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Sample records for acid transport activity

  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. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. 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

  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. Activation of the aryl hydrocarbon receptor induces hepatic steatosis via the upregulation of fatty acid transport.

    PubMed

    Kawano, Yuki; Nishiumi, Shin; Tanaka, Shinwa; Nobutani, Kentaro; Miki, Akira; Yano, Yoshihiko; Seo, Yasushi; Kutsumi, Hiromu; Ashida, Hitoshi; Azuma, Takeshi; Yoshida, Masaru

    2010-12-15

    The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix/Per-ARNT-Sim domain transcription factor, which is activated by various xenobiotic ligands. AHR is known to be abundant in liver tissue and to be associated with hepatic steatosis. However, it has not yet been elucidated how the activation of AHR promotes hepatic steatosis. The aim of this study is to clarify the role of AHR in hepatic steatosis. The intraperitoneal injection of 3-methylcholanthrene (3MC), a potent AHR ligand, into C57BL/6J mice significantly increased the levels of triglycerides and six long-chain monounsaturated fatty acids in the livers of mice, resulting in hepatic microvesicular steatosis. 3MC significantly enhanced the expression level of fatty acid translocase (FAT), a factor regulating the uptake of long-chain fatty acids into hepatocytes, in the liver. In an in vitro experiment using human hepatoma HepG2 cells, 3MC increased the expression level of FAT, and the downregulation of AHR by AHR siRNA led to the suppression of 3MC-induced FAT expression. In addition, the mRNA level of peroxisome proliferator-activated receptor (PPAR) α, an upstream factor of FAT, was increased in the livers of 3MC-treated mice. Taking together, AHR activation induces hepatic microvesicular steatosis by increasing the expression level of FAT.

  20. Riluzole attenuates excitatory amino acid transporter type 3 activity in Xenopus oocytes via protein kinase C inhibition.

    PubMed

    Choi, Jung-Seok; Ryu, Jung-Hee; Zuo, Zhiyi; Yang, Seong-Mi; Chang, Hye-Won; Do, Sang-Hwan

    2013-08-05

    This study aimed to evaluate the effect of riluzole on the activity of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, and to investigate the role of protein kinase C (PKC) in this effect. EAAT3 expression was induced in Xenopus oocytes by injecting EAAT3 mRNA. Using the two-electrode voltage clamping method, membrane currents were recorded before, during, and after applying l-glutamate (30 μM) in the absence and presence of prior incubation with riluzole (0.3-100 μM). To study the effect of PKC on the riluzole-induced change in EAAT3 activity, oocytes were preincubated with 100 μM phorbol-12-myristate-13-acetate (PMA), a PKC activator, or PKC inhibitors (2 µM staurosporine and 100 µM chelerythrine) before the recording. Responses were quantified by integrating current traces and are reported in microCoulombs (μC). Riluzole reduced EAAT3 activity in a concentration-dependent manner (0.3-100 μM). Treatment of oocytes with PMA significantly increased the baseline and riluzole-reduced EAAT activity (P<0.05). In addition, treatment of oocytes with PKC inhibitors reduced basal transporter currents, but did not show a further significant decrease in the riluzole-reduced EAAT3 activity. These results suggest that riluzole reduces EAAT3 activity through PKC inhibition.

  1. CP-MLR/PLS directed QSAR study on apical sodium-codependent bile acid transporter inhibition activity of benzothiepines.

    PubMed

    Sharma, Brij Kishore; Singh, Prithvi; Pilania, Pradeep; Sarbhai, Kirti; Prabhakar, Yenamandra S

    2011-02-01

    The apical sodium-codependent bile acid transporter (ASBT) inhibition activity of benzothiepine derivatives have been analyzed based on topological and molecular features. Analysis of the structural features in conjunction with the biological endpoints in Combinatorial Protocol in Multiple Linear Regression (CP-MLR) led to the identification of 21 descriptors for modeling the activity. The study clearly suggested that the role of Randic shape index (path/walk ratio 3) and topological charges of 2-, 5-, and 6-orders to optimize the ASBT inhibitory activity of titled compounds. The influence of atomic van der Waals volumes, masses, Sanderson electronegativities, and polarizabilities are indicated via different lags of Moran and Geary autocorrelations. Presence of tertiary aromatic amine functionality in molecular structure has also shown its relevance in rationalizing the biological actions of benzothiepines. The PLS analysis has confirmed the dominance of information content of CP-MLR identified descriptors for modeling the activity when compared to those of the leftover ones.

  2. 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

  3. 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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  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. Transport to Rhebpress activity.

    PubMed

    Garrido, Amanda; Brandt, Marta; Djouder, Nabil

    2016-01-01

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

  7. 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

  8. Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

    PubMed Central

    Lobaton, C D; Moreno, A; Oxender, D L

    1984-01-01

    We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20. Images PMID:6717430

  9. Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

    PubMed

    Lobaton, C D; Moreno, A; Oxender, D L

    1984-03-01

    We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.

  10. 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.

  11. Active transport of. gamma. -aminobutyric acid and glycine into synaptic vesicles

    SciTech Connect

    Kish, P.E.; Fischer-Bovenkerk, C.; Ueda, T. )

    1989-05-01

    Although {gamma}-aminobutyric acid (GABA) and glycine are recognized as major amino acid inhibitory neurotransmitters in the central nervous system, their storage is poorly understood. In this study the authors have characterized vesicular GABA and glycine uptakes in the cerebrum and spinal cord, respectively. They present evidence that GABA and glycine are each taken up into isolated synaptic vesicles in an ATP-dependent manner and that the uptake is driven by an electrochemical proton gradient. Uptake for both amino acids exhibited kinetics with low affinity similar to a vesicular glutamate uptake. The ATP-dependent GABA uptake was not inhibited by the putative amino acid neurotransmitters glycine, taurine, glutamate, or aspartate or by GABA analogs, agonists, and antagonists. Similarly, ATP-dependent glycine uptake was hardly affected by GABA, taurine, glutamate, or aspartate or by glycine analogs or antagonists. The GABA uptake was not affected by chloride, which is in contrast to the uptake of the excitatory neurotransmitter glutamate, whereas the glycine uptake was slightly stimulated by low concentrations of chloride. Tissue distribution studies indicate that the vesicular uptake systems for GABA, glycine, and glutamate are distributed in different proportions in the cerebrum and spinal cord. These results suggest that the vesicular uptake systems for GABA, glycine, and glutamate are distinct from each other.

  12. Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells.

    PubMed Central

    Maruyama, K; MacLennan, D H

    1988-01-01

    Full-length cDNAs encoding neonatal and adult isoforms of the Ca2+-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum were expressed transiently in COS-1 cells. The microsomal fraction isolated from transfected COS-1 cells contained immunoreactive Ca2+-ATPase and catalyzed Ca2+ transport at rates at least 15-fold above controls. No differences were observed in either the rates or Ca2+ dependency of Ca2+ transport catalyzed by the two isoforms. Aspartic acid-351, the site of formation of the catalytic acyl phosphate in the enzyme, was mutated to asparagine, glutamic acid, serine, threonine, histidine, or alanine. In every case, Ca2+ transport activity and Ca2+-dependent phosphorylation were eliminated. Ca2+ transport was also eliminated by mutation of lysine-352 to arginine, glutamine, or glutamic acid or by mutation of Asp351-Lys352 to Lys351-Asp352. Mutation of lysine-515, the site of fluorescein isothiocyanate modification in the enzyme, resulted in diminished Ca2+ transport activity as follows: arginine, 60%; glutamine, 25%; glutamic acid, 5%. These results demonstrate the absolute requirement of acylphosphate formation for the Ca2+ transport function and define a residue important for ATP binding. They also demonstrate the feasibility of a thorough analysis of active sites in the Ca2+-ATPase by expression and site-specific mutagenesis. Images PMID:2966962

  13. Characterization of reduced and oxidized dopamine and 3,4-dihydrophenylacetic acid, on brain mitochondrial electron transport chain activities.

    PubMed

    Gautam, Alpa H; Zeevalk, Gail D

    2011-07-01

    Loss of dopamine (DA) homeostasis may be a contributing factor to cell damage in Parkinson's disease (PD). Past studies showing deleterious effects of DA on mitochondrial function, however, have been inconsistent raising questions about mitochondria as a downstream target for DA. Issues such as the dopamine species i.e., reduced or oxidized, time of exposure and the effect of major metabolites such as 3,4-dihydrophenylacetic acid (DOPAC) may contribute to the disparate findings. The present study used isolated, lysed rat brain mitochondria to characterize the effects of oxidized or reduced DA and DOPAC on complex activities of the electron transport chain (ETC). Time of exposure and quantitation of reduced or oxidized catachols for DA and DOPAC were monitored for all experiments. Reduced DA and DOPAC with or without a 30min preincubation had no affect on NADH oxidase activity which monitors the activities of complexes I, III and IV. Complex II activity was inhibited by reduced DA (≥500μM), but not by reduced DOPAC and was significantly attenuated by SOD suggesting reactive oxygen species involvement. In contrast, fully oxidized DA and DOPAC dose dependently inhibited NADH oxidase, complex I and complex III activities with IC(50s) in the 50-200μM range. No preincubation was required for inhibition with the catechols when they were fully oxidized. Oxidized DA inhibited complex I only when exposure occurred during stimulated electron flow, suggesting covalent binding of quinones to proteins within active sites of the complex. In intact, well coupled mitochondria, extramitochondrial DA was shown to access the mitochondrial matrix in a dose, time and energy-dependent fashion. The findings suggest that many of the reported inconsistencies with regards to the effects of DA and DOPAC on ETC function can be attributed to the oxidized state of the catechol at the time of exposure. In addition, the findings provide possible downstream targets for DA that could contribute

  14. Fatty Acid Flippase Activity Of UCP2 Is Essential For Its Proton Transport In Mitochondria

    PubMed Central

    Berardi, Marcelo J.; Chou, James J.

    2014-01-01

    SUMMARY Modulation of cellular energy expenditure is fundamental to normal and pathological cell growth and differentiation. Mitochondria stores energy as a proton gradient across their inner membrane. Uncoupling proteins (UCPs) can dissipate the gradient to produce heat or regulate metabolite fluxes. UCP-mediated proton currents require fatty acids (FA) and are blocked by nucleotides, but the molecular basis of these processes is unknown. We find, by nuclear magnetic resonance and functional mutagenesis, that UCP2 can bind FA laterally thru its peripheral site, and this intramembrane molecular recognition is essential for UCP2-catalyzed FA flipping across the membrane, which in turn is essential for proton translocation. The antagonist GDP binds inside the UCP2 cavity and perturbs its conformation, which can displace FA from the peripheral site as a mean of inhibiting proton currents. Our data provide a biophysical perspective of the intricate interplay of UCPs, FA, and nucleotides in determining proton fluxes in mitochondria. PMID:25127353

  15. Volume-activated amino acid efflux from term human placental tissue: stimulation of efflux via a pathway sensitive to anion transport inhibitors.

    PubMed

    Shennan, D B; McNeillie, S A

    1995-04-01

    The effect of a hyposmotic challenge and hence cell-swelling upon the efflux of a variety of solutes from isolated human placental tissue has been examined. A hyposmotic shock increased the fractional release of taurine, the most abundant free amino acid in placental tissue, via a pathway sensitive to niflumic acid, DIDS (4,4'-Diisothiocyanatostilbene-2',2'-disulphonic acid,) NPPB (5-Nitro-2(3-phenylpropylamino)benzoic acid) and DIOA (R(+)[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden -5-y) oxy] acetic acid). In contrast, tamoxifen was without effect. The cell-swelling induced efflux of taurine was attenuated (40 per cent) by replacing external Cl- with NO3-. The efflux of glutamic acid was also markedly increased by a hyposmotic challenge. Niflumic acid inhibited both basal and volume-activated glutamic acid efflux. A hyposmotic shock also increased alpha-aminoisobutyric acid efflux but not that of 3-O-methylglucose and SO4(2)-. The results suggest that the human placenta can respond to cell-swelling by releasing organic osmolytes such as amino acids via a pathway which is sensitive to anion transport inhibitors. However, it appears that the volume-activated amino acid transport system is independent from the placental anion-exchange pathways. The efflux of these compounds may act with K+ and Cl- efflux to effect a regulatory volume decrease in placental tissue. In addition, volume-activated transport may play a role in transplacental amino acid transfer.

  16. 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.

  17. Involvement of vacuolar sequestration and active transport in tolerance of Saccharomyces cerevisiae to hop iso-alpha-acids.

    PubMed

    Hazelwood, Lucie A; Walsh, Michael C; Pronk, Jack T; Daran, Jean-Marc

    2010-01-01

    The hop plant, Humulus lupulus L., has an exceptionally high content of secondary metabolites, the hop alpha-acids, which possess a range of beneficial properties, including antiseptic action. Studies performed on the mode of action of hop iso-alpha-acids have hitherto been restricted to lactic acid bacteria. The present study investigated molecular mechanisms of hop iso-alpha-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-alpha-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-alpha-acid detoxification and tolerance. This screening and further analysis of deletion mutants confirmed that yeast tolerance to hop iso-alpha-acids involves three major processes, active proton pumping into the vacuole by the vacuolar-type ATPase to enable vacuolar sequestration of iso-alpha-acids and alteration of cell wall structure and, to a lesser extent, active export of iso-alpha-acids across the plasma membrane. Furthermore, iso-alpha-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelators.

  18. Block of ATP-binding cassette B19 ion channel activity by 5-nitro-2-(3-phenylpropylamino)-benzoic acid impairs polar auxin transport and root gravitropism.

    PubMed

    Cho, Misuk; Henry, Elizabeth M; Lewis, Daniel R; Wu, Guosheng; Muday, Gloria K; Spalding, Edgar P

    2014-12-01

    Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.

  19. 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

  20. Uric acid, indoxyl sulfate, and methylguanidine activate bulbospinal neurons in the RVLM via their specific transporters and by producing oxidative stress.

    PubMed

    Oshima, N; Onimaru, H; Matsubara, H; Uchida, T; Watanabe, A; Takechi, H; Nishida, Y; Kumagai, H

    2015-09-24

    Patients with chronic renal failure often have hypertension, but the cause of hypertension, other than an excess of body fluid, is not well known. We hypothesized that the bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are stimulated by uremic toxins in patients with chronic renal failure. To investigate whether RVLM neurons are sensitive to uremic toxins, such as uric acid, indoxyl sulfate, or methylguanidine, we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons of Wister rats using the whole-cell patch-clamp technique during superfusion with these toxins. A brainstem-spinal cord preparation that preserved the sympathetic nervous system was used for the experiments. During uric acid, indoxyl sulfate, or methylguanidine superfusion, almost all the RVLM neurons were depolarized. To examine the transporters for these toxins on RVLM neurons, histological examinations were performed. The uric acid-, indoxyl sulfate-, and methylguanidine-depolarized RVLM neurons showed the presence of urate transporter 1 (URAT 1), organic anion transporter (OAT)1 or OAT3, and organic cation transporter (OCT)3, respectively. Furthermore, the toxin-induced activities of the RVLM neurons were suppressed by the addition of an anti-oxidation drug (VAS2870, an NAD(P)H oxidase inhibitor), and a histological examination revealed the presence of NAD(P)H oxidase (nox)2 and nox4 in these RVLM neurons. The present results show that uric acid, indoxyl sulfate, and methylguanidine directly stimulate bulbospinal RVLM neurons via specific transporters on these neurons and by producing oxidative stress. These uremic toxins may cause hypertension by activating RVLM neurons.

