Effects of ribonuclease A on amino acid transport in Neurospora crassa.

PubMed

Stuart, W D; Woodward, D O

1975-04-01

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.

Recent advances on uric acid transporters

PubMed Central

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

2017-01-01

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

Monocarboxylate Transporter MCT1 Promotes Tumor Metastasis Independently of Its Activity as a Lactate Transporter.

PubMed

Payen, Valéry L; Hsu, Myriam Y; Rädecke, Kristin S; Wyart, Elisabeth; Vazeille, Thibaut; Bouzin, Caroline; Porporato, Paolo E; Sonveaux, Pierre

2017-10-15

Extracellular acidosis resulting from intense metabolic activities in tumors promotes cancer cell migration, invasion, and metastasis. Although host cells die at low extracellular pH, cancer cells resist, as they are well equipped with transporters and enzymes to regulate intracellular pH homeostasis. A low extracellular pH further activates proteolytic enzymes that remodel the extracellular matrix to facilitate cell migration and invasion. Monocarboxylate transporter MCT1 is a passive transporter of lactic acid that has attracted interest as a target for small-molecule drugs to prevent metastasis. In this study, we present evidence of a function for MCT1 in metastasis beyond its role as a transporter of lactic acid. MCT1 activates transcription factor NF-κB to promote cancer cell migration independently of MCT1 transporter activity. Although pharmacologic MCT1 inhibition did not modulate MCT1-dependent cancer cell migration, silencing or genetic deletion of MCT1 in vivo inhibited migration, invasion, and spontaneous metastasis. Our findings raise the possibility that pharmacologic inhibitors of MCT1-mediated lactic acid transport may not effectively prevent metastatic dissemination of cancer cells. Cancer Res; 77(20); 5591-601. ©2017 AACR . ©2017 American Association for Cancer Research.

Molecular characteristics of mammalian and insect amino acid transporters: implications for amino acid homeostasis.

PubMed

Castagna, M; Shayakul, C; Trotti, D; Sacchi, V F; Harvey, W R; Hediger, M A

1997-01-01

In mammalian cells, the uptake of amino acids is mediated by specialized, energy-dependent and passive transporters with overlapping substrate specificities. Most energy-dependent transporters are coupled either to the cotransport of Na+ or Cl- or to the countertransport of K+. Passive transporters are either facilitated transporters or channels. As a prelude to the molecular characterization of the different classes of transporters, we have isolated transporter cDNAs by expression-cloning with Xenopus laevis oocytes and we have characterized the cloned transporters functionally by uptake studies into oocytes using radiolabelled substrates and by electrophysiology to determine substrate-evoked currents. Mammalian transporters investigated include the dibasic and neutral amino acid transport protein D2/NBAT (system b0+) and the Na(+)- and K(+)-dependent neuronal and epithelial high-affinity glutamate transporter EAAC1 (system XAG-). A detailed characterization of these proteins has provided new information on transport characteristics and mechanisms for coupling to different inorganic ions. This work has furthermore advanced our understanding of the roles these transporters play in amino acid homeostasis and in various pathologies. For example, in the central nervous system, glutamate transporters are critically important in maintaining the extracellular glutamate concentration below neurotoxic levels, and defects of the human D2 gene have been shown to account for the formation of kidney stones in patients with cystinuria. Using similar approaches, we are investigating the molecular characteristics of K(+)-coupled amino acid transporters in the larval lepidopteran insect midgut. In the larval midgut, K+ is actively secreted into the lumen through the concerted action of an apical H+ V-ATPase and an apical K+/2H+ antiporter, thereby providing the driving force for absorption of amino acids. In vivo, the uptake occurs at extremely high pH (pH 10) and is driven by a

Modulation of ileal bile acid transporter (ASBT) activity by depletion of plasma membrane cholesterol: association with lipid rafts

PubMed Central

Annaba, Fadi; Sarwar, Zaheer; Kumar, Pradeep; Saksena, Seema; Turner, Jerrold R.; Dudeja, Pradeep K.; Gill, Ravinder K.; Alrefai, Waddah A.

2016-01-01

Apical sodium-dependent bile acid transporter (ASBT) represents a highly efficient conservation mechanism of bile acids via mediation of their active transport across the luminal membrane of terminal ileum. To gain insight into the cellular regulation of ASBT, we investigated the association of ASBT with cholesterol and sphingolipid-enriched specialized plasma membrane microdomains known as lipid rafts and examined the role of membrane cholesterol in maintaining ASBT function. Human embryonic kidney (HEK)-293 cells stably transfected with human ASBT, human ileal brush-border membrane vesicles, and human intestinal epithelial Caco-2 cells were utilized for these studies. Floatation experiments on Optiprep density gradients demonstrated the association of ASBT protein with lipid rafts. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-β-cyclodextrin (MβCD) significantly reduced the association of ASBT with lipid rafts, which was paralleled by a decrease in ASBT activity in Caco-2 and HEK-293 cells treated with MβCD. The inhibition in ASBT activity by MβCD was blocked in the cells treated with MβCD-cholesterol complexes. Kinetic analysis revealed that MβCD treatment decreased the Vmax of the transporter, which was not associated with alteration in the plasma membrane expression of ASBT. Our study illustrates that cholesterol content of lipid rafts is essential for the optimal activity of ASBT and support the association of ASBT with lipid rafts. These findings suggest a novel mechanism by which ASBT activity may be rapidly modulated by alterations in cholesterol content of plasma membrane and thus have important implications in processes related to maintenance of bile acid and cholesterol homeostasis. PMID:18063707

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

USDA-ARS?s Scientific Manuscript database

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...

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

PubMed

Ramesh, Sunita A; Kamran, Muhammad; Sullivan, Wendy; Chirkova, Larissa; Okamoto, Mamoru; Degryse, Fien; McLaughlin, Michael; Gilliham, Matthew; Tyerman, Stephen D

2018-05-01

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABA A receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA] i ) in both wheat ( Triticum aestivum ) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA] i because TaALMT1 facilitates GABA efflux but GABA does not complex Al 3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14 C-GABA uptake into TaALMT1 -expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1 F213C ) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA] i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status. © 2018 American Society of Plant Biologists. All rights reserved.

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

PubMed

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

2015-01-01

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

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

PubMed

Leonardi, M G; Casartelli, M; Fiandra, L; Parenti, P; Giordana, B

2001-12-01

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch. Copyright 2001 Wiley-Liss, Inc.

Redesigning Channel-Forming Peptides: Amino Acid Substitutions that Enhance Rates of Supramolecular Self-Assembly and Raise Ion Transport Activity

PubMed Central

Shank, Lalida P.; Broughman, James R.; Takeguchi, Wade; Cook, Gabriel; Robbins, Ashley S.; Hahn, Lindsey; Radke, Gary; Iwamoto, Takeo; Schultz, Bruce D.; Tomich, John M.

2006-01-01

Three series of 22-residue peptides derived from the transmembrane M2 segment of the glycine receptor α1-subunit (M2GlyR) have been designed, synthesized, and tested to determine the plasticity of a channel-forming sequence and to define whether channel pores with enhanced conductive properties could be created. Sixteen sequences were examined for aqueous solubility, solution-association tendency, secondary structure, and half-maximal concentration for supramolecular assembly, channel activity, and ion transport properties across epithelial monolayers. All peptides interact strongly with membranes: associating with, inserting across, and assembling to form homooligomeric bundles when in micromolar concentrations. Single and double amino acid replacements involving arginine and/or aromatic amino acids within the final five C-terminal residues of the peptide cause dramatic effects on the concentration dependence, yielding a range of K1/2 values from 36 ± 5 to 390 ± 220 μM for transport activity. New water/lipid interfacial boundaries were established for the transmembrane segment using charged or aromatic amino acids, thus limiting the peptides' ability to move perpendicularly to the plane of the bilayer. Formation of discrete water/lipid interfacial boundaries appears to be necessary for efficient supramolecular assembly and high anion transport activity. A peptide sequence is identified that may show efficacy in channel replacement therapy for channelopathies such as cystic fibrosis. PMID:16387776

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

PubMed

Larimore, F S; Roon, R J

1978-02-07

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

Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

PubMed Central

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

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

Roles of organic anion transporters in the renal excretion of perfluorooctanoic acid.

PubMed

Nakagawa, Hatsuki; Hirata, Taku; Terada, Tomohiro; Jutabha, Promsuk; Miura, Daisaku; Harada, Kouji H; Inoue, Kayoko; Anzai, Naohiko; Endou, Hitoshi; Inui, Ken-Ichi; Kanai, Yoshikatsu; Koizumi, Akio

2008-07-01

Perfluorooctanoic acid, an environmental contaminant, is found in both wild animals and human beings. There are large species and sex differences in the renal excretion of perfluorooctanoic acid. In the present study, we aimed to characterize organic anion transporters 1-3 (OAT1-3) in human beings and rats to investigate whether the species differences in the elimination kinetics of perfluorooctanoic acid from the kidneys can be attributed to differences in the affinities of these transporters for perfluorooctanoic acid. We used human (h) and rat (r) OAT transient expression cell systems and measured the [(14)C] perfluorooctanoic acid transport activities. Both human and rat OAT1 and OAT3 mediated perfluorooctanoic acid transport to similar degrees. Specifically, the kinetic parameters, K(m), were 48.0 +/- 6.4 microM for h OAT1; 51.0 +/- 12.0 microM for rOAT1; 49.1 +/- 21.4 microM for hOAT3 and 80.2 +/- 17.8 microM for rOAT3, respectively. These data indicate that both human and rat OAT1 and OAT3 have high affinities for perfluorooctanoic acid and that the species differences in its renal elimination are not attributable to affinity differences in these OATs between human beings and rats. In contrast, neither hOAT2 nor rOAT2 transported perfluorooctanoic acid. In conclusion, OAT1 and OAT3 mediated perfluorooctanoic acid transport in vitro, suggesting that these transporters also transport perfluorooctanoic acid through the basolateral membrane of proximal tubular cells in vivo in both human beings and rats. Neither human nor rat OAT2 mediated perfluorooctanoic acid transport. Collectively, the difference between the perfluorooctanoic acid half-lives in human beings and rats is not likely to be attributable to differences in the affinities of these transporters for perfluorooctanoic acid.

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.

Fatty acid transport and transporters in muscle are critically regulated by Akt2.

PubMed

Jain, Swati S; Luiken, Joost J F P; Snook, Laelie A; Han, Xiao Xia; Holloway, Graham P; Glatz, Jan F C; Bonen, Arend

2015-09-14

Muscle contains various fatty acid transporters (CD36, FABPpm, FATP1, FATP4). Physiological stimuli (insulin, contraction) induce the translocation of all four transporters to the sarcolemma to enhance fatty acid uptake similarly to glucose uptake stimulation via glucose transporter-4 (GLUT4) translocation. Akt2 mediates insulin-induced, but not contraction-induced, GLUT4 translocation, but its role in muscle fatty acid transporter translocation is unknown. In muscle from Akt2-knockout mice, we observed that Akt2 is critically involved in both insulin-induced and contraction-induced fatty acid transport and translocation of fatty acid translocase/CD36 (CD36) and FATP1, but not of translocation of fatty acid-binding protein (FABPpm) and FATP4. Instead, Akt2 mediates intracellular retention of both latter transporters. Collectively, our observations reveal novel complexities in signaling mechanisms regulating the translocation of fatty acid transporters in muscle. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Isolation of Plasma Membrane Vesicles from Mouse Placenta at Term and Measurement of System A and System β Amino Acid Transporter Activity

PubMed Central

Kusinski, L.C.; Jones, C.J.P.; Baker, P.N.; Sibley, C.P.; Glazier, J.D.

2010-01-01

Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and β, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl2 precipitation and centrifugation. Vesicles were enriched 11.3 ± 0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system β activity in mouse placental vesicles, measured as Na+-dependent uptake of 14C-methylaminoisobutyric acid (MeAIB) and 3H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system β activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function. PMID:19954844

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

PubMed Central

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

2014-01-01

Sodium glucose cotransporter 2 (SGLT2) inhibitors have been reported to lower the serum uric acid (SUA) level. To elucidate the mechanism responsible for this reduction, SUA and the urinary excretion rate of uric acid (UEUA) were analysed after the oral administration of luseogliflozin, a SGLT2 inhibitor, to healthy subjects. After dosing, SUA decreased, and a negative correlation was observed between the SUA level and the UEUA, suggesting that SUA decreased as a result of the increase in the UEUA. The increase in UEUA was correlated with an increase in urinary d-glucose excretion, but not with the plasma luseogliflozin concentration. Additionally, in vitro transport experiments showed that luseogliflozin had no direct effect on the transporters involved in renal UA reabsorption. To explain that the increase in UEUA is likely due to glycosuria, the study focused on the facilitative glucose transporter 9 isoform 2 (GLUT9ΔN, SLC2A9b), which is expressed at the apical membrane of the kidney tubular cells and transports both UA and d-glucose. It was observed that the efflux of [14C]UA in Xenopus oocytes expressing the GLUT9 isoform 2 was trans-stimulated by 10 mm d-glucose, a high concentration of glucose that existed under SGLT2 inhibition. On the other hand, the uptake of [14C]UA by oocytes was cis-inhibited by 100 mm d-glucose, a concentration assumed to exist in collecting ducts. In conclusion, it was demonstrated that the UEUA could potentially be increased by luseogliflozin-induced glycosuria, with alterations of UA transport activity because of urinary glucose. PMID:25044127

Exogenous FABP4 increases breast cancer cell proliferation and activates the expression of fatty acid transport proteins.

PubMed

Guaita-Esteruelas, Sandra; Bosquet, Alba; Saavedra, Paula; Gumà, Josep; Girona, Josefa; Lam, Eric W-F; Amillano, Kepa; Borràs, Joan; Masana, Lluís

2017-01-01

Adipose tissue plays an important role in tumor progression, because it provides nutrients and adipokines to proliferating cells. Fatty acid binding protein 4 (FABP4) is a key adipokine for fatty acid transport. In metabolic pathologies, plasma levels of FABP4 are increased. However, the role of this circulating protein is unknown. Recent studies have demonstrated that FABP4 might have a role in tumor progression, but the molecular mechanisms involved are still unclear. In this study, we analysed the role of eFABP4 (exogenous FABP4) in breast cancer progression. MCF-7 and MDA-MB-231 breast cancer cells did not express substantial levels of FABP4 protein, but intracellular FABP4 levels increased after eFABP4 incubation. Moreover, eFABP4 enhanced the proliferation of these breast cancer cells but did not have any effect on MCF-7 and MDA-MB-231 cell migration. Additionally, eFABP4 induced the AKT and MAPK signaling cascades in breast cancer cells, and the inhibition of these pathways reduced the eFBAP4-mediated cell proliferation. Interestingly, eFABP4 treatment in MCF-7 cells increased levels of the transcription factor FoxM1 and the fatty acid transport proteins CD36 and FABP5. In summary, we showed that eFABP4 plays a key role in tumor proliferation and activates the expression of fatty acid transport proteins in MCF-7 breast cancer cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Blood-brain barrier transport of the alpha-keto acid analogs of amino acids.

PubMed

Steele, R D

1986-06-01

A number of alpha-keto acid analogs of amino acids have been found to penetrate the blood-brain barrier (BBB). Pyruvate, alpha-ketobutyrate, alpha-ketoisocaproate, and alpha-keto-gamma-methiolbutyrate all cross the BBB by a carrier-mediated process and by simple diffusion. Under normal physiological conditions, diffusion accounts for roughly 15% or less of total transport. Aromatic alpha-keto acids, phenylpyruvate, and p-hydroxyphenylpyruvate do not penetrate the BBB, nor do they inhibit the transport of other alpha-keto acids. Evidence based primarily on inhibition studies indicates that the carrier-mediated transport of alpha-keto acids occurs via the same carrier demonstrated previously for propionate, acetoacetate, and beta-hydroxybutyrate transport, commonly referred to as the monocarboxylate carrier. As a group, the alpha-keto acid analogs of the amino acids have the highest affinity for the carrier, followed by propionate and beta-hydroxybutyrate. Starvation for 4 days induces transport of alpha-keto acids, but transport is suppressed in rats fed commercial laboratory rations and subjected to portacaval shunts. The mitochondrial pyruvate translocator inhibitor alpha-cyanocinnamate has no effect on the BBB transport of alpha-keto acids.

Effects of a Series of Acidic Drugs on L-Lactic Acid Transport by the Monocarboxylate Transporters MCT1 and MCT4.

PubMed

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

2017-01-01

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

Gastric acid secretion: activation and inhibition.

PubMed Central

Sachs, G.; Prinz, C.; Loo, D.; Bamberg, K.; Besancon, M.; Shin, J. M.

1994-01-01

Peripheral regulation of gastric acid secretion is initiated by the release of gastrin from the G cell. Gastrin then stimulates the cholecystokinin-B receptor on the enterochromaffin-like cell beginning a calcium signaling cascade. An exocytotic release of histamine follows with concomitant activation of a C1- current. The released histamine begins the H2-receptor mediated sequence of events in the parietal cell, which results in activation of the gastric H+/K+ - ATPase. This enzyme is the final common pathway of acid secretion. The H+/K+ - ATPase is composed of two subunits: the larger alpha-subunit couples ion transport to hydrolysis of ATP, the smaller beta-subunit is required for appropriate assembly of the holoenzyme. Both the membrane and extracytoplasmic domain contain the ion transport pathway, and therefore, this region is the target for the antisecretory drugs of the post-H2 era. The 100 kDa alpha-subunit has probably 10 membrane spanning segments with, therefore, five extracytoplasmic loops. The 35 kDA beta-subunit has a single membrane spanning segment, and most of this protein is extracytoplasmic with the six or seven N glycosylation consensus sequences occupied. Omeprazole is an acid-accumulated, acid-activated, prodrug that binds covalently to two cysteine residues at positions 813 (or 822) and 892, accessible from the acidic face of the pump. Lansoprazole binds to cys321, 813 (or 822) and 892; pantoprazole binds to cys813 and 822. The common binding site for these drugs (cys813 or 822) is responsible for the inhibition of acid transport. Covalent inhibition of the acid pump improves control of acid secretion, but since the effective half life of the inhibition in man is about 48 hr, full inhibition of acid secretion, perhaps necessary for eradication of Helicobacter pylori in combination with a single antibiotic, will require prolongation of the effect of this class of drug. PMID:7502535

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

PubMed Central

Dawson, Paul A.

2011-01-01

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

A branched-chain amino acid metabolite drives vascular fatty acid transport and causes insulin resistance.

PubMed

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

2016-04-01

Epidemiological and experimental data implicate branched-chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms that underlie this link remain unclear. Insulin resistance in skeletal muscle stems from the excess accumulation of lipid species, a process that requires blood-borne lipids to initially traverse the blood vessel wall. How this trans-endothelial transport occurs and how it is regulated are not well understood. Here we leveraged PPARGC1a (also known as PGC-1α; encoded by Ppargc1a), a transcriptional coactivator that regulates broad programs of fatty acid consumption, to identify 3-hydroxyisobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a new paracrine regulator of trans-endothelial fatty acid transport. We found that 3-HIB is secreted from muscle cells, activates endothelial fatty acid transport, stimulates muscle fatty acid uptake in vivo and promotes lipid accumulation in muscle, leading to insulin resistance in mice. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the ability of PGC-1α to promote endothelial fatty acid uptake. 3-HIB levels are elevated in muscle from db/db mice with diabetes and from human subjects with diabetes, as compared to those without diabetes. These data unveil a mechanism in which the metabolite 3-HIB, by regulating the trans-endothelial flux of fatty acids, links the regulation of fatty acid flux to BCAA catabolism, providing a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes.

Dicarboxylic acid transport in Bradyrhizobium japonicum: use of Rhizobium meliloti dct gene(s) to enhance nitrogen fixation.

PubMed Central

Birkenhead, K; Manian, S S; O'Gara, F

1988-01-01

A recombinant plasmid encoding Rhizobium meliloti sequences involved in dicarboxylic acid transport (plasmid pRK290:4:46) (E. Bolton, B. Higgisson, A. Harrington, and F. O'Gara, Arch. Microbiol. 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in Bradyrhizobium japonicum. The expression of the dct sequences on plasmid pRK290:4:46 in B. japonicum CJ1 resulted in increased growth rates in media containing dicarboxylic acids as the sole source of carbon. In addition, strain CJ1(pRK290:4:46) exhibited enhanced succinate uptake activity when grown on dicarboxylic acids under aerobic conditions. Under free-living nitrogen-fixing conditions, strain CJ1(pRK290:4:46) exhibited higher nitrogenase (acetylene reduction) activity compared with that of the wild-type strain. This increase in nitrogenase activity also correlated with an enhanced dicarboxylic acid uptake rate under these microaerobic conditions. The regulation of dicarboxylic acid transport by factors such as metabolic inhibitors and the presence of additional carbon sources was similar in both the wild-type and the engineered strains. The implications of increasing nitrogenase activity through alterations in the dicarboxylic acid transport system are discussed. PMID:3422072

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Melton, Elaina M.; Center for Cardiovascular Sciences, Albany Medical College, Albany, NY; Cerny, Ronald L.

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,more » 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

Expression and functional characterisation of System L amino acid transporters in the human term placenta.

PubMed

Gaccioli, Francesca; Aye, Irving L M H; Roos, Sara; Lager, Susanne; Ramirez, Vanessa I; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2015-06-09

System L transporters LAT1 (SLC7A5) and LAT2 (SLC7A8) mediate the uptake of large, neutral amino acids in the human placenta. Many System L substrates are essential amino acids, thus representing crucial nutrients for the growing fetus. Both LAT isoforms are expressed in the human placenta, but the relative contribution of LAT1 and LAT2 to placental System L transport and their subcellular localisation are not well established. Moreover, the influence of maternal body mass index (BMI) on placental System L amino acid transport is poorly understood. Therefore the aims of this study were to determine: i) the relative contribution of the LAT isoforms to System L transport activity in primary human trophoblast (PHT) cells isolated from term placenta; ii) the subcellular localisation of LAT transporters in human placenta; and iii) placental expression and activity of System L transporters in response to maternal overweight/obesity. System L mediated leucine uptake was measured in PHT cells after treatment with si-RNA targeting LAT1 and/or LAT2. The localisation of LAT isoforms was studied in isolated microvillous plasma membranes (MVM) and basal membranes (BM) by Western blot analysis. Results were confirmed by immunohistochemistry in sections of human term placenta. Expression and activity System L transporters was measured in isolated MVM from women with varying pre-pregnancy BMI. Both LAT1 and LAT2 isoforms contribute to System L transport activity in primary trophoblast cells from human term placenta. LAT1 and LAT2 transporters are highly expressed in the MVM of the syncytiotrophoblast layer at term. LAT2 is also localised in the basal membrane and in endothelial cells lining the fetal capillaries. Measurements in isolated MVM vesicles indicate that System L transporter expression and activity is not influenced by maternal BMI. LAT1 and LAT2 are present and functional in the syncytiotrophoblast MVM, whereas LAT2 is also expressed in the BM and in the fetal capillary

Down-Regulation of Placental Transport of Amino Acids Precedes the Development of Intrauterine Growth Restriction in Maternal Nutrient Restricted Baboons1

PubMed Central

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

2016-01-01

Intrauterine growth restriction (IUGR) is an important risk factor for perinatal complications and adult disease. IUGR is associated with down-regulation of placental amino acid transporter expression and activity at birth. It is unknown whether these changes are a cause or a consequence of human IUGR. We hypothesized that placental amino acid transport capacity is reduced prior to onset of reduced fetal growth in baboons with maternal nutrient restriction (MNR). Pregnant baboons were fed either a control (n = 8) or MNR diet (70% of control diet, n = 9) from Gestational Day 30. At Gestational Day 120 (0.65 of gestation), fetuses and placentas were collected. Microvillous (MVM) and basal (BM) plasma membrane vesicles were isolated. System A and system L transport activity was determined in MVM, and leucine transporter activity was assessed in BM using radiolabeled substrates. MVM amino acid transporter isoform expression (SNAT1, SNAT2, and SNAT4 and LAT1 and LAT2) was measured using Western blots. LAT1 and LAT2 expression were also determined in BM. Maternal and fetal plasma amino acids concentrations were determined using mass spectrometry. Fetal and placental weights were unaffected by MNR. MVM system A activity was decreased by 37% in MNR baboon placentas (P = 0.03); however MVM system A amino acid transporter protein expression was unchanged. MVM system L activity and BM leucine transporter activity were not altered by MNR. Fetal plasma concentrations of essential amino acids isoleucine and leucine were reduced, while citrulline increased (P < 0.05) in MNR fetuses compared to controls. In this primate model of IUGR, placental MVM system A amino acid transporter activity is decreased prior to the onset of reduction in the fetal growth trajectory. The reduction in plasma leucine and isoleucine in MNR fetuses may be caused by reduced activity of MVM system A, which is strongly coupled with system L essential amino acid uptake. Our findings indicate that reduced

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

PubMed

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

2016-11-01

Intrauterine growth restriction (IUGR) is an important risk factor for perinatal complications and adult disease. IUGR is associated with down-regulation of placental amino acid transporter expression and activity at birth. It is unknown whether these changes are a cause or a consequence of human IUGR. We hypothesized that placental amino acid transport capacity is reduced prior to onset of reduced fetal growth in baboons with maternal nutrient restriction (MNR). Pregnant baboons were fed either a control (n = 8) or MNR diet (70% of control diet, n = 9) from Gestational Day 30. At Gestational Day 120 (0.65 of gestation), fetuses and placentas were collected. Microvillous (MVM) and basal (BM) plasma membrane vesicles were isolated. System A and system L transport activity was determined in MVM, and leucine transporter activity was assessed in BM using radiolabeled substrates. MVM amino acid transporter isoform expression (SNAT1, SNAT2, and SNAT4 and LAT1 and LAT2) was measured using Western blots. LAT1 and LAT2 expression were also determined in BM. Maternal and fetal plasma amino acids concentrations were determined using mass spectrometry. Fetal and placental weights were unaffected by MNR. MVM system A activity was decreased by 37% in MNR baboon placentas (P = 0.03); however MVM system A amino acid transporter protein expression was unchanged. MVM system L activity and BM leucine transporter activity were not altered by MNR. Fetal plasma concentrations of essential amino acids isoleucine and leucine were reduced, while citrulline increased (P < 0.05) in MNR fetuses compared to controls. In this primate model of IUGR, placental MVM system A amino acid transporter activity is decreased prior to the onset of reduction in the fetal growth trajectory. The reduction in plasma leucine and isoleucine in MNR fetuses may be caused by reduced activity of MVM system A, which is strongly coupled with system L essential amino acid uptake. Our findings indicate that reduced

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.

Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

PubMed

Park, Yun-Yong; Sohn, Bo Hwa; Johnson, Randy L; Kang, Myoung-Hee; Kim, Sang Bae; Shim, Jae-Jun; Mangala, Lingegowda S; Kim, Ji Hoon; Yoo, Jeong Eun; Rodriguez-Aguayo, Cristian; Pradeep, Sunila; Hwang, Jun Eul; Jang, Hee-Jin; Lee, Hyun-Sung; Rupaimoole, Rajesha; Lopez-Berestein, Gabriel; Jeong, Woojin; Park, Inn Sun; Park, Young Nyun; Sood, Anil K; Mills, Gordon B; Lee, Ju-Seog

2016-01-01

Metabolic activation is a common feature of many cancer cells and is frequently associated with the clinical outcomes of various cancers, including hepatocellular carcinoma. Thus, aberrantly activated metabolic pathways in cancer cells are attractive targets for cancer therapy. Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ) are oncogenic downstream effectors of the Hippo tumor suppressor pathway, which is frequently inactivated in many cancers. Our study revealed that YAP1/TAZ regulates amino acid metabolism by up-regulating expression of the amino acid transporters solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5). Subsequently, increased uptake of amino acids by the transporters (SLC38A1 and SLC7A5) activates mammalian target of rapamycin complex 1 (mTORC1), a master regulator of cell growth, and stimulates cell proliferation. We also show that high expression of SLC38A1 and SLC7A5 is significantly associated with shorter survival in hepatocellular carcinoma patients. Furthermore, inhibition of the transporters and mTORC1 significantly blocks YAP1/TAZ-mediated tumorigenesis in the liver. These findings elucidate regulatory networks connecting the Hippo pathway to mTORC1 through amino acid metabolism and the mechanism's potential clinical implications for treating hepatocellular carcinoma. YAP1 and TAZ regulate cancer metabolism and mTORC1 through regulation of amino acid transportation, and two amino acid transporters, SLC38A1 and SLC7A5, might be important therapeutic targets. © 2015 by the American Association for the Study of Liver Diseases.

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

PubMed

Ganapathy, Vadivel

2009-01-15

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

Stimulation by epinephrine of the membrane transport of long chain fatty acid in the adipocyte

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abumrad, N.A.; Perry, P.R.; Whitesell, R.R.

1985-08-25

In isolated rat adipocytes, epinephrine rapidly stimulates the transport of long chain fatty acid across the plasma membrane. At a concentration of unbound oleate of 0.1 microM and 5 min exposure to the hormone, the minimal effective concentration of epinephrine is 0.03 and the optimal concentration 0.3 microM (0.01 and 0.1 microgram/ml). The stimulated rates are 5-10-fold the basal rate of influx or efflux. The hormone effect is on the transport process specifically as shown by isolation of the product of transport in either direction as unesterified fatty acid and inhibition by the transport inhibitors phloretin and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thismore » effect of epinephrine on transport coordinates physiologically with lipase activation to bring about fatty acid release from adipose tissue.« less

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

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

Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells.

PubMed

Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2013-02-01

Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

Electron transport chains of lactic acid bacteria - walking on crutches is part of their lifestyle

PubMed Central

Brooijmans, Rob; Hugenholtz, Jeroen

2009-01-01

A variety of lactic acid bacteria contain rudimentary electron transport chains that can be reconstituted by the addition of heme and menaquinone to the growth medium. These activated electron transport chains lead to higher biomass production and increased robustness, which is beneficial for industrial applications, but a major concern when dealing with pathogenic lactic acid bacteria. PMID:20948651

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

PubMed

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

1998-05-01

surface area between RG2 tumors and contralateral cortex. K1ACPC, deltaK1ACPC, and K DTPA were directly related to tumor cell density, were higher in regions of "impending" necrosis, and the tumor/contralateral brain ACPC radio-activity ratios (0 to 10 minutes) were very similar to that obtained with 0 to 60 minutes experiments. These results indicate that facilitated transport of ACPC is upregulated across C6 and RG2 glioma capillaries, and that tumors can induce upregulation of amino acid transporter expression in their supporting vasculature. They also suggest that early imaging (e.g., 0 to 20 minutes) with radiolabeled amino acids in a clinical setting may be optimal for defining brain tumors.

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

PubMed

Gordon, R; Burford, I 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 (1)C-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 NaK ATPase pump.

Intellectual disability and bleeding diathesis due to deficient CMP--sialic acid transport.

PubMed

Mohamed, Miski; Ashikov, Angel; Guillard, Mailys; Robben, Joris H; Schmidt, Samuel; van den Heuvel, B; de Brouwer, Arjan P M; Gerardy-Schahn, Rita; Deen, Peter M T; Wevers, Ron A; Lefeber, Dirk J; Morava, Eva

2013-08-13

To identify the underlying genetic defect in a patient with intellectual disability, seizures, ataxia, macrothrombocytopenia, renal and cardiac involvement, and abnormal protein glycosylation. Genetic studies involved homozygosity mapping by 250K single nucleotide polymorphism array and SLC35A1 sequencing. Functional studies included biochemical assays for N-glycosylation and mucin-type O-glycosylation and SLC35A1-encoded cytidine 5'-monophosphosialic acid (CMP-sialic acid) transport after heterologous expression in yeast. We performed biochemical analysis and found combined N- and O-glycosylation abnormalities and specific reduction in sialylation in this patient. Homozygosity mapping revealed homozygosity for the CMP-sialic acid transporter SLC35A1. Mutation analysis identified a homozygous c.303G > C (p.Gln101His) missense mutation that was heterozygous in both parents. Functional analysis of mutant SLC35A1 showed normal Golgi localization but 50% reduction in transport activity of CMP-sialic acid in vitro. We confirm an autosomal recessive, generalized sialylation defect due to mutations in SLC35A1. The primary neurologic presentation consisting of ataxia, intellectual disability, and seizures, in combination with bleeding diathesis and proteinuria, is discriminative from a previous case described with deficient sialic acid transporter. Our study underlines the importance of sialylation for normal CNS development and regular organ function.

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

PubMed

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

2010-03-01

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

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

PubMed

Horák, J; Kotyk, A

1993-04-01

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

Ammonia Transporters and Their Role in Acid-Base Balance

PubMed Central

2017-01-01

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. PMID:28151423

A vacuolar membrane protein Avt7p is involved in transport of amino acid and spore formation in Saccharomyces cerevisiae.

PubMed

Tone, Junichi; Yamanaka, Atsushi; Manabe, Kunio; Murao, Nami; Kawano-Kawada, Miyuki; Sekito, Takayuki; Kakinuma, Yoshimi

2015-01-01

Active transport systems for various amino acids operate in the vacuolar membrane of Saccharomyces cerevisiae. The gene families for vacuolar amino acid transporters were identified by reverse genetics experiments. In the AVT transporter family, Avt1p works for active uptake of amino acid into vacuole, and Avt3p, Avt4p, and Avt6p for active extrusion of amino acid from vacuole to cytosol. Here, we found green fluorescent protein-tagged Avt7p, an unidentified member of the AVT family, localized to the vacuolar membrane of S. cerevisiae. Disruption of the AVT7 gene enhanced both vacuolar contents of several amino acids and uptake activities of glutamine and proline by vacuolar membrane vesicles. Efficiency of spore formation was impaired by the disruption of the AVT7 gene, suggesting the physiological importance of Avt7p-dependent efflux of amino acid from vacuoles under nutrient-poor condition.

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

PubMed Central

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

2015-01-01

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

Intracellular pH regulation by acid-base transporters in mammalian neurons

PubMed Central

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

2014-01-01

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

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

Regulators of Slc4 bicarbonate transporter activity.

PubMed

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.

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

PubMed Central

Mulligan, Christopher; Mindell, Joseph A.

2013-01-01

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

Novel Lactate Transporters from Carboxylic Acid-Producing Rhizopus

USDA-ARS?s Scientific Manuscript database

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...

Neutral amino acid transport across brain microvessel endothelial cell monolayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

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,more » 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.« less

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

PubMed

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

2017-12-01

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

Regulation of renal amino acid transporters during metabolic acidosis.

PubMed

Moret, Caroline; Dave, Mital H; Schulz, Nicole; Jiang, Jean X; Verrey, Francois; Wagner, Carsten A

2007-02-01

The kidney plays a major role in acid-base homeostasis by adapting the excretion of acid equivalents to dietary intake and metabolism. Urinary acid excretion is mediated by the secretion of protons and titratable acids, particularly ammonia. NH(3) is synthesized in proximal tubule cells from glutamine taken up via specific amino acid transporters. We tested whether kidney amino acid transporters are regulated in mice in which metabolic acidosis was induced with NH(4)Cl. Blood gas and urine analysis confirmed metabolic acidosis. Real-time RT-PCR was performed to quantify the mRNAs of 16 amino acid transporters. The mRNA of phosphoenolpyruvate carboxykinase (PEPCK) was quantified as positive control for the regulation and that of GAPDH, as internal standard. In acidosis, the mRNA of kidney system N amino acid transporter SNAT3 (SLC38A3/SN1) showed a strong induction similar to that of PEPCK, whereas all other tested mRNAs encoding glutamine or glutamate transporters were unchanged or reduced in abundance. At the protein level, Western blotting and immunohistochemistry demonstrated an increased abundance of SNAT3 and reduced expression of the basolateral cationic amino acid/neutral amino acid exchanger subunit y(+)-LAT1 (SLC7A7). SNAT3 was localized to the basolateral membrane of the late proximal tubule S3 segment in control animals, whereas its expression was extended to the earlier S2 segment of the proximal tubule during acidosis. Our results suggest that the selective regulation of SNAT3 and y(+)LAT1 expression may serve a major role in the renal adaptation to acid secretion and thus for systemic acid-base balance.

Handling of the homocysteine S-conjugate of methylmercury by renal epithelial cells: role of organic anion transporter 1 and amino acid transporters.

PubMed

Zalups, Rudolfs K; Ahmad, Sarfaraz

2005-11-01

Recently, the activity of the organic anion transporter 1 (OAT1) protein has been implicated in the basolateral uptake of inorganic mercuric species in renal proximal tubular cells. Unfortunately, very little is known about the role of OAT1 in the renal epithelial transport of organic forms of mercury, such as methylmercury (CH(3)Hg(+)). Homocysteine (Hcy) S-conjugates of methylmercury [(S)-(3-amino-3-carboxypropylthio)(methyl)mercury (CH(3)Hg-Hcy)] have been identified recently as being potentially important biologically relevant forms of mercury. Thus, the present study was designed to characterize the transport of CH(3)Hg-Hcy in Madin-Darby canine kidney (MDCK) cells (which are derived from the distal nephron) that were transfected stably with the human isoform of OAT1 (hOAT1). Data on saturation kinetics, time dependence, substrate specificity, and temperature dependence demonstrated that CH(3)Hg-Hcy is a transportable substrate of hOAT1. However, substrate-specificity data from the control MDCK cells also showed that CH(3)Hg-Hcy is a substrate of one or more transporter(s) that is/are not hOAT1. Additional findings indicated that at least one amino acid transport system was probably responsible for this transport. It is noteworthy that the activity of amino acid transporters accounted for the greatest level of uptake of CH(3)Hg-Hcy in the hOAT1-expressing cells. Furthermore, rates of survival of the hOAT1-transfected MDCK cells were significantly lower than those of corresponding control MDCK cells when they were exposed to cytotoxic concentrations of CH(3)Hg-Hcy. Collectively, the present data indicate that CH(3)Hg-Hcy is a transportable substrate of OAT1 and amino acid transporters and, thus, is probably a transportable mercuric species taken up in vivo by proximal tubular epithelial cells.

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. © 2015 Authors; published by Portland Press Limited.

Transepithelial transport of alpha-lipoic acid across human intestinal Caco-2 cell monolayers.

PubMed

Takaishi, Naoki; Yoshida, Kazutaka; Satsu, Hideo; Shimizu, Makoto

2007-06-27

Alpha-lipoic acid (LA) is used in dietary supplements or food with antioxidative functions. The mechanism for the intestinal absorption of alpha-lipoic acid was investigated in this study by using human intestinal Caco-2 cell monolayers. LA was rapidly transported across the Caco-2 cell monolayers, this transport being energy-dependent, suggesting transporter-mediated transport to be the mechanism involved. The LA transport was strongly dependent on the pH value, being accelerated in the acidic pH range. Furthermore, such monocarboxylic acids as benzoic acid and medium-chain fatty acids significantly inhibited LA transport, suggesting that a proton-linked monocarboxylic acid transporter (MCT) was involved in the intestinal transport of LA. The conversion of LA to the more antioxidative dihydrolipoic acid was also apparent during the transport process.

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

PubMed

Weiner, I David; Verlander, Jill W

2011-01-01

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

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

PubMed Central

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

2015-01-01

Maternal obesity and gestational diabetes mellitus (GDM) increase the risk of delivering infants that are large for gestational age with greater adiposity, who are prone to the development of metabolic disease in childhood and beyond. These maternal conditions are also associated with increased levels of the proinflammatory cytokine TNF-α in maternal tissues and the placenta. Recent evidence suggests that changes in placental amino acid transport contribute to altered fetal growth. TNF-α was previously shown to stimulate System A amino acid transport in primary human trophoblasts (PHTs), however the molecular mechanisms remain unknown. In this study, we tested the hypothesis that TNF-α regulates amino acid uptake in cultured PHTs by a mitogen-activated protein kinase (MAPK)-dependent mechanism. Treatment of PHTs with TNF-α significantly increased System A amino acid transport, as well as Erk and p38 MAPK signaling. Pharmacological antagonism of p38, but not Erk MAPK activity, inhibited TNF-α stimulated System A activity. Silencing of p38 MAPK using siRNA transfections prevented TNF-α stimulated System A transport in PHTs. TNF-α significantly increased the protein expression of System A transporters SNAT1 and SNAT2, but did not affect their mRNA expression. The effects of TNF-α on SNAT1 and SNAT2 protein expression were reversed by p38 MAPK siRNA silencing. In conclusion, TNF-α regulates System A activity through increased SNAT1 and SNAT2 transporter protein expression in PHTs. These findings suggest that p38 MAPK may represent a critical mechanistic link between elevated proinflammatory cytokines and increased placental amino acid transport in obese and GDM pregnancies associated with fetal overgrowth. PMID:26508738

Naphthalenemethyl ester derivative of dihydroxyhydrocinnamic acid, a component of cinnamon, increases glucose disposal by enhancing translocation of glucose transporter 4.

PubMed

Kim, W; Khil, L Y; Clark, R; Bok, S H; Kim, E E; Lee, S; Jun, H S; Yoon, J W

2006-10-01

Cinnamon extracts have anti-diabetic effects. Phenolic acids, including hydrocinnamic acids, were identified as major components of cinnamon extracts. Against this background we sought to develop a new anti-diabetic compound using derivatives of hydroxycinnamic acids purified from cinnamon. We purified hydroxycinnamic acids from cinnamon, synthesised a series of derivatives, and screened them for glucose transport activity in vitro. We then selected the compound with the highest glucose transport activity in epididymal adipocytes isolated from male Sprague-Dawley rats in vitro, tested it for glucose-lowering activity in vivo, and studied the mechanisms involved. A naphthalenemethyl ester of 3,4-dihydroxyhydrocinnamic acid (DHH105) showed the highest glucose transport activity in vitro. Treatment of streptozotocin-induced diabetic C57BL/6 mice and spontaneously diabetic ob/ob mice with DHH105 decreased blood glucose levels to near normoglycaemia. Further studies revealed that DHH105 increased the maximum speed of glucose transport and the translocation of glucose transporter 4 (GLUT4, now known as solute carrier family 2 [facilitated glucose transporter], member 4 [SLC2A4]) in adipocytes, resulting in increased glucose uptake. In addition, DHH105 enhanced phosphorylation of the insulin receptor-beta subunit and insulin receptor substrate-1 in adipocytes, both in vitro and in vivo. This resulted in the activation of phosphatidylinositol 3-kinase and Akt/protein kinase B, contributing to the translocation of GLUT4 to the plasma membrane. We conclude that DHH105 lowers blood glucose levels through the enhancement of glucose transport, mediated by an increase in insulin-receptor signalling. DHH105 may be a valuable candidate for a new anti-diabetic drug.

The role of monocarboxylate transporters in uptake of lactic acid in HeLa cells.

PubMed

Cheeti, Sravanthi; Warrier, Bharat K; Lee, Chi H

2006-11-15

This study was aimed to identify the monocarboxylate transporters (MCTs) in HeLa cells and to delineate their role in transportation of L-lactic acid. The functional role of MCTs in lactic acid transport was evaluated at various mucosal pHs (4.5-7.4) or in the presence of various loading doses (0.2-2mM) of lactic acid, MCT substrates (nicotinic acid, n-butyric acid, etc.) and inhibitors (alpha-cyano-4-hydroxycinnamate and para-chloromercuribenzoic acid). The molecular properties of MCTs were characterized using reverse transcription-polymerase chain reaction (RT-PCR). The uptake rate of lactic acid by HeLa cells significantly increased from 0.353+/-0.052 to 1.103+/-0.196 micromol/mg protein as the extra-cellular pH changed from 7.4 to 4.5, indicating that activities of MCT were mediated through H(+)-linked mechanism. The uptake profile of lactic acid followed the saturable process with the K(m) value of 0.53 mM. The uptake rate of lactic acid is concentration dependent and is reduced in the presence of MCT inhibitors. MCT isoforms 1, 5 and 6 in HeLa cells were identified by RT-PCR. HeLa cell line can be used as an effective screening tool for intravaginally administered drugs targeted toward MCT.

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

PubMed Central

Zelcer, Noam; Saeki, Tohru; Bot, Ilse; Kuil, Annemieke; Borst, Piet

2003-01-01

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis. PMID:12220224

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

Cyclosporin derivatives inhibit hepatitis B virus entry without interfering with NTCP transporter activity.

PubMed

Shimura, Satomi; Watashi, Koichi; Fukano, Kento; Peel, Michael; Sluder, Ann; Kawai, Fumihiro; Iwamoto, Masashi; Tsukuda, Senko; Takeuchi, Junko S; Miyake, Takeshi; Sugiyama, Masaya; Ogasawara, Yuki; Park, Sam-Yong; Tanaka, Yasuhito; Kusuhara, Hiroyuki; Mizokami, Masashi; Sureau, Camille; Wakita, Takaji

2017-04-01

The sodium taurocholate co-transporting polypeptide (NTCP) is the main target of most hepatitis B virus (HBV) specific entry inhibitors. Unfortunately, these agents also block NTCP transport of bile acids into hepatocytes, and thus have the potential to cause adverse effects. We aimed to identify small molecules that inhibit HBV entry while maintaining NTCP transporter function. We characterized a series of cyclosporine (CsA) derivatives for their anti-HBV activity and NTCP binding specificity using HepG2 cells overexpressing NTCP and primary human hepatocytes. The four most potent derivatives were tested for their capacity to prevent HBV entry, but maintain NTCP transporter function. Their antiviral activity against different HBV genotypes was analysed. We identified several CsA derivatives that inhibited HBV infection with a sub-micromolar IC 50 . Among them, SCY446 and SCY450 showed low activity against calcineurin (CN) and cyclophilins (CyPs), two major CsA cellular targets. This suggested that instead, these compounds interacted directly with NTCP to inhibit viral attachment to host cells, and have no immunosuppressive function. Importantly, we found that SCY450 and SCY995 did not impair the NTCP-dependent uptake of bile acids, and inhibited multiple HBV genotypes including a clinically relevant nucleoside analog-resistant HBV isolate. This is the first example of small molecule selective inhibition of HBV entry with no decrease in NTCP transporter activity. It suggests that the anti-HBV activity can be functionally separated from bile acid transport. These broadly active anti-HBV molecules are potential candidates for developing new drugs with fewer adverse effects. In this study, we identified new compounds that selectively inhibited hepatitis B virus (HBV) entry, and did not impair bile acid uptake. Our evidence offers a new strategy for developing anti-HBV drugs with fewer side effects. Copyright © 2016 European Association for the Study of the Liver

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

PubMed

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

2015-10-01

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

Echinococcus granulosus: specificity of amino acid transport systems in protoscoleces.

PubMed

Jeffs, S A; Arme, C

1987-08-01

Protoscoleces of Echinococcus granulosus absorb the L-amino acids proline, methionine, leucine, alanine, serine, phenylalanine, lysine and glutamic acid by a combination of mediated transport and diffusion. All eight amino acids were accumulated against a concentration gradient. Comparison of Kt and Vmax values suggests that a low affinity for a particular compound is compensated for by a relatively larger number of transport sites for that compound. Four systems serve for the transport of the eight substrates studied: 2 for neutral (EgN1, EgN2) and 1 each for acidic (EgA) and basic (EgB) amino acids. All eight amino acids are incorporated into protein to varying degrees and substantial portions of absorbed L-alanine and L-methionine are metabolized into other compounds.

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

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids.

PubMed

Mathieu, Christoph; González Salgado, Amaia; Wirdnam, Corina; Meier, Stefan; Grotemeyer, Marianne Suter; Inbar, Ehud; Mäser, Pascal; Zilberstein, Dan; Sigel, Erwin; Bütikofer, Peter; Rentsch, Doris

2014-10-01

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.

Identification and functional characterization of a Na+-independent neutral amino acid transporter with broad substrate selectivity.

PubMed

Segawa, H; Fukasawa, Y; Miyamoto, K; Takeda, E; Endou, H; Kanai, Y

1999-07-09

We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the

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

PubMed Central

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

2012-01-01

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

Human cationic amino acid transporter hCAT-3 is preferentially expressed in peripheral tissues.

