermal load and the physiological variables were positive and significant ( P < 0.05). They reflect the degree of stress imposed by each THI value during the transportation, and may be used as recommended ranges and limit thermal load values in transported goats. The results demonstrated that administration of 100 mg/kg body weight of AA before road transportation mitigated the risk of adverse effects of high THI values and other stress factors due to road transportation in goats.

  20. Amino acid composition analysis of secondary transport proteins from Escherichia coli with relation to functional classification, ligand specificity and structure.

    PubMed

    Saidijam, Massoud; Patching, Simon G

    2015-01-01

    We have performed an amino acid composition (AAC) analysis of the complete sequences for 235 secondary transport proteins from Escherichia coli, which have functions in the uptake and export of organic and inorganic metabolites, efflux of drugs and in controlling membrane potential. This revealed the trends in content for specific amino acid types and for combinations of amino acids with similar physicochemical properties. In certain proteins or groups of proteins, the so-called spikes of high content for a specific amino acid type or combination of amino acids were identified and confirmed statistically, which in some cases could be directly related to function and ligand specificity. This was prevalent in proteins with a function of multidrug or metal ion efflux. Any tool that can help in identifying bacterial multidrug efflux proteins is important for a better understanding of this mechanism of antibiotic resistance. Phylogenetic analysis based on sequence alignments and comparison of sequences at the N- and C-terminal ends confirmed transporter Family classification. Locations of specific amino acid types in some of the proteins that have crystal structures (EmrE, LacY, AcrB) were also considered to help link amino acid content with protein function. Though there are limitations, this work has demonstrated that a basic analysis of AAC is a useful tool to use in combination with other computational and experimental methods for classifying and investigating function and ligand specificity in a large group of transport or other membrane proteins, including those that are molecular targets for development of new drugs.

  1. Association between SLC2A9 transporter gene variants and uric acid phenotypes in African American and white families

    PubMed Central

    de Andrade, Mariza; Matsumoto, Martha; Mosley, Tom H.; Kardia, Sharon; Turner, Stephen T.

    2011-01-01

    Objectives. SLC2A9 gene variants associate with serum uric acid in white populations, but little is known about African American populations. Since SLC2A9 is a transporter, gene variants may be expected to associate more closely with the fractional excretion of urate, a measure of renal tubular transport, than with serum uric acid, which is influenced by production and extrarenal clearance. Methods. Genotypes of single nucleotide polymorphisms (SNPs) distributed across the SLC2A9 gene were obtained in the Genetic Epidemiology Network of Arteriopathy cohorts. The associations of SNPs with serum uric acid, fractional excretion of urate and urine urate-to-creatinine ratio were assessed with adjustments for age, sex, diuretic use, BMI, homocysteine and triglycerides. Results. We identified SLC2A9 gene variants that were associated with serum uric acid in 1155 African American subjects (53 SNPs) and 1132 white subjects (63 SNPs). The most statistically significant SNPs in African American subjects (rs13113918) and white subjects (rs11723439) were in the latter half of the gene and explained 2.7 and 2.8% of the variation in serum uric acid, respectively. After adjustment for this SNP in African Americans, 0.9% of the variation in serum uric acid was explained by an SNP (rs1568318) in the first half of the gene. Unexpectedly, SLC2A9 gene variants had stronger associations with serum uric acid than with fractional excretion of urate. Conclusions. These findings support two different loci by which SLC2A9 variants affect uric acid levels in African Americans and suggest SLC2A9 variants affect serum uric acid level via renal and extrarenal clearance. PMID:21186168

  2. Inactivating Mutations in MFSD2A, Required for Omega-3 Fatty Acid Transport in Brain, Cause a Lethal Microcephaly Syndrome

    PubMed Central

    Guemez-Gamboa, Alicia; Nguyen, Long N.; Yang, Hongbo; Zaki, Maha S.; Kara, Majdi; Ben-Omran, Tawfeg; Akizu, Naiara; Rosti, Rasim Ozgur; Rosti, Basak; Scott, Eric; Schroth, Jana; Copeland, Brett; Vaux, Keith K.; Cazenave-Gassiot, Amaury; Quek, Debra Q.Y.; Wong, Bernice H.; Tan, Bryan C.; Wenk, Markus R.; Gunel, Murat; Gabriel, Stacey; Chi, Neil C.; Silver, David L.; Gleeson, Joseph G.

    2015-01-01

    Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and although considered essential, deficiency has not been linked to disease1,2. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the Major Facilitator Superfamily Domain 2a (Mfsd2a)3. Mfsd2a transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Patients exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa zebrafish morphants, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans. PMID:26005868

  3. HIV-Protease Inhibitors Suppress Skeletal Muscle Fatty Acid Oxidation by Reducing CD36 and CPT-I Fatty Acid Transporters

    PubMed Central

    Richmond, Scott R.; Carper, Michael J.; Lei, Xiaoyong; Zhang, Sheng; Yarasheski, Kevin E.; Ramanadham, Sasanka

    2010-01-01

    Infection with human immunodeficiency virus (HIV) and treatment with HIV-protease inhibitor (PI)-based highly active antiretroviral therapies (HAART) is associated with dysregulated fatty acid and lipid metabolism. Enhanced lipolysis, increased circulating fatty acid levels, and hepatic and intramuscular lipid accumulation appear to contribute to insulin resistance in HIV-infected people treated with PI-based HAART. However, it is unclear whether currently prescribed HIV-PIs directly alter skeletal muscle fatty acid transport, oxidation, and storage. We find that ritonavir (r, 5 μmol/l) plus 20 μmol/l of atazanavir (ATV), lopinavir (LPV), or darunavir (DRV) reduce palmitate oxidation(16-21%) in differentiated C2C12 myotubes. Palmitate oxidation was increased following exposure to high fatty acid media but this effect was blunted when myotubes were pre-exposed to the HIV-PIs. However, LPV/r and DRV/r, but not ATV/r suppressed palmitate uptake into myotubes. We found no effect of the HIV-PIs on FATP1, FATP4, or FABPpm but both CD36/FAT and carnitine palmitoyltransferase I (CPTI) were reduced by all three regimens though ATV/r caused only a small decrease in CPT1, relative to LPV/r or DRV/r. In contrast, sterol regulatory element binding protein-1 was increased by all 3 HIV-PIs. These findings suggest that HIV-PIs suppress fatty acid oxidation in murine skeletal muscle cells and that this may be related to decreases in cytosolic- and mitochondrial-associated fatty acid transporters. HIV-PIs may also directly impair fatty acid handling and partitioning in skeletal muscle, and this may contribute to the cluster of metabolic complications that occur in people living with HIV. PMID:20117238

  4. Particle-facilitated lead and arsenic transport in abandoned mine sites soil influenced by simulated acid rain.

    PubMed

    Shaoping, Hu; Xincai, Chen; Jiyan, Shi; Yingxu, Chen; Qi, Lin

    2008-05-01

    The role of acid rain in affecting Pb and As transport from mine tailings was investigated by pumping simulated acid rain at a infiltration rate of 10.2 cm/h through soil columns. Simulated acid rain with pH of 3.0, 4.5 and 5.6 were used as leaching solutions. Results showed that 86.9-95.9% of Pb and 90-91.8% of As eluted from the columns were adsorbed by particles in the leachates. Scanning electron microscopy (SEM) analysis showed that particles released from the columns were mainly composed of flocculated aggregates and plate or rod shaped discrete grains. Transmission electron microscopy (TEM) coupled with energy dispersive X-ray analysis (EDX) showed that these particles were predominantly silicate minerals. Results from our experiments demonstrated that when rapid infiltration conditions or a rainstorm exist, particle-facilitated transport of contaminants is likely to the dominant metal transport pathway influenced by acid rain.

  5. A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth.

    PubMed Central

    Verheul, A; Hagting, A; Amezaga, M R; Booth, I R; Rombouts, F M; Abee, T

    1995-01-01

    Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed. PMID:7887604

  6. Multi-component reactive transport modeling of natural attenuation of an acid groundwater plume at a uranium mill tailings site

    NASA Astrophysics Data System (ADS)

    Zhu, Chen; Hu, Fang Q.; Burden, David S.

    2001-11-01

    Natural attenuation of an acidic plume in the aquifer underneath a uranium mill tailings pond in Wyoming, USA was simulated using the multi-component reactive transport code PHREEQC. A one-dimensional model was constructed for the site and the model included advective-dispersive transport, aqueous speciation of 11 components, and precipitation-dissolution of six minerals. Transport simulation was performed for a reclamation scenario in which the source of acidic seepage will be terminated after 5 years and the plume will then be flushed by uncontaminated upgradient groundwater. Simulations show that successive pH buffer reactions with calcite, Al(OH) 3(a), and Fe(OH) 3(a) create distinct geochemical zones and most reactions occur at the boundaries of geochemical zones. The complex interplay of physical transport processes and chemical reactions produce multiple concentration waves. For SO 42- transport, the concentration waves are related to advection-dispersion, and gypsum precipitation and dissolution. Wave speeds from numerical simulations compare well to an analytical solution for wave propagation.