  1. 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

  2. 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

  3. Excitatory amino acid transporters tonically restrain nTS synaptic and neuronal activity to modulate cardiorespiratory function

    PubMed Central

    2015-01-01

    The nucleus tractus solitarii (nTS) is the initial central termination site for visceral afferents and is important for modulation and integration of multiple reflexes including cardiorespiratory reflexes. Glutamate is the primary excitatory neurotransmitter in the nTS and is removed from the extracellular milieu by excitatory amino acid transporters (EAATs). The goal of this study was to elucidate the role of EAATs in the nTS on basal synaptic and neuronal function and cardiorespiratory regulation. The majority of glutamate clearance in the central nervous system is believed to be mediated by astrocytic EAAT 1 and 2. We confirmed the presence of EAAT 1 and 2 within the nTS and their colocalization with astrocytic markers. EAAT blockade with dl-threo-β-benzyloxyaspartic acid (TBOA) produced a concentration-related depolarization, increased spontaneous excitatory postsynaptic current (EPSC) frequency, and enhanced action potential discharge in nTS neurons. Solitary tract-evoked EPSCs were significantly reduced by EAAT blockade. Microinjection of TBOA into the nTS of anesthetized rats induced apneic, sympathoinhibitory, depressor, and bradycardic responses. These effects mimicked the response to microinjection of exogenous glutamate, and glutamate responses were enhanced by EAAT blockade. Together these data indicate that EAATs tonically restrain nTS excitability to modulate cardiorespiratory function. PMID:26719090

  4. Loratadine and analogues: discovery and preliminary structure-activity relationship of inhibitors of the amino acid transporter B(0)AT2.

    PubMed

    Cuboni, Serena; Devigny, Christian; Hoogeland, Bastiaan; Strasser, Andrea; Pomplun, Sebastian; Hauger, Barbara; Höfner, Georg; Wanner, Klaus T; Eder, Matthias; Buschauer, Armin; Holsboer, Florian; Hausch, Felix

    2014-11-26

    B(0)AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B(0)AT2 with an IC50 of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B(0)AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure-activity relationships. Our studies provide the first chemical tool for B(0)AT2.

  5. A replacement of the active-site aspartic acid residue 293 in mouse cathepsin D affects its intracellular stability, processing and transport in HEK-293 cells.

    PubMed Central

    Partanen, Sanna; Storch, Stephan; Löffler, Hans-Gerhard; Hasilik, Andrej; Tyynelä, Jaana; Braulke, Thomas

    2003-01-01

    The substitution of an active-site aspartic acid residue by asparagine in the lysosomal protease cathepsin D (CTSD) results in a loss of enzyme activity and severe cerebrocortical atrophy in a novel form of neuronal ceroid lipofuscinosis in sheep [Tyynelä, Sohar, Sleat, Gin, Donnelly, Baumann, Haltia and Lobel (2000) EMBO J. 19, 2786-2792]. In the present study we have introduced the corresponding mutation by replacing aspartic acid residue 293 with asparagine (D293N) into the mouse CTSD cDNA to analyse its effect on synthesis, transport and stability in transfected HEK-293 cells. The complete inactivation of mutant D293N mouse CTSD was confirmed by a newly developed fluorimetric quantification system. Moreover, in the heterologous overexpression systems used, mutant D293N mouse CTSD was apparently unstable and proteolytically modified during early steps of the secretory pathway, resulting in a loss of mass by about 1 kDa. In the affected sheep, the endogenous mutant enzyme was stable but also showed the shift in its molecular mass. In HEK-293 cells, the transport of the mutant D293N mouse CTSD to the lysosome was delayed and associated with a low secretion rate compared with wild-type CTSD. These data suggest that the mutation may result in a conformational change which affects stability, processing and transport of the enzyme. PMID:12350228

  6. 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

  7. Epileptic Activity Increases Cerebral Amino Acid Transport Assessed by 18F-Fluoroethyl-l-Tyrosine Amino Acid PET: A Potential Brain Tumor Mimic.

    PubMed

    Hutterer, Markus; Ebner, Yvonne; Riemenschneider, Markus J; Willuweit, Antje; McCoy, Mark; Egger, Barbara; Schröder, Michael; Wendl, Christina; Hellwig, Dirk; Grosse, Jirka; Menhart, Karin; Proescholdt, Martin; Fritsch, Brita; Urbach, Horst; Stockhammer, Guenther; Roelcke, Ulrich; Galldiks, Norbert; Meyer, Philipp T; Langen, Karl-Josef; Hau, Peter; Trinka, Eugen

    2017-01-01

    O-(2-(18)F-fluoroethyl)-l-tyrosine ((18)F-FET) PET is a well-established method increasingly used for diagnosis, treatment planning, and monitoring in gliomas. Epileptic activity, frequently occurring in glioma patients, can influence MRI findings. Whether seizures also affect (18)F-FET PET imaging is currently unknown. The aim of this retrospective analysis was to investigate the brain amino acid metabolism during epileptic seizures by (18)F-FET PET and to elucidate the pathophysiologic background.

  8. 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.

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

    PubMed

    McGinley, Cian; Bishop, David J

    2016-12-01

    McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βmin vitro), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βmin vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

  10. 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.

  11. Bile acids alter the subcellular localization of CNT2 (concentrative nucleoside cotransporter) and increase CNT2-related transport activity in liver parenchymal cells

    PubMed Central

    Fernández-Veledo, Sonia; Huber-Ruano, Isabel; Aymerich, Ivette; Duflot, Sylvie; Casado, F. Javier; Pastor-Anglada, Marçal

    2006-01-01

    CNT2 (concentrative nucleoside cotransporter) is a plasma membrane high-affinity Na+-coupled adenosine transporter, also localized in intracellular structures. This transporter protein may play additional roles other than nucleoside salvage, since it has recently been shown to be under purinergic control via KATP channels, by a mechanism that does not seem to involve changes in its subcellular localization. In an attempt to identify the agents that promote CNT2 trafficking, bile acids were found to increase CNT2-related transport activity in a KATP channel-independent manner in both Fao hepatoma and rat liver parenchymal cells. A maximum effect was recorded after treatment with hydrophilic anions such as TCA (taurocholate). However, this effect did not involve changes in the amount of CNT2 protein, it was instead associated with a subcellular redistribution of CNT2, resulting in an accumulation of the transporter at the plasma membrane. This was deduced from subcellular fractionation studies, biotinylation of plasma membrane proteins and subsequent CNT2 detection in streptavidin precipitates and in vivo confocal microscopic analysis of the distribution of a YFP (yellow fluorescent protein)–CNT2 construct. The induction of CNT2 translocation, triggered by TCA, was inhibited by wortmannin, dibutyryl-AMPc, PD98059 and colchicine, thus suggesting the involvement of the PI3K/ERK (phosphoinositide 3-kinase/extracellular-signal related kinase) pathway in microtubule-dependent activation of recombinant CNT2. These are novel effects of bile-acid physiology and provide the first evidence for short-term regulation of CNT2 translocation into and from the plasma membrane. PMID:16390326

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

    PubMed

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

    2014-09-05

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

  13. The Next Generation Non-competitive Active Polyester Nanosystems for Transferrin Receptor-mediated Peroral Transport Utilizing Gambogic Acid as a Ligand

    PubMed Central

    Saini, P.; Ganugula, R.; Arora, M.; Kumar, M. N. V. Ravi

    2016-01-01

    The current methods for targeted drug delivery utilize ligands that must out-compete endogenous ligands in order to bind to the active site facilitating the transport. To address this limitation, we present a non-competitive active transport strategy to overcome intestinal barriers in the form of tunable nanosystems (NS) for transferrin receptor (TfR) utilizing gambogic acid (GA), a xanthanoid, as its ligand. The NS made using GA conjugated poly(lactide-co-glycolide) (PLGA) have shown non-competitive affinity to TfR evaluated in cell/cell-free systems. The fluorescent PLGA-GA NS exhibited significant intestinal transport and altered distribution profile compared to PLGA NS in vivo. The PLGA-GA NS loaded with cyclosporine A (CsA), a model peptide, upon peroral dosing to rodents led to maximum plasma concentration of CsA at 6 h as opposed to 24 h with PLGA-NS with at least 2-fold higher levels in brain at 72 h. The proposed approach offers new prospects for peroral drug delivery and beyond. PMID:27388994

  14. Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein.

    PubMed

    Kim, Yeun Ju; Guzman-Hernandez, Maria Luisa; Wisniewski, Eva; Echeverria, Nicolas; Balla, Tamas

    2016-02-01

    Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and-B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER-PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER-PM contact zones with a possible link to a devastating human disease.

  15. Effect of gossypol-acetic acid on calcium transport and ATPase activity in plasma membranes from ram and bull spermatozoa.

    PubMed

    Breitbart, H; Rubinstein, S; Nass-Arden, L

    1984-10-01

    The effects of gossypol acetic acid on the activity of Mg-ATPase and Ca-Mg-ATPase and on calcium uptake by plasma membranes from ram and bull spermatozoa were examined. The three parameters were almost completely inhibited by 10 microM gossypol for both ram and bull sperm. In order to assess the effects of higher gossypol concentrations isolated membrane vesicles were loaded with calcium by operating the ATP-dependent calcium pump after which gossypol was added and calcium uptake followed. At 10 microM gossypol, additional calcium uptake was 85% inhibited while at 40 microM a release of the accumulated calcium was observed. The inhibitory effect of 10 microM gossypol was almost completely reversible by simple dilution of gossypol-treated membranes, whilst at 40 microM the effect was only 50% reversible. The data show a high degree of similarity between bull and ram, suggesting minimal differences between the two species as far as the structure and function of the sperm plasma membrane is concerned.

  16. Dexmedetomidine increases the activity of excitatory amino acid transporter type 3 expressed in Xenopus oocytes: the involvement of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Do, Sang-Hwan; Park, Seong-Joo; Shin, Hyun-Jung; Paik, Hye-Sun; Zuo, Zhiyi; Yoon, Hea-Jo; Ryu, Jung-Hee

    2014-09-05

    Dexmedetomidine, an α2 adrenergic agonist, has neuroprotective and anticonvulsant properties in addition to its sedative and anxiolytic effects. We hypothesized that dexmedetomidine would increase the activity of excitatory amino acid transporter type 3 (EAAT3) and that this effect would involve protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K), two protein kinases known to regulate EAAT3 activity. EAAT3 was expressed in Xenopus oocytes by injecting its mRNA. Two-electrode voltage clamping was used to record membrane currents before, during, and after application of 30 μM l-glutamate in the presence of 0.1-30 nM dexmedetomidine. Dexmedetomidine-treated oocytes were also exposed to a PKC activator (phorbol-12-myristate-13-acetate [PMA]), PKC inhibitors (chelerythrine, staurosporine, and calphostin C), and PI3K inhibitors (wortmannin and LY294002) before current measurement. Dexmedetomidine application resulted in a concentration-dependent increase in the EAAT3 activity in response to l-glutamate. The kinetic study showed that dexmedetomidine significantly increased the Vmax without changing Km. Treatment of oocytes with PMA significantly increased transporter currents compared with controls, but treatment with dexmedetomidine plus PMA did not further increase the response compared with PMA or dexmedetomidine alone. In addition, pre-treatment of oocytes with PKC inhibitors and PI3K inhibitors significantly abolished the dexmedetomidine-enhanced EAAT3 activity. These results suggest that dexmedetomidine increases the activity of EAAT3 expressed in Xenopus oocytes. PKC and PI3K seem to mediate this effect. These findings may explain the neuroprotective and anticonvulsant effects of dexmedetomidine.

  17. Rest interval duration does not influence adaptations in acid/base transport proteins following 10 weeks of sprint-interval training in active women.

    PubMed

    McGinley, Cian; Bishop, David John

    2017-02-01

    The removal of protons (H(+)) produced during intense exercise is important for skeletal muscle function, yet it remains unclear how best to structure exercise training to improve muscle pH regulation. We investigated whether 4 weeks of work-matched, sprint-interval training (SIT), performed 3 days per week, with either 1 min (Rest-1; n = 7) or 5 min (Rest-5; n = 7) of rest between sprints, influenced adaptations in acid/base transport protein content, non-bicarbonate muscle buffer capacity (βmin vitro), and exercise capacity in active women. Following one week of post-testing, comprising a biopsy, a repeated-sprint ability (RSA) test, and a graded-exercise test, maintenance of adaptations was then studied by reducing SIT volume to one day per week for a further 5 weeks. After 4 weeks of SIT, there was increased protein abundance of monocarboxylate transporter (MCT)1, sodium/hydrogen exchanger (NHE)1, and carbonic anhydrase (CA)XIV for both groups, but rest interval duration did not influence the adaptive response. In contrast, greater improvements in total work performed during the RSA test after 4 weeks of SIT was evident for Rest-5 compared to Rest-1 [effect size (ES): 0.51; 90% confidence limits ±0.37), whereas both groups had similarly modest improvements in VO2peak When training volume was reduced to one day per week, enhanced acid/base transport protein abundance was maintained, although NHE1 content increased further for Rest-5 only. Finally, our data support intracellular lactate as a signaling molecule for inducing MCT1 expression, but neither lactate nor H(+) accumulation appear to be important signaling factors in MCT4 regulation.

  18. 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.

  19. Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids: Part II. Effects on intestinal histology and active nutrient transport.

    PubMed

    Walsh, M C; Rostagno, M H; Gardiner, G E; Sutton, A L; Richert, B T; Radcliffe, J S

    2012-08-01

    The objective of this study was to evaluate the effects of water-delivered, direct-fed microbials (DFM) or organic acids on intestinal morphology and active nutrient absorption in weanling pigs after deliberate Salmonella infection. Pigs (n = 88) were weaned at 19 ± 2 d of age and assigned to 1 of the following treatments, which were administered for 14 d: 1) control diet; 2) control diet + DFM (Enterococcus faecium, Bacillus subtilis, and Bacillus licheniformis) in drinking water at 10(9) cfu/L for each strain of bacteria; 3) control diet + organic acid-based blend (predominantly propionic, acetic, and benzoic acids) in drinking water at 2.58 mL/L; and 4) control diet + 55 mg/kg carbadox. Pigs were challenged with 10(10) cfu Salmonella enterica var Typhimurium 6 d after commencement of treatments. Pigs (n = 22/d) were harvested before Salmonella challenge and on d 2, 4, and 8 after challenge. Duodenal, jejunal, and ileal mucosal tissues were sampled for measurement of villus height and crypt depth. Jejunal tissue was sampled for determination of active nutrient absorption in modified Ussing chambers. Duodenal villus height was greater in pigs fed in-feed antibiotic before infection (P < 0.05). Jejunal crypts were deeper in DFM- and acid-treated pigs on d 4 after infection compared with all other treatments (P < 0.05). Salmonella infection resulted in a linear decrease in phosphorus (P < 0.001) and glucose (P < 0.05) active transport, and an increase (P < 0.001) in glutamine uptake immediately after challenge. Salmonella infection reduced basal short-circuit current (I(sc)); however, water-delivered DFM or organic acid treatments caused greater basal I(sc) on d 2 after challenge than did carbadox. Carbachol-induced chloride ion secretion was greatest in negative control pigs before infection (P < 0.01) and DFM-treated pigs (P < 0.05) after infection. In conclusion, both the DFM and acidification treatments induced increases in basal active ion movement and jejunal

  20. 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.

  1. 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

  2. Laboratory Exercise on Active Transport.

    ERIC Educational Resources Information Center

    Stalheim-Smith, Ann; Fitch, Greg K.