PubMed

Vékony, N; Wolf, S; Boissel, J P; Gnauert, K; Closs, E I

2001-10-16

At least five distinct carrier proteins form the family of mammalian cationic amino acid transporters (CATs). We have cloned a cDNA containing the complete coding region of human CAT-3. hCAT-3 is glycosylated and localized to the plasma membrane. Transport studies in Xenopus laevis oocytes revealed that hCAT-3 is selective for cationic L-amino acids and exhibits a maximal transport activity similar to other CAT proteins. The apparent substrate affinity and sensitivity to trans-stimulation of hCAT-3 resembles most closely hCAT-2B. This is in contrast to rat and murine CAT-3 proteins that have been reported to display a very low activity and to be inhibited by neutral and anionic L-amino acids as well as D-arginine (Hosokawa, H., et al. (1997) J. Biol. Chem. 272, 8717-8722; Ito, K., and Groudine, M. (1997) J. Biol. Chem. 272, 26780-26786). Also, in adult rat and mouse, CAT-3 has been found exclusively in central neurons. Human CAT-3 expression is not restricted to the brain, in fact, by far the highest expression was found in thymus. Also in other peripheral tissues, hCAT-3 expression was equal to or higher than in most brain regions, suggesting that hCAT-3 is not a neuron-specific transporter.

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

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.

Activation of classical protein kinase C reduces the expression of human cationic amino acid transporter 3 (hCAT-3) in the plasma membrane.

PubMed

Rotmann, Alexander; Vékony, Nicole; Gassner, Davina; Niegisch, Günter; Strand, Dennis; Martiné, Ursula; Closs, Ellen I

2006-04-01

We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185-54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCalpha-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect.

Activation of classical protein kinase C reduces the expression of human cationic amino acid transporter 3 (hCAT-3) in the plasma membrane

PubMed Central

Rotmann, Alexander; Vékony, Nicole; Gassner, Davina; Niegisch, Günter; Strand, Dennis; Martiné, Ursula; Closs, Ellen I.

2005-01-01

We have previously shown that activation of PKC (protein kinase C) results in internalization of hCAT-1 [human CAT-1 (cationic amino acid transporter 1)] and a decrease in arginine transport [Rotmann, Strand, Martiné and Closs (2004) J. Biol. Chem. 279, 54185–54192]. However, others found increased transport rates for arginine in response to PKC activation, suggesting a differential effect of PKC on different CAT isoforms. Therefore we investigated the effect of PKC on hCAT-3, an isoform expressed in thymus, brain, ovary, uterus and mammary gland. In Xenopus laevis oocytes and human U373MG glioblastoma cells, hCAT-3-mediated L-arginine transport was significantly reduced upon treatment with compounds that activate classical PKC. In contrast, inactive phorbol esters and an activator of novel PKC isoforms had no effect. PKC inhibitors (including the PKCα-preferring Ro 31-8280) reduced the inhibitory effect of the PKC-activating compounds. Microscopic analyses revealed a PMA-induced reduction in the cell-surface expression of fusion proteins between hCAT-3 and enhanced green fluorescent protein expressed in X. laevis oocytes and glioblastoma cells. Western-blot analysis of biotinylated surface proteins demonstrated a PMA-induced decrease in hCAT-3 in the plasma membrane, but not in total protein lysates. Pretreatment with a PKC inhibitor also reduced this PMA effect. It is concluded that similar to hCAT-1, hCAT-3 activity is decreased by PKC via reduction of transporter molecules in the plasma membrane. Classical PKC isoforms seem to be responsible for this effect. PMID:16332251

Novel families of vacuolar amino acid transporters.

PubMed

Sekito, Takayuki; Fujiki, Yuki; Ohsumi, Yoshinori; Kakinuma, Yoshimi

2008-08-01

Amino acids are compartmentalized in the vacuoles of microorganisms and plants. In Saccharomyces cerevisiae, basic amino acids accumulate preferentially into vacuoles but acidic amino acids are almost excluded from them. This indicates that selective machineries operate at the vacuolar membrane. The members of the amino acid/auxin permease family and the major facilitator superfamily involved in the vacuolar compartmentalization of amino acids have been recently identified in studies using S. cerevisiae. Homologous genes for these transporters are also found in plant and mammalian genomes. The physiological significance in response to nitrogen starvation can now be discussed. (c) 2008 IUBMB

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bridges, R.J.; Meister, A.

1985-06-25

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

Molecular transport machinery involved in orchestrating luminal acid-induced duodenal bicarbonate secretion in vivo

PubMed Central

Singh, Anurag Kumar; Liu, Yongjian; Riederer, Brigitte; Engelhardt, Regina; Thakur, Basant Kumar; Soleimani, Manoocher; Seidler, Ursula

2013-01-01

The duodenal villus brush border membrane expresses several ion transporters and/or channels, including the solute carrier 26 anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1), the Na+/H+ exchanger isoform 3 (NHE3), as well as the anion channels cystic fibrosis transmembrane conductance regulator (CFTR) and Slc26a9. Using genetically engineered mouse models lacking Scl26a3, Slc26a6, Slc26a9 or Slc9a3 (NHE3), the study was carried out to assess the role of these transporters in mediating the protective duodenal bicarbonate secretory response (DBS-R) to luminal acid; and to compare it to their role in DBS-R elicited by the adenylyl cyclase agonist forskolin. While basal DBS was reduced in the absence of any of the three Slc26 isoforms, the DBS-R to forskolin was not altered. In contrast, the DBS-R to a 5 min exposure to luminal acid (pH 2.5) was strongly reduced in the absence of Slc26a3 or Slc26a9, but not Slc26a6. CFTR inhibitor [CFTR(Inh)-172] reduced the first phase of the acid-induced DBS-R, while NHE3 inhibition (or knockout) abolished the sustained phase of the DBS-R. Luminal acid exposure resulted in the activation of multiple intracellular signalling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by endocytosis in the later stage. We conclude that the long-lasting DBS-R to luminal acid exposure activates multiple duodenocyte signalling pathways and involves changes in trafficking and/or activity of CFTR, Slc26 isoforms Slc26a3 and Slc26a9, and NHE3. PMID:24018950

Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

PubMed

Nagasawa, Kazuki; Nagai, Katsuhito; Ishimoto, Atsushi; Fujimoto, Sadaki

2003-08-27

We previously indicated that lovastatin acid, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, was transported by a monocarboxylate transporter (MCT) in cultured rat mesangial cells. In this study, to identify the MCT isoform(s) responsible for the lovastatin acid uptake, the transport mechanism was investigated using bovine kidney NBL-1 cells, which have been reported to express only MCT4 at the protein level. On RT-PCR analysis, the message of mRNAs for MCT1 and MCT4 was detected in the NBL-1 cells used in this study, which was confirmed by kinetic analysis of [14C]L-lactic acid uptake, consisting of high- and low-affinity components corresponding to MCT1 and MCT4, respectively. The lovastatin acid uptake depended on an inwardly directed H+-gradient, and was inhibited by representative monocarboxylates, but not by inhibitors/substrates for organic anion transporting polypeptides and organic anion transporters. In addition, L-lactic acid competitively inhibited the uptake of lovastatin acid and lovastatin acid inhibited the low affinity component of [14C]L-lactic acid uptake dose dependently. The inhibition constant of L-lactic acid for lovastatin acid uptake was almost the same as the Michaelis constant for [14C]L-lactic acid uptake by the low-affinity component. These kinetic evidences imply that lovastatin acid was taken up into NBL-1 cells via MCT4.

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

PubMed

Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

2015-02-01

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

Uric Acid Induces Renal Inflammation via Activating Tubular NF-κB Signaling Pathway

PubMed Central

Zhou, Yang; Fang, Li; Jiang, Lei; Wen, Ping; Cao, Hongdi; He, Weichun; Dai, Chunsun; Yang, Junwei

2012-01-01

Inflammation is a pathologic feature of hyperuricemia in clinical settings. However, the underlying mechanism remains unknown. Here, infiltration of T cells and macrophages were significantly increased in hyperuricemia mice kidneys. This infiltration of inflammatory cells was accompanied by an up-regulation of TNF-α, MCP-1 and RANTES expression. Further, infiltration was largely located in tubular interstitial spaces, suggesting a role for tubular cells in hyperuricemia-induced inflammation. In cultured tubular epithelial cells (NRK-52E), uric acid, probably transported via urate transporter, induced TNF-α, MCP-1 and RANTES mRNA as well as RANTES protein expression. Culture media of NRK-52E cells incubated with uric acid showed a chemo-attractive ability to recruit macrophage. Moreover uric acid activated NF-κB signaling. The uric acid-induced up-regulation of RANTES was blocked by SN 50, a specific NF-κB inhibitor. Activation of NF-κB signaling was also observed in tubule of hyperuricemia mice. These results suggest that uric acid induces renal inflammation via activation of NF-κB signaling. PMID:22761883

Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport.

PubMed

Fischer, C P; Bode, B P; Takahashi, K; Tanabe, K K; Souba, W W

1996-05-01

The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL-6 receptor expression on the cell surface. It is likely that this

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Rat Liver Canalicular Membrane Vesicles Contain an ATP-Dependent Bile Acid Transport System

NASA Astrophysics Data System (ADS)

Nishida, Toshirou; Gatmaitan, Zenaida; Che, Mingxin; Arias, Irwin M.

1991-08-01

The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature-dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side-out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP-dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders.

A role for gamma-glutamyl transpeptidase and the amino acid transport system xc- in cystine transport by a human pancreatic duct cell line.

PubMed Central

Sweiry, J H; Sastre, J; Viña, J; Elsässer, H P; Mann, G E

1995-01-01

1. The roles of the gamma-glutamyl cycle and the anionic amino acid transport system xc- in mediating L-cystine uptake were investigated in cultured human pancreatic duct PaTu 8902 cells. This cell line exhibits morphological features of normal pancreatic duct cells and expresses gamma-glutamyl transpeptidase (gamma-GT, EC 2.3.2.2), an enzyme involved in the metabolism and regulation of intracellular glutathione (GSH). 2. Uptake of L-cystine (10 microM) was linear for up to 10 min, temperature dependent, Na+ independent, saturable (Michaelis-Menten constant (Km), 86 +/- 25 microM; maximal velocity (Vmax), 109 +/- 33 nmol (mg protein)-1 h-1) and reduced by 80-90% by a 50-fold excess concentration of L-glutamate and L-homocysteic acid, but not L-aspartate. These transport properties resemble those described for system xc-, which exchanges cystine for intracellular glutamate. 3. Acivicin, a known inhibitor of gamma-GT, decreased gamma-GT activity from 2.58 +/- 0.96 to 0.97 +/- 0.11 mU (mg protein)-1 and decreased the initial rates of L-cystine and L-glutamine uptake by 41-55%. Anthglutin (1-gamma-L-glutamyl-2-(2-carboxyphenylhyl)hydrazine), a structurally different inhibitor of gamma-GT, also caused a concentration-dependent (0.01-1 mM) decrease in gamma-GT activity and L-cystine uptake. 4. Neither acivicin nor anthglutin inhibited the uptake of L-glutamate, a poor substrate for gamma-GT. 5. In the presence of a 500-fold excess concentration of glutamate, which should abolish entry of cystine via system xc-, the remaining fraction of cystine transport was inhibited by 50% by acivicin, suggesting that transport is, in part, dependent on the activity of gamma-GT. 6. Cystine transport was also 60-80% inhibited by a series of gamma-glutamyl amino acids (5 mM) including gamma-glutamyl-glutamate, gamma-glutamyl-glutamine and gamma-glutamyl-glycine. alpha-Dipeptides inhibited cystine transport by only 6-22%. 7. These findings demonstrate that in human pancreatic duct Pa

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

PubMed

Mackenzie, Bryan; Erickson, Jeffrey D

2004-02-01

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

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

PubMed Central

Heyne, R I; de Vrij, W; Crielaard, W; Konings, W N

1991-01-01

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism. PMID:1670936

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. Copyright © 2014 the American Physiological Society.

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

PubMed Central

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

2014-01-01

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

CSF/plasma ratios of amino acids: reference data and transports in children.

PubMed

Akiyama, Tomoyuki; Kobayashi, Katsuhiro; Higashikage, Akihito; Sato, Junko; Yoshinaga, Harumi

2014-01-01

We intended to investigate the effects of age, gender, and medications on amino acid cerebrospinal fluid (CSF)/plasma ratios in children, and to determine whether amino acid transports across the blood-CSF barrier in children differ from those in adults. Amino acid concentrations measured by ion-exchange high-performance liquid chromatography were used (CSF from 99 children, simultaneously collected plasma from 76 children). Influence of age, gender, and medications on the amino acid CSF concentrations and CSF/plasma ratios were analyzed by linear multiple regression. Interactions of amino acid transports were analyzed by correlation analysis of CSF/plasma ratios. CSF/plasma ratios of serine, valine, histidine, and arginine were higher in younger children. The glutamate CSF/plasma ratio was higher in older children. Serine, alanine, threonine, valine, and histidine CSF/plasma ratios were lower in females. Glutamine, methionine, tyrosine, and phenylalanine CSF/plasma ratios were elevated with valproate therapy. Serine, threonine, valine, leucine, and tyrosine CSF/plasma ratios were lower with clobazam therapy. The asparagine CSF/plasma ratio was elevated with pyridoxal phosphate therapy. Transports of most essential neutral amino acids interacted with each other, as did neutral amino acids with low molecular weights. Cationic amino acids interacted with each other and some essential neutral amino acids. Acidic amino acids had no interactions with other amino acids. Age, gender, and anti-epileptic drugs affect amino acid CSF/plasma ratios in children. Transport interactions between amino acids in children showed no remarkable difference from those of adults and generally followed the substrate specificities of multiple amino acid transport systems. Copyright © 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

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.

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

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.

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

In‐stream sorption of fulvic acid in an acidic stream: A stream‐scale transport experiment

USGS Publications Warehouse

McKnight, Diane M.; Hornberger, George M.; Bencala, Kenneth E.; Boyer, Elizabeth 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.

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

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

PubMed Central

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 via 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 re-secretion1. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-taurocholate co-transporting polypeptide (NTCP or SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT or SLC10A2) expressed on enterocytes in the terminal ileum2. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption3,4. However, a lack of 3-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 data2,5-8. The crystal structure of an ASBT homolog from Neisseria meningitidis (ASBTNM) in detergent was reported recently9, 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 better the structural changes associated with the coupled transport of Na+ and bile acids, we crystallized and solved two structures of a ASBT homolog from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives alternate accessibility to the highly conserved “crossover” region, where two discontinuous transmembrane helices cross each other. This result has implications for the location and

Glutathione affects the transport activity of Rhizobium leguminosarum 3841 and is essential for efficient nodulation.

PubMed

Cheng, Guojun; Karunakaran, Ramakrishnan; East, Alison K; Munoz-Azcarate, Olaya; Poole, Philip S

2017-04-01

As glutathione (GSH) plays an essential role in growth and symbiotic capacity of rhizobia, a glutathione synthetase (gshB) mutant of Rhizobium leguminosarum biovar viciae 3841 (Rlv3841) was characterised. It fails to efficiently utilise various compounds as a sole carbon source, including glucose, succinate, glutamine and histidine, and shows 60%-69% reduction in uptake rates of glucose, succinate and the non-metabolisable substrate α-amino isobutyric acid. The defect in glucose uptake can be overcome by addition of exogenous GSH, indicating GSH, but not its bacterial synthesis, is required for efficient transport. GSH is not involved in the regulation of the activity of Rlv3841's transporters via the global regulator of transport, PtsNTR. Although lack of GSH reduces transcription of the branched amino acid transporter, this was not the case for all uptake transport systems, for example, the amino acid permease. This suggests GSH alters activity and/or assembly of transport systems by an unknown mechanism. In interaction with plants, the gshB mutant is not only severely impaired in rhizosphere colonisation, but also shows a 50% reduction in dry weight of plants and nitrogen-fixation ability. This reveals that changes in GSH metabolism affect the bacterial-plant interactions required for symbiosis. © FEMS 2017.

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.

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

PubMed Central

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.; Gillham, Matthew

2015-01-01

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. PMID:26219411

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

PubMed

Escudero, Leticia; Mariscal, Vicente; Flores, Enrique

2015-08-01

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. Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in

Sialic acid catabolism and transport gene clusters are lineage specific in Vibrio vulnificus.

PubMed

Lubin, Jean-Bernard; Kingston, Joseph J; Chowdhury, Nityananda; Boyd, E Fidelma

2012-05-01

Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several Vibrio species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In Vibrio cholerae on chromosome I, these genes are carried on the Vibrio pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced Vibrio vulnificus clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within V. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of V. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in V. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in siaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus and that the TRAP transporter is essential for sialic acid uptake.

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

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.

Nano and Mesoscale Ion and Water Transport in Perfluorosulfonic AcidMembranes

DTIC Science & Technology

2017-10-01

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

Maternal protein restriction in the rat inhibits placental insulin, mTOR, and STAT3 signaling and down-regulates placental amino acid transporters.

PubMed

Rosario, Fredrick J; Jansson, Nina; Kanai, Yoshikatsu; Prasad, Puttur D; Powell, Theresa L; Jansson, Thomas

2011-03-01

The mechanisms underlying reduced fetal growth in response to maternal protein restriction are not well established. Maternal levels of insulin, IGF-I, and leptin are decreased in rats fed a low protein (LP) diet. Because these hormones stimulate placental amino acid transporters in vitro, we hypothesized that maternal protein restriction inhibits placental leptin, insulin/IGF-I, and mammalian target of rapamycin signaling and down-regulates the expression and activity of placental amino acid transporters. Pregnant rats were fed either an isocaloric low protein (LP, 4% protein) or control diet (18% protein) and studied at gestational day (GD)15, GD19, or GD21 (term 23). At GD19 and GD21, placental expression of phosphorylated eukaryotic initiation factor 4E binding protein 1 (Thr-36/46 or Thr-70) and phosphorylated S6 ribosomal protein (Ser-235/236) was decreased in the LP group. In addition, placental expression of phosphorylated S6 kinase 1 (Thr-389), phosphorylated Akt (Thr-308), and phosphorylated signal transducer and activator of transcription 3 (Tyr-705) was reduced at GD21. In microvillous plasma membranes (MVM) isolated from placentas of LP animals, protein expression of the sodium-coupled neutral amino acid transporter (SNAT)2 and the large neutral amino acid transporters 1 and 2 was reduced at GD19 and GD21. MVM SNAT1 protein expression was reduced at GD21 in LP rats. SNAT4 and 4F2 heavy chain expression in MVM was unaltered. System A and L amino acid transporter activity was decreased in MVM from LP animals at GD19 and GD21. In conclusion, maternal protein restriction inhibits placental insulin, mammalian target of rapamycin signaling, and signal transducer and activator of transcription 3 signaling, which is associated with a down-regulation of placental amino acid transporters. We speculate that maternal endocrine and metabolic control of placental nutrient transport reduces fetal growth in response to protein restriction.

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

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

PubMed

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

2017-04-01

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

Influence of rye flour enzymatic biotransformation on the antioxidant capacity and transepithelial transport of phenolic acids.

PubMed

de Lima, Fabíola Aliaga; Martins, Isabela Mateus; Faria, Ana; Calhau, Conceição; Azevedo, Joana; Fernandes, Iva; Mateus, Nuno; Macedo, Gabriela Alves

2018-03-01

Phenolic acids have been reported to play a role on the antioxidant activity and other important biological activities. However, as most polyphenolics in food products are either bound to cellular matrices or present as free polymeric forms, the way they are absorbed has not been totally clear until now. Hydrolytic enzymes may act to increase functionalities in polyphenolic-rich foods, enhancing the bioaccessibility of phenolic compounds and minerals from whole grains. The aim of this study was to evaluate the action of tannin acyl hydrolase (tannase) on the total phenols, phenolic acid profile, antioxidant capacity and in vitro bioaccessibility of phenolic acids found in whole rye flour (RF). Besides increasing total phenols and the antioxidant capacity, tannase treatment increased the amounts of ferulic, sinapic and vanillic acids identified in RF, evidencing a new type of feruloyl esterase catalytic action of tannase. Vanillic and sinapic acids in tannase-treated whole rye flour (RFT) were higher than RF after in vitro gastrointestinal digestion, and higher amounts of transported vanillic acid through the Caco-2 monolayer were detected in RFT. However, the bioaccessibility and the transport efficiency of RF phenolic acids were higher than RFT. Underutilized crops like rye and rye-derived products may be an important source of phenolic acids. The tannase biotransformation, even influencing the total phenolics and antioxidant capacity of RF, did not increase the bioaccessibility of phenolic acids under the experimental conditions of this study.

The SLC3 and SLC7 families of amino acid transporters.

PubMed

Fotiadis, Dimitrios; Kanai, Yoshikatsu; Palacín, Manuel

2013-01-01

Amino acids are necessary for all living cells and organisms. Specialized transporters mediate the transfer of amino acids across plasma membranes. Malfunction of these proteins can affect whole-body homoeostasis giving raise to diverse human diseases. Here, we review the main features of the SLC3 and SLC7 families of amino acid transporters. The SLC7 family is divided into two subfamilies, the cationic amino acid transporters (CATs), and the L-type amino acid transporters (LATs). The latter are the light or catalytic subunits of the heteromeric amino acid transporters (HATs), which are associated by a disulfide bridge with the heavy subunits 4F2hc or rBAT. These two subunits are glycoproteins and form the SLC3 family. Most CAT subfamily members were functionally characterized and shown to function as facilitated diffusers mediating the entry and efflux of cationic amino acids. In certain cells, CATs play an important role in the delivery of L-arginine for the synthesis of nitric oxide. HATs are mostly exchangers with a broad spectrum of substrates and are crucial in renal and intestinal re-absorption and cell redox balance. Furthermore, the role of the HAT 4F2hc/LAT1 in tumor growth and the application of LAT1 inhibitors and PET tracers for reduction of tumor progression and imaging of tumors are discussed. Finally, we describe the link between specific mutations in HATs and the primary inherited aminoacidurias, cystinuria and lysinuric protein intolerance. Copyright © 2012 Elsevier Ltd. All rights reserved.

Report membrane transport of lactic acid in the filamentous fungus Rhizopus

USDA-ARS?s Scientific Manuscript database

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...

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

Supplementation of Ascorbic Acid in Weanling Horses Following Prolonged Transportation

PubMed Central

Ralston, Sarah; Stives, Michelle

2012-01-01

Simple Summary Horses normally synthesize adequate amounts of ascorbic acid (vitamin C) in their liver to meet their needs for the vitamin. However, prolonged stress results in low plasma concentrations and reduced immune function. Weanling horses were supplemented with ascorbic acid for 5 or 10 days or no ascorbic acid (4 per group) following 50+ hours of transportation. Supplementation caused increases in plasma concentrations but both supplemented groups had decreased plasma ascorbic acid for 1 to 3 weeks following cessation of supplementation, possibly due to suppressed synthesis. Supplementation of ascorbic acid following prolonged stress will increase plasma concentrations, but prolonged supplementation should be avoided. Abstract Though horses synthesize ascorbic acid in their liver in amounts that meet their needs under normal circumstances, prolonged stress results in low plasma concentrations due to enhanced utilization and renal excretion and can reduce immune function. It was hypothesized that plasma ascorbic acid could be maintained in weanling horses by oral supplementation following prolonged transportation. Weanlings were supplemented with no ascorbic acid (Tx 0: n = 4), 5 grams ascorbic acid twice daily for 5 days (Tx 1: n = 4) or for 10 days (Tx 2: n = 4) following >50 hours of transportation. Supplementation caused slight (P < 0.2) increases in plasma ascorbic acid concentrations. Both supplemented groups had decreased (P < 0.05) plasma concentrations for 1 to 3 weeks following cessation of supplementation, possibly due to increased renal excretion or suppressed hepatic synthesis. Supplementation of ascorbic acid following prolonged stress will increase plasma concentrations, but prolonged supplementation should be avoided. PMID:26486916

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity

PubMed Central

Lin, Huilan; Burton, Damali; Li, Liangtao; Warner, David E.; Phillips, John D.; Ward, Diane McVEY; Kaplan, Jerry

2015-01-01

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865–33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains. PMID:19538181

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lasry, Inbal; Berman, Bluma; Glaser, Fabian

2009-08-28

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

Novel homologous lactate transporter improves L-lactic acid production from glycerol in recombinant strains of Pichia pastoris.