  7. Multi-component reactive transport modeling of natural attenuation of an acid groundwater plume at a uranium mill tailings site.

    PubMed

    Zhu, C; Hu, F Q; Burden, D S

    2001-11-01

    Natural attenuation of an acidic plume in the aquifer underneath a uranium mill tailings pond in Wyoming, USA was simulated using the multi-component reactive transport code PHREEQC. A one-dimensional model was constructed for the site and the model included advective-dispersive transport, aqueous speciation of 11 components, and precipitation-dissolution of six minerals. Transport simulation was performed for a reclamation scenario in which the source of acidic seepage will be terminated after 5 years and the plume will then be flushed by uncontaminated upgradient groundwater. Simulations show that successive pH buffer reactions with calcite, Al(OH)3(a), and Fe(OH)3(a) create distinct geochemical zones and most reactions occur at the boundaries of geochemical zones. The complex interplay of physical transport processes and chemical reactions produce multiple concentration waves. For SO4(2-) transport, the concentration waves are related to advection-dispersion, and gypsum precipitation and dissolution. Wave speeds from numerical simulations compare well to an analytical solution for wave propagation.

  8. Polyphenols and phenolic acids from strawberry and apple decrease glucose uptake and transport by human intestinal Caco-2 cells.

    PubMed

    Manzano, Susana; Williamson, Gary

    2010-12-01

    The effect of polyphenols, phenolic acids and tannins (PPTs) from strawberry and apple on uptake and apical to basolateral transport of glucose was investigated using Caco-2 intestinal cell monolayers. Substantial inhibition on both uptake and transport was observed by extracts from both strawberry and apple. Using sodium-containing (glucose transporters SGLT1 and GLUT2 both active) and sodium-free (only GLUT2 active) conditions, we show that the inhibition of GLUT2 was greater than that of SGLT1. The extracts were analyzed and some of the constituent PPTs were also tested. Quercetin-3-O-rhamnoside (IC₅₀ =31 μM), phloridzin (IC₅₀=146 μM), and 5-caffeoylquinic acid (IC₅₀=2570 μM) contributed 26, 52 and 12%, respectively, to the inhibitory activity of the apple extract, whereas pelargonidin-3-O-glucoside (IC₅₀=802 μM) contributed 26% to the total inhibition by the strawberry extract. For the strawberry extract, the inhibition of transport was non-competitive based on kinetic analysis, whereas the inhibition of cellular uptake was a mixed-type inhibition, with changes in both V(max) and apparent K(m) . The results in this assay show that some PPTs inhibit glucose transport from the intestinal lumen into cells and also the GLUT2-facilitated exit on the basolateral side.

  9. Effect of tachycardia on lipid metabolism and expression of fatty acid transporters in heart ventricles of the rat.

    PubMed

    Wojcik, B; Harasim, E; Zabielski, P; Chabowski, A; Gorski, J

    2015-10-01

    Tachycardia increases oxidation of the plasma-borne long chain fatty acids in the heart. The aim of the present study was to examine effect of tachycardia on: 1) the total level of free fatty acids, diacylglycerols, triacylglycerols and phospholipids in both heart ventricles; 2) (14)C-palmitate incorporation in the lipid fractions; 3) expression of fatty acid and glucose transporters in the ventricles. Tachycardia was induced in anesthetized rats by electrical atrial pacing at the rate of 600/min. Samples of the left (LV) and right (RV) ventricle were taken after 30 and 60 min pacing. The level free fatty acids, diacylglycerols, triacylglycerols and phospholipids was determined by means of gas-liquid chromatography and (14)C-palmitate incorporation by liquid scintillation counting, respectively. Expression of fatty acid- and glucose-transporters was determined using Western blot technique. In LV, 30min pacing increased the content of diacylglycerols whereas the content of other lipids remained stable. After 60 min of pacing the levels of the examined lipid fractions did not differ from the respective control values. In RV, the content of diacylglycerols and triacylglycerols was reduced both after 30 and 60 min pacing. Tachycardia also affected incorporation of (14)C-palmitate in lipid fractions of goth ventricles. 30 min pacing up-regulated plasmalemmal expression of FAT/CD36 (fatty acid translocase) in both ventricles and reduced its microsomal expression in LV. After 60 min pacing they did not differ from the respective control values. Plasmalemmal expression of FATP-1 (fatty acid transport protein 1) increased and its microsomal expression decreased in RV after 30 min pacing. After 60 min pacing the plasmalemmal FATP-1 expression remained elevated whereas the microsomal expression did not differ from the control value. Pacing did not affect or expression of FABPpm (plasma membrane associated fatty acid binding protein) in either plasma membranes and microsomal

  10. Pharmacology of Glutamate Transport in the CNS: Substrates and Inhibitors of Excitatory Amino Acid Transporters (EAATs) and the Glutamate/Cystine Exchanger System x c -

    NASA Astrophysics Data System (ADS)

    Bridges, Richard J.; Patel, Sarjubhai A.

    As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamate participates not only in standard fast synaptic communication, but also contributes to higher order signal processing, as well as neuropathology. Given this variety of functional roles, interest has been growing as to how the extracellular concentrations of l-glutamate surrounding neurons are regulated by cellular transporter proteins. This review focuses on two prominent systems, each of which appears capable of influencing both the signaling and pathological actions of l-glutamate within the CNS: the sodium-dependent excitatory amino acid transporters (EAATs) and the glutamate/cystine exchanger, system x c - (Sx c -). While the family of EAAT subtypes limit access to glutamate receptors by rapidly and efficiently sequestering l-glutamate in neurons and glia, Sxc - provides a route for the export of glutamate from cells into the extracellular environment. The primary intent of this work is to provide an overview of the inhibitors and substrates that have been developed to delineate the pharmacological specificity of these transport systems, as well as be exploited as probes with which to selectively investigate function. Particular attention is paid to the development of small molecule templates that mimic the structural properties of the endogenous substrates, l-glutamate, l-aspartate and l-cystine and how strategic control of functional group position and/or the introduction of lipophilic R-groups can impact multiple aspects of the transport process, including: subtype selectivity, inhibitory potency, and substrate activity.

  11. T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle.

    PubMed

    Hallerdei, Janine; Scheibe, Renate J; Parkkila, Seppo; Waheed, Abdul; Sly, William S; Gros, Gerolf; Wetzel, Petra; Endeward, Volker

    2010-12-13

    We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

  12. The Role of Vacuolar Malate-Transport Capacity in Crassulacean Acid Metabolism and Nitrate Nutrition. Higher Malate-Transport Capacity in Ice Plant after Crassulacean Acid Metabolism-Induction and in Tobacco under Nitrate Nutrition1

    PubMed Central

    Lüttge, Ulrich; Pfeifer, Tanja; Fischer-Schliebs, Elke; Ratajczak, Rafael

    2000-01-01

    Anion uptake by isolated tonoplast vesicles was recorded indirectly via increased H+-transport by H+-pumping of the V-ATPase due to dissipation of the electrical component of the electrochemical proton gradient, ΔμH+, across the membrane. ATP hydrolysis by the V-ATPase was measured simultaneously after the Palmgren test. Normalizing for ATP-hydrolysis and effects of chloride, which was added to the assays as a stimulating effector of the V-ATPase, a parameter, Jmalrel, of apparent ATP-dependent malate-stimulated H+-transport was worked out as an indirect measure of malate transport capacity. This allowed comparison of various species and physiological conditions. Jmalrel was high in the obligate crassulacean acid metabolism (CAM) species Kalanchoë daigremontiana Hamet et Perrier, it increased substantially after CAM induction in ice plant (Mesembryanthemum crystallinum), and it was positively correlated with NO3− nutrition in tobacco (Nicotiana tabacum). For tobacco this was confirmed by measurements of malate transport energized via the V-PPase. In ice plant a new polypeptide of 32-kD apparent molecular mass appeared, and a 33-kD polypeptide showed higher levels after CAM induction under conditions of higher Jmalrel. It is concluded that tonoplast malate transport capacity plays an important role in physiological regulation in CAM and NO3− nutrition and that a putative malate transporter must be within the 32- to 33-kD polypeptide fraction of tonoplast proteins. PMID:11080309

  13. Modeling of facilitated transport of phenylalanine by emulsion liquid membranes with di(2-ethylhexyl)phosphoric acid as a carrier

    SciTech Connect

    Liu, X.; Liu, D.

    1998-12-01

    A mathematical model is developed in this paper to simulate the facilitated transport of phenylalanine (Phe) in emulsion liquid membrane (ELM) systems with di(2-ethylhexyl)phosphoric acid as a carrier. The model takes into account the mass transfer in both the external aqueous phase and the organic membrane phase interfacial reaction as well as membrane breakage during agitation. The model is tested by comparing theoretical predications with experimental results using Phe extraction by ELM processes. It is found that the model is valid for simulating the facilitated transport of Phe with ELM under various experimental conditions.