    1985-01-01

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

  3. A new multicomponent salt of imidazole and tetrabromoterepthalic acid: Structural, optical, thermal, electrical transport properties and antibacterial activity along with Hirshfeld surface analysis

    NASA Astrophysics Data System (ADS)

    Dey, Sanjoy Kumar; Saha, Rajat; Singha, Soumen; Biswas, Susobhan; Layek, Animesh; Middya, Somnath; Ray, Partha Pratim; Bandhyopadhyay, Debasis; Kumar, Sanjay

    2015-06-01

    Herein, we report the structural, optical, thermal and electrical transport properties of a new multicomponent salt (TBTA2-)·2(IM+)·(water) [TBTA-IM] of tetrabromoterepthalic acid (TBTA) with imidazole (IM). The crystal structure of TBTA-IM is determined by both the single crystal and powder X-ray diffraction techniques. The structural analysis has revealed that the supramolecular charge assisted O-⋯Hsbnd N+ hydrogen bonding and Br⋯π interactions play the most vital role in formation of this multicomponent supramolecular assembly. The Hirshfeld surface analysis has been carried out to investigate supramolecular interactions and associated 2D fingerprint plots reveal the relative contribution of these interactions in the crystal structure quantitatively. According to theoretical analysis the HOMO-LUMO energy gap of the salt is 2.92 eV. The salt has been characterized by IR, UV-vis and photoluminescence spectroscopic studies. It shows direct optical transition with band gaps of 4.1 eV, which indicates that the salt is insulating in nature. The photoluminescence spectrum of the salt is significantly different from that of TBTA. Further, a comparative study on the antibacterial activity of the salt with respect to imidazole, Gatifloxacin and Ciprofloxacin has been performed. Moreover, the current-voltage (I-V) characteristic of ITO/TBTA-IM/Al sandwich structure exhibits good rectifying property and the electron tunneling process governs the electrical transport mechanism of the device.

  4. Activated transport in AMTEC electrodes

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  5. Regulators of Slc4 bicarbonate transporter activity

    PubMed Central

    Thornell, Ian M.; Bevensee, Mark O.

    2015-01-01

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

  6. Activated transport in AMTEC electrodes

    SciTech Connect

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

    1992-07-01

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

  7. Activated transport in AMTEC electrodes

    SciTech Connect

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

    1992-01-01

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

  8. 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

  9. 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

  10. Niflumic acid activates additional currents of the human glial L-glutamate transporter EAAT1 in a substrate-dependent manner.

    PubMed

    Takahashi, Kanako; Ishii-Nozawa, Reiko; Takeuchi, Koichi; Nakazawa, Ken; Sekino, Yuko; Sato, Kaoru

    2013-01-01

    The astrocytic L-glutamate (L-Glu) transporter EAAT1 participates in the removal of L-Glu from the synaptic cleft and maintenance of non-toxic concentrations in the extracellular fluid. We have shown that niflumic acid (NFA), a non-steroidal anti-inflammatory drug (NSAIDs), alters L-Glu-induced EAAT1 currents in a voltage-dependent manner using the two-electrode voltage clamp technique in Xenopus oocytes expressing EAAT1. In this study, we characterised the effects of NFA on each type of ion-flux through EAAT1. NFA modulated currents induced by both L-Glu and L-aspartate (L-Asp) in a voltage-dependent manner. Ion-substitution experiments revealed that the activation of additional H(+) conductance was involved in the modulation of currents induced by L-Asp and L-Glu, but Cl(-) was involved only with the L-Asp currents. NFA activated additional currents of EAAT1 in a substrate-dependent manner.

  11. 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.

  12. 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

  13. 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

  14. Focal Thalamic Degeneration from Ethanol and Thiamine Deficiency is Associated with Neuroimmune Gene Induction, Microglial Activation, and Lack of Monocarboxylic Acid Transporters

    PubMed Central

    Qin, Liya; Crews, Fulton T

    2014-01-01

    Background Wernicke's encephalopathy-Korsakoff syndrome (WE-KS) is common in alcoholics, caused by thiamine deficiency (TD; vitamin B1) and associated with lesions to the thalamus (THAL). Although TD alone can cause WE, the high incidence in alcoholism suggests that TD and ethanol (EtOH) interact. Methods Mice in control, TD, or EtOH groups alone or combined were studied after 5 or 10 days of treatment. THAL and entorhinal cortex (ENT) histochemistry and mRNA were assessed. Results Combined EtOH-TD treatment for 5 days (EtOH-TD5) showed activated microglia, proinflammatory gene induction and THAL neurodegeneration that was greater than that found with TD alone (TD5), whereas 10 days resulted in marked THAL degeneration and microglial-neuroimmune activation in both groups. In contrast, 10 days of TD did not cause ENT degeneration. Interestingly, in ENT, TD10 activated microglia and astrocytes more than EtOH-TD10. In THAL, multiple astrocytic markers were lost consistent with glial cell loss. TD blocks glucose metabolism more than acetate. Acetate derived from hepatic EtOH metabolism is transported by monocarboxylic acid transporters (MCT) into both neurons and astrocytes that use acetyl-CoA synthetase (AcCoAS) to generate cellular energy from acetate. MCT and AcCoAS expression in THAL is lower than ENT prompting the hypothesis that focal THAL degeneration is related to insufficient MCT and AcCoAS in THAL. To test this hypothesis, we administered glycerin triacetate (GTA) to increase blood acetate and found it protected the THAL from TD-induced degeneration. Conclusions Our findings suggest that EtOH potentiates TD-induced THAL degeneration through neuroimmune gene induction. The findings support the hypothesis that TD deficiency inhibits global glucose metabolism and that a reduced ability to process acetate for cellular energy results in THAL focal degeneration in alcoholics contributing to the high incidence of Wernicke-Korsakoff syndrome in alcoholism. PMID

  15. Differential activation of pregnane X receptor by carnosic acid, carnosol, ursolic acid, and rosmarinic acid.

    PubMed

    Seow, Chun Ling; Lau, Aik Jiang

    2017-03-10

    Pregnane X receptor (PXR) regulates the expression of many genes, including those involved in drug metabolism and transport, and has been linked to various diseases, including inflammatory bowel disease. In the present study, we determined whether carnosic acid and other chemicals in rosemary extract (carnosol, ursolic acid, and rosmarinic acid) are PXR activators. As assessed in dual-luciferase reporter gene assays, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, activated human PXR (hPXR) and mouse PXR (mPXR), whereas carnosol and ursolic acid, but not carnosic acid or rosmarinic acid, activated rat PXR (rPXR). Dose-response experiments indicated that carnosic acid, carnosol, and ursolic acid activated hPXR with EC50 values of 0.79, 2.22, and 10.77μM, respectively. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, transactivated the ligand-binding domain of hPXR and recruited steroid receptor coactivator-1 (SRC-1), SRC-2, and SRC-3 to the ligand-binding domain of hPXR. Carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, increased hPXR target gene expression, as shown by an increase in CYP3A4, UGT1A3, and ABCB1 mRNA expression in LS180 human colon adenocarcinoma cells. Rosmarinic acid did not attenuate the extent of hPXR activation by rifampicin, suggesting it is not an antagonist of hPXR. Overall, carnosic acid, carnosol, and ursolic acid, but not rosmarinic acid, are hPXR agonists, and carnosic acid shows species-dependent activation of hPXR and mPXR, but not rPXR. The findings provide new mechanistic insight on the effects of carnosic acid, carnosol, and ursolic acid on PXR-mediated biological effects.

  16. 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.

  17. Potent antimycobacterial activity of the pyridoxal isonicotinoyl hydrazone analog 2-pyridylcarboxaldehyde isonicotinoyl hydrazone: a lipophilic transport vehicle for isonicotinic acid hydrazide.

    PubMed

    Ellis, Samantha; Kalinowski, Danuta S; Leotta, Lisa; Huang, Michael L H; Jelfs, Peter; Sintchenko, Vitali; Richardson, Des R; Triccas, James A

    2014-02-01

    The rise in drug-resistant strains of Mycobacterium tuberculosis is a major threat to human health and highlights the need for new therapeutic strategies. In this study, we have assessed whether high-affinity iron chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class can restrict the growth of clinically significant mycobacteria. Screening a library of PIH derivatives revealed that one compound, namely, 2-pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH), exhibited nanomolar in vitro activity against Mycobacterium bovis bacille Calmette-Guérin and virulent M. tuberculosis. Interestingly, PCIH is derived from the condensation of 2-pyridylcarboxaldehyde with the first-line antituberculosis drug isoniazid [i.e., isonicotinic acid hydrazide (INH)]. PCIH displayed minimal host cell toxicity and was effective at inhibiting growth of M. tuberculosis within cultured macrophages and also in vivo in mice. Further, PCIH restricted mycobacterial growth at high bacterial loads in culture, a property not observed with INH, which shares the isonicotinoyl hydrazide moiety with PCIH. When tested against Mycobacterium avium, PCIH was more effective than INH at inhibiting bacterial growth in broth culture and in macrophages, and also reduced bacterial loads in vivo. Complexation of PCIH with iron decreased its effectiveness, suggesting that iron chelation may play some role in its antimycobacterial efficacy. However, this could not totally account for its potent efficacy, and structure-activity relationship studies suggest that PCIH acts as a lipophilic vehicle for the transport of its intact INH moiety into the mammalian cell and the mycobacterium. These results demonstrate that iron-chelating agents such as PCIH may be of benefit in the treatment and control of mycobacterial infection.

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

    PubMed

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

    2016-01-01

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

  19. 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.

  20. His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

    PubMed Central

    Lu, Jade M.-Y.; Bush, Daniel R.

    1998-01-01

    The proton–sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton–sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction. PMID:9671798

  1. 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.

  2. The Amino Acid Transporter JhI-21 Coevolves with Glutamate Receptors, Impacts NMJ Physiology, and Influences Locomotor Activity in Drosophila Larvae.

    PubMed

    Ziegler, Anna B; Augustin, Hrvoje; Clark, Nathan L; Berthelot-Grosjean, Martine; Simonnet, Mégane M; Steinert, Joern R; Geillon, Flore; Manière, Gérard; Featherstone, David E; Grosjean, Yael

    2016-01-25

    Changes in synaptic physiology underlie neuronal network plasticity and behavioral phenomena, which are adjusted during development. The Drosophila larval glutamatergic neuromuscular junction (NMJ) represents a powerful synaptic model to investigate factors impacting these processes. Amino acids such as glutamate have been shown to regulate Drosophila NMJ physiology by modulating the clustering of postsynaptic glutamate receptors and thereby regulating the strength of signal transmission from the motor neuron to the muscle cell. To identify amino acid transporters impacting glutmatergic signal transmission, we used Evolutionary Rate Covariation (ERC), a recently developed bioinformatic tool. Our screen identified ten proteins co-evolving with NMJ glutamate receptors. We selected one candidate transporter, the SLC7 (Solute Carrier) transporter family member JhI-21 (Juvenile hormone Inducible-21), which is expressed in Drosophila larval motor neurons. We show that JhI-21 suppresses postsynaptic muscle glutamate receptor abundance, and that JhI-21 expression in motor neurons regulates larval crawling behavior in a developmental stage-specific manner.

  3. 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

  4. 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.

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

    SciTech Connect

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

    2012-11-15

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

  6. Controlling Salmonella infection in weanling pigs through water delivery of direct-fed microbials or organic acids: Part II. Effects on intestinal histology and active nutrient transport

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate the effects of water-delivered direct-fed microbials (DFM) or organic acids on intestinal morphology and active nutrient absorption in weanling pigs following deliberate Salmonella infection. Pigs (n = 88) were weaned at 19 ± 2 d of age and assigned to one...

  7. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice

    PubMed Central

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

    2016-01-01

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  8. Dehydroeburicoic Acid from Antrodia camphorata Prevents the Diabetic and Dyslipidemic State via Modulation of Glucose Transporter 4, Peroxisome Proliferator-Activated Receptor α Expression and AMP-Activated Protein Kinase Phosphorylation in High-Fat-Fed Mice.

    PubMed

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

    2016-06-03

    This study investigated the potential effects of dehydroeburicoic acid (TT), a triterpenoid compound from Antrodia camphorata, in vitro and examined the effects and mechanisms of TT on glucose and lipid homeostasis in high-fat-diet (HFD)-fed mice. The in vitro study examined the effects of a MeOH crude extract (CruE) of A. camphorata and Antcin K (AnK; the main constituent of fruiting body of this mushroom) on membrane glucose transporter 4 (GLUT4) and phospho-Akt in C2C12 myoblasts cells. The in vitro study demonstrated that treatment with CruE, AnK and TT increased the membrane levels of glucose transporter 4 (GLUT4) and phospho-Akt at different concentrations. The animal experiments were performed for 12 weeks. Diabetic mice were randomly divided into six groups after 8 weeks of HFD-induction and treated with daily oral gavage doses of TT (at three dose levels), fenofibrate (Feno) (at 0.25 g/kg body weight), metformin (Metf) (at 0.3 g/kg body weight) or vehicle for another 4 weeks while on an HFD diet. HFD-fed mice exhibited increased blood glucose levels. TT treatment dramatically lowered blood glucose levels by 34.2%~43.4%, which was comparable to the antidiabetic agent-Metf (36.5%). TT-treated mice reduced the HFD-induced hyperglycemia, hypertriglyceridemia, hyperinsulinemia, hyperleptinemia, and hypercholesterolemia. Membrane levels of GLUT4 were significantly higher in CruE-treated groups in vitro. Skeletal muscle membrane levels of GLUT4 were significantly higher in TT-treated mice. These groups of mice also displayed lower mRNA levels of glucose-6-phosphatase (G6 Pase), an inhibitor of hepatic glucose production. The combination of these agents produced a net hypoglycemic effect in TT-treated mice. TT treatment enhanced the expressions of hepatic and skeletal muscle AMP-activated protein kinase (AMPK) phosphorylation in mice. TT-treated mice exhibited enhanced expression of hepatic fatty acid oxidation enzymes, including peroxisome proliferator-activated

  9. Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA c...

  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. 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.

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

    PubMed

    Shi, Yigong

    2013-01-01

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

  13. 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.

  14. 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

  15. 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.

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

    PubMed Central

    2011-01-01

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

  17. 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.

  18. Energetics of active transport processes.

    PubMed

    Essig, A; Caplan, S R

    1968-12-01

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

  19. Expression and/or activity of the SVCT2 ascorbate transporter may be decreased in many aggressive cancers, suggesting potential utility for sodium bicarbonate and dehydroascorbic acid in cancer therapy.

    PubMed

    McCarty, Mark F

    2013-10-01

    Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor whose elevated activity in many cancers helps them to survive under hypoxic conditions and enhances their capacity to grow invasively, establish metastases, and survive chemo- or radiotherapy. Optimal intracellular levels of ascorbate suppress the level and transcriptional activity of HIF-1under normoxic or mildly hypoxic conditions by supporting the activity of proly and asparagyl hydroxylases that target HIF-1alpha. High intracellular ascorbate can also work in various ways to down-regulate activation of NF-kappaB which, like HIF-1 is constitutively active in many cancers and promotes aggressive behavior - in part by promoting transcription of HIF-1alpha. Yet recent evidence suggests that, even in the context of adequate ascorbate nutrition, the intracellular ascorbate content of many aggressive cancers may be supoptimal for effective HIF-1 control. This likely reflects low expression or activity of the SVCT2 ascorbate transporter. The expression of SVCT2 in cancers has so far received little study; but the extracellular acidity characteristic of many tumors would be expected to reduce the activity of this transporter, which has a mildly alkaline pH optimum. Unfortunately, since SVCT2 has a high affinity for ascorbate, and its activity is nearly saturated at normal healthy serum levels of this vitamin, increased oral administration of ascorbate would be unlikely to have much impact on the intracellular ascorbate content of tumors. However, cancers in which HIF-1 is active express high levels of glucose transporters such as GLUT-1, and these transporters can promote influx of dehydroascorbic acid (DHA) via facilitated diffusion; once inside the cell, DHA is rapidly reduced to ascorbate, which effectively is "trapped" within the cell. Hence, episodic intravenous infusions of modest doses of DHA may have potential for optimizing the intracellular ascorbate content of cancers, potentially

  20. 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...