PubMed

de Lima, Pollyne Borborema Almeida; Mulder, Kelly Cristina Leite; Melo, Nadiele Tamires Moreira; Carvalho, Lucas Silva; Menino, Gisele Soares; Mulinari, Eduardo; de Castro, Virgilio H; Dos Reis, Thaila F; Goldman, Gustavo Henrique; Magalhães, Beatriz Simas; Parachin, Nádia Skorupa

2016-09-15

Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous L-lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris. Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for L-lactic acid production. Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. We showed that P. pastoris has a

Expression and regulation of the neutral amino acid transporter B0AT1 in rat small intestine

PubMed Central

Jando, Julia; Camargo, Simone M. R.; Herzog, Brigitte

2017-01-01

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B0AT1 (SLC6A19). Its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ACE2) and is additionally enhanced by aminopeptidase N (CD13). We investigated in this study the expression of B0AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible short- and long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B0AT1, ACE2 and CD13 co-localize on the luminal membrane of small intestinal villi and by Western blotting that their protein expression increases in distal direction. Furthermore, we observed an elevated transport activity of the neutral amino acid L-isoleucine during the nocturnal active phase compared to the inactive one. Gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of B0AT1 protein expression and L-isoleucine transport. Investigation of the chronic dietary regulation of B0AT1, ACE2 and CD13 by different diets revealed an increased B0AT1 protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ACE2 protein expression a reverse localization of the effect. Dietary regulation for CD13 protein expression was not as distinct as for the two other proteins. Ring uptake experiments showed a tendency for increased L-isoleucine uptake under amino acid-supplemented diet and in vivo L-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. Additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. Taken together, our experiments did not reveal an acute amino acid-induced regulation of B0AT1 but revealed a chronic dietary

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. © 2016 Authors; published by Portland Press Limited.

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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Stimulation of the amino acid transporter SLC6A19 by JAK2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhavsar, Shefalee K.; Hosseinzadeh, Zohreh; Merches, Katja

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 myeloproliferativemore » 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

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. © 2014 American Society of Plant Biologists. All Rights Reserved.

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

PubMed

Tu, Hongbin; Wang, Yu; Li, Hongyan; Brinster, Lauren R; Levine, Mark

2017-09-01

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

Prenatal physical activity and diet composition affect the expression of nutrient transporters and mTOR signaling molecules in the human placenta.

PubMed

Brett, K E; Ferraro, Z M; Holcik, M; Adamo, K B

2015-02-01

Adequate nutrient delivery to the fetus is essential for optimal growth. Differences in prenatal physical activity level and diet quality influence maternal energy balance and these factors may alter placental nutrient transport. We investigated the associations between meeting physical activity guidelines and the quality of maternal diet on the expression of genes involved in fatty acid, amino acid and glucose transport, and mammalian target of rapamycin (mTOR) and insulin signaling in the placenta from 16 term pregnancies. Physical activity was directly measured with accelerometry, diet composition was assessed with 24 h dietary recalls, and gene expression was measured with custom polymerase chain reaction (PCR) arrays. Women who met physical activity guidelines had lower gene expression of fatty acid transport protein 4 (FATP4), insulin-like growth factor 1 (IGF1), and the beta non-catalytic subunit of AMP-activated protein kinase (AMPK), and a higher expression of SNAT2. There was a strong positive correlation observed between total sugar intake and glucose transporter 1 (GLUT1) (r = 0.897, p = 0.000, n = 12), and inverse correlations between total sugar and mTOR and IGF1 expression. Percentage of total calories from protein was inversely related to insulin-like growth factor 1 receptor (IGF1R) (r = -0.605, p = 0.028, n = 13). Variations in maternal physical activity and diet composition altered the expression of genes involved in fatty acid, amino acid and glucose transport and mTOR signaling. Future research on placental nutrient transport should include direct measures of maternal PA and dietary habits to help eliminate confounding factors. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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

PubMed

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

2017-03-01

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

Arabidopsis thaliana NIP7;1: An Anther-Specific Boric Acid Transporter of the Aquaporin Superfamily Regulated by an Unusual Tyrosine in Helix 2 of the Transport Pore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Tian; Choi, Won-Gyu; Baudry, Jerome Y

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1more » forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9 11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte

Arabidopsis thaliana NIP7;1: an anther-specific boric acid transporter of the aquaporin superfamily regulated by an unusual tyrosine in helix 2 of the transport pore.

PubMed

Li, Tian; Choi, Won-Gyu; Wallace, Ian S; Baudry, Jerome; Roberts, Daniel M

2011-08-09

Plant nodulin-26 intrinsic proteins (NIPs) are members of the aquaporin superfamily that serve as multifunctional transporters of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as the NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which encode channel proteins expressed in roots and leaf nodes, respectively, that participate in the transport of the critical cell wall nutrient boric acid. Modeling of the protein encoded by the AtNIP7;1 gene shows that it is a third member of the NIP II pore subclass in Arabidopsis. However, unlike AtNIP5;1 and AtNIP6;1 proteins, which form constitutive boric acid channels, AtNIP7;1 forms a channel with an extremely low intrinsic boric acid transport activity. Molecular modeling and molecular dynamics simulations of AtNIP7;1 suggest that a conserved tyrosine residue (Tyr81) located in transmembrane helix 2 adjacent to the aromatic arginine (ar/R) pore selectivity region stabilizes a closed pore conformation through interaction with the canonical Arg220 in ar/R region. Substitution of Tyr81 with a Cys residue, characteristic of established NIP boric acid channels, results in opening of the AtNIP7;1 pore that acquires a robust, transport activity for boric acid as well as other NIP II test solutes (glycerol and urea). Substitution of a Phe for Tyr81 also opens the channel, supporting the prediction from MD simulations that hydrogen bond interaction between the Tyr81 phenol group and the ar/R Arg may contribute to the stabilization of a closed pore state. Expression analyses show that AtNIP7;1 is selectively expressed in developing anther tissues of young floral buds of A. thaliana, principally in developing pollen grains of stage 9-11 anthers. Because boric acid is both an essential nutrient as well as a toxic compound at high concentrations, it is proposed that Tyr81 modulates transport and may provide an additional level of regulation for this transporter in male gametophyte development

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

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.

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

USDA-ARS?s Scientific Manuscript database

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...

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.

INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE

PubMed Central

Grzegorzewicz, Anna E.; Pham, Ha; Gundi, Vijay A. K. B.; Scherman, Michael S.; North, Elton J.; Hess, Tamara; Jones, Victoria; Gruppo, Veronica; Born, Sarah E. M.; Korduláková, Jana; Chavadi, Sivagami Sundaram; Morisseau, Christophe; Lenaerts, Anne J.; Lee, Richard E.; McNeil, Michael R.; Jackson, Mary

2011-01-01

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. PMID:22344175

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

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saini, Nipun; Black, Paul N.; Montefusco, David

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 formore » 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.« less

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis

PubMed Central

Illing, Elisa; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F.; Dunlap, Quinn A.; Sorscher, Eric J.; Woodworth, Bradford A.

2016-01-01

Objectives Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl- transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl- secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Study Design Basic research Setting Laboratory Subjects and Methods Chlorogenic acid was tested on primary murine nasal septal epithelial(MNSE)[CFTR+/+ and transgenic CFTR-/-] and human sinonasal epithelial(HSNE)[CFTR+/+ and F508del/F508del] cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl- transport. Cellular cAMP, phosphorylation of the CFTR regulatory domain(R-D), and CFTR mRNA transcription were also measured. Results Chlorogenic acid stimulated transepithelial Cl- secretion [(change in short-circuit current(ΔISC=μA/cm2)] in MNSE(13.1+/-0.9 vs. 0.1+/-0.1, p<0.05) and HSNE(34.3+/-0.9 vs. 0.0+/-0.1, p<0.05). The drug had a long duration until peak effect at 15-30 minutes after application. Significant inhibition with INH-172, as well as absent stimulation in cultures lacking functional CFTR, suggests effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Conclusion Chlorogenic acid is a water soluble agent that promotes CFTR-mediated Cl- transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. PMID:26019132

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis.

PubMed

Illing, Elisa A; Cho, Do-Yeon; Zhang, Shaoyan; Skinner, Daniel F; Dunlap, Quinn A; Sorscher, Eric J; Woodworth, Bradford A

2015-08-01

Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl(-) transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl(-) secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Basic research. Laboratory. Chlorogenic acid was tested on primary murine nasal septal epithelial (MNSE) (CFTR(+/+) and transgenic CFTR(-/-)) and human sinonasal epithelial (HSNE) (CFTR(+/+) and F508del/F508del) cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl(-) transport. Cellular cyclic adenosine monophosphate (cAMP), phosphorylation of the CFTR regulatory domain (R-D), and CFTR mRNA transcription were also measured. Chlorogenic acid stimulated transepithelial Cl(-) secretion (change in short-circuit current [ΔISC = µA/cm(2)]) in MNSE (13.1 ± 0.9 vs 0.1 ± 0.1; P < .05) and HSNE (34.3 ± 0.9 vs 0.0 ± 0.1; P < .05). The drug had a long duration until peak effect at 15 to 30 minutes after application. Significant inhibition with INH-172 as well as absent stimulation in cultures lacking functional CFTR suggest effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Chlorogenic acid is a water-soluble agent that promotes CFTR-mediated Cl(-) transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2015.

Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism1[OPEN

PubMed Central

Cho, Misuk; Henry, Elizabeth M.; Lewis, Daniel R.; Wu, Guosheng; Muday, Gloria K.

2014-01-01

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. PMID:25324509

Dietary fish oil regulates gene expression of cholesterol and bile acid transporters in mice.

PubMed

Kamisako, Toshinori; Tanaka, Yuji; Ikeda, Takanori; Yamamoto, Kazuo; Ogawa, Hiroshi

2012-03-01

  Fish oil rich in n-3 polyunsaturated fatty acids is known to affect hepatic lipid metabolism. Several studies have demonstrated that fish oil may affect the bile acid metabolism as well as lipid metabolism, whereas only scarce data are available. The aim of this study was to investigate the effect of fish oil on the gene expression of the transporters and enzymes related to bile acid as well as lipid metabolism in the liver and small intestine.   Seven-week old male C57BL/6 mice were fed diets enriched in 10% soybean oil or 10% fish oil for 4 weeks. After 4 weeks, blood, liver and small intestine were obtained.   Hepatic mRNA expression of lipids (Abcg5/8, multidrug resistance gene product 2) and bile acids transporters (bile salt export pump, multidrug resistance associated protein 2 and 3, organic solute transporter α) was induced in fish oil-fed mice. Hepatic Cyp8b1, Cyp27a1 and bile acid CoA : amino acid N-acyltransferase were increased in fish oil-fed mice compared with soybean-oil fed mice. Besides, intestinal cholesterol (Abcg5/8) and bile acid transporters (multidrug resistance associated protein 2 and organic solute transporter α) were induced in fish oil-fed mice.   Fish oil induced the expression of cholesterol and bile acid transporters not only in liver but in intestine. The upregulation of Abcg5/g8 by fish oil is caused by an increase in cellular 27-HOC through Cyp27a1 induction. The hepatic induction of bile acid synthesis through Cyp27a1 may upregulate expression of bile acid transporters in both organs. © 2012 The Japan Society of Hepatology.

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

PubMed Central

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

2014-01-01

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues. PMID:25019617

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

PubMed

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

2014-01-01

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

Role for ion transport in porcine vocal fold epithelial defense to acid challenge.

PubMed

Erickson-Levendoski, Elizabeth; Sivasankar, M Preeti

2012-02-01

The vocal fold epithelium is routinely exposed to gastric contents, including acid and pepsin, during laryngopharyngeal reflux events. The epithelium may possess intrinsic defenses to reflux. The first objective of the current study was to examine whether vocal fold epithelial ion transport is one potential mechanism of defense to gastric contents. The second objective was to determine whether ion transport in response to gastric contents is associated with the secretion of bicarbonate. Prospective design in excised porcine larynges. Laboratory. Porcine vocal folds (N = 56) were exposed on the luminal surface to acid, pepsin, or sham challenges. Ion transport at baseline and following challenge exposure was measured using electrophysiological techniques. To examine specific ion transport mechanisms, vocal folds were pretreated with either a sodium channel blocker or bicarbonate channel blocker. Within 60 seconds of acid but not pepsin exposure, there was a significant increase in ion transport. This rapid increase in ion transport was transient and related to bicarbonate secretion. The current data suggest that porcine vocal folds immediately increase bicarbonate secretion following exposure to acid. Bicarbonate secretion may act to neutralize acid. These findings contribute to the identification of the mechanisms underlying vocal fold defense to reflux and offer implications for the development of treatments for reflux-induced vocal fold injury.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oppedisano, Francesca; Catto, Marco; Koutentis, Panayiotis A.

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 tomore » 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.« less

Docosahexaenoic Acid Supplementation in Pregnancy Modulates Placental Cellular Signaling and Nutrient Transport Capacity in Obese Women.

PubMed

Lager, Susanne; Ramirez, Vanessa I; Acosta, Ometeotl; Meireles, Christiane; Miller, Evelyn; Gaccioli, Francesca; Rosario, Fredrick J; Gelfond, Jonathan A L; Hakala, Kevin; Weintraub, Susan T; Krummel, Debra A; Powell, Theresa L

2017-12-01

Maternal obesity in pregnancy has profound impacts on maternal metabolism and promotes placental nutrient transport, which may contribute to fetal overgrowth in these pregnancies. The fatty acid docosahexaenoic acid (DHA) has bioactive properties that may improve outcomes in obese pregnant women by modulating placental function. To determine the effects of DHA supplementation in obese pregnant women on maternal metabolism and placental function. Pregnant women were supplemented with DHA or placebo. Maternal fasting blood was collected at 26 and 36 weeks' gestation, and placentas were collected at term. Academic health care institution. Thirty-eight pregnant women with pregravid body mass index ≥30 kg/m2. DHA (800 mg, algal oil) or placebo (corn/soy oil) daily from 26 weeks to term. DHA content of maternal erythrocyte and placental membranes, maternal fasting blood glucose, cytokines, metabolic hormones, and circulating lipids were determined. Insulin, mTOR, and inflammatory signaling were assessed in placental homogenates, and nutrient transport capacity was determined in isolated syncytiotrophoblast plasma membranes. DHA supplementation increased erythrocyte (P < 0.0001) and placental membrane DHA levels (P < 0.0001) but did not influence maternal inflammatory status, insulin sensitivity, or lipids. DHA supplementation decreased placental inflammation, amino acid transporter expression, and activity (P < 0.01) and increased placental protein expression of fatty acid transporting protein 4 (P < 0.05). Maternal DHA supplementation in pregnancy decreases placental inflammation and differentially modulates placental nutrient transport capacity and may mitigate adverse effects of maternal obesity on placental function. Copyright © 2017 Endocrine Society

New insights into the roles of proteins and lipids in membrane transport of fatty acids.

PubMed

Hamilton, James A

2007-01-01

Recent calculations of the apparent permeability coefficients for long-chain fatty acids (LCFA) in phospholipid bilayers provide a new perspective on their transport in a membrane. LCFA have permeabilities that are many orders of magnitude higher than glucose, amino acids, and ions. Transport of LCFA through membranes must therefore be considered to be much different from these nutrients, and there is no a priori requirement for catalysis by a membrane protein. New evidence indicates that the plasma membrane proteins postulated as catalysts for transporting LCFA into the cell fall into three categories. Some act as enzymes, mainly for the activation of LCFA to the acyl CoA, which is required for subsequent intracellular metabolism of LCFA. Other proteins appear to participate in sequestering and trafficking of LCFA. Finally, some proteins have undefined mechanisms. The established mechanisms are consistent with biophysical properties of LCFA in membranes, including fast free diffusion by "flip-flop" in the phospholipid bilayer.

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.

Active Transportation Surveillance - United States, 1999-2012.

PubMed

Whitfield, Geoffrey P; Paul, Prabasaj; Wendel, Arthur M

2015-08-28

Physical activity is a health-enhancing behavior, and most U.S. adults do not meet the 2008 Physical Activity Guidelines for Americans. Active transportation, such as by walking or bicycling, is one way that persons can be physically active. No comprehensive, multiyear assessments of active transportation surveillance in the United States have been conducted. 1999-2012. Five surveillance systems assess one or more components of active transportation. The American Community Survey and the National Household Travel Survey (NHTS) both assess the mode of transportation to work in the past week. From these systems, the proportion of respondents who reported walking or bicycling to work can be calculated. NHTS and the American Time Use Survey include 1-day assessments of trips or activities. With that information, the proportion of respondents who report any walking or bicycling for transportation can be calculated. The National Health and Nutrition Examination Survey and the National Health Interview Survey both assess recent (i.e., in the past week or past month) habitual physical activity behaviors, including those performed during active travel. From these systems, the proportion of respondents who report any recent habitual active transportation can be calculated. The prevalence of active transportation as the primary commute mode to work in the past week ranged from 2.6% to 3.4%. The 1-day assessment indicated that the prevalence of any active transportation ranged from 10.5% to 18.5%. The prevalence of any habitual active transportation ranged from 23.9% to 31.4%. No consistent trends in active transportation across time periods and surveillance systems were identified. Among systems, active transportation was usually more common among men, younger respondents, and minority racial/ethnic groups. Among education groups, the highest prevalence of active transportation was usually among the least or most educated groups, and active transportation tended to be more

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT, a Member of the l-Amino Acid Transporter (LAT) Family*

PubMed Central

Bartoccioni, Paola; del Rio, César; Ratera, Merce; Kowalczyk, Lukasz; Baldwin, Jocelyn M.; Zorzano, Antonio; Quick, Matthias; Baldwin, Stephen A.; Vázquez-Ibar, José Luis; Palacín, Manuel

2010-01-01

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC50 similar to the apparent KM of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT. PMID:20610400

Intestinal absorption and activation of decitabine amino acid ester prodrugs mediated by peptide transporter PEPT1 and enterocyte enzymes.

PubMed

Tao, Wenhui; Zhao, Dongyang; Sun, Mengchi; Wang, Ziyu; Lin, Bin; Bao, Yu; Li, Yingying; He, Zhonggui; Sun, Yinghua; Sun, Jin

2018-04-25

Decitabine (DAC), a potent DNA methyltransferase (DNMT) inhibitor, has a limited oral bioavailability. Its 5'-amino acid ester prodrugs could improve its oral delivery but the specific absorption mechanism is not yet fully understood. The aim of this present study was to investigate the in vivo absorption and activation mechanism of these prodrugs using in situ intestinal perfusion and pharmacokinetics studies in rats. Although PEPT1 transporter is pH dependent, there appeared to be no proton cotransport in the perfusion experiment with a preferable transport at pH 7.4 rather than pH 6.5. This suggested that the transport was mostly dependent on the dissociated state of the prodrugs and the proton gradient might play only a limited role. In pH 7.4 HEPES buffer, an increase in P eff was observed for L-val-DAC, D-val-DAC, L-phe-DAC and L-trp-DAC (2.89-fold, 1.2-fold, 2.73-fold, and 1.90-fold, respectively), compared with the parent drug. When co-perfusing the prodrug with Glysar, a known substrate of PEPT1, the permeabilities of the prodrugs were significantly inhibited compared with the control. To further investigate the absorption of the prodrugs, L-val-DAC was selected and found to be concentration-dependent and saturable, suggesting a carrier-mediated process (intrinsic K m : 7.80 ± 2.61 mM) along with passive transport. Determination of drug in intestinal homogenate after perfusion further confirmed that the metabolic activation mainly involved an intestinal first-pass effect. In a pharmacokinetic evaluation, the oral bioavailability of L-val-DAC, L-phe-DAC and L-trp-DAC were nearly 1.74-fold, 1.69-fold and 1.49-fold greater than that of DAC. The differences in membrane permeability and oral bioavailability might be due to the different stability in the intestinal lumen and the distinct PEPT1 affinity which is mainly caused by the stereochemistry, hydrophobicity and steric hindrance of the side chains. In summary, the detailed investigation of the

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

PubMed Central

Longnecker, K.; Sherr, B. F.; Sherr, E. B.

2005-01-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea. PMID:16332746

Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem.

PubMed

Longnecker, K; Sherr, B F; Sherr, E B

2005-12-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

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.

Placental fatty acid transport in maternal obesity.

PubMed

Cetin, I; Parisi, F; Berti, C; Mandò, C; Desoye, G

2012-12-01

Pregestational obesity is a significant risk factor for adverse pregnancy outcomes. Maternal obesity is associated with a specific proinflammatory, endocrine and metabolic phenotype that may lead to higher supply of nutrients to the feto-placental unit and to excessive fetal fat accumulation. In particular, obesity may influence placental fatty acid (FA) transport in several ways, leading to increased diffusion driving force across the placenta, and to altered placental development, size and exchange surface area. Animal models show that maternal obesity is associated with increased expression of specific FA carriers and inflammatory signaling molecules in placental cotyledonary tissue, resulting in enhanced lipid transfer across the placenta, dislipidemia, fat accumulation and possibly altered development in fetuses. Cell culture experiments confirmed that inflammatory molecules, adipokines and FA, all significantly altered in obesity, are important regulators of placental lipid exchange. Expression studies in placentas of obese-diabetic women found a significant increase in FA binding protein-4 expression and in cellular triglyceride content, resulting in increased triglyceride cord blood concentrations. The expression and activity of carriers involved in placental lipid transport are influenced by the endocrine, inflammatory and metabolic milieu of obesity, and further studies are needed to elucidate the strong association between maternal obesity and fetal overgrowth.

Evaluation of active and passive transport processes in corneas extracted from preserved rabbit eyes.

PubMed

Majumdar, Soumyajit; Hingorani, Tushar; Srirangam, Ramesh

2010-04-01

In vitro transcorneal permeability studies are an important screening tool in drug development. The objective of this research is to examine the feasibility of using corneas isolated from preserved rabbit eyes as a model for permeability evaluation. Eyes from male New Zealand White rabbits were used immediately or were stored overnight in phosphate-buffered saline (PBS) or Hanks balanced salt solution (HBSS) over wet ice. Integrity of isolated corneas was evaluated by measuring the TEER and by determining the permeability of paracellular and transcellular markers. Active transport was assessed by measuring transcorneal permeability of selected amino acids. Esterase activity was estimated using p-nitrophenyl assay. In all cases, corneas from freshly enucleated eyes were compared to those isolated from the day-old preserved eyes. Transcellular and paracellular passive diffusion was not affected by the storage medium and observed to be similar in the fresh and preserved eye models. However, amino acid transporters demonstrated lower functional activity in corneas excised from eyes preserved in PBS. Moreover, preserved eyes displayed almost 1.5-fold lower esterase activity in the corneal tissue. Thus, corneas isolated from day-old eyes, preserved in HBSS, closely mimics freshly excised rabbit corneas in terms of both active and passive transport characteristics but possesses slightly reduced enzymatic activity. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Role for Ion Transport in Porcine Vocal Fold Epithelial Defense to Acid Challenge

PubMed Central

Erickson-Levendoski, Elizabeth; Sivasankar, M. Preeti

2012-01-01

Objective The vocal fold epithelium is routinely exposed to gastric contents, including acid and pepsin, during laryngopharyngeal reflux events. The epithelium may possess intrinsic defenses to reflux. The first objective of the current study was to examine whether vocal fold epithelial ion transport is one potential mechanism of defense to gastric contents. The second objective was to determine whether ion transport in response to gastric contents is associated with the secretion of bicarbonate. Study Design Prospective design in excised porcine larynges. Setting Laboratory. Subjects and Methods Porcine vocal folds (N = 56) were exposed on the luminal surface to acid, pepsin, or sham challenges. Ion transport at baseline and following challenge exposure was measured using electrophysiological techniques. To examine specific ion transport mechanisms, vocal folds were pretreated with either a sodium channel blocker or bicarbonate channel blocker. Results Within 60 seconds of acid but not pepsin exposure, there was a significant increase in ion transport. This rapid increase in ion transport was transient and related to bicarbonate secretion. Conclusion The current data suggest that porcine vocal folds immediately increase bicarbonate secretion following exposure to acid. Bicarbonate secretion may act to neutralize acid. These findings contribute to the identification of the mechanisms underlying vocal fold defense to reflux and offer implications for the development of treatments for reflux-induced vocal fold injury. PMID:22086905

The systems biology of uric acid transporters: the role of remote sensing and signaling.