  14. Promoter Analysis of the Human Ascorbic Acid Transporters SVCT1 & 2: Mechanisms of Adaptive Regulation in Liver Epithelial Cells

    PubMed Central

    Reidling, Jack C.; Rubin, Stanley A.

    2010-01-01

    Ascorbic acid, the active form of vitamin C, is a vital antioxidant in the human liver, yet the molecular mechanisms involved in the regulation of ascorbic acid transporters (hSVCT1 and hSVCT2) in liver cells are poorly understood. Therefore, we characterized the minimal promoter regions of hSVCT1 & 2 in cultured human liver epithelial cells (HepG2) and examined the effects of ascorbic acid deprivation and supplementation on activity and regulation of the transport systems. Identified minimal promoters required for basal activity were found to include multiple cis-regulatory elements, whereas mutational analysis demonstrated that HNF-1 sites in the hSVCT1 promoter and KLF/Sp1 sites in the hSVCT2 promoter were essential for activities. When cultured in ascorbic acid deficient or supplemented media, HepG2 cells demonstrated significant (P < 0.01) and specific reciprocal changes in [14C]-Ascorbic acid uptake, and in hSVCT1 mRNA and protein levels as well as hSVCT1 promoter activity. However, no significant changes in hSVCT2 expression or promoter activity were observed during ascorbic acid deficient or supplemented conditions. We mapped the ascorbic acid responsive region in the hSVCT1 promoter and determined that HNF-1 sites are important for the adaptive regulation response. The results of these studies further characterize the hSVCT1 and 2 promoters, establish that ascorbic acid uptake by human liver epithelial cells is adaptively regulated, and show that transcriptional mechanisms via HNF-1 in the hSVCT1 promoter may, in part, be involved in this regulation. PMID:20471816

  15. Recent Advances in Understanding Amino Acid Sensing Mechanisms that Regulate mTORC1

    PubMed Central

    Zheng, Liufeng; Zhang, Wei; Zhou, Yuanfei; Li, Fengna; Wei, Hongkui; Peng, Jian

    2016-01-01

    The mammalian target of rapamycin (mTOR) is the central regulator of mammalian cell growth, and is essential for the formation of two structurally and functionally distinct complexes: mTORC1 and mTORC2. mTORC1 can sense multiple cues such as nutrients, energy status, growth factors and hormones to control cell growth and proliferation, angiogenesis, autophagy, and metabolism. As one of the key environmental stimuli, amino acids (AAs), especially leucine, glutamine and arginine, play a crucial role in mTORC1 activation, but where and how AAs are sensed and signal to mTORC1 are not fully understood. Classically, AAs activate mTORC1 by Rag GTPases which recruit mTORC1 to lysosomes, where AA signaling initiates. Plasma membrane transceptor L amino acid transporter 1 (LAT1)-4F2hc has dual transporter-receptor function that can sense extracellular AA availability upstream of mTORC1. The lysosomal AA sensors (PAT1 and SLC38A9) and cytoplasmic AA sensors (LRS, Sestrin2 and CASTOR1) also participate in regulating mTORC1 activation. Importantly, AAs can be sensed by plasma membrane receptors, like G protein-coupled receptor (GPCR) T1R1/T1R3, and regulate mTORC1 without being transported into the cells. Furthermore, AA-dependent mTORC1 activation also initiates within Golgi, which is regulated by Golgi-localized AA transporter PAT4. This review provides an overview of the research progress of the AA sensing mechanisms that regulate mTORC1 activity. PMID:27690010

  16. Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

    PubMed Central

    Lennen, Rebecca M.; Politz, Mark G.; Kruziki, Max A.

    2013-01-01

    Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB+ strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts. PMID:23104810

  17. Ondansetron attenuates the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes.

    PubMed

    Hur, Wonseok; Lee, Mi Kyoung; Park, Hee-Pyeong; Kim, Chong-Sung; Yoon, Hea-Jo; Zuo, Zhiyi; Do, Sang-Hwan

    2014-06-15

    The purpose of this study was to evaluate the effect of ondansetron on excitatory amino acid transporter type 3 (EAAT3) and to elucidate the roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in the effect. EAAT3 was expressed in Xenopus oocytes following the injection of rat EAAT3 mRNAs. Using the two-electrode voltage clamping method, the inward currents induced by L-glutamate were measured for 1 min in the presence and absence of ondansetron (1-1000 μM). Different concentrations of L-glutamate (3-300 μM) were used to determine the kinetic characteristics of EAAT3. To identify the involvement of PKC and PI3K in the effect, oocytes were exposed to a PKC activator and to PKC inhibitors and PI3K inhibitors, and L-glutamate-induced currents were recorded. Ondansetron decreased EAAT3 activity in a dose-dependent manner. In a kinetic study, ondansetron (10 μM for 3 min) reduced Vmax, but not Km compared with the control group. The PKC activator abolished the ondansetron-induced decrease in EAAT3 activity. The PKC inhibitors (staurosporine and chelerythrine) and ondansetron had not additive or synergistic effects on EAAT3 activity. The PI3K inhibitors (wortmannin and LY294002) decreased the EAAT3 response, although there were no differences among the groups comprising ondansetron, PI3K inhibitors, and PI3K inhibitors plus ondansetron. Our results demonstrate that ondansetron attenuates EAAT3 activity and this effect seems to be mediated by PKC and PI3K.

  18. A novel monocarboxylate transporter inhibitor as a potential treatment strategy for γ-hydroxybutyric acid overdose

    PubMed Central

    Vijay, Nisha; Morse, Bridget L.; Morris, Marilyn E.

    2014-01-01

    Purpose Monocarboxylate transporter (MCT) inhibition represents a potential treatment strategy for γ-hydroxybutyric acid (GHB) overdose by blocking its renal reabsorption in the kidney. This study further evaluated the effects of a novel, highly potent MCT inhibitor, AR-C155858, on GHB toxicokinetics/toxicodynamics (TK/TD). Methods Rats were administered GHB (200, 600 or 1500 mg/kg i.v. or 1500 mg/kg po) with and without AR-C155858. Breathing frequency was continuously monitored using whole-body plethysmography. Plasma and urine samples were collected up to 8 hours. The effect of AR-C155858 on GHB brain/plasma partitioning was also assessed. Results AR-C155858 treatment significantly increased GHB renal and total clearance after intravenous GHB administration at all the GHB doses used in this study. GHB-induced respiratory depression was significantly improved by AR-C155858 as demonstrated by an improvement in the respiratory rate. AR-C155858 treatment also resulted in a significant reduction in brain/plasma partitioning of GHB (0.1 ± 0.03) when compared to GHB alone (0.25 ± 0.02). GHB CLR and CLoral (CL/F) following oral administration were also significantly increased following AR-C155858 treatment (from 1.82 ± 0.63 to 5.74 ± 0.86 and 6.52 ± 0.88 to 10.2 ± 0.75 ml/min/kg, respectively). Conclusion The novel and highly potent MCT inhibitor represents a potential treatment option for GHB overdose. PMID:25480120

  19. Manifestation of Preferential Flow and Nitrate Transport in Central European Soils on Acid Crystalline Rocks

    NASA Astrophysics Data System (ADS)

    Dolezal, F.; Cislerova, M.; Vogel, T.; Zavadil, J.; Vacek, J.; Kvitek, T.; Prazak, P.; Nechvatal, M.; Bayer, T.

    2006-12-01

    Large areas of Central Europe are occupied by highlands and peneplains of medium altitudes, built by acid crystalline rocks. The soils overlying them are typically of medium textures. They are neither markedly water- repellent nor greatly swelling and shrinking. These landscapes are characterized by high vulnerability of water bodies, both surface and subsurface. The existing methodologies of vulnerability assessment regard the heavier among these soils as little vulnerable to diffuse pollution, while in reality they may be virtually equally vulnerable, because of the short-circuiting effect of preferential flow and transport. Our experiment site was Valeèov (49° 38' 40" N, 14° 30' 25" E, 461 m a.s.l.) in the Bohemo-Moravian highland, with average annual precipitation 660 mm and average annual air temperature 7.2 ° C. The field trials, starting from 2001, were focused on growing potato under different conditions. Soil moisture content was measured by Theta- probe capacitance sensors, soil water suction by Watermark sensors and tensiometers. Nitrate leaching was monitored by soil solution sampling with ceramic suction cups and zero-tension lysimeters. The hydraulic conductivity of the soil was measured on small cores and by suction and pressure infiltrometers. The following preferential flow manifestations are analyzed and quantified: a) the spatial variability of soil moisture content and suction after rainstorms, b) the spatial and temporal variability of soil's hydraulic conductivity and its dependence on soil moisture content, c) the spatial variability of percolation volumes in parallel lysimeters, d) the variability of nitrate concentrations in the lysimeter leachate, e) the apparent absence of correlation between leachate volumes and leachate concentrations in lysimeters, f) the lower mean and higher variance of leachate concentrations in lysimeters, in comparison with those in suction cups.