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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

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

    PubMed

    Ozcan, Nuran; Krämer, Reinhard; Morbach, Susanne

    2005-07-01

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

  19. The transport properties of activated carbon fibers

    SciTech Connect

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

    1990-07-01

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

  20. The Transport Properties of Activated Carbon Fibers

    DOE R&D Accomplishments Database

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

    1990-07-01

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

  1. 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.

  2. 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

  3. Resynthesis of phosphatidylinositol in permeabilized neutrophils following phospholipase Cbeta activation: transport of the intermediate, phosphatidic acid, from the plasma membrane to the endoplasmic reticulum for phosphatidylinositol resynthesis is not dependent on soluble lipid carriers or vesicular transport.

    PubMed Central

    Whatmore, J; Wiedemann, C; Somerharju, P; Swigart, P; Cockcroft, S

    1999-01-01

    Receptor-mediated phospholipase C (PLC) hydrolysis of phosphoinositides is accompanied by the resynthesis of phosphatidylinositol (PI). Hydrolysis of phosphoinositides occurs at the plasma membrane, and the resulting diacylglycerol (DG) is converted into phosphatidate (PA). Two enzymes located at the endoplasmic reticulum (ER) function sequentially to convert PA back into PI. We have established an assay whereby the resynthesis of PI could be followed in permeabilized cells. In the presence of [gamma-32P]ATP, DG generated by PLC activation accumulates label when converted into PA. The 32P-labelled PA is subsequently converted into labelled PI. The formation of labelled PI reports the arrival of labelled PA from the plasma membrane to the ER. Cytosol-depleted, permeabilized human neutrophils are capable of PI resynthesis following stimulation of PLCbeta (in the presence of phosphatidylinositol-transfer protein), provided that CTP and inositol are also present. We also found that wortmannin, an inhibitor of endocytosis, or cooling the cells to 15 degrees C did not stop PI resynthesis. We conclude that PI resynthesis is dependent neither on vesicular transport mechanisms nor on freely diffusible, soluble transport proteins. Phosphatidylcholine-derived PA generated by the ADP-ribosylation-factor-stimulated phospholipase D pathway was found to accumulate label, reflecting the rapid cycling of PA to DG, and back. This labelled PA was not converted into PI. We conclude that PA derived from the PLC pathway is selected for PI resynthesis, and its transfer to the ER could be membrane-protein-mediated at sites of close membrane contact. PMID:10393103

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. LAT1 activity of carboxylic acid bioisosteres: Evaluation of hydroxamic acids as substrates.

    PubMed

    Zur, Arik A; Chien, Huan-Chieh; Augustyn, Evan; Flint, Andrew; Heeren, Nathan; Finke, Karissa; Hernandez, Christopher; Hansen, Logan; Miller, Sydney; Lin, Lawrence; Giacomini, Kathleen M; Colas, Claire; Schlessinger, Avner; Thomas, Allen A

    2016-10-15

    Large neutral amino acid transporter 1 (LAT1) is a solute carrier protein located primarily in the blood-brain barrier (BBB) that offers the potential to deliver drugs to the brain. It is also up-regulated in cancer cells, as part of a tumor's increased metabolic demands. Previously, amino acid prodrugs have been shown to be transported by LAT1. Carboxylic acid bioisosteres may afford prodrugs with an altered physicochemical and pharmacokinetic profile than those derived from natural amino acids, allowing for higher brain or tumor levels of drug and/or lower toxicity. The effect of replacing phenylalanine's carboxylic acid with a tetrazole, acylsulfonamide and hydroxamic acid (HA) bioisostere was examined. Compounds were tested for their ability to be LAT1 substrates using both cis-inhibition and trans-stimulation cell assays. As HA-Phe demonstrated weak substrate activity, its structure-activity relationship (SAR) was further explored by synthesis and testing of HA derivatives of other LAT1 amino acid substrates (i.e., Tyr, Leu, Ile, and Met). The potential for a false positive in the trans-stimulation assay caused by parent amino acid was evaluated by conducting compound stability experiments for both HA-Leu and the corresponding methyl ester derivative. We concluded that HA's are transported by LAT1. In addition, our results lend support to a recent account that amino acid esters are LAT1 substrates, and that hydrogen bonding may be as important as charge for interaction with the transporter binding site.

  10. Antiprotozoal activity of betulinic acid derivatives.

    PubMed

    Domínguez-Carmona, D B; Escalante-Erosa, F; García-Sosa, K; Ruiz-Pinell, G; Gutierrez-Yapu, D; Chan-Bacab, M J; Giménez-Turba, A; Peña-Rodríguez, L M

    2010-04-01

    Betulinic acid (1), isolated from the crude extract of the leaves of Pentalinon andrieuxii (Apocynaceae), together with betulinic acid acetate (2), betulonic acid (3), betulinic acid methyl ester (4), and betulin (5) were evaluated for their antiprotozoal activity. The results showed that modifying the C-3 position increases leishmanicidal activity while modification of the C-3 and C-28 positions decreases trypanocidal activity.

  11. 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.

  12. Ratchet transport powered by chiral active particles

    PubMed Central

    Ai, Bao-quan

    2016-01-01

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

  13. 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.

  14. 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.

  15. Stochastic steps in secondary active sugar transport.

    PubMed

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

    2016-07-05

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

  16. Stochastic steps in secondary active sugar transport

    PubMed Central

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

    2016-01-01

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

  17. 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.

  18. 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

  19. 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 +/.

  20. 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

  1. Development of novel active transport membrande devices

    SciTech Connect

    Laciak, D.V.

    1994-11-01

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

  2. Health Impacts of Active Transportation in Europe

    PubMed Central

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

    2016-01-01

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

  3. Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula.

    PubMed

    Yang, Chengbo; Yang, Xiaojian; Lackeyram, Dale; Rideout, Todd C; Wang, Zirong; Stoll, Barbara; Yin, Yulong; Burrin, Douglas G; Fan, Ming Z

    2016-06-01

    Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA co-transporter (B(0)AT1, encoded by the SLC6A19 gene) plays a dominant role for apical uptake of large neutral AA including L-Gln, we hypothesized that high apical Na(+)-Gln co-transport activity, and B(0)AT1 (SLC6A19) in co-expression with angiotensin-converting enzyme 2 (ACE2) were expressed along the entire small intestinal crypt-villus axis in young animals via unique control mechanisms. Kinetics of Na(+)-Gln co-transport activity in the apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from liquid formula-fed young pigs, were measured with the membrane potential being clamped to zero using thiocyanate. Apical maximal Na(+)-Gln co-transport activity was much higher (p < 0.05) in the upper villus cells than in the middle villus (by 29 %) and the crypt (by 30 %) cells, whereas Na(+)-Gln co-transport affinity was lower (p < 0.05) in the upper villus cells than in the middle villus and the crypt cells. The B(0)AT1 (SLC6A19) mRNA abundance was lower (p < 0.05) in the crypt (by 40-47 %) than in the villus cells. There were no significant differences in B(0)AT1 and ACE2 protein abundances on the apical membrane among the upper villus, the middle villus and the crypt cells. Our study suggests that piglet fast growth is associated with very high intestinal apical Na(+)-neutral AA uptake activities via abundantly co-expressing B(0)AT1 and ACE2 proteins in the apical membrane and by transcribing the B(0)AT1 (SLC6A19) gene in the epithelia along the entire crypt-villus axis.

  4. Vitamin D Transport Proteins Megalin and Disabled-2 Are Expressed in Prostate and Colon Epithelial Cells and Are Induced and Activated by All-Trans-Retinoic Acid

    PubMed Central

    Ternes, Shantel B.; Rowling, Matthew J.

    2014-01-01

    Megalin and disabled-2 (Dab2) are essential for uptake of the 25-hydroxycholecalciferol (25D3)-vitamin D binding protein (DBP) complex in tissues. In the kidney, this mechanism regulates serum 25D3 levels and production of 1,25-dihydroxycholecalciferol (1,25D3) by CYP27B1 for systemic use. Previously, we showed that mammary epithelial cells expressing CYP27B1 express megalin and Dab2 and internalize DBP by endocytosis, indicating 25D3 was accessible for conversion to 1,25D3 in extra-renal tissues. Moreover, induction of megalin and Dab2 (protein and mRNA abundance) by all-trans-retinoic acid (RA) enhanced DBP uptake. This suggests megalin and Dab2 play a central role in uptake of vitamin D and may predict actions of vitamin D in extra-renal tissues. Here, we characterized megalin and Dab2 expression and uptake of DBP in transformed human prostate and colon epithelial cells. Megalin and Dab2 were expressed in prostate and colon epithelial cells, which was markedly enhanced following treatment with RA. Furthermore, DBP uptake was stimulated by low-dose RA supplementation in LNCaP, PC-3, and Caco-2 cells. Taken together, these are the first studies to our knowledge that have demonstrated modulated expression of megalin and Dab2, as well as an association between increased expression of endocytic proteins with DBP uptake in prostate and colon cells. PMID:23909735

  5. 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

  6. 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.

  7. 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.

  8. Fluid transport by active elastic membranes

    NASA Astrophysics Data System (ADS)

    Evans, Arthur A.; Lauga, Eric

    2011-09-01

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

  9. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

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

    2016-01-01

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

  10. 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

  11. 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.

  12. Vesicles protect activated acetic acid.

    PubMed

    Todd, Zoe R; House, Christopher H

    2014-10-01

    Abstract Methyl thioacetate, or activated acetic acid, has been proposed to be central to the origin of life and an important energy currency molecule in early cellular evolution. We have investigated the hydrolysis of methyl thioacetate under various conditions. Its uncatalyzed rate of hydrolysis is about 3 orders of magnitude faster (K=0.00663 s(-1); 100°C, pH 7.5, concentration=0.33 mM) than published rates for its catalyzed production, making it unlikely to accumulate under prebiotic conditions. However, our experiments showed that methyl thioacetate was protected from hydrolysis when inside its own hydrophobic droplets. Further, we found that methyl thioacetate protection from hydrolysis was also possible in droplets of hexane and in the membranes of nonanoic acid vesicles. Thus, the hydrophobic regions of prebiotic vesicles and early cell membranes could have offered a refuge for this energetic molecule, increasing its lifetime in close proximity to the reactions for which it would be needed. This model of early energy storage evokes an additional critical function for the earliest cell membranes.

  13. 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.

  14. Histone H3K4 trimethylation by MLL3 as part of ASCOM complex is critical for NR activation of bile acid transporter genes and is downregulated in cholestasis

    PubMed Central

    Li, Yanfeng; Surapureddi, S.; Balasubramaniyan, N.; Ahn, Jaeyong; Goldstein, J. A.; Suchy, Frederick J.

    2011-01-01

    The nuclear receptor Farnesoid x receptor (FXR) is a critical regulator of multiple genes involved in bile acid homeostasis. The coactivators attracted to promoters of FXR target genes and epigenetic modifications that occur after ligand binding to FXR have not been completely defined, and it is unknown whether these processes are disrupted during cholestasis. Using a microarray, we identified decreased expression of mixed lineage leukemia 3 (MLL3), a histone H3 lysine 4 (H3K4) lysine methyl transferase at 1 and 3 days of post-common bile duct ligation (CBDL) in mice. Chromatin immunoprecipitation analysis (ChIP) analysis revealed that H3K4me3 of transporter promoters by MLL3 as part of activating signal cointegrator-2 -containing complex (ASCOM) is essential for activation of bile salt export pump (BSEP), multidrug resistance associated protein 2 (MRP2), and sodium taurocholate cotransporting polypeptide (NTCP) genes by FXR and glucocorticoid receptor (GR). Knockdown of nuclear receptor coactivator 6 (NCOA6) or MLL3/MLL4 mRNAs by small interfering RNA treatment led to a decrease in BSEP and NTCP mRNA levels in hepatoma cells. Human BSEP promoter transactivation by FXR/RXR was enhanced in a dose-dependent fashion by NCOA6 cDNA coexpression and decreased by AdsiNCOA6 infection in HepG2 cells. GST-pull down assays showed that domain 3 and 5 of NCOA6 (LXXLL motifs) interacted with FXR and that the interaction with domain 5 was enhanced by chenodeoxycholic acid. In vivo ChIP assays in HepG2 cells revealed ligand-dependent recruitment of ASCOM complex to FXR element in BSEP and GR element in NTCP promoters, respectively. ChIP analysis demonstrated significantly diminished recruitment of ASCOM complex components and H3K4me3 to Bsep and Mrp2 promoter FXR elements in mouse livers after CBDL. Taken together, these data show that the “H3K4me3” epigenetic mark is essential to activation of BSEP, NTCP, and MRP2 genes by nuclear receptors and is downregulated in cholestasis

  15. Bursts of Active Transport in Living Cells

    NASA Astrophysics Data System (ADS)

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-01

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

  16. Bursts of active transport in living cells.

    PubMed

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-15

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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Acid Rain: Activities for Science Teachers.

    ERIC Educational Resources Information Center

    Johnson, Eric; And Others

    1983-01-01

    Seven complete secondary/college level acid rain activities are provided. Activities include overview; background information and societal implications; major concepts; student objectives; vocabulary/material lists; procedures; instructional strategies; and questions/discussion and extension suggestions. Activities consider effects of acid rain on…

  4. 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.

  5. 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.

  6. 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.

  7. Active learning in transportation engineering education

    NASA Astrophysics Data System (ADS)

    Weir, Jennifer Anne

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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. Activation of carboxylic acids in asymmetric organocatalysis.

    PubMed

    Monaco, Mattia Riccardo; Poladura, Belén; Diaz de Los Bernardos, Miriam; Leutzsch, Markus; Goddard, Richard; List, Benjamin

    2014-07-01

    Organocatalysis, catalysis using small organic molecules, has recently evolved into a general approach for asymmetric synthesis, complementing both metal catalysis and biocatalysis. Its success relies to a large extent upon the introduction of novel and generic activation modes. Remarkably though, while carboxylic acids have been used as catalyst directing groups in supramolecular transition-metal catalysis, a general and well-defined activation mode for this useful and abundant substance class is still lacking. Herein we propose the heterodimeric association of carboxylic acids with chiral phosphoric acid catalysts as a new activation principle for organocatalysis. This self-assembly increases both the acidity of the phosphoric acid catalyst and the reactivity of the carboxylic acid. To illustrate this principle, we apply our concept in a general and highly enantioselective catalytic aziridine-opening reaction with carboxylic acids as nucleophiles.

  13. 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.

  14. Single-molecule detection with active transport

    NASA Astrophysics Data System (ADS)

    Ball, David Allan

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

  15. 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

  16. 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.

  17. 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).

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. Transport in active systems crowded by obstacles

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  3. 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.

  4. 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.

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

    PubMed

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

    2017-01-25

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

  6. 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.

  7. 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.

  8. 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.

  9. Building biologically active nucleic acid nanocomplexes.

    PubMed

    Smith, C I Edvard; Lundin, Karin E; Simonson, Oscar E; Moreno, Pedro M D; Svahn, Mathias G; Wenska, Malgorzata; Strömberg, Roger

    2008-01-01

    The Bioplex technology allows the hybridization of functional entities to various forms of nucleic acids by the use of synthetic nucleic acid analogs. Such supramolecular assemblies can be made in a predetermined fashion and can confer new properties. The Zorro technology is based on a novel construct generated to simultaneously bind to both DNA strands. Such compounds may have gene silencing activity.

  10. Primary cultures of rat cortical microglia treated with nicotine increases in the expression of excitatory amino acid transporter 1 (GLAST) via the activation of the α7 nicotinic acetylcholine receptor.