PubMed

Nigam, Sanjay K; Bhatnagar, Vibha

2018-07-01

Uric acid homeostasis in the body is mediated by a number of SLC and ABC transporters in the kidney and intestine, including several multispecific 'drug' transporters (e.g., OAT1, OAT3, and ABCG2). Optimization of uric acid levels can be viewed as a 'systems biology' problem. Here, we consider uric acid transporters from a systems physiology perspective using the framework of the 'Remote Sensing and Signaling Hypothesis.' This hypothesis explains how SLC and ABC 'drug' and other transporters mediate interorgan and interorganismal communication (e.g., gut microbiome and host) via small molecules (e.g., metabolites, antioxidants signaling molecules) through transporters expressed in tissues lining body fluid compartments (e.g., blood, urine, cerebrospinal fluid). The list of uric acid transporters includes: SLC2A9, ABCG2, URAT1 (SLC22A12), OAT1 (SLC22A6), OAT3 (SLC22A8), OAT4 (SLC22A11), OAT10 (SLC22A13), NPT1 (SLC17A1), NPT4 (SLC17A3), MRP2 (ABCC2), MRP4 (ABCC4). Normally, SLC2A9, - along with URAT1, OAT1 and OAT3, - appear to be the main transporters regulating renal urate handling, while ABCG2 appears to regulate intestinal transport. In chronic kidney disease (CKD), intestinal ABCG2 becomes much more important, suggesting remote organ communication between the injured kidney and the intestine. The remote sensing and signaling hypothesis provides a useful systems-level framework for understanding the complex interplay of uric acid transporters expressed in different tissues involved in optimizing uric acid levels under normal and diseased (e.g., CKD, gut microflora dysbiosis) conditions.

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney.

PubMed

Pinho, Maria João; Pinto, Vanda; Serrão, Maria Paula; Jose, Pedro A; Soares-da-Silva, Patrício

2007-07-01

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Fatty acid-amino acid conjugates are essential for systemic activation of salicylic acid-induced protein kinase and accumulation of jasmonic acid in Nicotiana attenuata.

PubMed

Hettenhausen, Christian; Heinrich, Maria; Baldwin, Ian T; Wu, Jianqiang

2014-11-28

Herbivory induces the activation of mitogen-activated protein kinases (MAPKs), the accumulation of jasmonates and defensive metabolites in damaged leaves and in distal undamaged leaves. Previous studies mainly focused on individual responses and a limited number of systemic leaves, and more research is needed for a better understanding of how different plant parts respond to herbivory. In the wild tobacco Nicotiana attenuata, FACs (fatty acid-amino acid conjugates) in Manduca sexta oral secretions (OS) are the major elicitors that induce herbivory-specific signaling but their role in systemic signaling is largely unknown. Here, we show that simulated herbivory (adding M. sexta OS to fresh wounds) dramatically increased SIPK (salicylic acid-induced protein kinase) activity and jasmonic acid (JA) levels in damaged leaves and in certain (but not all) undamaged systemic leaves, whereas wounding alone had no detectable systemic effects; importantly, FACs and wounding are both required for activating these systemic responses. In contrast to the activation of SIPK and elevation of JA in specific systemic leaves, increases in the activity of an important anti-herbivore defense, trypsin proteinase inhibitor (TPI), were observed in all systemic leaves after simulated herbivory, suggesting that systemic TPI induction does not require SIPK activation and JA increases. Leaf ablation experiments demonstrated that within 10 minutes after simulated herbivory, a signal (or signals) was produced and transported out of the treated leaves, and subsequently activated systemic responses. Our results reveal that N. attenuata specifically recognizes herbivore-derived FACs in damaged leaves and rapidly send out a long-distance signal to phylotactically connected leaves to activate MAPK and JA signaling, and we propose that FACs that penetrated into wounds rapidly induce the production of another long-distance signal(s) which travels to all systemic leaves and activates TPI defense.

Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice.

PubMed

Di Giacopo, Andrea; Rubio-Aliaga, Isabel; Cantone, Alessandra; Artunc, Ferruh; Rexhepaj, Rexhep; Frey-Wagner, Isabelle; Font-Llitjós, Mariona; Gehring, Nicole; Stange, Gerti; Jaenecke, Isabel; Mohebbi, Nilufar; Closs, Ellen I; Palacín, Manuel; Nunes, Virginia; Daniel, Hannelore; Lang, Florian; Capasso, Giovambattista; Wagner, Carsten A

2013-12-15

Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b(0,+)AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b(0,+). No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (∼100%), but fractional excretions of lysine (∼35%), ornithine (∼16%), and cystine (∼11%) were less affected. Loss of function of b(0,+)AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.

Histidine residues in the Na+-coupled ascorbic acid transporter-2 (SVCT2) are central regulators of SVCT2 function, modulating pH sensitivity, transporter kinetics, Na+ cooperativity, conformational stability, and subcellular localization.

PubMed

Ormazabal, Valeska; Zuñiga, Felipe A; Escobar, Elizabeth; Aylwin, Carlos; Salas-Burgos, Alexis; Godoy, Alejandro; Reyes, Alejandro M; Vera, Juan Carlos; Rivas, Coralia I

2010-11-19

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

Heteromeric amino acid transporters. In search of the molecular bases of transport cycle mechanisms.

PubMed

Palacín, Manuel; Errasti-Murugarren, Ekaitz; Rosell, Albert

2016-06-15

Heteromeric amino acid transporters (HATs) are relevant targets for structural studies. On the one hand, HATs are involved in inherited and acquired human pathologies. On the other hand, these molecules are the only known examples of solute transporters composed of two subunits (heavy and light) linked by a disulfide bridge. Unfortunately, structural knowledge of HATs is scarce and limited to the atomic structure of the ectodomain of a heavy subunit (human 4F2hc-ED) and distant prokaryotic homologues of the light subunits that share a LeuT-fold. Recent data on human 4F2hc/LAT2 at nanometer resolution revealed 4F2hc-ED positioned on top of the external loops of the light subunit LAT2. Improved resolution of the structure of HATs, combined with conformational studies, is essential to establish the structural bases for light subunit recognition and to evaluate the functional relevance of heavy and light subunit interactions for the amino acid transport cycle. © 2016 Authors; published by Portland Press Limited.

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.

Cationic amino acid transporter 1-mediated L-arginine transport at the inner blood-retinal barrier.

PubMed

Tomi, Masatoshi; Kitade, Naohisa; Hirose, Shirou; Yokota, Noriko; Akanuma, Shin-Ichi; Tachikawa, Masanori; Hosoya, Ken-ichi

2009-11-01

The purpose of this study was to identify the transporter mediating l-arginine transport at the inner blood-retinal barrier (BRB). The apparent uptake clearance of [(3)H]L-arginine into the rat retina was found to be 118 microL/(min.g retina), supporting a carrier-mediated influx transport of L-arginine at the BRB. [(3)H]L-arginine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), used as an in vitro model of the inner BRB, was primarily an Na(+)-independent and saturable process with Michaelis-Menten constants of 11.2 microM and 530 microM. This process was inhibited by rat cationic amino acid transporter (CAT) 1-specific small interfering RNA as well as substrates of CATs, L-arginine, L-lysine, and L-ornithine. The expression of cationic amino acid transporter (CAT) 1 mRNA was 25.9- and 796-fold greater than that of CAT3 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of CAT1 protein was detected in TR-iBRB2 cells and immunostaining of CAT1 was observed along the rat retinal capillaries. In conclusion, CAT1 is localized in retinal capillary endothelial cells and at least in part mediates L-arginine transport at the inner BRB. This process seems to be closely involved in visual functions by supplying precursors of biologically important molecules like nitric oxide in the neural retina.

Exogenous sialic acid transport contributes to group B streptococcus infection of mucosal surfaces.

PubMed

Pezzicoli, Alfredo; Ruggiero, Paolo; Amerighi, Fulvia; Telford, John L; Soriani, Marco

2012-09-15

By sequence analysis of available group B streptococcus (GBS) genomes, we discovered a conserved putative operon involved in the catabolism of sialic acid, containing a tripartite transporter formed by two integral membrane components and a sugar-binding unit, named SAL0039. Expression analysis in the presence of different substrates revealed that SAL0039 was specifically upregulated by the presence of sialic acid and downregulated when bacteria were grown in human blood or in the presence of a high concentration of glucose. The role of SAL0039 in sugar transport was supported by the inability of the sal0039 deletion mutant strain to import exogenous sialic acid and to grow in semidefined medium supplemented with this sugar. Furthermore, in vivo evidence showed that the presence of exogenous sialic acid significantly increased the capacity of GBS to infect mice at the mucosal level. These findings suggest that transport of sialic acid may also contribute to GBS infections.

The γ-Glutamyl Cycle in the Choroid Plexus: Its Possible Function in Amino Acid Transport

PubMed Central

Tate, Suresh S.; Ross, Leonard L.; Meister, Alton

1973-01-01

Various anatomic regions of rabbit brain have been examined for activities of the enzymes of the γ-glutamyl cycle. While these enzyme activities were widely distributed in the brain, they are present in much higher concentrations in the choroid plexus than in other parts of the brain. The activities observed are of about the same order of magnitude as found in the kidney. These observations and other considerations suggest that the γ-glutamyl cycle may play a significant role in the transport of amino acids between blood and cerebrospinal fluid. PMID:4145786

Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist.

PubMed

Januszewicz, Elzbieta; Pajak, Beata; Gajkowska, Barbara; Samluk, Lukasz; Djavadian, Rouzanna L; Hinton, Barry T; Nałecz, Katarzyna A

2009-12-01

In the brain beta-oxidation, which takes place in astrocytes, is not a major process of energy supply. Astrocytes synthesize important lipid metabolites, mainly due to the processes taking place in peroxisomes. One of the compounds necessary in the process of mitochondrial beta-oxidation and export of acyl moieties from peroxisomes is l-carnitine. Two Na-dependent plasma membrane carnitine transporters were shown previously to be present in astrocytes: a low affinity amino acid transporter B(0,+) and a high affinity cation/carnitine transporter OCTN2. The expression of OCTN2 is known to increase in peripheral tissues upon the stimulation of peroxisome proliferators-activator receptor alpha (PPARalpha), a nuclear receptor known to up-regulate several enzymes involved in fatty acid metabolism. The present study was focused on another high affinity carnitine transporter-OCTN3, its presence, regulation and activity in astrocytes. Experiments using the techniques of real-time PCR, Western blot and immunocytochemistry analysis demonstrated the expression of octn3 in rat astrocytes and, out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found in these cells. PPARalpha activator-2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid (WY-14,643) stimulated by 50% expression of octn3, while, on the contrary to peripheral tissues, it did not change the expression of octn2. This observation was correlated with an increased Na-independent activity of carnitine transport. Analysis by transmission electron microscopy showed an augmented intracellular localization of OCTN3 upon PPARalpha stimulation, mainly in peroxisomes, indicating a physiological role of OCTN3 as peroxisomal membrane transporter. These observations point to an important role of OCTN3 in peroxisomal fatty acid metabolism in astrocytes.

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. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome.

PubMed

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

2015-07-01

Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and, although it is considered essential, deficiency has not been linked to disease. 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-containing 2a (MFSD2A) protein. 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. Affected individuals 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-morphant zebrafish, 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.

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. © 2015 Wiley Publishing Asia Pty Ltd.

Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex

PubMed Central

Sage, Jay M.

2014-01-01

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l−1·min−1, respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex. PMID:24598365

Colloidal transport by active filaments

NASA Astrophysics Data System (ADS)

Manna, Raj Kumar; Kumar, P. B. Sunil; Adhikari, R.

2017-01-01

Enhanced colloidal transport beyond the limit imposed by diffusion is usually achieved through external fields. Here, we demonstrate the ballistic transport of a colloidal sphere using internal sources of energy provided by an attached active filament. The latter is modeled as a chain of chemo-mechanically active beads connected by potentials that enforce semi-flexibility and self-avoidance. The fluid flow produced by the active beads and the forces they mediate are explicitly taken into account in the overdamped equations of motion describing the colloid-filament assembly. The speed and efficiency of transport depend on the dynamical conformational states of the filament. We characterize these states using filament writhe as an order parameter and identify ones yielding maxima in speed and efficiency of transport. The transport mechanism reported here has a remarkable resemblance to the flagellar propulsion of microorganisms which suggests its utility in biomimetic systems.

Complementary stimulation of hepatobiliary transport and detoxification systems by rifampicin and ursodeoxycholic acid in humans.

PubMed

Marschall, Hanns-Ulrich; Wagner, Martin; Zollner, Gernot; Fickert, Peter; Diczfalusy, Ulf; Gumhold, Judith; Silbert, Dagmar; Fuchsbichler, Andrea; Benthin, Lisbet; Grundström, Rosita; Gustafsson, Ulf; Sahlin, Staffan; Einarsson, Curt; Trauner, Michael

2005-08-01

Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Design of a New Glutamine-Fipronil Conjugate with α-Amino Acid Function and its Uptake by A. thaliana Lysine Histidine Transporter 1 ( AtLHT1).

PubMed

Jiang, Xunyuan; Xie, Yun; Ren, Zhanfu; Ganeteg, Ulrika; Lin, Fei; Zhao, Chen; Xu, Hanhong

2018-06-26

Creating novel pesticides with phloem-mobility is essential for controlling insects in vascular tissue and root, and conjugating existing pesticides with amino acid is an effective approach. In order to obtain highly phloem-mobile candidate for efficient pesticide, an electro-neutral L-glutamine-fipronil conjugate (L-GlnF) retaining α-amino acid function was designed and synthesized to fit the substrate specificity of amino acid transporter. Cotyledon uptake and phloem loading tests with Ricinus communis have verified that L-GlnF was phloem mobile, and its phloem mobility was higher than its enantiomer D-GlnF and other previously reported amino acid-fipronil conjugates. Inhibition experiments then suggested that the uptake of L-GlnF was, at least partially, mediated by active transport mechanism. This inference was further strengthened by assimilation experiments with Xenopus oocytes and genetically modified Arabidopsis thaliana, which showed direct correlation between the uptake of L-GlnF and expression of amino acid transporter AtLHT1. Thus, conjugation with L-Gln appears to be a potential strategy to ensure the uptake of pesticides via endogenous amino acid transport system.

Study of Tranexamic Acid during Air Medical Prehospital Transport (STAAMP) Trial

DTIC Science & Technology

2014-10-01

AD______________ AWARD NUMBER: W81XWH-13-2-0080 TITLE: Study of Tranexamic acid ... Tranexamic acid during Air Medical Prehospital transport (STAAMP) trial 5b. GRANT NUMBER W81XWH-13-2-0080 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...and explained the purpose of this study to Pittsburgh local and surrounding area. 15. SUBJECT TERMS Prehospital ; Tranexamic acid 16

[Lipoproteins as a specific circulatory transport system].

PubMed

Titov, V N

1998-01-01

-ligand-receptor interaction, which is considered to be a key stage in the multistage process of active transport to the cells of polyenic fatty acids. However, the significant differences of active and inactive transport of polyenic fatty acids in the blood stream await a separate consideration.

Vitamin C transporter gene polymorphisms, dietary vitamin C and serum ascorbic acid.

PubMed

Cahill, Leah E; El-Sohemy, Ahmed

2009-01-01

Vitamin C transporter proteins SVCT1 and SVCT2 are required for the absorption and transport of vitamin C in humans. This study aims to determine whether common SVCT genotypes modify the association between dietary vitamin C and serum ascorbic acid. Non-smoking men and women (n=1,046) aged 20-29 were participants of the Toronto Nutrigenomics and Health Study. Overnight fasting blood samples were collected to determine serum ascorbic acid concentrations by HPLC and to genotype for two SVCT1 (rs4257763 and rs6596473) and two SVCT2 (rs6139591 and rs2681116) polymorphisms. No diet-gene interactions were observed for the vitamin C transporter polymorphisms, however, the average (mean+/-SE) serum ascorbic acid concentrations differed between rs4257763 genotypes (GG: 24.4+/-1.3, GA: 26.8+/-1.1, AA: 29.7+/-1.4 micromol/l; p=0.002). For this polymorphism, the correlation between dietary vitamin C and serum ascorbic acid was only significant in subjects with a G allele. The SVCT2 polymorphisms also appeared to modify the strength of the diet-serum correlation. Our findings demonstrate that genetic variation in SVCT1 can influence serum ascorbic acid concentrations and that SVCT1 and SVCT2 genotypes modify the strength of the correlation between dietary vitamin C and serum ascorbic acid. Copyright © 2010 S. Karger AG, Basel.

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

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

NASA Astrophysics Data System (ADS)

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

2017-10-01

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

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

PubMed

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

2014-09-01

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

Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex.

PubMed

Sage, Jay M; Carruthers, Anthony

2014-05-15

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l(-1)·min(-1), respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex. Copyright © 2014 the American Physiological Society.

Active transportation and public transportation use to achieve physical activity recommendations? A combined GPS, accelerometer, and mobility survey study.

PubMed

Chaix, Basile; Kestens, Yan; Duncan, Scott; Merrien, Claire; Thierry, Benoît; Pannier, Bruno; Brondeel, Ruben; Lewin, Antoine; Karusisi, Noëlla; Perchoux, Camille; Thomas, Frédérique; Méline, Julie

2014-09-27

Accurate information is lacking on the extent of transportation as a source of physical activity, on the physical activity gains from public transportation use, and on the extent to which population shifts in the use of transportation modes could increase the percentage of people reaching official physical activity recommendations. In 2012-2013, 234 participants of the RECORD GPS Study (French Paris region, median age = 58) wore a portable GPS receiver and an accelerometer for 7 consecutive days and completed a 7-day GPS-based mobility survey (participation rate = 57.1%). Information on transportation modes and accelerometry data aggregated at the trip level [number of steps taken, energy expended, moderate to vigorous physical activity (MVPA), and sedentary time] were available for 7,644 trips. Associations between transportation modes and accelerometer-derived physical activity were estimated at the trip level with multilevel linear models. Participants spent a median of 1 h 58 min per day in transportation (8.2% of total time). Thirty-eight per-cent of steps taken, 31% of energy expended, and 33% of MVPA over 7 days were attributable to transportation. Walking and biking trips but also public transportation trips with all four transit modes examined were associated with greater steps, MVPA, and energy expenditure when compared to trips by personal motorized vehicle. Two simulated scenarios, implying a shift of approximately 14% and 33% of all motorized trips to public transportation or walking, were associated with a predicted 6 point and 13 point increase in the percentage of participants achieving the current physical activity recommendation. Collecting data with GPS receivers, accelerometers, and a GPS-based electronic mobility survey of activities and transportation modes allowed us to investigate relationships between transportation modes and physical activity at the trip level. Our findings suggest that an increase in active transportation

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

USDA-ARS?s Scientific Manuscript database

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,...

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. Copyright © 2016 Carvalho et al.

Active transport and heat.

PubMed

Tait, Peter W

2011-07-01

Increasing heat may impede peoples' ability to be active outdoors thus limiting active transport options. Co-benefits from mitigation of and adaptation to global warming should not be assumed but need to be actively designed into strategies.

Towards bridging the gap between acid-base transporters and neuronal excitability modulation

PubMed Central

Liu, Ying; Chen, Li-Ming

2014-01-01

pH homeostasis is a fundamental regulator of the function of the central nervous system. Dysfunction of acid-base transporters often results in disturbance of neuronal excitability. In a latest issue of Journal of Neuroscience, Jones et al. report that increasing intracellular bicarbonate concentration substantially stimulates the excitability of pyramidal neurons from mouse hippocampus by inhibiting KCNQ potassium channel. The finding shed important new light in understanding the molecular mechanism underlying the regulation of neuronal excitability by acid-base transporters. PMID:25755844

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

PubMed

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

2015-07-01

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

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development*

PubMed Central

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.

2016-01-01

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. PMID:27008858

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

PubMed

Martin, Audrey; Daniel, Jaiyanth

2018-02-05

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

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.

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

PubMed Central

Casal, Margarida; Cardoso, Helena; Leão, Cecília

1998-01-01

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of Vmax versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol. PMID:9464405

Active transportation environments surrounding Canadian schools.

PubMed

O'Loghlen, Sean; Pickett, J William; Janssen, Ian

2011-01-01

Walking or cycling to school represents an opportunity for children to engage in physical activity. The study objectives were to: 1) describe active transportation policies, programs, and built environments of Canadian schools and their surrounding neighbourhoods, and 2) document variations based on urban-rural location and school type (primary vs. secondary vs. mixed primary/secondary schools). 397 schools from across Canada were studied. A school administrator completed a questionnaire and responses were used to assess schools' policies and programs related to active transportation and the safety and aesthetics of their respective neighbourhoods. Built environment features in a 1 km-radius circular buffer around each school were measured using geographic information systems. Greater than 70% of schools had passive policies (e.g., skateboards permitted on school grounds) and facilities (e.g., bicycle racks in secure area to avoid theft) to encourage bicycle and small-wheeled vehicle use. Less than 40% of schools had active programs designed to encourage active transportation, such as organized 'walk to school' days. Garbage in the streets, crime and substance abuse were barriers in most school neighbourhoods. Approximately 42% of schools were located on high-speed roads not amenable to active transportation and 14% did not have a sidewalk leading to the school. Secondary schools had less favourable active transportation policies/programs and neighbourhood safety/aesthetics compared to primary schools. Rural schools had less favourable built environments than urban schools. Canadian children, particularly those from rural areas, face a number of impediments to active transportation as a method of travelling to school.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2015-02-15

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

Membrane transport of amino acid enantiomers in protoscoleces of Echinococcus granulosus (Cestoda).

PubMed

Allen, J T; Arme, C

1991-02-01

Protoscoleces of Echinococcus granulosus absorb both L- and D-alanine. Concentration ratios exceed 1 with values for D-alanine exceeding those for the L-isomer, suggesting that both are absorbed by active mechanisms. Uptake of both isomers involves both diffusion and carrier-mediated components. Values for the diffusion component (Kd) for L- and D-alanine were 0.21 and 0.38 nmol mg-1 protein/1.5 min mM-1 respectively, and values for Kt, the transport constants, 0.17 mM and 0.21 mM respectively. Uptake of both isomers was inhibited competitively by a number of other amino acids.

Gene expression of amino acid transporter in pigeon (Columbia livia) intestine during post-hatch development and its correlation with amino acid in pigeon milk.

PubMed

Zhang, X Y; Zhang, N N; Wan, X P; Li, L L; Zou, X T

2017-05-01

This study was conducted to evaluate gene expression of the amino acid transporter in post-hatch pigeon small intestine and the association of pigeon milk amino acid with the above transporter's gene expression. A total of 48 pigeon breeding families were randomly allocated to 8 groups of 6 replicates of one parental pigeon pair and 2 squabs. Samples of pigeon milk and duodenum, jejunum, and ileum were collected on d 1, 2, 3, 4, 6, 8, 10, and 14 post hatch. The results showed that levels of crude protein (8.93 to 15.56%) were highest in pigeon milk on an air-dry basis. Amino acid content in pigeon milk remained constant in the first 4 d, declined abruptly at d 6, then increased dramatically from d 8 to 14. There was a significant effect of interaction between age and intestinal segments on those amino acid transporters gene expression. mRNA abundance of ATB0'+, SNAT-2, LAT-4, rBAT, b0'+AT, EAAT-3 and PAT-1 was highest in the ileum; B0AT1, asc-1, and IMINO were predominate in the jejunum; and CAT-1 and y+LAT2 were greatest in the duodenum. Age-related changes of amino acid transporter mRNA was inconsistent. mRNA levels of SNAT-2, rBAT, y+LAT2, b0'+AT, and EAAT-3 ascended with age, whereas that of asc-1, CAT-1, and IMINO diminished significantly. Levels of B0AT1 and PAT-1 mRNA abundance were minimized at d 6. However, few correlations were found between pigeon milk amino acid and the amino acid transporter gene expressions in squab small intestine. Our findings provide a comprehensive elaboration on ontogeny of the amino acid transporter in post-hatch pigeon intestine. © 2016 Poultry Science Association Inc.