  20. Potential application of a glucose-transport-deficient mutant of Schizosaccharomyces pombe for removing gluconic acid from grape must.

    PubMed

    Peinado, Rafael A; Moreno, Juan J; Medina, Manuel; Mauricio, Juan C

    2005-02-23

    Musts from rotten grapes typically contain high levels of gluconic acid, which can raise severe problems in winemaking processes. In this work, the ability of the glucose-transport-deficient mutant YGS-5 of Schizosaccharomyces pombe to completely or partly remove gluconic acid from a synthetic glucose-containing medium and the potential use of this yeast strain for the same purpose in musts and wines were examined. Surprisingly, the S. pombe YGS-5 strain successfully removed 93% of the initial gluconic acid (2.5 gL(-1)) and 80% of the initial malic acid (1.0 gL(-1)) within 30 h after inoculation. Also, the yeast strain produced no volatile compounds other than those obtained in fermentations conducted with the wine yeast Saccharomyces cerevisiae. S. pombe YGS-5 could thus be used to remove gluconic acid present in musts from rotten grapes. On the basis of these results, various ways of using S. pombe YGS-5 to treat musts containing gluconic acid in order to solve the problems due to the high gluconic acid concentrations in botrytized grape must are proposed.

  1. Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae.

    PubMed

    Sekito, Takayuki; Chardwiriyapreecha, Soracom; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

    2014-01-01

    Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

  2. Transport, distribution space and intracellular concentration of the anti-inflammatory drug niflumic acid in the perfused rat liver.

    PubMed

    Kelmer-Bracht, A M; Ishii-Iwamoto, E L; Bracht, A

    1993-05-05

    Transport and distribution space of niflumic acid in the perfused rat liver were investigated employing the multiple-indicator dilution technique with constant infusion of the drug (step input). Niflumic acid permeated the cell membrane in both directions at very high rates and its distribution in the cellular space was flow-limited; at least at 37 degrees, the rates of influx and efflux could not be measured. Dissociation of the niflumic acid-albumin complex also occurred at very high rates. The apparent space of distribution of niflumic acid in the liver depended on the concentration of the drug and varied between 4.37 (1 mM) and 43.5 (10 microM) times the water space; even with 90% extracellular binding to albumin, the apparent space of distribution of niflumic acid was 5.1 times greater than the water space. The high apparent spaces of distribution reflected the high intracellular concentrations. The ratio of intracellular bound plus free concentration to the extracellular bound plus free concentration (Ci/Ce) varied between 6.62 (1 mM portal niflumic acid) and 71.0 (10 microM portal niflumic acid). Metabolic transformation depended on the concentration of the free form. Intracellular binding is probably the major reason for the high concentration of the drug in the hepatic tissue.

  3. Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

    PubMed

    de la Ballina, Laura R; Cano-Crespo, Sara; González-Muñoz, Elena; Bial, Susanna; Estrach, Soline; Cailleteau, Laurence; Tissot, Floriane; Daniel, Hannelore; Zorzano, Antonio; Ginsberg, Mark H; Palacín, Manuel; Féral, Chloé C

    2016-04-29

    CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with β-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.

  4. Salicylic acid transport in Ricinus communis involves a pH-dependent carrier system in addition to diffusion.

    PubMed

    Rocher, Françoise; Chollet, Jean-François; Legros, Sandrine; Jousse, Cyril; Lemoine, Rémi; Faucher, Mireille; Bush, Daniel R; Bonnemain, Jean-Louis

    2009-08-01

    Despite its important functions in plant physiology and defense, the membrane transport mechanism of salicylic acid (SA) is poorly documented due to the general assumption that SA is taken up by plant cells via the ion trap mechanism. Using Ricinus communis seedlings and modeling tools (ACD LogD and Vega ZZ softwares), we show that phloem accumulation of SA and hydroxylated analogs is completely uncorrelated with the physicochemical parameters suitable for diffusion (number of hydrogen bond donors, polar surface area, and, especially, LogD values at apoplastic pHs and Delta LogD between apoplast and phloem sap pH values). These and other data (such as accumulation in phloem sap of the poorly permeant dissociated form of monohalogen derivatives from apoplast and inhibition of SA transport by the thiol reagent p-chloromercuribenzenesulfonic acid [pCMBS]) lead to the following conclusions. As in intestinal cells, SA transport in Ricinus involves a pH-dependent carrier system sensitive to pCMBS; this carrier can translocate monohalogen analogs in the anionic form; the efficiency of phloem transport of hydroxylated benzoic acid derivatives is tightly dependent on the position of the hydroxyl group on the aromatic ring (SA corresponds to the optimal position) but moderately affected by halogen addition in position 5, which is known to increase plant defense. Furthermore, combining time-course experiments and pCMBS used as a tool, we give information about the localization of the SA carrier. SA uptake by epidermal cells (i.e. the step preceding the symplastic transport to veins) insensitive to pCMBS occurs via the ion-trap mechanism, whereas apoplastic vein loading involves a carrier-mediated mechanism (which is targeted by pCMBS) in addition to diffusion.

  5. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids.

    PubMed

    Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J; Kwatra, Deep; Paturi, Kalyani D; Mitra, Ashim K

    2010-04-05

    HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the growth and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of P(appB-A) to P(appA-B) in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged, glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake; elevation of cyclosporine uptake in the presence of 5 muM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories.

  6. Modulation of fatty acid transport and metabolism by maternal obesity in the human full-term placenta.

    PubMed

    Dubé, Evemie; Gravel, Ariane; Martin, Coralie; Desparois, Guillaume; Moussa, Issa; Ethier-Chiasson, Maude; Forest, Jean-Claude; Giguère, Yves; Masse, André; Lafond, Julie

    2012-07-01

    Knowledge of the consequences of maternal obesity in human placental fatty acids (FA) transport and metabolism is limited. Animal studies suggest that placental uptake of maternal FA is altered by maternal overnutrition. We hypothesized that high maternal body mass index (BMI) affects human placental FA transport by modifying expression of key transporters. Full-term placentas were obtained by vaginal delivery from normal weight (BMI, 18.5-24.9 kg/m(2)) and obese (BMI > 30 kg/m(2)) women. Blood samples were collected from the mother at each trimester and from cord blood at delivery. mRNA and protein expression levels were evaluated with real-time RT-PCR and Western blotting. Lipoprotein lipase (LPL) activity was evaluated using enzyme fluorescence. In vitro linoleic acid transport was studied with isolated trophoblasts. Our results demonstrated that maternal obesity is associated with increased placental weight, decreased gestational age, decreased maternal high-density lipoprotein (HDL) levels during the first and third trimesters, increased maternal triglyceride levels during the second and third trimesters, and increased maternal T3 levels during all trimesters, and decreased maternal cholesterol (CHOL) and low-density lipoprotein (LDL) levels during the third trimester; and increased newborn CHOL, LDL, apolipoprotein B100, and T3 levels. Increases in placental CD36 mRNA and protein expression levels, decreased SLC27A4 and FABP1 mRNA and protein and FABP3 protein expression, and increased LPL activity and decreased villus cytotrophoblast linoleic acid transport were also observed. No changes were seen in expression of PPARA, PPARD, or PPARG mRNA and protein. Overall this study demonstrated that maternal obesity impacts placental FA uptake without affecting fetal growth. These changes, however, could modify the fetus metabolism and its predisposition to develop diseases later in life.

  7. Effect of HEPES buffer on the uptake and transport of P-glycoprotein substrates and large neutral amino acids

    PubMed Central

    Luo, Shuanghui; Pal, Dhananjay; Shah, Sujay J.; Kwatra, Deep; Paturi, Kalyani D.; Mitra, Ashim. K.