    PubMed

    Morioka, N; Tokuhara, M; Nakamura, Y; Idenoshita, Y; Harano, S; Zhang, F F; Hisaoka-Nakashima, K; Nakata, Y

    2014-01-31

    Although the clearance of glutamate from the synapse under physiological conditions is performed by astrocytic glutamate transporters, their expression might be diminished under pathological conditions. Microglia glutamate transporters, however, might serve as a back-up system when astrocytic glutamate uptake is impaired, and could have a prominent neuroprotective function under pathological conditions. In the current study, the effect of nicotine, well known as a neuroprotective molecule, on the function of glutamate transporters in cultured rat cortical microglia was examined. Reverse transcription polymerase chain reaction and pharmacological approaches demonstrated that, glutamate/aspartate transporter (GLAST), not glutamate transporter 1 (GLT-1), is the major functional glutamate transporter in cultured cortical microglia. Furthermore, the α7 subunit was demonstrated to be the key subunit comprising nicotinic acetylcholine (nACh) receptors in these cells. Treatment of cortical microglia with nicotine led to a significant increase of GLAST mRNA expression and (14)C-glutamate uptake in a concentration- and time-dependent manner, which were markedly inhibited by pretreatment with methyllycaconitine, a selective α7 nACh receptor antagonist. The nicotine-induced expression of GLAST mRNA and protein is mediated through an inositol trisphosphate (IP3) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) depend intracellular pathway, since pretreatment with either xestospongin C, an IP3 receptor antagonist, or KN-93, a CaMKII inhibitor, blocked GLAST expression. Together, these findings indicate that activation of nACh receptors, specifically those expressing the α7 subunit, on cortical microglia could be a key mechanism of the neuroprotective effect of nACh receptor ligands such as nicotine.

  11. 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.

  12. 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.

  13. 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.

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

    PubMed

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

    2011-07-01

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

  15. Comparative Transport Activity of Intact Cells, Membrane Vesicles, and Mesosomes of Bacillus licheniformis

    PubMed Central

    MacLeod, Robert A.; Thurman, Paul; Rogers, H. J.

    1973-01-01

    Sodium ion was shown to stimulate strongly the transport of l-glutamic acid into cells of Bacillus licheniformis 6346 His−. Lithium ion had a slight capacity to replace Na+ in this capacity, but K+ was without effect. Three of five amino acids tested. l-glutamic acid, l-aspartic acid, and l-alanine, were concentrated against a gradient in the cells. Intracellular pools of these amino acids were extractable with 5% trichloroacetic acid. Pools of l-histidine and l-lysine could not be detected. No evidence of active transport of lysine into cells could be detected, and histidine was taken up in the absence of chloramphenicol but not in its presence. The uptake of glutamic acid by membrane vesicle preparations was strongly stimulated by reduced nicotinamide adenine dinucleotide (NADH) and to a lesser extent by succinate. The presence of phenazine methosulfate increased uptake in the presence of succinate. Either l- or d-lactate and adenosine triphosphate were without effect. None of these compounds stimulated the uptake of glutamic acid by mesosomes, although some mesosome preparations contained separable membrane which was very active. NADH strongly stimulated the uptake of aspartic acid and alanine by membrane vesicles but had only a slight effect on the uptake of histidine and lysine. No evidence of active transport of any of the amino acids into mesosomes could be detected either in the presence or absence of NADH. NADH stimulation of the uptake of glutamic acid by membrane vesicles was destroyed by exposure to light of 360 nm; this inactivation was reversible by vitamin K2(5) or K2(10). Sodium ion stimulated transport of glutamic acid by membrane vesicles. PMID:4347247

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

    PubMed

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

    2017-01-01

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

  17. 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.

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

    PubMed

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

    2013-08-01

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

  19. Chloride transport in functionally active phagosomes isolated from Human neutrophils

    PubMed Central

    Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

    2012-01-01

    Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport

  20. 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.

  1. [Measurement of the transport activities of bile salt export pump using chemiluminescence detection method].

    PubMed

    Yamaguchi, Kana; Murai, Tsuyoshi; Yabuuchi, Hikaru; Hui, Shu-Ping; Kurosawa, Takao

    2010-05-01

    Monovalent bile acids, such as taurine- and glycine-conjugated bile acids, are excreted into bile by bile salt export pumps (BSEP, ABCB11). Human BSEP (hBSEP) is physiologically important because it was identified as the gene responsible for the genetic disease: progressive familial intrahepatic cholestasis type 2 (PFIC-2). The evaluation of the inhibitory effect of hBSEP transport activity provides significant information for predicting toxic potential in the early phase of drug development. The role and function of hBSEP have been investigated by the examination of the ATP-dependent transport of radioactive isotopically (RI)-labeled bile acid such as a tritium labeled taurocholic acid, in membrane vesicles obtained from hBSEP-expressing cells. The chemiluminescence detection method using 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) had been developed for a simple analysis of bile acids in human biological fluids. This method is extremely sensitive and it may be applicable for the measurements of bile acid transport activities by hBSEP vesicles without using RI-labeled bile acid. The present paper deals with an application of the chemiluminescence detection method using 3alpha-HSD with enzyme cycling method to the measurement of ATP-dependent transport activities of taurocholic acid (T-CA) in membrane vesicles obtained from hBSEP-expressing Sf9 cells. Calibration curves for T-CA was linear over the range from 10 to 400 pmol/ml. The values of the kinetic parameters for hBSEP vesicles obtained by the chemiluminescence detection method were comparable with the values of that obtained by liquid chromatography-mass spectrometry method. This assay method was highly useful for the measurements of bile acid transport activities.

  2. Zymographic detection of cinnamic acid decarboxylase activity.

    PubMed

    Prim, Núria; Pastor, F I Javier; Diaz, Pilar

    2002-11-01

    The manuscript includes a concise description of a new, fast and simple method for detection of cinnamic acid decarboxylase activity. The method is based on a color shift caused a by pH change and may be an excellent procedure for large screenings of samples from natural sources, as it involves no complex sample processing or purification. The method developed can be used in preliminary approaches to biotransformation processes involving detection of hydroxycinnamic acid decarboxylase activity.

  3. 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

  4. 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.

  5. 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.

  6. The cytotoxic activity of ursolic acid derivatives.

    PubMed

    Ma, Chao-Mei; Cai, Shao-Qing; Cui, Jing-Rong; Wang, Rui-Qing; Tu, Peng-Fei; Hattori, Masao; Daneshtalab, Mohsen

    2005-06-01

    Ursolic acid and 2alpha-hydroxyursolic acid isolated from apple peels were found to show growth inhibitory activity against four tumor cell lines, HL-60, BGC, Bel-7402 and Hela. Structural modifications were performed on the C-3, C-28 and C-11 positions of ursolic acid and the cytotoxicity of the derivatives was evaluated. The SAR revealed that the triterpenes possessing two hydrogen-bond forming groups (an H-donor and a carbonyl group) at positions 3 and 28 exhibit cytotoxic activity. The configuration at C-3 was found to be important for the activity. Introduction of an amino group increased the cytotoxicity greatly. A 3beta-amino derivative was 20 times more potent than the parent ursolic acid. The 28-aminoalkyl dimer compounds showed selective cytotoxicity.

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

    PubMed

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

    2015-07-29

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

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

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

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

  9. 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

  10. 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

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

    ERIC Educational Resources Information Center

    Missouri Univ., Columbia. Missouri LINC.

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

  12. 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.

  13. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  15. 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

  16. 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

  17. Active urea transport independent of H+ and Na+ transport in frog skin epithelium.

    PubMed

    Lacoste, I; Dunel-Erb, S; Harvey, B J; Laurent, P; Ehrenfeld, J

    1991-10-01

    We investigated the relationship between H+ secretion (JH), Na+ absorption (JNa), and urea transport (Ju) in skin of frogs (Rana esculenta) adapted to running tap water, NaCl (100 mM), and KCl (100 mM). In addition, cell morphological changes, particularly in the mitochondria-rich cells (MRC), were followed. NaCl adaptation stimulated an active Ju, reduced JNa and JH, and caused a decrease in the apical surface of MRC. After KCl adaptation, JNa and JH were increased and highly correlated, with a twofold increase in Ju, whereas the numerous MRC developed infoldings on their apical membranes. No correlation was found between JH and Ju. Clamping the skins in a range of +/- 50 mV or changing the external pH from 7.4 to 5.4 (at high cellular buffering power) had no effect on Ju. Depolarization of the basolateral membranes (serosal KCl-Ringer) had no effect on Ju. Ju was reversibly blocked by acidification of the cells by oxygen-free solution and sulfhydryl reagents (Hg2+, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide). Diethylstilbestrol, a proton transport blocker, had no effect on Ju. Apical addition of amiloride and derivatives (phenamil and ethylisopropyl amiloride) reversibly blocked Ju, whereas ouabain had no effect. We conclude that a cation (Na+ or H+)-dependent process is unlikely to exist in R. esculenta skin. A primary active transport in a two-step process is the simplest hypothesis to account for the energy-dependent Ju that develops in NaCl-adapted frogs.

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

    PubMed Central

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

    2000-01-01

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

  19. 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.

  20. Compromising KCC2 transporter activity enhances the development of continuous seizure activity

    PubMed Central

    Kelley, Matthew R.; Deeb, Tarek Z.; Brandon, Nicholas J.; Dunlop, John; Davies, Paul A.; Moss, Stephen J.

    2016-01-01

    Impaired neuronal inhibition has long been associated with the increased probability of seizure occurrence and heightened seizure severity. Fast synaptic inhibition in the brain is primarily mediated by the type A γ-aminobutyric acid receptors (GABAARs), ligand-gated ion channels that can mediate Cl− influx resulting in membrane hyperpolarization and the restriction of neuronal firing. In most adult brain neurons, the K+/Cl− co-transporter-2 (KCC2) establishes hyperpolarizing GABAergic inhibition by maintaining low [Cl−]i. In this study, we sought to understand how decreased KCC2 transport function affects seizure event severity. We impaired KCC2 transport in the 0-Mg2+ ACSF and 4-aminopyridine in vitro models of epileptiform activity in acute mouse brain slices. Experiments with the selective KCC2 inhibitor VU0463271 demonstrated that reduced KCC2 transport increased the duration of SLEs, resulting in non-terminating discharges of clonic-like activity. We also investigated slices obtained from the KCC2-Ser940Ala (S940A) point-mutant mouse, which has a mutation at a known functional phosphorylation site causing behavioral and cellular deficits under hyperexcitable conditions. We recorded from the entorhinal cortex of S940A mouse brain slices in both 0-Mg2+ ACSF and 4-aminopyridine, and demonstrated that loss of the S940 residue increased the susceptibility of continuous clonic-like discharges, an in vitro form of status epilepticus. Our experiments revealed KCC2 transport activity is a critical factor in seizure event duration and mechanisms of termination. Our results highlight the need for therapeutic strategies that potentiate KCC2 transport function in order to decrease seizure event severity and prevent the development of status epilepticus. PMID:27108931

  1. 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

  2. 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

  3. Methane activation and oxidation in sulfuric acid.

    PubMed

    Goeppert, Alain; Dinér, Peter; Ahlberg, Per; Sommer, Jean

    2002-07-15

    The H/D exchange observed when methane is contacted with D(2)SO(4) at 270-330 degrees C shows that the alkane behaves as a sigma base and undergoes rapid and reversible protonation at this temperature. DFT studies of the hydrogen exchange between a monomer and a dimer of sulfuric acid and methane show that the transition states involved in the exchange are bifunctional, that is one hydrogen atom is transferred from a hydroxy group in sulfuric acid to methane, while one hydrogen atom is abstracted from methane by a non-hydroxy oxygen atom in sulfuric acid. All the transition states include a CH(5) moiety, which shows similarities to the methanium ion CH(5) (+). The calculated potential activation energy of the hydrogen exchange for the monomer is 174 kJ mol(-1), which is close to the experimental value (176 kJ mol(-1)). Solvation of the monomer and the transition state of the monomer with an extra sulfuric acid molecule, decrease the potential activation energy by 6 kJ mol(-1). The acid-base process is in competition, however, with an oxidative process involving methane and sulfuric acid which leads to CO(2), SO(2), and water, and thus to a decrease of acidity and loss of reactivity of the medium.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  13. 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

  14. 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.

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

    ERIC Educational Resources Information Center

    HAZWRAP, The Hazardous Waste Remedial Actions Program.

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

  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. 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,...

  18. Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity.

    PubMed

    Korosh, Travis; Jordan, Kelsey D; Wu, Ja-Shin; Yarlett, Nigel; Upmacis, Rita K

    2016-01-01

    Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 μM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 μM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole-resistant T. vaginalis.

  19. 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

  20. 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.

  1. 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

  2. Nicotine decreases the activity of glutamate transporter type 3.

    PubMed

    Yoon, Hea-Jo; Lim, Young-Jin; Zuo, Zhiyi; Hur, Wonseok; Do, Sang-Hwan

    2014-02-10

    Nicotine, the main ingredient of tobacco, elicits seizures in animal models and cigarette smoking is regarded as a behavioral risk factor associated with epilepsy or seizures. In the hippocampus, the origin of nicotine-induced seizures, most glutamate uptake could be performed primarily by excitatory amino acid transporter type 3 (EAAT3). An association between temporal lobe epilepsy and EAAT3 downregulation has been reported. Therefore, we hypothesized that nicotine may elicit seizures through the attenuation of EAAT3 activity. We investigated chronic nicotine exposure (72 h) cause reduction of the activity of EAAT3 in a Xenopus oocyte expression system using a two-electrode voltage clamp. The roles of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) were also determined. Nicotine (0.001-1 μM) resulted in a time- and dose-dependent decrease in EAAT3 activity with maximal inhibition at nicotine concentrations of 0.03 μM or higher and at an exposure time of 72 h. Vmax on the glutamate response was significantly reduced in the nicotine group (0.03 μM for 72 h), but the Km value of EAAT3 for glutamate was not altered. When nicotine-exposed oocytes (0.03 μM for 72 h) were pretreated with phorbol-12-myristate-13-acetate (PMA, a PKC activator), the nicotine-induced reduction in EAAT3 activity was abolished. PKC inhibitors (staurosporine, chelerythrine, and calphostin C) significantly reduced basal EAAT3 activity, but there were no significant differences among the PKC inhibitors, nicotine, and PKC inhibitors+nicotine groups. Similar response patterns were observed among PI3K inhibitors (wortmannin and LY294002), nicotine, and PI3K inhibitors+nicotine. In conclusion, this study suggests that nicotine decreases EAAT3 activity, and that this inhibition seems to be dependent on PKC and PI3K. Our results may provide an additional mechanism for nicotine-induced seizure.

  3. 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...

  4. 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

  5. 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.

  6. Heat transport in active harmonic chains

    SciTech Connect

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

    2011-08-15

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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. A natural mutation led truncation in Ma1, one of the two aluminum-activated malate transporter like genes at the Ma locus, is associated with low fruit acidity in apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acidity levels greatly affect fruit taste and flavor, thereby the consumer’s acceptance and market value. In mature apple fruit, malic acid is the nearly exclusive organic acid. Several studies have confirmed that the major quantitative trait locus Ma largely controls the variation of fruit acidity ...

  14. 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.

  15. Microbial killing activity of peracetic acid.

    PubMed

    Thamlikitkul, V; Trakulsomboon, S; Louisirirotchanakul, S; Chaiprasert, A; Foongladda, S; Thipsuvan, K; Arjratanakool, W; Kunyok, R; Wasi, C; Santiprasitkul, S; Danchaivijitr, S

    2001-10-01

    In vitro killing activity of peracetic acid (Perasafe) at a concentration of 0.26 per cent w/v was tested against Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi A, Acinetobacter baumannii, Sternotrophomonas maltophilia, Enterococcus faecium, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus subtilis spore, Mycobacterium tuberculosis and human immuno-deficiency virus type I. Exposure to Peracetic acid (0.26% w/v) for 10 minutes resulted in massive killing of all the aforementioned organisms and spore.