Kinetics of dodecanoic acid adsorption from caustic solution by activated carbon.

PubMed

Pendleton, Phillip; Wu, Sophie Hua

2003-10-15

This study examines the influences of adsorbent porosity and surface chemistry and of carbon dosage on dodecanoic acid adsorption kinetics from aqueous and 2 M NaOH solutions as batch adsorption processes. Both adsorbents are steam-activated carbons prepared from either coconut or coal precursors. Prior to use the adsorbents were washed in deionized water or 2 M NaOH. Mass transfer coefficients and effective overall diffusion coefficients indicate a minor contribution from adsorbent porosity. In contrast, high surface oxygen content impedes transport to and into the adsorbent structure. Carbon dosage shows a proportional increase in transport coefficients with increasing mass; these coefficients are constant when normalized per unit mass. Neither water nor NaOH treatment of the adsorbents has a significant influence on dodecanoic acid adsorption kinetics. Molecular and Knudsen diffusion coefficients are defined to demonstrate that the overall effective diffusion coefficient values and the diffusion process are controlled by surface diffusion.

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.

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

PubMed

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

2018-07-01

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

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. © 2016 Federation of European Biochemical Societies.

The effects of reduced dietary protein level on amino acid transporters and mTOR signaling pathway in pigs.

PubMed

Wang, Dan; Wan, Xuebin; Peng, Jian; Xiong, Qi; Niu, Hongdan; Li, Huanan; Chai, Jin; Jiang, Siwen

2017-04-01

Amino acid transporter plays an important role in regulating mTOR signaling pathway. This study investigated the effects of reduced dietary protein levels on amino acid transporters and mTOR signaling pathway. A total of 54 weaning pigs were randomly allocated into a 3 × 3 factorial design, followed by slaughtering the pigs separately after 10-, 25- and 45-day feeding, with 18 pigs from each feeding period divided into three subgroups for treatment with three different protein-level diets: 20% crude protein (CP) diet (normal recommended, high protein, HP), 17% CP diet (medium protein, MP) and 14% CP diet (low protein, LP). The results indicated that reduced dietary protein level decreased the weight of longissimus dorsi. Additionally, quantitative PCR chip analysis showed that mRNA expression of amino acid transporters SLC38A2, SLC1A7, SLC7A1, SLC7A5, SLC16A10 and SLC3A2 in the LP group were significantly (P < 0.05) higher than those in the MP or HP group, and the phosphorylation of mTOR and S6K1 decreased in the LP group after 25-day feeding. Furthermore, the vitro experimental results further confirmed that the mRNA levels for SLC7A1, SLC7A5, SLC3A2, SLC38A2 and SLC36A1 were increased and the phosphorylation of mTOR and S6K1 was decreased when the concentration of amino acids in C2C12 myoblasts was reduced. All these results indicated that the LP diet induced a high expression of amino acid transporters and the inhibition of the mTOR activity, which resulting in restriction on protein synthesis and longissimus dorsi growth. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed

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

2012-06-01

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

Synthesis and biological activity of amino acid conjugates of abscisic acid.

PubMed

Todoroki, Yasushi; Narita, Kenta; Muramatsu, Taku; Shimomura, Hajime; Ohnishi, Toshiyuki; Mizutani, Masaharu; Ueno, Kotomi; Hirai, Nobuhiro

2011-03-01

We prepared 19 amino acid conjugates of the plant hormone abscisic acid (ABA) and investigated their biological activity, enzymatic hydrolysis by a recombinant Arabidopsis amidohydrolases GST-ILR1 and GST-IAR3, and metabolic fate in rice seedlings. Different sets of ABA-amino acids induced ABA-like responses in different plants. Some ABA-amino acids, including some that were active in bioassays, were hydrolyzed by recombinant Arabidopsis GST-IAR3, although GST-ILR1 did not show hydrolysis activity for any of the ABA-amino acids. ABA-L-Ala, which was active in all the bioassays, an Arabidopsis seed germination, spinach seed germination, and rice seedling elongation assays, except in a lettuce seed germination assay and was hydrolyzed by GST-IAR3, was hydrolyzed to free ABA in rice seedlings. These findings suggest that some plant amidohydrolases hydrolyze some ABA-amino acid conjugates. Because our study indicates the possibility that different plants have hydrolyzing activity toward different ABA-amino acids, an ABA-amino acid may function as a species-selective pro-hormone of ABA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Differential impact of amino acids on OXPHOS system activity following carbohydrate starvation in Arabidopsis cell suspensions.

PubMed

Cavalcanti, João Henrique F; Quinhones, Carla G S; Schertl, Peter; Brito, Danielle S; Eubel, Holger; Hildebrandt, Tatjana; Nunes-Nesi, Adriano; Braun, Hans-Peter; Araújo, Wagner L

2017-12-01

Plant respiration mostly depends on the activity of glycolysis and the oxidation of organic acids in the tricarboxylic acid cycle to synthesize ATP. However, during stress situations plant cells also use amino acids as alternative substrates to donate electrons through the electron-transfer flavoprotein (ETF)/ETF:ubiquinone oxidoreductase (ETF/ETFQO) complex to the mitochondrial electron transport chain (mETC). Given this, we investigated changes of the oxidative phosphorylation (OXPHOS) system in Arabidopsis thaliana cell culture under carbohydrate starvation supplied with a range of amino acids. Induction of isovaleryl-CoA dehydrogenase (IVDH) activity was observed under carbohydrate starvation which was associated with increased amounts of IVDH protein detected by immunoblotting. Furthermore, activities of the protein complexes of the mETC were reduced under carbohydrate starvation. We also observed that OXPHOS system activity behavior is differently affected by different amino acids and that proteins associated with amino acids catabolism are upregulated in cells following carbohydrate starvation. Collectively, our results support the contention that ETF/ETFQO is an essential pathway to donate electrons to the mETC and that amino acids are alternative substrates to maintain respiration under carbohydrate starvation. © 2017 Scandinavian Plant Physiology Society.

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

NASA Astrophysics Data System (ADS)

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

2013-07-01

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

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter

PubMed Central

Rizzello, Antonia; Romano, Alessandro; Kottra, Gabor; Acierno, Raffaele; Storelli, Carlo; Verri, Tiziano; Daniel, Hannelore; Maffia, Michele

2013-01-01

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications. PMID:23569229

The role of L-type amino acid transporters in the uptake of glyphosate across mammalian epithelial tissues.

PubMed

Xu, Jiaqiang; Li, Gao; Wang, Zhuoyi; Si, Luqin; He, Sijie; Cai, Jialing; Huang, Jiangeng; Donovan, Maureen D

2016-02-01

Glyphosate is one of the most commonly used herbicides worldwide due to its broad spectrum of activity and reported low toxicity to humans. Glyphosate has an amino acid-like structure that is highly polar and shows low bioavailability following oral ingestion and low systemic toxicity following intravenous exposures. Spray applications of glyphosate in agricultural or residential settings can result in topical or inhalation exposures to the herbicide. Limited systemic exposure to glyphosate occurs following skin contact, and pulmonary exposure has also been reported to be low. The results of nasal inhalation exposures, however, have not been evaluated. To investigate the mechanisms of glyphosate absorption across epithelial tissues, the permeation of glyphosate across Caco-2 cells, a gastrointestinal epithelium model, was compared with permeation across nasal respiratory and olfactory tissues excised from cows. Saturable glyphosate uptake was seen in all three tissues, indicating the activity of epithelial transporters. The uptake was shown to be ATP and Na(+) independent, and glyphosate permeability could be significantly reduced by the inclusion of competitive amino acids or specific LAT1/LAT2 transporter inhibitors. The pattern of inhibition of glyphosate permeability across Caco-2 and nasal mucosal tissues suggests that LAT1/2 play major roles in the transport of this amino-acid-like herbicide. Enhanced uptake into the epithelial cells at barrier mucosae, including the respiratory and gastrointestinal tracts, may result in more significant local and systemic effects than predicted from glyphosate's passive permeability, and enhanced uptake by the olfactory mucosa may result in further CNS disposition, potentially increasing the risk for brain-related toxicities. Copyright © 2015 Elsevier Ltd. All rights reserved.

Activation of peroxisome proliferator-activated receptor δ induces fatty acid β-oxidation in skeletal muscle and attenuates metabolic syndrome

PubMed Central

Tanaka, Toshiya; Yamamoto, Joji; Iwasaki, Satoshi; Asaba, Hiroshi; Hamura, Hiroki; Ikeda, Yukio; Watanabe, Mitsuhiro; Magoori, Kenta; Ioka, Ryoichi X.; Tachibana, Keisuke; Watanabe, Yuichiro; Uchiyama, Yasutoshi; Sumi, Koichi; Iguchi, Haruhisa; Ito, Sadayoshi; Doi, Takefumi; Hamakubo, Takao; Naito, Makoto; Auwerx, Johan; Yanagisawa, Masashi; Kodama, Tatsuhiko; Sakai, Juro

2003-01-01

In this study, we defined the role of peroxisome proliferator-activated receptor β/δ (PPARδ) in metabolic homeostasis by using subtype selective agonists. Analysis of rat L6 myotubes treated with the PPARδ subtype-selective agonist, GW501516, by the Affymetrix oligonucleotide microarrays revealed that PPARδ controls fatty acid oxidation by regulating genes involved in fatty acid transport, β-oxidation, and mitochondrial respiration. Similar PPARδ-mediated gene activation was observed in the skeletal muscle of GW501516-treated mice. Accordingly, GW501516 treatment induced fatty acid β-oxidation in L6 myotubes as well as in mouse skeletal muscles. Administration of GW501516 to mice fed a high-fat diet ameliorated diet-induced obesity and insulin resistance, an effect accompanied by enhanced metabolic rate and fatty acid β-oxidation, proliferation of mitochondria, and a marked reduction of lipid droplets in skeletal muscles. Despite a modest body weight change relative to vehicle-treated mice, GW501516 treatment also markedly improved diabetes as revealed by the decrease in plasma glucose and blood insulin levels in genetically obese ob/ob mice. These data suggest that PPARδ is pivotal to control the program for fatty acid oxidation in the skeletal muscle, thereby ameliorating obesity and insulin resistance through its activation in obese animals. PMID:14676330

Na+/H+ exchanger 3 inhibitor diminishes the amino-acid-enhanced transepithelial calcium transport across the rat duodenum.

PubMed

Thammayon, Nithipak; Wongdee, Kannikar; Lertsuwan, Kornkamon; Suntornsaratoon, Panan; Thongbunchoo, Jirawan; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

2017-04-01

Na + /H + exchanger (NHE)-3 is important for intestinal absorption of nutrients and minerals, including calcium. The previous investigations have shown that the intestinal calcium absorption is also dependent on luminal nutrients, but whether aliphatic amino acids and glucose, which are abundant in the luminal fluid during a meal, similarly enhance calcium transport remains elusive. Herein, we used the in vitro Ussing chamber technique to determine epithelial electrical parameters, i.e., potential difference (PD), short-circuit current (Isc), and transepithelial resistance, as well as 45 Ca flux in the rat duodenum directly exposed on the mucosal side to glucose or various amino acids. We found that mucosal glucose exposure led to the enhanced calcium transport, PD, and Isc, all of which were insensitive to NHE3 inhibitor (100 nM tenapanor). In the absence of mucosal glucose, several amino acids (12 mM in the mucosal side), i.e., alanine, isoleucine, leucine, proline, and hydroxyproline, markedly increased the duodenal calcium transport. An inhibitor for NHE3 exposure on the mucosal side completely abolished proline- and leucine-enhanced calcium transport, but not transepithelial transport of both amino acids themselves. In conclusion, glucose and certain amino acids in the mucosal side were potent stimulators of the duodenal calcium absorption, but only amino-acid-enhanced calcium transport was NHE3-dependent.

Role of positively charged residues of the second transmembrane domain in the ion transport activity and conformation of human uncoupling protein-2.

PubMed

Hoang, Tuan; Matovic, Tijana; Parker, James; Smith, Matthew D; Jelokhani-Niaraki, Masoud

2015-04-14

Residing at the inner mitochondrial membrane, uncoupling protein-2 (UCP2) mediates proton transport from the intermembrane space (IMS) to the mitochondrial matrix and consequently reduces the rate of ATP synthesis in the mitochondria. The ubiquitous expression of UCP2 in humans can be attributed to the protein's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, as well as glucose and lipid metabolisms. Currently, the structural properties and ion transport mechanism of UCP2 and other UCP homologues remain poorly understood. UCP2-mediated proton transport is activated by fatty acids and inhibited by di- and triphosphate purine nucleotides. UCP2 also transports chloride and some other small anions. Identification of key amino acid residues of UCP2 in its ion transport pathway can shed light on the protein's ion transport function. On the basis of our previous studies, the second transmembrane helix segment (TM2) of UCP2 exhibited chloride channel activity. In addition, it was suggested that the positively charged residues on TM2 domains of UCPs 1 and 2 were important for their chloride transport activity. On this basis, to further understand the role of these positively charged residues on the ion transport activity of UCP2, we recombinantly expressed four TM2 mutants: R76Q, R88Q, R96Q, and K104Q. The wild type UCP2 and its mutants were purified and reconstituted into liposomes, and their conformation and ion (proton and chloride) transport activity were studied. TM2 Arg residues at the matrix interface of UCP2 proved to be crucial for the protein's anion transport function, and their absence resulted in highly diminished Cl(-) transport rates. On the other hand, the two other positively charged residues of TM2, located at the UCP2-IMS interface, could participate in the salt-bridge formation in the protein and promote the interhelical tight packing in the UCP2. Absence of these residues did not

Overexpression of a C4-dicarboxylate transporter is the key for rerouting citric acid to C4-dicarboxylic acid production in Aspergillus carbonarius.

PubMed

Yang, Lei; Christakou, Eleni; Vang, Jesper; Lübeck, Mette; Lübeck, Peter Stephensen

2017-03-14

C 4 -dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varieties carbon sources, have revealed its potential as a fungal cell factory. Earlier attempts to reroute citric acid production into C 4 -dicarboxylic acids have been with limited success. In this study, a glucose oxidase deficient strain of A. carbonarius was used as the parental strain to overexpress a native C 4 -dicarboxylate transporter and the gene frd encoding fumarate reductase from Trypanosoma brucei individually and in combination. Impacts of the introduced genetic modifications on organic acid production were investigated in a defined medium and in a hydrolysate of wheat straw containing high concentrations of glucose and xylose. In the defined medium, overexpression of the C 4 -dicarboxylate transporter alone and in combination with the frd gene significantly increased the production of C 4 -dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the frd gene alone did not result in any significant change of organic acid production profile. In the wheat straw hydrolysate after 9 days of cultivation, similar results were obtained as in the defined medium. High amounts of malic acid and succinic acid were produced by the same strains. This study demonstrates that the key to change the citric acid production into production of C 4 -dicarboxylic acids in A. carbonarius is the C 4 -dicarboxylate transporter. Furthermore it shows that the C 4 -dicarboxylic acid production by A. carbonarius can be further increased via metabolic engineering and also shows the potential of A. carbonarius to utilize lignocellulosic biomass as substrates for C 4 -dicarboxylic acid production.

SNPs Altering Ammonium Transport Activity of Human Rhesus Factors Characterized by a Yeast-Based Functional Assay

PubMed Central

Deschuyteneer, Aude; Boeckstaens, Mélanie; De Mees, Christelle; Van Vooren, Pascale; Wintjens, René; Marini, Anna Maria

2013-01-01

Proteins of the conserved Mep-Amt-Rh family, including mammalian Rhesus factors, mediate transmembrane ammonium transport. Ammonium is an important nitrogen source for the biosynthesis of amino acids but is also a metabolic waste product. Its disposal in urine plays a critical role in the regulation of the acid/base homeostasis, especially with an acid diet, a trait of Western countries. Ammonium accumulation above a certain concentration is however pathologic, the cytotoxicity causing fatal cerebral paralysis in acute cases. Alteration in ammonium transport via human Rh proteins could have clinical outcomes. We used a yeast-based expression assay to characterize human Rh variants resulting from non synonymous single nucleotide polymorphisms (nsSNPs) with known or unknown clinical phenotypes and assessed their ammonium transport efficiency, protein level, localization and potential trans-dominant impact. The HsRhAG variants (I61R, F65S) associated to overhydrated hereditary stomatocytosis (OHSt), a disease affecting erythrocytes, proved affected in intrinsic bidirectional ammonium transport. Moreover, this study reveals that the R202C variant of HsRhCG, the orthologue of mouse MmRhcg required for optimal urinary ammonium excretion and blood pH control, shows an impaired inherent ammonium transport activity. Urinary ammonium excretion was RHcg gene-dose dependent in mouse, highlighting MmRhcg as a limiting factor. HsRhCGR202C may confer susceptibility to disorders leading to metabolic acidosis for instance. Finally, the analogous R211C mutation in the yeast ScMep2 homologue also impaired intrinsic activity consistent with a conserved functional role of the preserved arginine residue. The yeast expression assay used here constitutes an inexpensive, fast and easy tool to screen nsSNPs reported by high throughput sequencing or individual cases for functional alterations in Rh factors revealing potential causal variants. PMID:23967154

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.

Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/anion transporter, TaALMT1

USDA-ARS?s Scientific Manuscript database

TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

Reduced placental amino acid transport in response to maternal nutrient restriction in the baboon.

PubMed

Pantham, Priyadarshini; Rosario, Fredrick J; Nijland, Mark; Cheung, Alex; Nathanielsz, Peter W; Powell, Theresa L; Galan, Henry L; Li, Cun; Jansson, Thomas

2015-10-01

Intrauterine growth restriction increases the risk of perinatal complications and predisposes the infant to diabetes and cardiovascular disease in later life. Mechanisms by which maternal nutrient restriction (MNR) reduces fetal growth are poorly understood. We hypothesized that MNR decreases placental amino acid (AA) transporter activity, leading to reduced transplacental transfer of AAs. Pregnant baboons were fed either a control (ad libitum, n = 7), or MNR diet (70% of control diet, n = 7) from gestational day (GD) 30. At GD 165 (0.9 gestation), placentas (n = 7 in each group) were collected, and microvillous plasma membrane vesicles (MVM) isolated. MVM system A and system L AA transport was determined in vitro using radiolabeled substrates and rapid filtration techniques. In vivo transplacental AA transport was assessed by infusing nine (13)C- or (2)H-labeled essential AA as a bolus into the maternal circulation (n = 5 control, n = 4 MNR) at cesarean section. A fetal vein-to-maternal artery mole percent excess ratio for each essential AA was calculated. Fetal and placental weights were significantly reduced in the MNR group compared with controls (P < 0.01). The activity of system A and system L was markedly reduced by 73 and 84%, respectively, in MVM isolated from baboon placentas at GD 165 following MNR (P < 0.01). In vivo, the fetal vein-to-maternal artery mole percent excess ratio was significantly reduced for leucine, isoleucine, methionine, phenylalanine, threonine, and tryptophan in MNR baboons (P < 0.05). This is the first study to investigate placental AA transport in a nonhuman primate model of MNR. We demonstrate that the downregulation of system A and system L activity in syncytiotrophoblast MVM in MNR leads to decreased transplacental AA transport and, consequently, reduced circulating fetal AA concentrations, a potential mechanism linking maternal undernutrition to reduced fetal growth. Copyright © 2015 the American Physiological Society.

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.

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

PubMed

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

2016-06-16

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

Fatty acids activate a chimera of the clofibric acid-activated receptor and the glucocorticoid receptor.

PubMed Central

Göttlicher, M; Widmark, E; Li, Q; Gustafsson, J A

1992-01-01

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Nature (London) 347, 645-650], which encodes a 97% similar protein with a particularly well-conserved putative ligand-binding domain. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. Testing of compounds related to lipid metabolism or peroxisomal proliferation revealed that 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activate the receptor chimera. In addition, saturated fatty acids induce the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. In conclusion, the present results indicate that fatty acids can regulate gene expression mediated by a member of the steroid nuclear receptor superfamily. Images PMID:1316614

Regulation of transmural transport of amino acid/metal conjugates by dietary calcium in crustacean digestive tract.

PubMed

Abdel-Malak, Rania; Ahearn, Gregory A

2014-03-01

Effects of luminal Ca(2+) and Mn(2+) on transmural mucosal to serosal (MS) transport of (3) H-L-leucine were characterized in the isolated and perfused intestine of the American lobster, Homarus americanus. (3) H-L-leucine MS transport in the presence of 20 µM Mn(2+) was a sigmoidal function of luminal amino acid concentration, following the Hill equation for multisite cooperative, carrier-mediated, transport. Luminal Ca(2+) was a non-competitive inhibitor of Mn(2+) -stimulated (3) H-L-leucine MS flux. Amino acid transport was hyperbolically stimulated by luminal Ca(2+) or Mn(2+). During 20 µM Mn(2+) -stimulation of (3) H-L-leucine MS flux, addition of 25 mM Ca(2+) strongly reduced amino acid transport Jmax , without affecting amino acid binding properties. Hyperbolic luminal Mn(2+) stimulation of 20 µM (3) H-L-leucine MS flux was also strongly inhibited by 25 mM luminal Ca(2+) , significantly reducing 20 µM (3) H-L-leucine Jmax . Increasing the luminal concentration of verapamil, a calcium channel blocker, significantly increased MS transport of 20 µM (3) H-L-leucine in the presence of 100 nM Mn(2+) by reducing diffusional Ca(2+) uptake into intestinal epithelial cells through verapamil-sensitive channels. A model is proposed supporting the concept of molecular mimicry, whereby (3) H-L-leucine enters lobster intestinal epithelial cells by one or more amino acid-specific transporters and by a dipeptide-like transporter that is capable of binding and transporting peptide molecular mimics (bis-complexes) between Ca(2+) or Mn(2+) and (3) H-L-leucine using the membrane potential as a major driving force for the transport event. According to the model, Ca(2+) entry through apical Ca(2+) channels regulates the magnitude of the membrane potential and therefore the size of the driving force for bis-complex uptake. © 2013 Wiley Periodicals, Inc.

Identification and characterization of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit corneal epithelial cell culture and rabbit cornea.

PubMed

Katragadda, Suresh; Talluri, Ravi Sankar; Pal, Dhananjay; Mitra, Ashim K

2005-11-01

The aim of this study was to investigate the presence of a Na+-dependent neutral amino acid transporter, ASCT1, in rabbit primary corneal epithelial cell culture and rabbit cornea. Uptake studies were carried out on rabbit primary corneal epithelial culture (rPCEC) cells using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Uptake and transport of L-alanine was determined at various concentrations. Inhibition studies were conducted in presence of various L- and D-amino acids, metabolic inhibitors like ouabain and sodium azide, and in the absence of sodium to delineate the functional characteristics of L-alanine uptake and transport. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA harvested from rabbit cornea and rPCEC cells for identification of ASCT1. Uptake of L-Ala was found to be saturable with a Km of 0.71 mM and a Vmax value of 0.84 micromoles min(-1) mg(-1) protein. Uptake was independent of pH and energy but depends on sodium. It was inhibited by serine, threonine, cysteine, and glutamine but did not respond to BCH (2-aminobicyclo [2,2,1] heptane-2-carboxylic acid) and MeAIB (alpha -methylaminoisobutyric acid). Transport of L-Ala across rabbit cornea was also saturable (Km 6.52 mM and Vmax 1.09 x 10(-2) micromoles min(-1) cm(-2)), energy independent, and subject to similar competitive inhibition. Presence of ASCT1 on rPCEC and on rabbit cornea was identified by RT-PCR. L-Alanine, the chosen model substrate, was actively transported by Na+-dependent, neutral amino acid exchanger ASCT1, which was identified and functionally characterized on rPCEC cells and rabbit cornea.