    2010-01-01

    HEPES has been widely employed as an organic buffer agent in cell culture medium as well as uptake and transport experiments in vitro. However, concentrations of HEPES used in such studies vary from one laboratory to another. In this study, we investigated the effect of HEPES on the uptake and bidirectional transport of P-gp substrates employing both Caco-2 and MDCK-MDR1 cells. ATP-dependent uptake of glutamic acid was also examined. ATP production was further quantified applying ATP Determination Kit. An addition of HEPES to the cellular washing and incubation media significantly altered the uptake and transport of P-gp substrates in both Caco-2 and MDCK-MDR1 cells. Uptake of P-gp substrates substantially diminished as the HEPES concentration was raised to 25 mM. Bidirectional (A-B and B-A) transport studies revealed that permeability ratio of PappB-A to PappA-B in the presence of 25 mM HEPES was significantly higher than control. The uptake of phenylalanine is an ATP-independent process, whereas the accumulation of glutamic acid is ATP-dependent. While phenylalanine uptake remained unchanged glutamic acid uptake was elevated with the addition of HEPES. Verapamil is an inhibitor of P-gp mediated uptake, elevation of cyclosporine uptake in the presence of 5 μM verapamil was compromised by the presence of 25 mM HEPES. The results of ATP assay indicated that HEPES stimulated the production of ATP. This study suggests that the addition of HEPES in the medium modulated the energy dependent efflux and uptake processes. The effect of HEPES on P-gp mediated drug efflux and transport may provide some mechanistic insight into possible reasons for inconsistencies in the results reported from various laboratories. PMID:20163160

  8. Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

    PubMed

    Xie, Yun; Zhao, Jun-Long; Wang, Chuan-Wei; Yu, Ai-Xin; Liu, Niu; Chen, Li; Lin, Fei; Xu, Han-Hong

    2016-05-18

    Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values <27) were analyzed at 1, 3, and 6 h after GlyF treatment by RT-qPCR. The results demonstrated that expression levels of four AAT genes, RcLHT6, RcANT15, RcProT2, and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional

  9. Transport of poly(acrylic acid) coated 2-line ferrihydrite nanoparticles in saturated aquifer sediments for environmental remediation

    NASA Astrophysics Data System (ADS)

    Xiang, Aishuang; Zhou, Sheng; Koel, Bruce E.; Jaffé, Peter R.

    2014-04-01

    Groundwater remediation using iron oxide and zero-valent iron nanoparticles (NPs) can be effective, but is limited in many applications due to the NP strong retention in groundwater-saturated porous media after injection, the passivation of the porous surface, and the high cost of nanomaterials versus macro scale iron. In this study, we investigated transport of bare and polymer-coated 2-line ferrihydrite NPs (30-300 nm) in saturated aquifer sediments. The influence of poly(acrylic acid) (PAA) polymer coatings was studied on the colloidal stability and transport in sediments packed column tests simulating groundwater flow in saturated sediments. In addition, the influence of calcium cations was investigated by transport measurements using sediments with calcium concentrations in the aqueous phase ranging from 0.5 (typical for most sediments) to 2 mM. Measurements were also made of zeta potential, hydrodynamic diameter, polymer adsorption and desorption properties, and bio-availability of PAA-coated NPs. We found that NP transport through the saturated aquifer sediments was improved by PAA coating and that the transport properties could be tuned by adjusting the polymer concentration. We further discovered that PAA coatings enhanced NP transport, compared to bare NPs, in all calcium-containing experiments tested, however, the presence of calcium always exhibited a negative effect on NP transport. In tests of bioavailability, the iron reduction rate of the coated and bare NPs by Geobacter sulfurreducens was the same, which shows that the PAA coating does not significantly reduce NP Fe(III) bioavailability. Our results demonstrate that much improved transport of iron oxide NP can be achieved in saturated aquifer sediments by introducing negatively charged polyelectrolytes and optimizing polymer concentrations, and furthermore, these coated NPs retain their bioavailability that is needed for applications in bio-environmental remediation.

  10. Role of a major facilitator superfamily transporter in adaptation capacity of Penicillium funiculosum under extreme acidic stress.

    PubMed

    Xu, Xiaoxue; Chen, Jinyin; Xu, Houjuan; Li, Duochuan

    2014-08-01

    Fungal species present in extreme low pH environments are expected to have adapted for tolerance to high H(+) concentrations. However, their adaptability mechanism is unclear. In this study, we isolated an acid-tolerant strain of Penicillium funiculosum, which can grow actively at pH 1.0 and thrived in pH 0.6. A major facilitator superfamily transporter (PfMFS) was isolated from an acid-sensitive random insertional mutant (M4) of the fungus. It encodes a putative protein of 551 residues and contains 14 transmembrane-spanning segments. A targeted mutant (M7) carrying an inactivated copy of PfMFS showed an obvious reduction of growth compared with the wild type (WT) and complementation of M7 with PfMFS restored the wild-type level of growth at pH 1.0. Further data showed that the wild-type showed higher intracellular pH than M7 in response to pH 1. Subcellular localization showed that PfMFS was a cell membrane protein. Homology modeling showed structural similarity with an MFS transporter EmrD from Escherichiacoli. These results demonstrate that the PfMFS transporter is involved in the acid resistance and intracellular pH homeostasis of P. funiculosum.

  11. Functional specialization and differential regulation of short-chain carboxylic acid transporters in the pathogen Candida albicans

    PubMed Central

    Vieira, Neide; Casal, Margarida; Johansson, Björn; MacCallum, Donna M; Brown, Alistair JP; Paiva, Sandra

    2010-01-01

    The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1-GFP (green fluorescent protein) and Jen2-GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1-GFP fusion. In the murine model of systemic candidiasis approximately 20–25% of C. albicans cells infecting the kidney expressed Jen1-GFP and Jen2-GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose-poor niches within the host, and that these short-chain carboxylic acid transporters may be important in the early stages of infection. PMID:19968788

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

    SciTech Connect

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

    1982-07-01

    A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of (/sup 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.

  13. Novel insights in transport mechanisms and kinetics of phenylacetic acid and penicillin-G in Penicillium chrysogenum.

    PubMed

    Douma, Rutger D; Deshmukh, Amit T; de Jonge, Lodewijk P; de Jong, Bouke W; Seifar, Reza M; Heijnen, Joseph J; van Gulik, Walter M

    2012-01-01

    Although penicillin-G (PenG) production by the fungus Penicillium chrysogenum is a well-studied process, little is known about the mechanisms of transport of the precursor phenylacetic acid (PAA) and the product PenG over the cell membrane. To obtain more insight in the nature of these mechanisms, in vivo stimulus response experiments were performed with PAA and PenG in chemostat cultures of P. chrysogenum at time scales of seconds to minutes. The results indicated that PAA is able to enter the cell by passive diffusion of the undissociated acid at a high rate, but is at the same time actively excreted, possibly by an ATP-binding cassette transporter. This results in a futile cycle, dissipating a significant amount of metabolic energy, which was confirmed by increased rates of substrate and oxygen consumption, and carbon dioxide production. To estimate the kinetic properties of passive import and active export of PAA over the cell membrane, a dynamic mathematical model was constructed. With this model, a good description of the dynamic data could be obtained. Also, PenG was found to be rapidly taken up by the cells upon extracellular addition, indicating that PenG transport is reversible. The measured concentration gradient of PenG over the cell membrane corresponded well with facilitated transport. Also, for PenG transport, a dynamic model was constructed and validated with experimental data. The outcome of the model simulations was in agreement with the presence of a facilitated transport system for PenG.

  14. Pharmacokinetics of amino acid ester prodrugs of acyclovir after oral administration: interaction with the transporters on Caco-2 cells.

    PubMed

    Katragadda, Suresh; Jain, Ritesh; Kwatra, Deep; Hariharan, Sudharshan; Mitra, Ashim K

    2008-10-01

    In vivo systemic absorption of the amino acid prodrugs of acyclovir (ACV) after oral administration was evaluated in rats. Stability of the prodrugs, L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and L-valine-ACV (VACV) was evaluated in various tissues. Interaction of these prodrugs with the transporters on Caco-2 cells was studied. In vivo systemic bioavailability of these prodrugs upon oral administration was evaluated in jugular vein cannulated rats. The amino acid ester prodrugs showed affinity towards various amino acid transporters as well as the peptide transporter on the Caco-2 cells. In terms of stability, EACV was most enzymatically stable compared to other prodrugs especially in liver homogenate. In oral absorption studies, ACV and AACV showed high terminal elimination rate constants (lambda(z)). SACV and VACV exhibited approximately five-fold increase in area under the curve (AUC) values relative to ACV (p<0.05). C(max(T)) (maximum concentration) of SACV was observed to be 39+/-22 microM in plasma which is 2 times better than VACV and 15 times better than ACV. C(last(T)) (concentration at the last time point) of SACV was observed to be 0.18+/-0.06 microM in plasma which is two times better than VACV and three times better than ACV. Amino acid ester prodrugs of ACV were absorbed at varying amounts (C(max)) and eliminated at varying rates (lambda(z)) thereby leading to varying extents (AUC). The amino acid ester prodrug SACV owing to its enhanced stability, higher AUC and better concentration at last time point seems to be a promising candidate for the oral treatment of herpes infections.

  15. Molecular features of the L-type amino acid transporter 2 determine different import and export profiles for thyroid hormones and amino acids.