  16. 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

  17. Study of active cooling for supersonic transports

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

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

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

    PubMed

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

    2010-05-01

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

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

    PubMed

    Klochkov, B N; Romanov, A S

    2013-01-01

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

  20. [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.

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

    PubMed Central

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

    2016-01-01

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

  2. Two Members of the Aluminum-Activated Malate Transporter Family, SlALMT4 and SlALMT5, are Expressed during Fruit Development, and the Overexpression of SlALMT5 Alters Organic Acid Contents in Seeds in Tomato (Solanum lycopersicum).

    PubMed

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Nakano, Ryohei; Ushijima, Koichiro; Kubo, Yasutaka; Mori, Izumi C; Higashiizumi, Emi; Galis, Ivan; Yamamoto, Yoko

    2016-11-01

    The aluminum-activated malate transporter (ALMT) family of proteins transports malate and/or inorganic anions across plant membranes. To demonstrate the possible role of ALMT genes in tomato fruit development, we focused on SlALMT4 and SlALMT5, the two major genes expressed during fruit development. Predicted proteins were classified into clade 2 of the family, many members of which localize to endomembranes. Tissue-specific gene expression was determined using transgenic tomato expressing the β-glucuronidase reporter gene controlled by their own promoters. Both the genes were expressed in vascular bundles connecting to developing seeds in fruit and in the embryo of mature seeds. Further, SlALMT5 was expressed in embryo in developing seeds in fruit. Subcellular localization of both proteins to the endoplasmic reticulum (ER) was established by transiently expressing the green fluorescent protein fusions in plant protoplasts. SlALMT5 probably localized to other endomembranes as well. Localization of SlALMT5 to the ER was also confirmed by immunoblot analysis. The transport function of both SlALMT proteins was investigated electrophysiologically in Xenopus oocytes. SlALMT5 transported malate and inorganic anions such as nitrate and chloride, but not citrate. SlALMT4 also transported malate, but the results were less consistent perhaps because it did not localize strongly to the plasma membrane. To elucidate the physiological role of SlALMT5 further, we overexpressed SlALMT5 in tomato. Compared with the wild type, overexpressors exhibited higher malate and citrate contents in mature seeds, but not in fruit. We conclude that the malate transport function of SlALMT5 expressed in developing fruit influences the organic acid contents in mature seeds.

  3. Antidiabetic Activity from Gallic Acid Encapsulated Nanochitosan

    NASA Astrophysics Data System (ADS)

    Purbowatiningrum; Ngadiwiyana; Ismiyarto; Fachriyah, E.; Eviana, I.; Eldiana, O.; Amaliyah, N.; Sektianingrum, A. N.

    2017-02-01

    Diabetes mellitus (DM) has become a health problem in the world because it causes death. One of the phenolic compounds that have antidiabetic activity is gallic acid. However, the use of this compound still provides unsatisfactory results due to its degradation during the absorption process. The solution offered to solve the problem is by encapsulated it within chitosan nanoparticles that serve to protect the bioactive compound from degradation, increases of solubility and delivery of a bioactive compound to the target site by using freeze-drying technique. The result of chitosan nanoparticle’s Scanning Electron Microscopy (SEM) showed that chitosan nanoparticle’s size is uniform and it is smaller than chitosan. The value of encapsulation efficiency (EE) of gallic acid which encapsulated within chitosan nanoparticles is about 50.76%. Inhibition test result showed that gallic acid-chitosan nanoparticles at 50 ppm could inhibite α-glucosidase activity in 28.87% with 54.94 in IC50. So it can be concluded that gallic acid can be encapsulated in nanoparticles of chitosan and proved that it could inhibit α-glucosidase.

  4. 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

  5. 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

  6. An Abiotic Glass-Bead Collector Exhibiting Active Transport

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  7. Fatty acid transport by vectorial acylation in mammals: roles played by different isoforms of rat long-chain acyl-CoA synthetases.

    PubMed

    Tong, Fumin; Black, Paul N; Coleman, Rosalind A; DiRusso, Concetta C

    2006-03-01

    Mammals express multiple isoforms of acyl-CoA synthetase (ACSL1 and ACSL3-6) in various tissues. These enzymes are essential for fatty acid metabolism providing activated intermediates for complex lipid synthesis, protein modification, and beta-oxidation. Yeast in contrast express four major ACSLs, which have well-defined functions. Two, Faa1p and Faa4p, are specifically required for fatty acid transport by vectorial acylation. Four ACSLs from the rat were expressed in a yeast faa1delta faa4delta strain and their roles in fatty acid transport and trafficking characterized. All four restored ACS activity yet varied in substrate preference. ACSL1, 4, and 6 were able to rescue fatty acid transport activity and triglyceride synthesis. ACSL5, however, was unable to facilitate fatty acid transport despite conferring robust oleoyl-CoA synthetase activity. This is the first study evaluating the role of the mammalian ACSLs in fatty acid transport and supports a role for ACSL1, 4, and 6 in transport by vectorial acylation.

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

    PubMed

    Tarran, Robert

    2004-01-01

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

  9. 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

    inhibition (more than 95%), which does not allow a uniform tracer distribution over the whole cellular space during a single passage through the liver. The degree of inhibition of sulfate transport by 100 microM flufenamic acid was a function of the concentration of nontracer sulfate. With sulfate in the range between 1.2 and 25 mM, the inhibition degree increased linearly with the concentration. In the presence of flufenamic acid, the saturation curve of equilibrium exchange showed a substrate inhibition-like phenomenon, which was absent in the control curve. As inhibitors of sulfate transport in hepatocytes, flufenamic and niflumic acids are less active than in erythrocytes by a factor of 10(2). This observation is most probably indicative of structural differences between the hepatic sulfate carrier and the anion carrier of erythrocytes. It is unlikely that the action of flufenamic acid and its analogs on sulfate transport is a consequence of energy metabolism inhibition. Nimesulide is as active as flufenamic or niflumic acid in inhibiting energy metabolism but considerably less efficient as an inhibitor of sulfate transport. Our results as well as literature data reveal that the interactions of the nonsteroidal anti-inflammatories with the liver membranes and intracellular structures are ample and complex. Even at high concentrations, however, these interactions are not so intense as to change the vascular and cellular spaces.

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

    PubMed Central

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

    2017-01-01

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

  11. Mouse SGLT3a generates proton-activated currents but does not transport sugar.

    PubMed

    Barcelona, Stephanie; Menegaz, Danusa; Díez-Sampedro, Ana

    2012-04-15

    Sodium-glucose cotransporters (SGLTs) are secondary active transporters belonging to the SLC5 gene family. SGLT1, a well-characterized member of this family, electrogenically transports glucose and galactose. Human SGLT3 (hSGLT3), despite sharing a high amino acid identity with human SGLT1 (hSGLT1), does not transport sugar, although functions as a sugar sensor. In contrast to humans, two different genes in mice and rats code for two different SGLT3 proteins, SGLT3a and SGLT3b. We previously cloned and characterized mouse SGLT3b (mSGLT3b) and showed that, while it does transport sugar like SGLT1, it likely functions as a physiological sugar sensor like hSGLT3. In this study, we cloned mouse SGLT3a (mSGLT3a) and characterized it by expressing it in Xenopus laevis oocytes and performing electrophysiology and sugar transport assays. mSGLT3a did not transport sugar, and sugars did not induce currents at pH 7.4, though acidic pH induced inward currents that increased in the presence of sugar. Moreover, mutation of residue 457 from glutamate to glutamine resulted in a Na(+)-dependent transport of sugar that was inhibited by phlorizin. To corroborate our results in oocytes, we expressed and characterized mSGLT3a in mammalian cells and confirmed our findings. In addition, we cloned, expressed, and characterized rat SGLT3a in oocytes and found characteristics similar to mSGLT3a. In summary, acidic pH induces currents in mSGLT3a, and sugar-induced currents are increased at acidic pH, but wild-type SGLT3a does not transport sugar.

  12. 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.

  13. 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

  14. 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

  15. Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase

    SciTech Connect

    Sode, Olaseni; Voth, Gregory A.

    2014-12-14

    Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A{sup 2−}, a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A{sup 2-} by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.

  16. Active transportation safety features around schools in Canada.

    PubMed

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

    2013-10-31

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

  17. Active Transportation Safety Features around Schools in Canada

    PubMed Central

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

    2013-01-01

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

  18. Entropic Ratchet transport of interacting active Brownian particles

    SciTech Connect

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

    2014-11-21

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

  19. Active urea transport in lower vertebrates and mammals.

    PubMed

    Bankir, Lise

    2014-01-01

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

  20. 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.

  1. Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

    PubMed Central

    Echtay, Karim S.; Winkler, Edith; Frischmuth, Karina; Klingenberg, Martin

    2001-01-01

    Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609–613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2′-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl− but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters. PMID:11171965

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Antineoplastic activity of zoledronic acid and denosumab.

    PubMed

    Zwolak, Pawel; Dudek, Arkadiusz Z

    2013-08-01

    Cancer patients suffer from cancer-induced bone pain, hypercalcemia, and reduced quality of life caused by pathological fractures. Many of these complications related to cancer can be treated, or at least controlled, using new anticancer agents. Recently, two agents used initially to treat osteoporosis demonstrated direct and indirect anticancer activity. In this review, we summarize current knowledge about direct and indirect anticancer activity of zoledronic acid (a third-generation bisphosphonate), and denosumab antibody against RANKL. Zoledronic acid influences the proliferation and viability of tumor cells in vitro, and effectively reduces tumor burden, tumor-induced pain, and tumor growth in vivo. Denosumab is a fully human monoclonal antibody preventing the binding of RANKL to its receptor on osteoclasts' membrane, and through this mechanism inhibits the resorption of the bone. Furthermore, this agent demonstrates direct anticancer activity through the RANKL signaling pathway. Because of these features both drugs may gain broader application for the treatment of cancer patients. However, further pre-clinical and clinical evaluation is needed for both agents to fully assess the antineoplastic mechanisms of activity of both agents.

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

    PubMed

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

    2009-08-01

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

  8. Classroom Activities in Transportation: Technology Education.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison.

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

  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. The connexion between active cation transport and metabolism in erythrocytes

    PubMed Central

    Whittam, R.; Ager, Margaret E.

    1965-01-01

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

  11. 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.

  12. Critical role of s465 in protein kinase C-increased rat glutamate transporter type 3 activity.

    PubMed

    Baik, Hee Jung; Huang, Yueming; Washington, Jacqueline M; Zuo, Zhiyi

    2009-01-01

    Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.

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

    PubMed

    Maibach, Edward; Steg, Linda; Anable, Jillian

    2009-10-01

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

  14. 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.

  15. 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

  16. 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.

  17. 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.

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

    SciTech Connect

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

    2013-11-01

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

  19. 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

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

    ERIC Educational Resources Information Center

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

    2005-01-01

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

  1. 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

  2. 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.

  3. 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.

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

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  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. Substrate regulation of ascorbate transport activity in astrocytes

    SciTech Connect

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

    1990-10-01

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

  7. Isoxazole analogues bind the System xc− Transporter: Structure-activity Relationship and Pharmacophore Model

    PubMed Central

    Patel, Sarjubhai A.; Rajale, Trideep; O’Brien, Erin; Burkhart, David J.; Nelson, Jared K.; Twamley, Brendan; Blumenfeld, Alex; Szabon-Watola, Monika I.; Gerdes, John M.; Bridges, Richard J.; Natale, Nicholas R.

    2009-01-01

    Analogues of amino methylisoxazole propionic acid (AMPA), were prepared from a common intermediate 12, including lipophilic analogues using lateral metalation and electrophilic quenching, and were evaluated at System xc−. Both the 5-naphthylethyl-(16) and 5-naphthylmethoxymethyl-(17) analogues adopt an E-conformation in the solid state, yet while the former has robust binding at System xc−, the latter is virtually devoid of activity. The most potent analogues were amino acid naphthyl-ACPA 7g, and hydrazone carboxylic acid, 11e Y=Y′=3,5-(CF3)2, which both inhibited glutamate up-take by the System xc− transporter with comparable potency to the endogenous substrate cystine, whereas in contrast the closed isoxazolo[3,4-d] pyridazinones 13 have significantly lower activity. A preliminary pharmacophore model has been constructed to provide insight into the analogue structure-activity relationships. PMID:19932968

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  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. 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.

  18. 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.

  19. 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

  20. 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.

  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. 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

  3. 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.

  4. Advocacy for active transport: advocate and city council perspectives

    PubMed Central

    2010-01-01

    Background Effective advocacy is an important part of efforts to increase population participation in physical activity. Research about effective health advocacy is scarce, however, the health sector can learn from the experiences and knowledge of community advocates and those who are on the receiving end of this advocacy. The aim of this study is to explore advocacy for active transport from the perspectives of community advocates and representatives from City councils. Methods Cycling and walking advocates were identified from the local contact list of Cycling Advocates Network and Living Streets Aotearoa. Semi-structured telephone interviews were conducted with cycle and walking advocates from throughout New Zealand. Advocates also nominated a suitable council officer at their local City council to be interviewed. Interviews were recorded and transcribed and categories of responses for each of the questions created. Results Several processes were used by advocates to engage with council staff, including formal council submissions, meetings, stakeholder forums and partnership in running community events promoting active transport. Several other agencies were identified as being influential for active transport, some as potential coalition partners and others as potential adversaries. Barriers to improving conditions for active transport included a lack of funding, a lack of will-power among either council staff or councillors, limited council staff capacity (time or training) and a culture of providing infrastructure for motor vehicles instead of people. Several suggestions were made about how the health sector could contribute to advocacy efforts, including encouraging political commitment, engaging the media, communicating the potential health benefits of active transport to the general public and being role models in terms of personal travel mode choice and having workplaces that support participation in active transport. Conclusions There is potential for the

  5. Triboelectrification of active pharmaceutical ingredients: week acids and their salts.

    PubMed

    Fujinuma, Kenta; Ishii, Yuji; Yashihashi, Yasuo; Yonemochi, Estuo; Sugano, Kiyohiko; Tarada, Katsuhide

    2015-09-30

    The effect of salt formulation on the electrostatic property of active pharmaceutical ingredients was investigated. The electrostatic property of weak acids (carboxylic acids and amide-enole type acid) and their sodium salts was evaluated by a suction-type Faraday cage meter. Free carboxylic acids showed negative chargeability, whereas their sodium salts showed more positive chargeability than the free acids. However, no such trend was observed for amide-enole type acids.

  6. 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

  7. 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.

  8. 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

  9. 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.

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

    PubMed Central

    Lang, Florian; Föller, Michael

    2014-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. 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.

  13. 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

  14. 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.

  15. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells.

    PubMed

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin.

  16. Activation of AMP-Activated Protein Kinase and Stimulation of Energy Metabolism by Acetic Acid in L6 Myotube Cells

    PubMed Central

    Maruta, Hitomi; Yoshimura, Yukihiro; Araki, Aya; Kimoto, Masumi; Takahashi, Yoshitaka; Yamashita, Hiromi

    2016-01-01

    Previously, we found that orally administered acetic acid decreased lipogenesis in the liver and suppressed lipid accumulation in adipose tissue of Otsuka Long-Evans Tokushima Fatty rats, which exhibit hyperglycemic obesity with hyperinsulinemia and insulin resistance. Administered acetic acid led to increased phosphorylation of AMP-activated protein kinase (AMPK) in both liver and skeletal muscle cells, and increased transcripts of myoglobin and glucose transporter 4 (GLUT4) genes in skeletal muscle of the rats. It was suggested that acetic acid improved the lipid metabolism in skeletal muscles. In this study, we examined the activation of AMPK and the stimulation of GLUT4 and myoglobin expression by acetic acid in skeletal muscle cells to clarify the physiological function of acetic acid in skeletal muscle cells. Acetic acid added to culture medium was taken up rapidly by L6 cells, and AMPK was phosphorylated upon treatment with acetic acid. We observed increased gene and protein expression of GLUT4 and myoglobin. Uptake of glucose and fatty acids by L6 cells were increased, while triglyceride accumulation was lower in treated cells compared to untreated cells. Furthermore, treated cells also showed increased gene and protein expression of myocyte enhancer factor 2A (MEF2A), which is a well-known transcription factor involved in the expression of myoglobin and GLUT4 genes. These results indicate that acetic acid enhances glucose uptake and fatty acid metabolism through the activation of AMPK, and increases expression of GLUT4 and myoglobin. PMID:27348124

  17. 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.