Recessive constitutive mutant Chinese hamster ovary cells (CHO-K1) with an altered A system for amino acid transport and the mechanism of gene regulation of the A system.

PubMed Central

Moffett, J; Englesberg, E

1984-01-01

Chinese hamster ovary cells (CHO-K1) starved for 24 h for amino acids show a severalfold increase in velocity of proline transport through the A system (Vmax is five times that of unstarved cells). This increase is inhibited by cycloheximide, actinomycin D, N-methyl-alpha-amino isobutyric acid (MeAIB, a non-metabolizable specific A system amino acid analog), and by other amino acids that are generally transported by the A system. However, transport by the A system is not a prerequisite for this repression, and all compounds that have affinity for the A system do not necessarily act as "co-repressors." The addition of proline, MeAIB, or other amino acids, as described above, to derepressed cells results in a rapid decrease in A system activity. As shown with proline and MeAIB, this decrease in activity is in part due to a rapid trans-inhibition and a slow, irreversible inactivation of the A system. Neither process is inhibited by cycloheximide or actinomycin D. Alanine antagonizes the growth of CHO-K1 pro cells by preventing proline transport, and alanine-resistant mutants (alar) have been isolated (Moffett et al., Somatic Cell Genet. 9:189-213, 1983). alar2 and alar4 are partial and full constitutive mutants for the A system and have two and six times the Vmax for proline uptake by the A system, respectively. The A system in alar4 is also immune to the co-repressor-induced inactivation. Both alar2 and alar4 phenotypes are recessive. Alar3 shows an increase in Vmax and Km for proline transport through the A system, and this phenotype is codominant. All three mutants have a pleiotropic effect, producing increases in activity of the ASC and P systems of amino acid transport. This increase is not due to an increase in the Na+ gradient. The ASC and P phenotypes behave similarly to the A system in hybrids. A model has been proposed incorporating these results. PMID:6538929

How a microbial drug transporter became essential for crop cultivation on acid soils: aluminium tolerance conferred by the multidrug and toxic compound extrusion (MATE) family

PubMed Central

Magalhaes, Jurandir V.

2010-01-01

Background Aluminium (Al) toxicity is a major agricultural constraint for crop cultivation on acid soils, which comprise a large portion of the world's arable land. One of the most widely accepted mechanisms of Al tolerance in plants is based on Al-activated organic acid release into the rhizosphere, with organic acids forming stable, non-toxic complexes with Al. This mechanism has recently been validated by the isolation of bona-fide Al-tolerance genes in crop species, which encode membrane transporters that mediate Al-activated organic acid release leading to Al exclusion from root apices. In crop species such as sorghum and barley, members in the multidrug and toxic compound extrusion (MATE) family underlie Al tolerance by a mechanism based on Al-activated citrate release. Scope and Conclusions The study of Al tolerance in plants as conferred by MATE family members is in its infancy. Therefore, much is yet to be discovered about the functional diversity and evolutionary dynamics that led MATE proteins to acquire transport properties conducive to Al tolerance in plants. In this paper we review the major characteristics of transporters in the MATE family and will relate this knowledge to Al tolerance in plants. The MATE family is clearly extremely flexible with respect to substrate specificity, which raises the possibility that Al tolerance as encoded by MATE proteins may not be restricted to Al-activated citrate release in plant species. There are also indications that regulatory loci may be of pivotal importance to fully explore the potential for Al-tolerance improvement based on MATE genes. PMID:20511585

Comparative phytotoxicity of usnic acid, salicylic acid, cinnamic acid and benzoic acid on photosynthetic apparatus of Chlamydomonas reinhardtii.

PubMed

Gao, Yazhi; Liu, Wei; Wang, Xiaoxiong; Yang, Lihua; Han, Su; Chen, Shiguo; Strasser, Reto Jörg; Valverde, Bernal E; Qiang, Sheng

2018-07-01

The effects of four phytotoxins usnic acid (UA), salicylic acid (SA), cinnamic acid (CA) and benzoic acid (BA) on photosynthesis of Chlamydomonas reinhardtii were studied in vivo to identify and localise their initial action sites on two photosystems. Our experimental evidence shows that the four phytotoxins have multiple targets in chloroplasts, which mainly lie in photosystem II (PSII), not photosystem I (PSI). They share an original action site by blocking electron transport beyond Q A (primary plastoquinone acceptor) at PSII acceptor side since a fast increase of the J-step level is the greatest change in chlorophyll a fluorescence induction kinetics OJIP in C. reinhardtii cells treated with the phytotoxins. UA decreases photosynthetic activity by reducing O 2 evolution rate, interrupting PSII electron transport at both the donor and acceptor sides, inactivating the PSII reaction centers (RCs), reducing the content of chlorophylls and carotenoids, destroying the conformation of antenna pigment assemblies, and casuing the degradation of D1/D2 proteins. SA damage to photosynthetic machinery is mainly attributed to inhibition of PSII electron transport beyond Q A at the acceptor side, inactivation of the PSII RCs, reduction of chlorophyll content, digestion of thylakoid ploypeptides and destabilization of thylakoid membranes. Both CA and BA affect the photosynthetic process by decreasing PSII electron transport efficiency at the acceptor side and the amount of active PSII RCs. Besides, the initial cause of BA-inhibiting photosynthesis is also assocaited with the O 2 evolution rate and the disconnection of some antenna molecules from PSII RCs. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Amino acid carryover in the subzonal space of mouse fertilized ova affects subsequent transport kinetics.

PubMed

Rudraraju, Nirmala; Baltz, Jay M

2009-11-01

SummaryWe have investigated whether culture in glycine-containing medium affects subsequent glycine transport by the specific transport system, GLYT1, which is the sole glycine transporter in fertilized mouse ova. When fertilized ova were maintained for 6 h in culture with a physiological level of glycine (1 mM), subsequent transport of radiolabelled glycine was decreased by 40% compared with fertilized ova that had been maintained in glycine-free medium. Kinetic measurements showed that the apparent glycine affinity was decreased after culture with glycine (Km increased from 0.20 to 0.41 mM), but maximal transport rate was unchanged (similar Vmax of 20 and 23 fmol/fertilized ovum/min). These findings could have reflected activation of GLYT1 by prolonged substrate starvation, similar to some other amino acid transport systems. However, our findings were instead consistent with the alteration in glycine transport being due to trapping of glycine within the zona pellucida resulting in competitive transport inhibition even after ova were removed from glycine-containing media. First, even very brief exposures to glycine resulted in decreased subsequent glycine transport rates, with a maximal effect apparent within ~6 min. Second, extensive washing (at least six) reversed the effect. Third, the effect was absent when zona-free fertilized ova were used. Thus, it appears that components of the external environment of preimplantation embryos may continue to affect transport kinetics for a period even after embryos are removed from environments that contain them.

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

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

PubMed

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

2015-02-01

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

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.

A novel TctA citrate transporter from an activated sludge metagenome: structural and mechanistic predictions for the TTT family.

PubMed

Batista-García, Ramón Alberto; Sánchez-Reyes, Ayixon; Millán-Pacheco, César; González-Zuñiga, Víctor Manuel; Juárez, Soledad; Folch-Mallol, Jorge Luis; Pastor, Nina

2014-09-01

We isolated a putative citrate transporter of the tripartite tricarboxylate transporter (TTT) class from a metagenomic library of activated sludge from a sewage treatment plant. The transporter, dubbed TctA_ar, shares ∼50% sequence identity with TctA of Comamonas testosteroni (TctA_ct) and other β-Proteobacteria, and contains two 20-amino acid repeat signature sequences, considered a hallmark of this particular transporter class. The structures for both TctA_ar and TctA_ct were modeled with I-TASSER and two possible structures for this transporter family were proposed. Docking assays with citrate resulted in the corresponding sets of proposed critical residues for function. These models suggest functions for the 20-amino acid repeats in the context of the two different architectures. This constitutes the first attempt at structure modeling of the TTT family, to the best of our knowledge, and could aid functional understanding of this little-studied family. © 2014 Wiley Periodicals, Inc.

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

PubMed Central

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

2012-01-01

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

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…

Toxicity of cephaloridine to carnitine transport and fatty acid metabolism in rabbit renal cortical mitochondria: structure-activity relationships.

PubMed

Tune, B M; Hsu, C Y

1994-09-01

Cephaloridine (Cld), the most widely studied nephrotoxic cephalosporin, has significant structural homology with carnitine, which facilitates the transport of long-chain fatty acids into the mitochondrial inner matrix. Because of this homology, and evidence of a role of lipids in cephaloglycin (Cgl) nephrotoxicity, protocols were designed to compare the effects of Cld and Cgl on renal cortical mitochondrial carnitine transport, on long-chain fatty acylcarnitine-mediated respiration and on the in situ mitochondrial pools and urinary excretion of carnitine and acylcarnitines. The following was found: 1) both cephalosporins reduced carnitine-facilitated pyruvate oxidation (CFPO) and palmitoylcarnitine-mediated respiration (PCMR) by 40 to 50% in mitochondria exposed in vivo (300 mg/kg b.wt., 1 hr). CFPO could be decreased by reduction of carnitine uptake, pyruvate oxidation or carnitine acetyltransferase activity; 2) neither cephalosporin reduced mitochondrial carnitine acetyltransferase or carnitine palmitoyltransferase; 3) with in vitro exposure (2000 micrograms/ml, immediate effect) Cgl had no significant toxicity to mitochondrial CFPO. Cld inhibited CFPO in a dose-dependent manner, up to 100% at 2000 micrograms/ml; this effect was reduced by increasing carnitine concentrations; 4) in vitro Cld prevented the potentiation of PCMR by preloading with carnitine, reduced mitochondrial acetylcarnitine/carnitine exchange by 70% and reduced PCMR by 30%; 5) in vivo Cld increased mitochondrial-free carnitine in the in situ kidney by 100%; and 6) in vivo Cld increased the fractional renal excretion of carnitine from 0 +/- 0 to 0.29 +/- 0.03 and the fractional excretion of long-chain acylcarnitines from 0.06 +/- 0.01 to 0.79 +/- 0.17.(ABSTRACT TRUNCATED AT 250 WORDS)

Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant.

PubMed Central

Rentsch, D; Hirner, B; Schmelzer, E; Frommer, W B

1996-01-01

A yeast mutant lacking SHR3, a protein specifically required for correct targeting of plasma membrane amino acid permeases, was used to study the targeting of plant transporters and as a tool to isolate new SHR3-independent amino acid transporters. For this purpose, an shr3 mutant was transformed with an Arabidopsis cDNA library. Thirty-four clones were capable of growth under selective conditions, but none showed homology with SHR3. However, genes encoding eight different amino acid transporters belonging to three different transporter families were isolated. Five of these are members of the general amino acid permease (AAP) gene family, one is a member of the NTR family, encoding an oligopeptide transporter, and two belong to a new class of transporter genes. A functional analysis of the latter two genes revealed that they encode specific proline transporters (ProT) that are distantly related to the AAP gene family. ProT1 was found to be expressed in all organs, but highest levels were found in roots, stems, and flowers. Expression in flowers was highest in the floral stalk phloem that enters the carpels and was downregulated after fertilization, indicating a specific role in supplying the ovules with proline. ProT2 transcripts were found ubiquitously throughout the plant, but expression was strongly induced under water or salt stress, implying that ProT2 plays an important role in nitrogen distribution during water stress, unlike members of the AAP gene family whose expression was repressed under the same conditions. These results corroborate the general finding that under water stress, amino acid export is impaired whereas proline export is increased. PMID:8776904

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…

Transport of biologically active material in laser cutting.

PubMed

Frenz, M; Mathezloic, F; Stoffel, M H; Zweig, A D; Romano, V; Weber, H P

1988-01-01

The transport of biologically active material during laser cutting with CO2 and Er lasers is demonstrated. This transport mechanism removes particles from the surface of gelatin, agar, and liver samples into the depth of the laser-formed craters. The transport phenomenon is explained by a contraction and condensation of enclosed hot water vapor. We show by cultivating transported bacteria in agar that biological particles can survive the shock of the transport. Determination of the numbers of active cells evidences a more pronounced activity of the cultivated bacteria after impact with an Er laser than with a CO2 laser.

A model for the topology of excitatory amino acid transporters determined by the extracellular accessibility of substituted cysteines.

PubMed

Seal, R P; Leighton, B H; Amara, S G

2000-03-01

Excitatory amino acid transporters (EAATs) function as both substrate transporters and ligand-gated anion channels. Characterization of the transporter's general topology is the first requisite step in defining the structural bases for these distinct activities. While the first six hydrophobic domains can be readily modeled as conventional transmembrane segments, the organization of the C-terminal hydrophobic domains, which have been implicated in both substrate and ion interactions, has been controversial. Here, we report the results of a comprehensive evaluation of the C-terminal topology of EAAT1 determined by the chemical modification of introduced cysteine residues. Our data support a model in which two membrane-spanning domains flank a central region that is highly accessible to the extracellular milieu and contains at least one reentrant loop domain.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukumoto, Shinya; Hanazono, Kiwamu; Fu, Dah-Renn

2013-09-13

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

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

PubMed

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

2017-07-01

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

Bicarbonate, NBCe1, NHE, and Carbonic Anhydrase Activity Enhance Lactate-H+ Transport in Bovine Corneal Endothelium

PubMed Central

Nguyen, Tracy T.

2011-01-01

Purpose. To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO3−, sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux. Methods. MCT1–4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pHi were measured in BCEC in HCO3−-free or HCO3−-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na+/H+ exchange blocker), disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells. Results. MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pHi measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpHi/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO3− but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO3−, was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA. Conclusions. MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H+ transport. Lactate dependent proton flux can activate Na+/H+ exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO3−, HCO3− transport, NHE

L-leucine, L-methionine, and L-phenylalanine share a Na(+)/K (+)-dependent amino acid transporter in shrimp hepatopancreas.

PubMed

Duka, Ada; Ahearn, Gregory A

2013-08-01

max in the same medium. These results suggest that shrimp BBMV transport (3)H-L-leucine by a single L-methionine- and L-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na(+) or K(+) acting as co-transport drivers binding to shared activator sites.

Molecular Determinants for Functional Differences between Alanine-Serine-Cysteine Transporter 1 and Other Glutamate Transporter Family Members*

PubMed Central

Scopelliti, Amanda J.; Ryan, Renae M.; Vandenberg, Robert J.

2013-01-01

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter GltPh. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and GltPh. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and GltPh, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and GltPh. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of GltPh, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family. PMID:23393130

Characterisation and cloning of a Na(+)-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B(0) transporter family.

PubMed

Pollard, Matthew; Meredith, David; McGivan, John D

2002-04-12

Na(+)-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B(0). This transporter is shown to differ in specificity from the B(0) transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B(0) transporter family we have isolated a cDNA encoding the NBL-1 cell System B(0) transporter. When expressed in Xenopus oocytes the clone catalysed Na(+)-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na(+)-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B(0)/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B(0) transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.

Dimethyl sulfoxide attenuates nitric oxide generation via modulation of cationic amino acid transporter-1 in human umbilical vein endothelial cells.

PubMed

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

2016-10-01

Dimethyl sulfoxide (DMSO) is a solvent that is commonly used in medicine. Conflicting data exist as to its effects on endothelial function. Endothelial cell dysfunction (ECD) is characterized by decreased endothelial nitric oxide synthase (eNOS) activity. Cationic amino acid transporter-1 (CAT-1), the specific arginine transporter for eNOS, has been shown to modulate eNOS activity. We hypothesize that DMSO inhibits eNOS activity through modulation of its selective arginine supplier CAT-1. We studied the effect of DMSO on arginine transport, NO2/NO3 generation as an index of NO production, as well as CAT-1 and Protein Kinase C alpha (PKC-α) (CAT-1 inhibitor) protein expression in human umbilical vein endothelial cell cultures (HUVECs). DMSO 2.5% and 3.5% (v/v) significantly attenuated arginine transport, a phenomenon which was prevented by co-incubation with l-arginine (1 mM). The aforementioned findings were accompanied by a decrease in NO2/NO3 generation. DMSO significantly increased the abundance of phosphorylated CAT-1 (the inactive form) and phosphorylated PKC-α protein, an effect that was attenuated by l-arginine. GO 6976 (PKC-α antagonist) prevented the decrease in arginine transport caused by DMSO. DMSO also induced profound transient morphological changes in HUVECs' structure but these were not related to its effect on arginine transport. In conclusion, DMSO inhibits NO generation by endothelial cells through modulation of CAT-1 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed Central

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

2015-01-01

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. PMID:25971966

Molecular Expression and Functional Activity of Efflux and Influx Transporters in Hypoxia Induced Retinal Pigment Epithelial Cells

PubMed Central

Vadlapatla, Ramya; Vadlapudi, Aswani Dutt; Ponnaluri, VK Chaithanya; Pal, Dhananjay; Mukherji, Mridul; Mitra, Ashim K.

2013-01-01

A decrease in tissue oxygen levels (aka hypoxia) mediates a number of vascular retinal diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. To date, the differential effect of hypoxia on expression of efflux and influx transporters in retinal cells has not been studied. Therefore, the objective of this study was to delineate molecular and functional expression of membrane transporters in human retinal pigment epithelial (RPE) cells cultured under normoxic and hypoxic conditions. Quantitative real time polymerase chain reaction (qPCR), ELISA and immunoblot analysis were performed to examine the RNA and protein expression levels of transporters. Further, functional activity was evaluated by performing the uptake of various substrates in both normoxic and hypoxic conditions. qPCR analysis showed elevated expression of efflux transporters (P-glycoprotein, multidrug resistant protein 2, breast cancer resistant protein) and influx transporters (folate receptor-α, cationic and neutral amino acid transporter, sodium dependent multivitamin transporter) in a time dependent manner. Immunoblot analysis further confirmed elevated expression of breast cancer resistant protein and sodium dependent multivitamin transporter. A decrease in the uptake of efflux transporter substrates (digoxin, lopinavir and abacavir) and enhanced uptake of influx transporter substrates (arginine, folic acid and biotin) in hypoxia relative to normoxia further confirmed elevated expression of transporters, respectively. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in RPE cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs. PMID:23827654

Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

PubMed

Erickson, Jeffrey D

2017-07-01

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC 50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K + -stimulated MeAIB transport displays an affinity (K m ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca 2+ , and is blocked by inhibition of voltage-gated Ca 2+ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca 2+ and voltage-gated calcium channels, but is also blocked by the Na + channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca 2+ , but on Na + ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K + -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017 International

Kinetics of the bile acid transporter and hepatitis B virus receptor Na+/taurocholate cotransporting polypeptide (NTCP) in hepatocytes.

PubMed

König, Alexander; Döring, Barbara; Mohr, Christina; Geipel, Andreas; Geyer, Joachim; Glebe, Dieter

2014-10-01

The human liver bile acid transporter Na(+)/taurocholate cotransporting polypeptide (NTCP) has recently been identified as liver-specific receptor for infection of hepatitis B virus (HBV), which attaches via the myristoylated preS1 (myr-preS1) peptide domain of its large surface protein to NTCP. Since binding of the myr-preS1 peptide to NTCP is an initiating step of HBV infection, we investigated if this process interferes with the physiological bile acid transport function of NTCP. HBV infection, myr-preS1 peptide binding, and bile acid transport assays were performed with primary Tupaia belangeri (PTH) and human (PHH) hepatocytes as well as NTCP-transfected human hepatoma HepG2 cells allowing regulated NTCP expression, in the presence of various bile acids, ezetimibe, and myr-preS1 peptides. The myr-preS1 peptide of HBV inhibited bile acid transport in PTH and PHH as well as in NTCP-expressing HEK293 and HepG2 cells. Inversely, HBV infection of PTH, PHH, and NTCP-transfected HepG2 cells was inhibited in a concentration-dependent manner by taurine and glycine conjugates of cholic acid and ursodeoxycholic acid as well as by ezetimibe. In NTCP-HepG2 cells and PTH, NTCP expression, NTCP transport function, myr-preS1 peptide binding, and HBV infection followed comparable kinetics. Myr-preS1 virus binding to NTCP, necessary for productive HBV infection, interferes with the physiological bile acid transport function of NTCP. Therefore, HBV infection via NTCP may be lockable by NTCP substrates and NTCP-inhibiting drugs. This opens a completely new way for an efficient management of HBV infection by the use of NTCP-directed drugs. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast

PubMed Central

Cleal, J K; Glazier, J D; Ntani, G; Crozier, S R; Day, P E; Harvey, N C; Robinson, S M; Cooper, C; Godfrey, K M; Hanson, M A; Lewis, R M

2011-01-01

Fetal growth depends on placental transfer of amino acids from maternal to fetal blood. The mechanisms of net amino acid efflux across the basal membrane (BM) of the placental syncytiotrophoblast to the fetus, although vital for amino acid transport, are poorly understood. We examined the hypothesis that facilitated diffusion by the amino acid transporters TAT1, LAT3 and LAT4 plays an important role in this process, with possible effects on fetal growth. Amino acid transfer was measured in isolated perfused human placental cotyledons (n= 5 per experiment) using techniques which distinguish between different transport processes. Placental TAT1, LAT3 and LAT4 proteins were measured, and mRNA expression levels (measured using real-time quantitative-PCR) were related to fetal and neonatal anthropometry and dual-energy X-ray absorptiometry measurements of neonatal lean mass in 102 Southampton Women's Survey (SWS) infants. Under conditions preventing transport by amino acid exchangers, all amino acids appearing in the fetal circulation were substrates of TAT1, LAT3 or LAT4. Western blots demonstrated the presence of TAT1, LAT3 and LAT4 in placental BM preparations. Placental TAT1 and LAT3 mRNA expression were positively associated with measures of fetal growth in SWS infants (P < 0.05). We provide evidence that the efflux transporters TAT1, LAT3 and LAT4 are present in the human placental BM, and may play an important role in the net efflux of amino acids to the fetus. Unlike other transporters they can increase fetal amino acid concentrations. Consistent with a role in placental amino acid transfer capacity and fetal growth TAT1 and LAT3 mRNA expression showed positive associations with infant size at birth. PMID:21224231

Omega 3 Fatty Acids Promote Macrophage Reverse Cholesterol Transport in Hamster Fed High Fat Diet

PubMed Central

Kasbi Chadli, Fatima; Nazih, Hassane; Krempf, Michel; Nguyen, Patrick; Ouguerram, Khadija

2013-01-01

The aim of this study was to investigate macrophage reverse cholesterol transport (RCT) in hamster, a CETP-expressing species, fed omega 3 fatty acids (ω3PUFA) supplemented high fat diet (HFD). Three groups of hamsters (n = 6/group) were studied for 20 weeks: 1) control diet: Control, 2) HFD group: HF and 3) HFD group supplemented with ω3PUFA (EPA and DHA): HFω3. In vivo macrophage-to-feces RCT was assessed after an intraperitoneal injection of 3H-cholesterol-labelled hamster primary macrophages. Compared to Control, HF presented significant (p<0.05) increase in body weight, plasma TG (p<0.01) and cholesterol (p<0.001) with an increase in VLDL TG and in VLDL and LDL cholesterol (p<0.001). Compared to HF, HFω3 presented significant decrease in body weight. HFω3 showed less plasma TG (p<0.001) and cholesterol (p<0.001) related to a decrease in VLDL TG and HDL cholesterol respectively and higher LCAT activity (p<0.05) compared to HF. HFω3 showed a higher fecal bile acid excretion (p<0.05) compared to Control and HF groups and higher fecal cholesterol excretion (p<0.05) compared to HF. This increase was related to higher gene expression of ABCG5, ABCA1 and SR-B1 in HFω3 compared to Control and HF groups (<0.05) and in ABCG1 and CYP7A1 compared to HF group (p<0.05). A higher plasma efflux capacity was also measured in HFω3 using 3H- cholesterol labeled Fu5AH cells. In conclusion, EPA and DHA supplementation improved macrophage to feces reverse cholesterol transport in hamster fed HFD. This change was related to the higher cholesterol and fecal bile acids excretion and to the activation of major genes involved in RCT. PMID:23613796

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state.