    PubMed

    Hinz, Katrin M; Neef, Dominik; Rutz, Claudia; Furkert, Jens; Köhrle, Josef; Schülein, Ralf; Krause, Gerd

    2017-03-05

    The L-type amino acid transporter 2 (LAT2) imports amino acids (AA) and also certain thyroid hormones (TH), e.g. 3,3'-T2 and T3, but not rT3 and T4. We utilized LAT2 mutations (Y130A, N133S, F242W) that increase 3,3'-T2 import and focus here on import and export capacity for AA, T4, T3, BCH and derivatives thereof to delineate molecular features. Transport studies and analysis of competitive inhibition of import by radiolabelled TH and AA were performed in Xenopus laevis oocytes. Only Y130A, a pocket widening mutation, enabled import for T4 and increased it for T3. Mutant F242W showed increased 3,3'-T2 import but no import rates for other TH derivatives. No export was detected for any TH by LAT2-wild type (WT). Mutations Y130A and N133S enabled only the export of 3,3'-T2, while N133S also increased AA export. Thus, distinct molecular LAT2-features determine bidirectional AA transport but only an unidirectional 3,3'-T2 and T3 import.

  16. Hepatic alterations are accompanied by changes to bile acid transporter-expressing neurons in the hypothalamus after traumatic brain injury

    PubMed Central

    Nizamutdinov, Damir; DeMorrow, Sharon; McMillin, Matthew; Kain, Jessica; Mukherjee, Sanjib; Zeitouni, Suzanne; Frampton, Gabriel; Bricker, Paul Clint S.; Hurst, Jacob; Shapiro, Lee A.

    2017-01-01

    Annually, there are over 2 million incidents of traumatic brain injury (TBI) and treatment options are non-existent. While many TBI studies have focused on the brain, peripheral contributions involving the digestive and immune systems are emerging as factors involved in the various symptomology associated with TBI. We hypothesized that TBI would alter hepatic function, including bile acid system machinery in the liver and brain. The results show activation of the hepatic acute phase response by 2 hours after TBI, hepatic inflammation by 6 hours after TBI and a decrease in hepatic transcription factors, Gli 1, Gli 2, Gli 3 at 2 and 24 hrs after TBI. Bile acid receptors and transporters were decreased as early as 2 hrs after TBI until at least 24 hrs after TBI. Quantification of bile acid transporter, ASBT-expressing neurons in the hypothalamus, revealed a significant decrease following TBI. These results are the first to show such changes following a TBI, and are compatible with previous studies of the bile acid system in stroke models. The data support the emerging idea of a systemic influence to neurological disorders and point to the need for future studies to better define specific mechanisms of action. PMID:28106051

  17. Inhibitory effect of caffeic acid on human organic anion transporters hOAT1 and hOAT3: a novel candidate for food-drug interaction.

    PubMed

    Uwai, Yuichi; Ozeki, Yukihiro; Isaka, Tomonori; Honjo, Hiroaki; Iwamoto, Kikuo

    2011-01-01

    Several kinds of food have been shown to influence the absorption and metabolism of drugs, although there is little information about their effect on the renal excretion of drugs. In this study, we performed uptake experiments using Xenopus laevis oocytes to assess the inhibitory effects of chlorogenic acid, caffeic acid and quinic acid, which are contained in coffee, fruits and vegetables, on human organic anion transporters hOAT1 and hOAT3; these transporters mediate renal tubular uptake of anionic drugs from blood. Injection of hOAT1 and hOAT3 cRNA into oocytes stimulated uptake of typical substrates of hOAT1 and hOAT3 (p-aminohippurate and estrone sulfate, respectively); among the three compounds tested, caffeic acid most strongly inhibited these transporters. The apparent 50% inhibitory concentrations of caffeic acid were estimated to be 16.6 µM for hOAT1 and 5.4 µM for hOAT3. Eadie-Hofstee plot analysis showed that caffeic acid inhibited both transporters in a competitive manner. In addition to the transport of p-aminohippurate and estrone sulfate, that of antifolates and antivirals was inhibited by caffeic acid. These findings show that caffeic acid has inhibitory potential against hOAT1 and hOAT3, suggesting that renal excretion of their substrates could be affected in patients consuming a diet including caffeic acid.

  18. Interaction of Peptide Transporter 1 With D-Glucose and L-Glutamic Acid; Possible Involvement of Taste Receptors.

    PubMed

    Arakawa, Hiroshi; Ohmachi, Taichi; Ichiba, Kiko; Kamioka, Hiroki; Tomono, Takumi; Kanagawa, Masahiko; Idota, Yoko; Hatano, Yasuko; Yano, Kentaro; Morimoto, Kaori; Ogihara, Takuo

    2016-01-01

    We investigated the influence of sweet and umami (savory) tastants on the intestinal absorption of cephalexin (CEX), a substrate of peptide transporter 1 (PEPT1, SLC15A1) in rats. After oral administration of glucose or mannitol to rats, CEX was administered together with a second dose of glucose or mannitol. Western blot analysis indicated that expression of PEPT1 in rat jejunum membrane was decreased by glucose, compared to mannitol. Furthermore, the maximum plasma concentration (Cmax) of orally administered CEX was reduced by glucose compared to mannitol. The effect of glucose was diminished by nifedipine, a L-type Ca(2+) channel blocker. We also found that Cmax of orally administered CEX was reduced by treatment with L-glutamic acid, compared to D-glutamic acid. Thus, excessive intake of glucose and L-glutamic acid may impair oral absorption of PEPT1 substrates.

  19. Fate and transport of glyphosate and aminomethylphosphonic acid in surface waters of agricultural basins

    USGS Publications Warehouse

    Gregoire, Caroline; Capel, Paul D.; Coupe, Richard H.; Kalkhoff, Stephen J.

    2011-01-01

    CONCLUSIONS: Glyphosate use in a watershed results in some occurrence in surface water; however, the watersheds most at risk for the offsite transport of glyphosate are those with high application rates, rainfall that results in overland runoff and a flow route that does not include transport through the soil.

  20. Differential usage of the transport systems for folic acid and methotrexate in normal human T-lymphocytes and leukemic cells.

    PubMed

    Biswal, Bijesh Kumar; Verma, Rama Shanker

    2009-11-01

    Methotrexate (MTX) has been used as an effective anti-cancer drug for a long time. Conceptually, it is accepted that MTX and folic acid are transported by folate receptors (FRs) in cancerous cells, but the exact mechanism of MTX uptake in human leukemia is unknown. The objective of this study was to investigate different transport systems for FA and MTX, and to delineate their uptake mechanism in MOLT4, K562, Hut78 leukemia cells and normal human T cells. In MOLT4, uptake of MTX was higher than FA, similar to that of K562, Hut78 and normal T cells. In MOLT4 cells, MTX uptake was maximum at pH 7.4 whereas FA uptake was maximum at pH 4.5. Uptake of FA and MTX was significantly inhibited by anions, suggesting anion-dependent transport system. FA uptake was found to be energy dependent whereas MTX uptake was energy independent. RT-PCR and immunofluorescence results demonstrated the presence of reduced folate carrier as well as proton coupled folate transporter and absence of FR in MOLT4 and normal T cells. These data suggest the existence of two separate and independent carrier-mediated transport systems for the uptake of FA and MTX in normal and leukemic human T cells.

  1. In vitro bioacessibility and transport across Caco-2 monolayers of haloacetic acids in drinking water.

    PubMed

    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.

  2. Neuronal transport of acid hydrolases and peroxidase within the lysosomal system or organelles: involvement of agranular reticulum-like cisterns.

    PubMed

    Broadwell, R D; Oliver, C; Brightman, M W

    1980-04-01

    Neurosecretory neurons of the hyperosmotically stressed hypothalamo-neurohypophysial system have been a useful model with which to demonstrate interrelationships among perikaryal lysosomes, agranular reticulum-like cisterns, endocytotic vacuoles, and the axoplasmic transport of acid hydrolases and horseradish peroxidase. Supraoptic neurons from normal mice and mice given 2% salt water to drink for 5--8 days have been studied using enzyme cytochemical techniques for peroxidase and lysosomal acid hydrolases. Peroxidase-labeling of these neurons was accomplished by intravenous injection or cerebral ventriculocisternal perfusion of the protein as previously reported (Broadwell and Brightman, '79). Compared to normal controls, supraoptic cell bodies from hyperosmotically stimulated mice contained elevated concentrations of peroxidase-labeled dense bodies demonstrated to be secondary lysosomes and acid hydrolase-positive and peroxidase-positive cisterns either attached or unattached to secondary lysosomes. These cisterns were smooth-surfaced and 400--1,000 A wide. Their morphology was similar to that of the agranular reticulum. Some of the cisterns contained both peroxidase and acid hydrolase activities. The cisterns probably represent an elongated form of lysosome and, therefore, are not elements of the agranular reticulum per se. By virtue of their direct connections with perikaryal secondary lysosomes, these cisterns may provide the route by which acid hydrolases and exogenous macromolecules can leave perikaryal secondary lysosomes for anterograde flow down the axon. Very few smooth-surfaced cisterns were involved in the retrograde transport of peroxidase within pituitary stalk axons from normal and salt-treated mice injected intravenously with peroxidase. Peroxidase undergoing retrograde transport was predominantly in endocytotic structures such as vacuoles and cup-shaped organelles, which deliver this exogenous macromolecule directly to secondary lysosomes for

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

    SciTech Connect

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

    1991-09-01

    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 {plus minus} 1.87 mM, the maximum uptake rate, Jmax, was 144.7 {plus minus} 55.7 nmol/mg of protein/min and the nonsaturable first-order rate constant, Kd, was 6.66 {plus minus} 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{prime}-diisothiocyanostilben-2,2{prime}-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.