  18. Neuroinflammation activates efflux transport by NFκB

    PubMed Central

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

    2009-01-01

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

  19. 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

  20. Activation of ion transport systems during cell volume regulation

    SciTech Connect

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

    1987-01-01

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

  1. Dopamine transporter occupancy by RTI-55, inhibition of dopamine transport and stimulation of locomotor activity

    SciTech Connect

    Gatley, S.J.; Gifford, A.N.; Volkow, N.D.

    1997-05-01

    Cocaine analogs such as RTI-55 (or {beta}CIT) with a higher affinity for the DAT are potentially useful as therapeutic drugs in cocaine abuse as well as for radiopharmaceutical use. Previously we showed that in mice RTI-55 (2 mg/Kg, i/p) reduced H-3 cocaine striatum-to-cerebellum ratios (St/Cb, {lg_bullet}) from 1.6 to 1.2 at 3 h after administration, with recovery by 12 h. In the present study we demonstrate a very similar time-course for transport {triangle} measured in striatal homo within 2 min of sacrifice. The maximum inhibition of uptake at about 1 h corresponded to about 80% of the control uptake rate, similar to the percent reduction in St/Cb. The time-course of the effect of this dose of RTI-55 on locomotor activity ({sq_bullet}) was complex, with a drop in the activity measure at 7 h, after a further injection of RTI-55, but activity remained higher than in saline controls. In spite of this complexity, which may be associated with stereotypies and/or exhaustion, the duration of increased activity is consistent with the duration of transporter blockade. These experiments support the notion that PET/SPECT measures of transporter occupancy accurately reflect transporter inhibition.

  2. Micelles Protect and Concentrate Activated Acetic Acid

    NASA Astrophysics Data System (ADS)

    Todd, Zoe; House, C.

    2014-01-01

    As more and more exoplanets are discovered and the habitability of such planets is considered, one can turn to searching for the origin of life on Earth in order to better understand what makes a habitable planet. Activated acetic acid, or methyl thioacetate, has been proposed to be central to the origin of life on Earth, and also as an important energy currency molecule in early cellular evolution. We have investigated the hydrolysis of methyl thioacetate under various conditions. Its uncatalyzed rate of hydrolysis is about three orders of magnitude faster (K = 0.00663 s^-1; 100°C, pH 7.5, concentration = 0.33mM) than published rates for its catalyzed production making it unlikely to accumulate under prebiotic conditions. However, we also observed that methyl thioacetate was protected from hydrolysis when inside its own hydrophobic droplets. We found that methyl thioacetate protection from hydrolysis was also possible in droplets of hexane and in the membranes of nonanoic acid micelles. Thus, the hydrophobic regions of prebiotic micelles and early cell membranes could have offered a refuge for this energetic molecule increasing its lifetime in close proximity to the reactions for which it would be needed. Methyl thioacetate could thus be important for the origin of life on Earth and perhaps for better understanding the potential habitability of other planets.

  3. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1990-01-01

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

  4. Active and passive calcium transport systems in plant cells

    SciTech Connect

    Sze, H.

    1991-01-01

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

  5. Transport of active ellipsoidal particles in ratchet potentials

    SciTech Connect

    Ai, Bao-Quan Wu, Jian-Chun

    2014-03-07

    Rectified transport of active ellipsoidal particles is numerically investigated in a two-dimensional asymmetric potential. The out-of-equilibrium condition for the active particle is an intrinsic property, which can break thermodynamical equilibrium and induce the directed transport. It is found that the perfect sphere particle can facilitate the rectification, while the needlelike particle destroys the directed transport. There exist optimized values of the parameters (the self-propelled velocity, the torque acting on the body) at which the average velocity takes its maximal value. For the ellipsoidal particle with not large asymmetric parameter, the average velocity decreases with increasing the rotational diffusion rate, while for the needlelike particle (very large asymmetric parameter), the average velocity is a peaked function of the rotational diffusion rate. By introducing a finite load, particles with different shapes (or different self-propelled velocities) will move to the opposite directions, which is able to separate particles of different shapes (or different self-propelled velocities)

  6. 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.

  7. 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.

  8. 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.

  9. "JCE" Classroom Activity #109: My Acid Can Beat Up Your Acid!

    ERIC Educational Resources Information Center

    Putti, Alice

    2011-01-01

    In this guided-inquiry activity, students investigate the ionization of strong and weak acids. Bead models are used to study acid ionization on a particulate level. Students analyze seven strong and weak acid models and make generalizations about the relationship between acid strength and dissociation. (Contains 1 table and 2 figures.)

  10. 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

  11. 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.

  12. The relationship between the acidity and the hydrocarbon cracking activity of ultrastable H-Y zeolite

    NASA Astrophysics Data System (ADS)

    Kuehne, Mark Andrew

    high heat of NH3 adsorption. However, if cracking is diffusion-limited, then the mesopores and fractures formed during steam dealumination may increase its observed cracking activity by increasing mass transport rates to acid sites located near the mesopores.

  13. 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

  14. 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

  15. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  16. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  17. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  18. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  19. 49 CFR 37.61 - Public transportation programs and activities in existing facilities.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Public transportation programs and activities in existing facilities. 37.61 Section 37.61 Transportation Office of the Secretary of Transportation TRANSPORTATION SERVICES FOR INDIVIDUALS WITH DISABILITIES (ADA) Transportation Facilities § 37.61...

  20. Regional differences in rat conjunctival ion transport activities.

    PubMed

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

    2012-10-01

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

  1. Regional differences in rat conjunctival ion transport activities

    PubMed Central

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

    2012-01-01

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

  2. 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.

  3. 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.

  4. Lipophilic pyrazinoic acid amide and ester prodrugs stability, activation and activity against M. tuberculosis.

    PubMed

    Simões, Marta Filipa; Valente, Emília; Gómez, M José Rodríguez; Anes, Elsa; Constantino, Luís

    2009-06-28

    Pyrazinamide (PZA) is active against M. tuberculosis and is a first line agent for the treatment of human tuberculosis. PZA is itself a prodrug that requires activation by a pyrazinamidase to form its active metabolite pyrazinoic acid (POA). Since the specificity of cleavage is dependent on a single bacterial enzyme, resistance to PZA is often found in tuberculosis patients. Esters of POA have been proposed in the past as alternatives to PZA however the most promising compounds were rapidly degraded in the presence of serum. In order to obtain compounds that could survive during the transport phase, we synthesized lipophilic ester and amide POA derivatives, studied their activity against M. tuberculosis, their stability in plasma and rat liver homogenate and also their activation by a mycobacterial homogenate. The new lipophilic ester prodrugs were found to be active in concentrations 10-fold lower than those needed for PZA to kill sensitive M. tuberculosis and also have a suitable stability in the presence of plasma. Amides of POA although more stable in plasma have lower activity. The reason can probably be found in the rate of activation of both types of prodrugs; while esters are easily activated by mycobacterial esterases, amides are resistant to activation and are not transformed into POA at a suitable rate.

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

    SciTech Connect

    Bachand, George David; Carroll-Portillo, Amanda

    2006-11-01

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

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

    PubMed Central

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

    2008-01-01

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

  7. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  8. 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.

  9. 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

  10. 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

  11. Acaricidal activity of usnic acid and sodium usnic acid against Psoroptes cuniculi in vitro.

    PubMed

    Shang, Xiaofei; Miao, Xiaolou; Lv, Huiping; Wang, Dongsheng; Zhang, Jiqin; He, Hua; Yang, Zhiqiang; Pan, Hu

    2014-06-01

    Usnic acid, a major active compound in lichens, was first isolated in 1884. Since then, usnic acid and its sodium salt (sodium usnic acid) have been used in medicine, perfumery, cosmetics, and other industries due to its extensive biological activities. However, its acaricidal activity has not been studied. In this paper, we investigated the acaricidal activity of usnic acid and sodium usnic acid against Psoroptes cuniculi in vitro. After evaluating the acaricidal activity and toxicity of usnic acid and sodium usnic acid in vitro, the results showed that at doses of 250, 125, and 62.5 mg/ml, usnic acid and sodium usnic acid can kill mites with 91.67, 85.00, and 55.00% and 100, 100, and 60.00% mortality after treatment 24 h. The LT50 values were 4.208, 8.249, and 16.950 h and 3.712, 7.339, and 15.773 h for usnic acid and sodium usnic acid, respectively. Sodium usnic acid has a higher acaricidal activity than usnic acid, which may be related to the difference in their structures.

  12. 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.

  13. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  14. 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

  15. Identification of OpuC as a Chill-Activated and Osmotically Activated Carnitine Transporter in Listeria monocytogenes

    PubMed Central

    Angelidis, Apostolos S.; Smith, Linda Tombras; Hoffman, Les M.; Smith, Gary M.

    2002-01-01

    The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress. PMID:12039715

  16. Identification of opuC as a chill-activated and osmotically activated carnitine transporter in Listeria monocytogenes.

    PubMed

    Angelidis, Apostolos S; Smith, Linda Tombras; Hoffman, Les M; Smith, Gary M

    2002-06-01

    The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress.

  17. Overexpression of the Aspergillus niger GatA transporter leads to preferential use of D-galacturonic acid over D-xylose

    PubMed Central

    2014-01-01

    Pectin is a structural heteropolysaccharide of the primary cell walls of plants and as such is a significant fraction of agricultural waste residues that is currently insufficiently used. Its main component, D-galacturonic acid, is an attractive substrate for bioconversion. The complete metabolic pathway is present in the genome of Aspergillus niger, that is used in this study. The objective was to identify the D-galacturonic acid transporter in A. niger and to use this transporter to study D-galacturonic acid metabolism. We have functionally characterized the gene An14g04280 that encodes the D-galacturonic acid transporter in A. niger. In a mixed sugar fermentation it was found that the An14g04280 overexpression strain, in contrast to the parent control strain, has a preference for D-galacturonic acid over D-xylose as substrate. Overexpression of this transporter in A. niger resulted in a strong increase of D-galacturonic acid uptake and induction of the D-galacturonic acid reductase activity, suggesting a metabolite controlled regulation of the endogenous D-galacturonic acid catabolic pathway. PMID:25177540

  18. Fluctuation driven active molecular transport in passive channel proteins

    NASA Astrophysics Data System (ADS)

    Kosztin, Ioan

    2006-03-01

    Living cells interact with their extracellular environment through the cell membrane, which acts as a protective permeability barrier for preserving the internal integrity of the cell. However, cell metabolism requires controlled molecular transport across the cell membrane, a function that is fulfilled by a wide variety of transmembrane proteins, acting as either passive or active transporters. In this talk it is argued that, contrary to the general belief, in active cell membranes passive and spatially asymmetric channel proteins can act as active transporters by consuming energy from nonequilibrium fluctuations fueled by cell metabolism. This assertion is demonstrated in the case of the E. coli aquaglyceroporin GlpF channel protein, whose high resolution crystal structure is manifestly asymmetric. By calculating the glycerol flux through GlpF within the framework of a stochastic model, it is found that, as a result of channel asymmetry, glycerol uptake driven by a concentration gradient is enhanced significantly in the presence of non-equilibrium fluctuations. Furthermore, the enhancement caused by a ratchet-like mechanism is larger for the outward, i.e., from the cytoplasm to the periplasm, flux than for the inward one, suggesting that the same non-equilibrium fluctuations also play an important role in protecting the interior of the cell against poisoning by excess uptake of glycerol. Preliminary data on water and sugar transport through aquaporin and maltoporin channels, respectively, are indicative of the universality of the proposed nonequilibrium-fluctuation-driven active transport mechanism. This work was supported by grants from the Univ. of Missouri Research Board, the Institute for Theoretical Sciences and the Department of Energy (DOE Contract W-7405-ENG-36), and the National Science Foundation (FIBR-0526854).

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  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. 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.

  9. 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.

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

    PubMed

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

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  11. Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

    PubMed Central

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

    2014-01-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  12. Motualevic Acids and Analogs: Synthesis and Antimicrobial Structure Activity Relationships

    PubMed Central

    Cheruku, Pradeep; Keffer, Jessica L.; Dogo-Isonagie, Cajetan; Bewley, Carole A.

    2010-01-01

    Synthesis of the marine natural products motualevic acids A, E, and analogs in which modifications have been made to the ω-brominated lipid (E)-14,14-dibromotetra-deca-2,13-dienoic acid or amino acid unit are reported, together with antimicrobial activities against Staphylococcus aureus, methicillin-resistant S. aureus, Enterococcus faecium, and vancomycin-resistant Enterococcus. PMID:20538459

  13. [Effect of nitrates on active transport of iodine].

    PubMed

    Szökeová, E; Tajtáková, M; Mirossay, L; Mojzis, J; Langer, P; Marcinová, E; Petrovicová, J; Zemberová, E; Bodnár, J

    2001-11-01

    Active iodine transport into the thyrocyte is catalyzed by the transmembrane transport protein Na+/J- symport (NIS) Nitrates can expel iodine from the bond with this transport protein which was found not only in the thyrocyte membrane but also in the cell membrane of the gastric mucosa. The weight of the thyroid gland in mg was significantly greater even when calculated in relation to body weight in the NIT group of rats who were given for 6 days nitrate by gastric tube (100 mg/kg/day) as compared with controls (CON) 17.56 +/- 8.4, 0.07 +/- 0.03/12.10 +/- 9.57, 0.05 +/- 0.03, P < or = 0.01. A lower thyroid activity in per cent calculated per 1 mg of its weight (1.39 +/- 1.0/2.22 +/- 0.9, P < or = 0.01), a higher activity in blood before removal of the thyroid gland (8.54 +/- 4.09/5.45 +/- 2.78) and a lower one after removal of the thyroid gland (1.09 +/- 0.05/0.21 +/- 0.10) before oral administration of I131 in group NIT, suggests a negative effect of nitrates on active iodine transport not only at the level of the thyrocyte but also possible interaction with iodine at the level of the digestive tract. A significantly higher serum level of TT3 in group NIT (0.66 +/- 0.27/0.44 +/- 0.21, P < or = 0.01 regardless of the TSH serum level (2.31 +/- 1.83/2.64 +/- 1.52) and T4 (22.72 +/- 8.2/25 +/- 11.0) suggests a qualitative change in thyroid hormone production in favour of T3 caused even by short-term nitrate administration.

  14. The Asymmetric Active Coupler: Stable Nonlinear Supermodes and Directed Transport

    PubMed Central

    Kominis, Yannis; Bountis, Tassos; Flach, Sergej

    2016-01-01

    We consider the asymmetric active coupler (AAC) consisting of two coupled dissimilar waveguides with gain and loss. We show that under generic conditions, not restricted by parity-time symmetry, there exist finite-power, constant-intensity nonlinear supermodes (NS), resulting from the balance between gain, loss, nonlinearity, coupling and dissimilarity. The system is shown to possess non-reciprocal dynamics enabling directed power transport functionality. PMID:27640818

  15. 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

  16. 77 FR 71430 - New Agency Information Collection Activity Under OMB Review: Public Transportation Baseline...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-30

    ... SECURITY Transportation Security Administration New Agency Information Collection Activity Under OMB Review: Public Transportation Baseline Assessment for Security Enhancement (BASE) Program AGENCY: Transportation Security Administration, DHS. ACTION: 30-day notice. SUMMARY: This notice announces that the...

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

    PubMed

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

    2009-05-26

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

  18. Physiological activities of hydroxyl fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the search of value-added products from surplus soybean oil, we produced many new hydroxy fatty acids through microbial bioconversion. Hydroxy fatty acids are used in a wide range of industrial products, such as resins, waxes, nylons plastics, lubricants, cosmetics, and additives in coatings and...

  19. 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

  20. 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.

  1. Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter

    PubMed Central

    Escudero, Leticia; Mariscal, Vicente

    2015-01-01

    ABSTRACT In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth. IMPORTANCE Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular

  2. 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

  3. Activation of glutamate transport evokes rapid glutamine release from perisynaptic astrocytes

    PubMed Central

    Uwechue, Nneka M; Marx, Mari-Carmen; Chevy, Quentin; Billups, Brian

    2012-01-01

    Stimulation of astrocytes by neuronal activity and the subsequent release of neuromodulators is thought to be an important regulator of synaptic communication. In this study we show that astrocytes juxtaposed to the glutamatergic calyx of Held synapse in the rat medial nucleus of the trapezoid body (MNTB) are stimulated by the activation of glutamate transporters and consequently release glutamine on a very rapid timescale. MNTB principal neurones express electrogenic system A glutamine transporters, and were exploited as glutamine sensors in this study. By simultaneous whole-cell voltage clamping astrocytes and neighbouring MNTB neurones in brainstem slices, we show that application of the excitatory amino acid transporter (EAAT) substrate d-aspartate stimulates astrocytes to rapidly release glutamine, which is detected by nearby MNTB neurones. This release is significantly reduced by the toxins l-methionine sulfoximine and fluoroacetate, which reduce glutamine concentrations specifically in glial cells. Similarly, glutamine release was also inhibited by localised inactivation of EAATs in individual astrocytes, using internal dl-threo-β-benzyloxyaspartic acid (TBOA) or dissipating the driving force by modifying the patch-pipette solution. These results demonstrate that astrocytes adjacent to glutamatergic synapses can release glutamine in a temporally precise, controlled manner in response to glial glutamate transporter activation. Since glutamine can be used by neurones as a precursor for glutamate and GABA synthesis, this represents a potential feedback mechanism by which astrocytes can respond to synaptic activation and react in a way that sustains or enhances further communication. This would therefore represent an additional manifestation of the tripartite relationship between synapses and astrocytes. PMID:22411007

  4. Glucose elevates NITRATE TRANSPORTER2.1 protein levels and nitrate transport activity independently of its HEXOKINASE1-mediated stimulation of NITRATE TRANSPORTER2.1 expression.

    PubMed

    de Jong, Femke; Thodey, Kate; Lejay, Laurence V; Bevan, Michael W

    2014-01-01

    Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth.

  5. 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.

  6. 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.

  7. 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.

  8. Sodium-coupled and electrogenic transport of B-complex vitamin nicotinic acid by slc5a8, a member of the Na/glucose co-transporter gene family

    PubMed Central

    Gopal, Elangovan; Fei, You-Jun; Miyauchi, Seiji; Zhuang, Lina; Prasad, Puttur D.; Ganapathy, Vadivel

    2005-01-01

    SMCT (sodium-coupled monocarboxylate transporter; slc5a8) is a Na+-coupled transporter for lactate, pyruvate and short-chain fatty acids. Similar to these already known substrates of SMCT, the water-soluble B-complex vitamin nicotinic acid also exists as a monocarboxylate anion (nicotinate) under physiological conditions. Therefore we evaluated the ability of SMCT to mediate the uptake of nicotinate. In mammalian cells, the cloned mouse SMCT (slc5a8) induced the uptake of nicotinate. The SMCT-induced uptake was Na+-dependent. The Michaelis constant for the uptake process was 296±88 μM. The Na+-activation kinetics indicated that at least two Na+ ions are involved in the process. Among the various structural analogues tested, nicotinate was the most effective substrate. Nicotinamide and methylnicotinate were not recognized by the transporter. 2-Pyrazine carboxylate and isonicotinate interacted with the transporter to a moderate extent. SMCT-mediated uptake of nicotinate was inhibited by lactate and pyruvate. In the Xenopus laevis oocyte expression system, SMCT-mediated nicotinate transport was electrogenic, as evident from the nicotinate-induced inward currents under voltage-clamp conditions. Substrate-induced currents in this expression system corroborated the substrate specificity determined in the mammalian cell expression system. The kinetic parameters with regard to the affinity of the transporter for nicotinate and the Hill coefficient for Na+ activation, determined by using the oocyte expression system, were also similar to those obtained from the mammalian cell expression system. We conclude that SMCT functions not only as a Na+-coupled transporter for short-chain fatty acids and lactate but also as a Na+-coupled transporter for the water-soluble vitamin nicotinic acid. PMID:15651982

  9. 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.

  10. 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

  11. 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

  12. Examining Changes in Radioxenon Isotope Activity Ratios during Subsurface Transport

    NASA Astrophysics Data System (ADS)

    Annewandter, Robert

    2014-05-01

    The Non-Proliferation Experiment (NPE) has demonstrated and modelled the usefulness of barometric pumping induced gas transport and subsequent soil gas sampling during On-Site inspections. Generally, gas transport has been widely studied with different numerical codes. However, gas transport of radioxenons and radioiodines in the post-detonation regime and their possible fractionation is still neglected in the open peer-reviewed literature. Atmospheric concentrations of the radioxenons Xe-135, Xe-133m, Xe-133 and Xe-131m can be used to discriminate between civilian releases (nuclear power plants or medical isotope facilities), and nuclear explosion sources. It is based on the multiple isotopic activity ratio method. Yet it is not clear whether subsurface migration of the radionuclides, with eventual release into the atmosphere, can affect the activity ratios due to fractionation. Fractionation can be caused by different mass diffusivities due to mass differences between the radionuclides. Cyclical changes in atmospheric pressure can drive subsurface gas transport. This barometric pumping phenomenon causes an oscillatoric flow in upward trending fractures or highly conductive faults which, combined with diffusion into the porous matrix, leads to a net transport of gaseous components - a so-called ratcheting effect. We use a general purpose reservoir simulator (Complex System Modelling Platform, CSMP++) which is recognized by the oil industry as leading in Discrete Fracture-Matrix (DFM) simulations. It has been applied in a range of fields such as deep geothermal systems, three-phase black oil simulations, fracture propagation in fractured, porous media, and Navier-Stokes pore-scale modelling among others. It is specifically designed to account for structurally complex geologic situation of fractured, porous media. Parabolic differential equations are solved by a continuous Galerkin finite-element method, hyperbolic differential equations by a complementary finite

  13. Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

    PubMed Central

    Sainz, M B; Goff, S A; Chandler, V L

    1997-01-01

    C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains. PMID:8972191

  14. 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

  15. 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.

  16. Differences in associations between active transportation and built environmental exposures when expressed using different components of individual activity spaces.

    PubMed

    van Heeswijck, Torbjorn; Paquet, Catherine; Kestens, Yan; Thierry, Benoit; Morency, Catherine; Daniel, Mark

    2015-05-01

    This study assessed relationships between built environmental exposures measured within components of individual activity spaces (i.e., travel origins, destinations and paths in-between), and use of active transportation in a metropolitan setting. Individuals (n=37,165) were categorised as using active or sedentary transportation based on travel survey data. Generalised Estimating Equations analysis was used to test relationships with active transportation. Strength and significance of relationships between exposures and active transportation varied for different components of the activity space. Associations were strongest when including travel paths in expression of the built environment. Land use mix and greenness were negatively related to active transportation.

  17. Antifeedant activity of anticopalic acid isolated from Vitex hemsleyi.

    PubMed

    Villegas Gómez, Clarisa; Martínez-Vázquez, Mariano; Esquivel, Baldomero

    2009-01-01

    The known labdane-type diterpenoids anticopalic acid (1) and 3 beta-hydroxyanticopalic acid (2) were isolated from extracts of the aerial parts of Vitex hemsleyi Briq. (Labiatae). The acid 1 showed an antifeedant, dose-dependent activity against Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae). To our knowledge this is the first report on the antifeedant activity of a labdane-type diterpene against S. frugiperda.

  18. Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol

    PubMed Central

    Dickens, David; Chiduza, George N.; Wright, Gareth S. A.; Pirmohamed, Munir; Antonyuk, Svetlana V.; Hasnain, S. Samar

    2017-01-01

    LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmax but not the Km of the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold. PMID:28272458

  19. Possible expression of a particular gamma-aminobutyric acid transporter isoform responsive to upregulation by hyperosmolarity in rat calvarial osteoblasts.

    PubMed

    Fujimori, Sayumi; Hinoi, Eiichi; Takarada, Takeshi; Iemata, Mika; Takahata, Yoshifumi; Yoneda, Yukio

    2006-11-21

    Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain, but widely distributed in different peripheral organs. We have previously shown the functional expression of GABA(B) receptors required for GABAergic signal input by cultured rat calvarial osteoblasts. This study focused on the possible functional expression of the machinery required for GABAergic signal termination such as GABA transporters. In rat calvarial osteoblasts cultured for 7 days, [(3)H]GABA accumulation was observed in a temperature-, sodium- and chloride-dependent manner, consisting of a single component with a K(m) value of 789.6+/-9.0 microM and a V(max) value of 4.4+/-0.1 nmol/min/mg protein, respectively. Both nipecotic and L-2,4-diaminobutyric acids significantly inhibited [(3)H]GABA accumulation in a concentration-dependent manner. Constitutive expression was seen with mRNA for the betaine/GABA transporter-1 (BGT-1) and taurine transporter (TauT), while hyperosmotic cultivation led to significant increases in both [(3)H]GABA accumulation and BGT-1 mRNA expression without affecting TauT mRNA expression. Highly immunoreactive cells were detected for the BGT-1 isoform at the surface of trabecular bone of neonatal rat tibias. Sustained exposure to GABA significantly inhibited alkaline phosphatase (ALP) activity, but not cellular viability, at concentrations above 0.1 mM in osteoblasts cultured for 3 to 28 days. Nipecotic acid not only decreased ALP activity alone, but also further decreased ALP activity in osteoblasts cultured in the presence of GABA. These results suggest that the BGT-1 isoform may be functionally expressed by rat calvarial osteoblasts to play a hitherto unidentified role in mechanisms underlying hyperosmotic regulation of osteoblastogenesis.

  20. Active and passive transport of drugs in the human placenta.

    PubMed

    Włoch, Stanisław; Pałasz, Artur; Kamiński, Marcin

    2009-10-01

    The human placenta, characterized by the processes of passive transport and facilitated diffusion, contains numerous active transport proteins, usually located in the microvilli of the syncytiotrophoblast or in the endothelium of the capillaries of the villi. These proteins use either the energy from ATP hydrolysis or other mechanisms resulting, among others, from the formation of the maternofetal ion gradient, which facilitates the transfer of various endogenous substances or xenobiotics across the body membranes. The proteins either trigger the efflux of these substances from the fetal tissues via the placenta into the maternal bloodstream, or conversely they accumulate them in the fetal tissues. Both the placenta and the fetus are equipped with independent systems of enzymes of 1st and 2nd phase of substrate metabolism, such as CYP450, glucuronyltransferase or sulphatase. An active therapy with a wide range of drugs, often at high toxicity levels, either shortly before or during pregnancy, has naturally posed a question concerning the degree of impermeability of the placental barrier and how effectively it can be crossed, including any possible negative embryotoxic or teratogenic consequences. Such hazards seem to be quite real, as many drugs are substrates for ABC transporters. Also the placenta itself, including its structure, is subject to vast transformations during pregnancy which may be observed as the thinning of the barrier separating the maternal blood from the fetal one, from 20-30 microm in the first trimester of gestation down to 2-4 microm in the third trimester of gestation.

  1. 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

  2. Acid activated montmorillonite as catalysts in methyl esterification reactions of lauric acid.

    PubMed

    Zatta, Leandro; Ramos, Luiz Pereira; Wypych, Fernando

    2012-01-01

    The catalytic activity of acid activated montmorillonite in the esterification of free fatty acids (FFA) is reported. Standard Montmorillonite (MMT) type STx-1 provided by the Clay Mineral Society repository was activated using phosphoric, nitric and sulphuric acids under different conditions and the resulting materials were characterized and evaluated as catalysts in the methyl esterification of lauric acid. Blank reactions carried out in the absence of any added catalyst presented conversions of 32.64, 69.79 and 79.23%, for alcohol:lauric acid molar ratios of 60:1, 12:1 and 6:1, respectively. In the presence of the untreated clay and using molar ratios of 12:1 and 6:1 with 12% of catalyst, conversions of 70.92 and 82.30% were obtained, respectively. For the acid activated clays, conversions up to 93.08% of lauric acid to methyl laurate were obtained, much higher than those observed for the thermal conversion or using untreated montmorillonite. Relative good correlations were observed between the catalytic activity and the development of acid sites and textural properties of the resulting materials. Therefore, a simple acid activation was able to improve the catalytic activity and produce clay catalysts that are environmental friendly, cost effective, noncorrosive and reusable.

  3. Chemoenzymatic synthesis of new 2,4-syn-functionalized (S)-glutamate analogues and structure-activity relationship studies at ionotropic glutamate receptors and excitatory amino acid transporters.

    PubMed

    Assaf, Zeinab; Larsen, Anja P; Venskutonytė, Raminta; Han, Liwei; Abrahamsen, Bjarke; Nielsen, Birgitte; Gajhede, Michael; Kastrup, Jette S; Jensen, Anders A; Pickering, Darryl S; Frydenvang, Karla; Gefflaut, Thierry; Bunch, Lennart

    2013-02-28

    In the mammalian central nervous system, (S)-glutamate (Glu) is released from the presynaptic neuron where it activates a plethora of pre- and postsynaptic Glu receptors. The fast acting ionotropic Glu receptors (iGluRs) are ligand gated ion channels and are believed to be involved in a vast number of neurological functions such as memory and learning, synaptic plasticity, and motor function. The synthesis of 14 enantiopure 2,4-syn-Glu analogues 2b-p is accessed by a short and efficient chemoenzymatic approach starting from readily available cyclohexanone 3. Pharmacological characterization at the iGluRs and EAAT1-3 subtypes revealed analogue 2i as a selective GluK1 ligand with low nanomolar affinity. Two X-ray crystal structures of the key analogue 2i in the ligand-binding domain (LBD) of GluA2 and GluK3 were determined. Partial domain closure was seen in the GluA2-LBD complex with 2i comparable to that induced by kainate. In contrast, full domain closure was observed in the GluK3-LBD complex with 2i, similar to that of GluK3-LBD with glutamate bound.

  4. 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).

  5. Natural cinnamic acids, synthetic derivatives and hybrids with antimicrobial activity.

    PubMed

    Guzman, Juan David

    2014-11-25

    Antimicrobial natural preparations involving cinnamon, storax and propolis have been long used topically for treating infections. Cinnamic acids and related molecules are partly responsible for the therapeutic effects observed in these preparations. Most of the cinnamic acids, their esters, amides, aldehydes and alcohols, show significant growth inhibition against one or several bacterial and fungal species. Of particular interest is the potent antitubercular activity observed for some of these cinnamic derivatives, which may be amenable as future drugs for treating tuberculosis. This review intends to summarize the literature data on the antimicrobial activity of the natural cinnamic acids and related derivatives. In addition, selected hybrids between cinnamic acids and biologically active scaffolds with antimicrobial activity were also included. A comprehensive literature search was performed collating the minimum inhibitory concentration (MIC) of each cinnamic acid or derivative against the reported microorganisms. The MIC data allows the relative comparison between series of molecules and the derivation of structure-activity relationships.

  6. 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

  7. 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.

  8. Functional, structural and phylogenetic analys