  4. Defining membrane spanning domains and crucial membrane-localized acidic amino acid residues for K⁺ transport of a Kup/HAK/KT-type Escherichia coli potassium transporter.

    PubMed

    Sato, Yoko; Nanatani, Kei; Hamamoto, Shin; Shimizu, Makoto; Takahashi, Miho; Tabuchi-Kobayashi, Mayumi; Mizutani, Akifumi; Schroeder, Julian I; Souma, Satoshi; Uozumi, Nobuyuki

    2014-05-01

    Potassium (K(+))-uptake transport proteins present in prokaryote and eukaryote cells are categorized into two classes; Trk/Ktr/HKT, K(+) channel, and Kdp belong to the same superfamily, whereas the remaining K(+)-uptake family, Kup/HAK/KT has no homology to the others, and neither its membrane topology nor crucial residues for K(+) uptake have been identified. We examined the topology of Kup from Escherichia coli. Results from the reporter fusion and cysteine labeling assays support a model with 12 membrane-spanning domains. A model for proton-coupled K(+) uptake mediated by Kup has been proposed. However, this study did not show any stimulation of Kup activity at low pH and any evidence of involvement of the three His in Kup-mediated K(+) uptake. Moreover, replacement of all four cysteines of Kup with serine did not abolish K(+) transport activity. To gain insight on crucial residues of Kup-mediated K(+) uptake activity, we focused on acidic residues in the predicted external and transmembrane regions, and identified four residues in the membrane regions required for K(+) uptake activity. This is different from no membrane-localized acidic residues essential for Trk/Ktr/HKTs, K(+) channels and Kdp. Taken together, these results demonstrate that Kup belongs to a distinct type of K(+) transport system.

  5. Transmembrane domains I and II of the gamma-aminobutyric acid transporter GAT-4 contain molecular determinants of substrate specificity.

    PubMed

    Melamed, Nir; Kanner, Baruch I

    2004-06-01

    The sodium- and chloride-dependent GABA transporters GABA transporter (GAT) 1 to 4 in the central nervous system enable efficient synaptic transmission by removing the neurotransmitter from the cleft. Taurine interacts only weakly with the GABA transporter GAT-4 (IC50 approximately 1.6 mM). Glutamate-61 is located in the conserved transmembrane domain I of GAT-4, whereas in the related taurine-transporter taurine transporter (TAUT), glycine occupies the equivalent position. [3H]GABA uptake by the GAT-4 E61G mutant becomes markedly more sensitive to inhibition by taurine (IC50 approximately 0.26 mM). Replacement of cysteine-94, located in the conserved transmembrane domain II of GAT-4, to its TAUT counterpart serine, results only in a modest increase in the ability of taurine to inhibit GABA uptake. However, introduction of glycine at this position decreases the IC50 for taurine by approximately 8-fold (IC50 approximately 0.20 mM). The inhibitory potency of taurine is inversely correlated with the volume of the side chain of the amino acid residue introduced at positions 61 and 94. It is striking that the IC50 for taurine of the E61G/C94G double mutant is decreased by approximately 35-fold (IC50 approximately 0.05 mM), and this inhibition of GABA transport is competitive. Changes in the inhibitory potency of the mutants described are also observed with beta-ala-nine and GABA, although they are much less pronounced. Our results suggest that determinants on transmembrane domains I and II can influence the specificity of the substrate binding pocket. The size of the side chain at positions 61 and 94 seems to determine the ability of substrate and substrate analogs to interact with the transporter.

  6. Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

    PubMed Central

    Clémençon, Benjamin; Lüscher, Benjamin P.; Fine, Michael; Baumann, Marc U.; Surbek, Daniel V.; Bonny, Olivier; Hediger, Matthias A.

    2014-01-01

    The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies. PMID:25286413

  7. Inhibition by Levorphanol and Related Drugs of Amino Acid Transport by Isolated Membrane Vesicles from Escherichia coli

    PubMed Central

    Holland, Mary J. C.; Simon, Eric J.

    1975-01-01

    Levorphanol inhibits the transport of the amino acids proline and lysine by cytoplasmic membrane vesicles derived from Escherichia coli. The degree of inhibition increases with increasing levorphanol concentration and ranges from 26% at 10−6 M levorphanol to 92% at 10−3 M levorphanol. The effect is independent of the energy source, since levorphanol inhibits proline uptake to the same extent in the presence of 20 mM d-lactate or 20 mM succinate and in the absence of an exogenous energy source. Levorphanol does not irreversibly alter the ability of membrane vesicles to transport proline, since incubation of membrane vesicles for 15 min in the presence of 0.25 mM levorphanol, a concentration which inhibits proline transport by more than 75%, has no effect on the rate of proline transport by these vesicles once the drug is removed. Both the maximum velocity and the Km of proline transport are modified by levorphanol, hence, the type of inhibition produced by levorphanol is mixed. The inhibitor constant (Ki) for levorphanol inhibition of proline transport is approximately 3 × 10−4 M. Membrane vesicles incubated in the presence of levorphanol accumulate much less proline at the steady state than do control vesicles. Furthermore, the addition of levorphanol to membrane vesicles preloaded to the steady state with proline produces a marked net efflux of proline. Levorphanol does not block either temperature-induced efflux or exchange of external proline with [14C]proline present in the intravesicular pool. Dextrorphan, the enantiomorph of levorphanol, and levallorphan, the N-allyl analogue of levorphanol, inhibit proline and lysine transport in a similar manner. Possible mechanisms of the effects of these drugs on cell membranes are discussed. PMID:1096802

  8. The mRNA expression of amino acid transporters, aminopeptidase N, and the di- and tri- peptide transporter PepT1 in the embryo of the domesticated chicken (Gallus gallus) shows developmental regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mRNA expression profile for ten amino acid transporters (AAT), the di-and tri- peptide transporter (Pept1), and aminopeptidase N (APN) during chick embryogenesis was determined. Fertilized eggs were sampled at days 9, 11, 15, 17, 19, and 20, post fertilization. Three to four embryos were sampl...

  9. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    PubMed

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  10. Expression of the surface antigen 4F2hc affects system-L-like neutral-amino-acid-transport activity in mammalian cells.

    PubMed Central

    Bröer, S; Bröer, A; Hamprecht, B

    1997-01-01

    Mammalian cells possess a variety of amino acid-transport systems with overlapping substrate specificity. System L is one of the major amino acid-transport systems of non-epithelial cells. By expression cloning we have recently demonstrated that the surface antigen 4F2hc (CD98) is a necessary component for expression of system-L-like amino acid-transport activity in C6-BU-1 rat glioma cells [Bröer, Bröer and Hamprecht (1995) Biochem. J. 312, 863-870]. 4F2hc mRNA was detected in CHO cells, COS cells, activated lymphocytes isolated from mouse spleen and primary cultures of astrocytes. In all these cell types, Na+-independent isoleucine transport was mediated by system L. No contribution of system y+L to isoleucine or arginine transport was detected in C6-BU-1 cells. In lymphocytes, both system-L-like amino acid-transport activity and 4F2hc mRNA levels increased after treatment with phorbol ester plus ionomycin. Antisense oligonucleotides caused modest inhibition of Na+-independent isoleucine transport in C6-BU-1 cells and primary cultures of astroglial cells, whereas arginine transport was unaffected. Overexpression of 4F2hc cDNA in CHO cells resulted in an increase in Na+-independent isoleucine transport. PMID:9182715

  11. Organic anion-transporting polypeptide 1a4 (Oatp1a4) is important for secondary bile acid metabolism

    PubMed Central

    Zhang, Youcai; Csanaky, Iván L.; Selwyn, Felcy Pavithra; Lehman-McKeeman, Lois D.; Klaassen, Curtis D.

    2013-01-01

    Organic anion transporting polypeptides (human: OATPs; rodent: Oatps) were thought to have important functions in bile acid (BA) transport. Oatp1a1, 1a4, and 1b2 are the three major Oatp1 family members in rodent liver. Our previous studies have characterized the BA homeostasis in Oatp1a1-null and Oatp1b2-null mice. The present study investigated the physiological role of Oatp1a4 in BA homeostasis by using Oatp1a4-null mice. Oatp1a4 expression is female-predominant in livers of mice, and thereby it was expected that female Oatp1a4-null mice will have more prominent changes than males. Interestingly, the present study demonstrated that female Oatp1a4-null mice had no significant alterations in BA concentrations in serum or liver, though they had increased mRNA of hepatic BA efflux transporters (Mrp4 and Ostα/β) and ileal BA transporters (Asbt and Ostα/β). In contrast, male Oatp1a4-null mice showed significantly altered BA homeostasis, including increased concentrations of deoxycholic acid (DCA) in serum, liver and intestinal contents. After feeding a DCA-supplemented diet, male but not female Oatp1a4-null mice had higher concentrations of DCA in serum and livers than their WT controls. This suggested that Oatp1a4 is important for intestinal absorption of secondary BAs in male mice. Furthermore, loss of Oatp1a4 function did not decrease BA accumulation in serum or livers of bile-ductligated mice, suggesting that Oatp1a4 is not likely a BA uptake transporter. In summary, the present study for the first time demonstrates that Oatp1a4 does not appear to mediate the hepatic uptake of BAs, but plays an important male-predominant role in secondary BA metabolism in mice. PMID:23747753

  12. Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification

    PubMed Central

    Cai, Ruiping; Yuan, Yanmeng; Guo, Zhanyun; Grewer, Christof; Zhang, Zhou

    2016-01-01

    Sodium-coupled neutral amino acid transporter 2 (SNAT2) belongs to solute carrier 38 (SLC38) family of transporters, which is ubiquitously expressed in mammalian tissues and mediates transport of small, neutral amino acids, exemplified by alanine(Ala, A). Yet structural data on SNAT2, including the relevance of intrinsic cysteine residues on structure and function, is scarce, in spite of its essential roles in many tissues. To better define the potential of intrinsic cysteines to form disulfide bonds in SNAT2, mutagenesis experiments and thiol-specific chemical modifications by N-ethylmaleimide (NEM) and methoxy-polyethylene glycol maleimide (mPEG-Mal, MW 5000) were performed, with or without the reducing regent dithiothreitol (DTT) treatment. Seven single mutant transporters with various cysteine (Cys, C) to alanine (Ala, A) substitutions, and a C245,279A double mutant were introduced to SNAT2 with a hemagglutinin (HA) tag at the C-terminus. The results showed that the cells expressing C245A or C279A were labeled by one equivalent of mPEG-Mal in the presence of DTT, while wild-type or all the other single Cys to Ala mutants were modified by two equivalents of mPEG-Mal. Furthermore, the molecular weight of C245,279A was not changed in the presence or absence of DTT treatment. The results suggest a disulfide bond between Cys245 and Cys279 in SNAT2 which has no effect on cell surface trafficking, as well as transporter function. The proposed disulfide bond may be important to delineate proximity in the extracellular domain of SNAT2 and related proteins. PMID:27355203

  13. Back to Acid Soil Fields: The Citrate Transporter SbMATE Is a Major Asset for Sustainable Grain Yield for Sorghum Cultivated on Acid Soils

    PubMed Central

    Carvalho, Geraldo; Schaffert, Robert Eugene; Malosetti, Marcos; Viana, Joao Herbert Moreira; Menezes, Cicero Bezerra; Silva, Lidianne Assis; Guimaraes, Claudia Teixeira; Coelho, Antonio Marcos; Kochian, Leon V.; van Eeuwijk, Fred A.; Magalhaes, Jurandir Vieira

    2015-01-01

    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. PMID:26681519

  14. Back to Acid Soil Fields: The Citrate Transporter SbMATE Is a Major Asset for Sustainable Grain Yield for Sorghum Cultivated on Acid Soils.

    PubMed

    Carvalho, Geraldo; Schaffert, Robert Eugene; Malosetti, Marcos; Viana, Joao Herbert Moreira; Menezes, Cicero Bezerra; Silva, Lidianne Assis; Guimaraes, Claudia Teixeira; Coelho, Antonio Marcos; Kochian, Leon V; van Eeuwijk, Fred A; Magalhaes, Jurandir Vieira

    2015-12-17

    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.

  15. Conserved Aspartic Acid Residues Lining the Extracellular Loop I of Sodium-coupled Bile Acid Transporter ASBT Interact with Na+ and 7α-OH Moieties on the Ligand Cholestane Skeleton*

    PubMed Central

    Hussainzada, Naissan; Da Silva, Tatiana Claro; Zhang, Eric Y.; Swaan, Peter W.

    2008-01-01

    Functional contributions of residues Val-99—Ser-126 lining extracellular loop (EL) 1 of the apical sodium-dependent bile acid transporter were determined via cysteine-scanning mutagenesis, thiol modification, and in silico interpretation. Despite membrane expression for all but three constructs (S112C, Y117C, S126C), most EL1 mutants (64%) were inactivated by cysteine mutation, suggesting a functional role during sodium/bile acid co-transport. A negative charge at conserved residues Asp-120 and Asp-122 is required for transport function, whereas neutralization of charge at Asp-124 yields a functionally active transporter. D124A exerts low affinity for common bile acids except deoxycholic acid, which uniquely lacks a 7α-hydroxyl (OH) group. Overall, we conclude that (i) Asp-122 functions as a Na+ sensor, binding one of two co-transported Na+ ions, (ii) Asp-124 interacts with 7α-OH groups of bile acids, and (iii) apolar EL1 residues map to hydrophobic ligand pharmacophore features. Based on these data, we propose a comprehensive mechanistic model involving dynamic salt bridge pairs and hydrogen bonding involving multiple residues to describe sodium-dependent bile acid transporter-mediated bile acid and cation translocation. PMID:18508772

  16. Protein Restriction with Amino Acid-Balanced Diets Shrinks Circulating Pool Size of Amino Acid by Decreasing Expression of Specific Transporters in the Small Intestine

    PubMed Central

    Luo, Min; Zhang, Xin; Sun, Wen Juan; Jiao, Ning; Li, De Fa; Yin, Jing Dong

    2016-01-01

    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. PMID:27611307

  17. Supplementation with branched-chain amino acids to a low-protein diet regulates intestinal expression of amino acid and peptide transporters in weanling pigs.

    PubMed

    Zhang, Shihai; Qiao, Shiyan; Ren, Man; Zeng, Xiangfang; Ma, Xi; Wu, Zhenlong; Thacker, Philip; Wu, Guoyao

    2013-11-01

    This study determined the effects of dietary branched-chain amino acids (AA) (BCAA) on growth performance, expression of jejunal AA and peptide transporters, and the colonic microflora of weanling piglets fed a low-protein (LP) diet. One hundred and eight Large White × Landrace × Duroc piglets (weaned at 28 days of age) were fed a normal protein diet (NP, 20.9 % crude protein), an LP diet (LP, 17.1 % crude protein), or an LP diet supplemented with BCAA (LP + BCAA, 17.9 % crude protein) for 14 days. Dietary protein restriction reduced piglet growth performance and small-intestinal villous height, which were restored by BCAA supplementation to the LP diet to values for the NP diet. Serum concentrations of BCAA were reduced in piglets fed the LP diet while those in piglets fed the LP + BCAA diet were similar to values for the NP group. mRNA levels for Na(+)-neutral AA exchanger-2, cationic AA transporter-1, b(0,+) AA transporter, and 4F2 heavy chain were more abundant in piglets fed the LP + BCAA diet than the LP diet. However, mRNA and protein levels for peptide transporter-1 were lower in piglets fed the LP + BCAA diet as compared to the LP diet. The colonic microflora did not differ among the three groups of pigs. In conclusion, growth performance, intestinal development, and intestinal expression of AA transporters in weanling piglets are enhanced by BCAA supplementation to LP diets. Our findings provide a new molecular basis for further understanding of BCAA as functional AA in animal nutrition.

  18. Genome-wide survey and expression analysis of the amino acid transporter superfamily in potato (Solanum tuberosum L.).

    PubMed

    Ma, Haoli; Cao, Xiaoli; Shi, Shandang; Li, Silu; Gao, Junpeng; Ma, Yuling; Zhao, Qin; Chen, Qin

    2016-10-01

    Amino acid transporters (AATs) are integral membrane proteins responsible for the transmembrane transport of amino acids and play important roles in various physiological processes of plants. However, there has not yet been a genome-wide overview of the StAAT gene family to date and only StAAP1 has been previously studied in potato. In this paper, a total of 72 StAATs were identified using a series of bioinformatics searches and classified into 12 subfamilies based on their phylogenetic relationship with known Arabidopsis and rice AATs. Chromosomal localization revealed their distribution on all 12 chromosomes. Nearly one-third of StAAT genes (23 of 72) were derived from gene duplication, among which tandem duplication made the greatest contribution to the expansion of the StAAT family. Motif analysis showed that the same subfamily had similar conserved motifs in both numbers and varieties. Moreover, high-throughput sequencing data was used to analyze the expression patterns of StAAT genes and was verified by quantitative real-time RT-PCR. The expression of StAAT genes exhibited both abundant and tissue-specific expression patterns, which might be connected to their functional roles in long- and short-distance transport. This study provided a comprehensive survey of the StAAT gene family, and could serve as a theoretical foundation for the further functional identification and utilization of family members.

  19. A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and o