PubMed

Aduri, Nanda G; Ernst, Heidi A; Prabhala, Bala K; Bhatt, Shweta; Boesen, Thomas; Gajhede, Michael; Mirza, Osman

2018-01-08

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2013-08-15

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

Resistin Regulates Fatty Acid Β Oxidation by Suppressing Expression of Peroxisome Proliferator Activator Receptor Gamma-Coactivator 1α (PGC-1α).

PubMed

He, Fang; Jin, Jie-Qiong; Qin, Qing-Qing; Zheng, Yong-Qin; Li, Ting-Ting; Zhang, Yun; He, Jun-Dong

2018-01-01

Abnormal fatty acid β oxidation has been associated with obesity and type 2 diabetes. Resistin is an adipokine that has been considered as a potential factor in obesity-mediated insulin resistance and type 2 diabetes. However, the effect of resistin on fatty acid β oxidation needs to be elucidated. We detected the effects of resistin on the expression of fatty acid oxidation (FAO) transcriptional regulatory genes, the fatty acid transport gene, and mitochondrial β-oxidation genes using real-time PCR. The rate of FAO was measured using 14C-palmitate. Immunofluorescence assay and western blot analysis were used to explore the underlying molecular mechanisms. Resistin leads to a reduction in expression of the FAO transcriptional regulatory genes ERRα and NOR1, the fatty acid transport gene CD36, and the mitochondrial β-oxidation genes CPT1, MCAD, and ACO. Importantly, treatment with resistin led to a reduction in the rate of cellular fatty acid oxidation. In addition, treatment with resistin reduced phosphorylation of acetyl CoA carboxylase (ACC) (inhibitory). Mechanistically, resistin inhibited the activation of CREB, resulting in suppression of PGC-1α. Importantly, overexpressing PGC-1α can rescue the inhibitory effects of resistin on fatty acid β oxidation. Activating the transcriptional activity of CREB using small molecular chemicals is a potential pharmacological strategy for preventing the inhibitory effects of resistin on fatty acid β oxidation. © 2018 The Author(s). Published by S. Karger AG, Basel.

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

NASA Astrophysics Data System (ADS)

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

2014-08-01

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

Activity-dependent formation of a vesicular inhibitory amino acid transporter gradient in the superior olivary complex of NMRI mice.

PubMed

Ebbers, Lena; Weber, Maren; Nothwang, Hans Gerd

2017-10-26

In the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem. Immunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14 -/- mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient. Our results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular

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

PubMed

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

2008-08-01

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

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

PubMed Central

2015-01-01

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

Change in active transportation and weight gain in pregnancy.

PubMed

Skreden, Marianne; Øverby, Nina C; Sagedal, Linda R; Vistad, Ingvild; Torstveit, Monica K; Lohne-Seiler, Hilde; Bere, Elling

2016-01-27

Pregnancy is characterised by large weight gain over a short period, and often a notable change in mode of transportation. This makes pregnancy suitable for examining the plausible, but in the scientific literature still unclear, association between active transportation and weight gain. We hypothesize that women continuing an active mode of transportation to work or school from pre- to early pregnancy will have a lower gestational weight gain (GWG) than those who change to a less active mode of transportation. We analysed prospective data from the Norwegian Fit for Delivery (NFFD) trial. Between September 2009 and February 2013 606 women were consecutively enrolled in median gestational week 16 (range; 8-20). Of 219 women who used an active mode of transportation (biking, walking, public transportation) pre-pregnancy, 66 (30%) converted to a less active mode in early pregnancy ("active-less active" group), and 153 (70%) continued with active transportation ("active-active" group). Pre-pregnancy weight was self-reported. Weight at gestational (GA) weeks 16, 30, 36, and at term delivery was objectively measured. Weight gain was compared between the two groups. Linear mixed effects analysis of the repeated weight measures was performed including the group*time interaction. A significant overall group effect was observed for the four time points together ("active-active" group: 77.3 kg vs. "active-less active" group: 78.8 kg, p = 0.008). The interaction term group*time was significant indicating different weight gain throughout pregnancy for the two groups; the mean differences between the groups were 0.7 kg at week 16, 1.4 kg at week 30, 2.1 kg at week 36, and 2.2 kg at term delivery, respectively. The findings indicate that active transportation is one possible approach to prevent excessive weight gain in pregnancy.

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.

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

Antioxidant and antimicrobial activities of cinnamic acid derivatives.

PubMed

Sova, M

2012-07-01

Cinnamic acid is an organic acid occurring naturally in plants that has low toxicity and a broad spectrum of biological activities. In the search for novel pharmacologically active compounds, cinnamic acid derivatives are important and promising compounds with high potential for development into drugs. Many cinnamic acid derivatives, especially those with the phenolic hydroxyl group, are well-known antioxidants and are supposed to have several health benefits due to their strong free radical scavenging properties. It is also well known that cinnamic acid has antimicrobial activity. Cinnamic acid derivatives, both isolated from plant material and synthesized, have been reported to have antibacterial, antiviral and antifungal properties. Acids, esters, amides, hydrazides and related derivatives of cinnamic acid with such activities are here reviewed.

Hepatic alterations are accompanied by changes to bile acid transporter-expressing neurons in the hypothalamus after traumatic brain injury.

PubMed

Nizamutdinov, Damir; DeMorrow, Sharon; McMillin, Matthew; Kain, Jessica; Mukherjee, Sanjib; Zeitouni, Suzanne; Frampton, Gabriel; Bricker, Paul Clint S; Hurst, Jacob; Shapiro, Lee A

2017-01-20

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.

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

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

PubMed

Wang, Li; Sweet, Douglas H

2012-10-15

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

Intracellular acidification reduces l-arginine transport via system y+L but not via system y+/CATs and nitric oxide synthase activity in human umbilical vein endothelial cells.

PubMed

Ramírez, Marco A; Morales, Jorge; Cornejo, Marcelo; Blanco, Elias H; Mancilla-Sierpe, Edgardo; Toledo, Fernando; Beltrán, Ana R; Sobrevia, Luis

2018-04-01

l-Arginine is taken up via the cationic amino acid transporters (system y + /CATs) and system y + L in human umbilical vein endothelial cells (HUVECs). l-Arginine is the substrate for endothelial NO synthase (eNOS) which is activated by intracellular alkalization, but nothing is known regarding modulation of system y + /CATs and system y + L activity, and eNOS activity by the pHi in HUVECs. We studied whether an acidic pHi modulates l-arginine transport and eNOS activity in HUVECs. Cells loaded with a pH-sensitive probe were subjected to 0.1-20 mmol/L NH 4 Cl pulse assay to generate pHi 7.13-6.55. Before pHi started to recover, l-arginine transport (0-20 or 0-1000 μmol/L, 10 s, 37 °C) in the absence or presence of 200 μmol/L N-ethylmaleimide (NEM) (system y + /CATs inhibitor) or 2 mmol/L l-leucine (systemy + L substrate) was measured. Protein abundance for eNOS and serine 1177 or threonine 495 phosphorylated eNOS was determined. The results show that intracellular acidification reduced system y + L but not system y + /CATs mediated l-arginine maximal transport capacity due to reduced maximal velocity. Acidic pHi reduced NO synthesis and eNOS serine 1177 phosphorylation. Thus, system y + L activity is downregulated by an acidic pHi, a phenomenon that may result in reduced NO synthesis in HUVECs. Copyright © 2018 Elsevier B.V. All rights reserved.

Alterations to N-linked oligosaccharides which affect intracellular transport rates and regulated secretion but not sorting of lysosomal acid phosphatase in Dictyostelium discoideum.

PubMed

Bush, J M; Ebert, D L; Cardelli, J A

1990-11-15

The importance of N-linked oligosaccharides and their associated modifications in the transport, sorting, and secretion of lysosomal acid phosphatase was investigated using three mutant Dictyostelium cell lines. These mutants synthesize altered N-linked oligosaccharides with the following properties: (i) in strain HL244 carbohydrate side chains lack mannose 6-sulfate residues, (ii) in strain M31 the side chains retain the two alpha-1,3-linked glucose residues resulting in less sulfate and methylphosphate modifications, and (iii) in strain HL243 the nonglucosylated branches are missing three of the outer mannose sugars and the oligosaccharides contain fewer sulfate and phosphate modifications. Lysosomal enzymes in both HL243 and HL244 are also missing a shared epitope termed common antigen-1 (CA-1), which consists in part of mannose 6-sulfate moieties. No increases were observed in the secretion of radiolabeled acid phosphatase or acid phosphatase activity during growth in any of the mutant cell lines, suggesting that the enzyme was correctly sorted to lysosomes. In support of this, Percoll gradient fractionations and indirect immunofluorescence microscopy indicated that acid phosphatase was transported to lysosomes in all cell lines. However, radiolabel pulse chase protocols indicated that newly synthesized acid phosphatase was transported out of the endoplasmic reticulum (ER) and into lysosomes at a two- to threefold slower rate in HL243 and at a sixfold slower rate in M31. The rate of transport of acid phosphatase from the ER to the Golgi was reduced only twofold in M31 as determined by digestion of newly synthesized enzyme with endoglycosidose H. This suggests that certain alterations in carbohydrate structure may only slightly affect transport of the enzyme from the ER to the Golgi but these alterations may greatly delay transport from the Golgi or post-Golgi compartments to lysosomes. Finally all three mutants secreted acid phosphatase at significantly lower

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

PubMed

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

2010-07-27

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

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.

Effects of clofibric acid on the activity and activity state of the hepatic branched-chain 2-oxo acid dehydrogenase complex.

PubMed Central

Zhao, Y; Jaskiewicz, J; Harris, R A

1992-01-01

Feeding clofibric acid to rats caused little or no change in total activity of the liver branched-chain 2-oxo acid dehydrogenase complex (BCODC). No change in mass of liver BCODC was detected by immunoblot analysis in response to dietary clofibric acid. No changes in abundance of mRNAs for the BCODC E1 alpha, E1 beta and E2 subunits were detected by Northern-blot analysis. Likewise, dietary clofibric acid had no effect on the activity state of liver BCODC (percentage of enzyme in the dephosphorylated, active, form) of rats fed on a chow diet. However, dietary clofibric acid greatly increased the activity state of liver BCODC of rats fed on a diet deficient in protein. No stable change in liver BCODC kinase activity was found in response to clofibric acid in either chow-fed or low-protein-fed rats. Clofibric acid had a biphasic effect on flux through BCODC in hepatocytes prepared from low-protein-fed rats. Stimulation of BCODC flux at low concentrations was due to clofibric acid inhibition of BCODC kinase, which in turn allowed activation of BCODC by BCODC phosphatase. Inhibition of BCODC flux at high concentrations was due to direct inhibition of BCODC by clofibric acid. The results suggest that the effects of clofibric acid in vivo on branched-chain amino acid metabolism can be explained by the inhibitory effects of this drug on BCODC kinase. Images Fig. 2. Fig. 3. PMID:1637295

Regulation of amino acid transport across intestines of goldfish acclimatized to different environmental temperatures

PubMed Central

Mepham, T. B.; Smith, M. W.

1966-01-01

1. Serosal transfers of valine and threonine were measured using everted sacs of anterior intestine taken from goldfish acclimatized to different temperatures. 2. Both valine and threonine were actively transported at incubation temperatures equal to or greater than the previous environmental temperature of the fish. There was also a positive serosal transfer of valine, but not threonine, at incubation temperatures below the previous environmental temperature of the fish. 3. The mean stable transmural potentials and amino-acid-evoked potentials depended both on the temperature to which the fish had been acclimatized and on the temperature at which the sacs were incubated. 4. There was a linear relation between the transmural potential and the serosal transfer of amino acid, one additional μmole of valine or threonine being transferred/2 hr incubation period for each 3 mV rise in potential. There was a less obvious correlation between the amino-acid-evoked potential and on serosal transfer of amino acid. 5. Acclimatization of the goldfish intestine from 8 to 25° C, assessed by changes occurring in the transmural potential and serosal transfer of amino acids, tended to stabilize both parameters, but the compensation in each case was only partial. 6. It is possible that the imbalance in transfer of valine-like and threonine-like amino acids, seen at incubation temperatures below the previous acclimatization temperature of the fish, has a special function in initiating the process of acclimatization to the new environmental temperature. PMID:5972157

mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

PubMed

Wyant, Gregory A; Abu-Remaileh, Monther; Wolfson, Rachel L; Chen, Walter W; Freinkman, Elizaveta; Danai, Laura V; Vander Heiden, Matthew G; Sabatini, David M

2017-10-19

The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Branched-chain amino acid transport in Streptococcus mutans Ingbritt.

PubMed

Dashper, S G; Reynolds, E C

1993-06-01

Leucine transport in glucose-energized cells of Streptococcus mutans exhibited Michaelis-Menten-type kinetics at low extracellular concentrations, with a K1 of 15.3 microM and a Vmax of 6.1 nmol/mg dry weight/min. At high extracellular leucine concentrations, the transmembrane diffusion of leucine was not saturable, indicating that passive diffusion becomes a significant mechanism of leucine transmembrane movement under these conditions. The proton motive force (PMF) was measured in glucose-energized cells of S. mutans and was found to have a maximum value of 126 mV at an extracellular pH (pH0) of 5.0; this decreased to 45 mV at pH0 8.0. The intracellular accumulation of leucine was significantly correlated with the magnitude of the PMF. The addition of excess isoleucine or valine caused a marked decrease in the leucine transport rate. Maximal rates of leucine transport occurred at pH0 6.0, and the rate of leucine transport was independent of the growth medium. The results suggest that there is a PMF-driven, branched-chain amino acid carrier in S. mutans with a proton: substrate stoichiometry of 1.

Regulation of hepatic bile acid transporters Ntcp and Bsep expression.

PubMed

Cheng, Xingguo; Buckley, David; Klaassen, Curtis D

2007-12-03

Sodium-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) are two key transporters for hepatic bile acid uptake and excretion. Alterations in Ntcp and Bsep expression have been reported in pathophysiological conditions. In the present study, the effects of age, gender, and various chemicals on the regulation of these two transporters were characterized in mice. Ntcp and Bsep mRNA levels in mouse liver were low in the fetus, but increased to its highest expression at parturition. After birth, mouse Ntcp and Bsep mRNA decreased by more than 50%, and then gradually increased to adult levels by day 30. Expression of mouse Ntcp mRNA and protein exhibit higher levels in female than male livers. No gender difference exists in BSEP/Bsep expression in human and mouse livers. Hormone replacements conducted in gonadectomized, hypophysectomized, and lit/lit mice indicate that female-predominant Ntcp expression in mouse liver is due to the inhibitory effect of male-pattern GH secretion, but not sex hormones. Ntcp and Bsep expression are in general resistant to induction by a large battery of microsomal enzyme inducers. Administration of cholestyramine increased Ntcp, whereas chenodeoxycholic acid (CDCA) increased Bsep mRNA expression. In conclusion, mouse Ntcp and Bsep are regulated by age, gender, cholestyramine, and bile acid, but resistant to induction by most microsomal enzyme inducers.

Vacuolar transport of the glutathione conjugate of trans-cinnamic acid.

PubMed

Walczak, H A; Dean, J V

2000-02-01

Red beet (Beta vulgaris L.) tonoplast membrane vesicles and [14C]trans-cinnamic acid-glutatione were used to study the vacuolar transport of phynylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [14C]trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [14C]trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoblast H+-electrochemical gradient, because uptake was not affected by the addition of NH4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 microM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 microM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated Km and Vmax values for uptake were 142 microM amd 5.95 nmol mg(-1) min(-1), respectively. Based on these results, phenylpropanoid-glutation conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.

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

Choline Transport Activity Regulates Phosphatidylcholine Synthesis through Choline Transporter Hnm1 Stability*

PubMed Central

Fernández-Murray, J. Pedro; Ngo, Michael H.; McMaster, Christopher R.

2013-01-01

Choline is a precursor for the synthesis of phosphatidylcholine through the CDP-choline pathway. Saccharomyces cerevisiae expresses a single high affinity choline transporter at the plasma membrane, encoded by the HNM1 gene. We show that exposing cells to increasing levels of choline results in two different regulatory mechanisms impacting Hnm1 activity. Initial exposure to choline results in a rapid decrease in Hnm1-mediated transport at the level of transporter activity, whereas chronic exposure results in Hnm1 degradation through an endocytic mechanism that depends on the ubiquitin ligase Rsp5 and the casein kinase 1 redundant pair Yck1/Yck2. We present details of how the choline transporter is a major regulator of phosphatidylcholine synthesis. PMID:24187140

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.

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

PubMed

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

2011-01-01

Absorption at the level of the intestine is likely a primary regulatory mechanism for the deposition of dietary supplemented folic acid into the chicken egg. Therefore, factors affecting the intestinal transport of folic acid in the laying hen may influence the level of egg folate concentrations. To this end, a series of experiments using intestinal everted sacs were conducted to characterize intestinal folic acid absorption processes in laying hens. Effects of naturally occurring folate derivatives (5-methyl and 10-formyltetrahydrofolate) as well as heme on folic acid absorption were also investigated. Folic acid absorption was measured based on the rate of uptake of (3)H-labeled folic acid in the everted sac from various segments of the small and large intestines. Folic acid concentration, incubation length, and pH condition were optimized before the performance of uptake experiments. The distribution profile of folic acid transport along the intestine was highest in the upper half of the small intestine. Maximum uptake rate (nmol·100 g tissue(-1)·min(-1)) was observed in the duodenum (20.6 ± 1.9) and jejunum (22.3 ± 2.0) and decreased significantly in the ileum (15.3 ± 1.1) and cecum (9.3 ± 0.9). Transport increased proportionately (P < 0.05) between 0.0001 and 0.1 µM folic acid. Above 0.1 µM, the slope of the regression line was not significantly different from zero (P < 0.137). Folic acid uptake in the jejunum showed a maximum rate of transport at pH 6.0, but was lowest at pH 7.5. The presence of 5-methyl and 10-formyltetrahydrofolate as well as heme impeded folic acid uptake, reducing intestinal folic acid absorption when added at concentrations ranging from 0 to 100 µM. Overall, these data indicated the presence of a folic acid transport system in the entire intestine of the laying hen. Uptake of folic acid in the cecum raises the likelihood of absorption of bacterial-derived folate.

Uptake mechanism of valproic acid in human placental choriocarcinoma cell line (BeWo).

PubMed

Ushigome, F; Takanaga, H; Matsuo, H; Tsukimori, K; Nakano, H; Ohtani, H; Sawada, Y

2001-04-13

Valproic acid is an anticonvulsant widely used for the treatment of epilepsy. However, valproic acid is known to show fetal toxicity, including teratogenicity. In the present study, to elucidate the mechanisms of valproic acid transport across the blood-placental barrier, we carried out transcellular transport and uptake experiments with human placental choriocarcinoma epithelial cells (BeWo cells) in culture. The permeability coefficient of [3H]valproic acid in BeWo cells for the apical-to-basolateral flux was greater than that for the opposite flux, suggesting a higher unidirectional transport in the fetal direction. The uptake of [3H]valproic acid from the apical side was temperature-dependent and enhanced under acidic pH. In the presence of 50 microM carbonyl cyanide p-trifluoromethoxylhydrazone, the uptake of [3H]valproic acid was significantly reduced. A metabolic inhibitor, 10 mM sodium azide, also significantly reduced the uptake of [3H]valproic acid. Therefore, valproic acid is actively transported in a pH-dependent manner on the brush-border membrane of BeWo cells. Kinetic analysis of valproic acid uptake revealed the involvement of a non-saturable component and a saturable component. The Michaelis constant for the saturable transport (K(t)) was smaller under acidic pH, suggesting a proton-linked active transport mechanism for valproic acid in BeWo cells. In the inhibitory experiments, some short-chain fatty acids, such as acetic acid, lactic acid, propanoic acid and butyric acid, and medium-chain fatty acids, such as hexanoic acid and octanoic acid, inhibited the uptake of [3H]valproic acid. The uptake of [3H]valproic acid was also significantly decreased in the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, salicylic acid and furosemide, which are well-known inhibitors of the anion exchange system. Moreover, p-aminohippuric acid significantly reduced the uptake of [3H]valproic acid. These results suggest that an active transport

Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2010-01-01

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.

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

PubMed

Shibutani, Shusaku; Okazaki, Hana; Iwata, Hiroyuki

2017-11-03

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

Modulation of the cationic amino acid transport system y+L by surface potential, ouabain and thrombin in human platelets: effects of uremia.

PubMed

Alves de Sá Siqueira, Mariana; Martins, Marcela Anjos; Rodrigues Pereira, Natália; Bandeira Moss, Monique; Santos, Sérgio F F; Mann, Giovanni E; Mendes-Ribeiro, Antônio C; Brunini, Tatiana M C

2007-01-01

Nitric oxide (NO), a key endogenous mediator involved in the maintenance of platelet function, is synthesized from the amino acid L-arginine. We have shown that L-arginine transport in platelets is rate-limiting for NO synthesis. A disturbance in the L-arginine-NO pathway in platelets was previously described in chronic renal failure (CRF) patients. Detailed kinetic studies were performed in platelets from controls (n = 60) and hemodialysis patients (n = 26). The transport of L-arginine in platelets is mediated via system y+L, which is competitively inhibited by L-leucine in the presence of Na+ and by the irreversible inhibitor pCMB. In platelets, system y+L is markedly stimulated by an Na+/K+-ATPase inhibitor, ouabain, and by changes in surface potential, while it is downregulated by intraplatelet amino acid depletion (zero-trans) and by thrombin. In CRF patients, activation of L-arginine transport was limited to well-nourished patients compared to malnourished patients and controls, where it was reduced and did not differ significantly among the groups under zero-trans conditions. Our results provide the first evidence that system y+L in platelets is modulated by zero-trans conditions, surface potential, thrombin and intraplatelet Na+ concentration. Our findings suggest that enhanced transport in CRF involves increased L-arginine exchange with intraplatelet neutral amino acids.

Transepithelial transport of flavanone in intestinal Caco-2 cell monolayers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kobayashi, Shoko; Konishi, Yutaka

2008-03-28

Our recent study [S. Kobayashi, S. Tanabe, M. Sugiyama, Y. Konishi, Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers, Biochim. Biophys. Acta, 1778 (2008) 33-41] shows that the mechanism of absorption of hesperetin involves both proton-coupled active transport and transcellular passive diffusion. Here, as well as analyzing the cell permeability of hesperetin, we also study the transport of other flavanones, naringenin and eriodictyol, using Caco-2 cell monolayers. Similar to hesperetin mentioned, naringenin and eriodictyol showed proton-coupled polarized transport in apical-to-basolateral direction in non-saturable manner, constant permeation in the apical-to-basolateral direction (J{sub ap{yields}}{sub bl}) irrespective of the transepithelialmore » electrical resistance (TER), and preferable distribution into the basolateral side after apical loading in the presence of a proton gradient. Furthermore, the proton-coupled J{sub ap{yields}}{sub bl} of hesperetin, naringenin and eriodictyol, were inhibited by substrates of the monocarboxylic acid transporter (MCT), such as benzoic acid, but not by ferulic acid. In contrast, both benzoic and ferulic acids have no stimulatory effect on J{sub ap{yields}}{sub bl} of each flavanone by trans-stimulation analysis. These results indicates that proton-driven active transport is commonly participated in the absorption of flavanone in general, and that its transport is presumed to be unique other than MCT-mediated transport for absorption of phenolic acids (PAs), sodium-dependent MCT (SMCT) nor anion exchanger-mediated transport.« less

Benzylserine inhibits breast cancer cell growth by disrupting intracellular amino acid homeostasis and triggering amino acid response pathways.

PubMed

van Geldermalsen, Michelle; Quek, Lake-Ee; Turner, Nigel; Freidman, Natasha; Pang, Angel; Guan, Yi Fang; Krycer, James R; Ryan, Renae; Wang, Qian; Holst, Jeff

2018-06-26

Cancer cells require increased levels of nutrients such as amino acids to sustain their rapid growth. In particular, leucine and glutamine have been shown to be important for growth and proliferation of some breast cancers, and therefore targeting the primary cell-surface transporters that mediate their uptake, L-type amino acid transporter 1 (LAT1) and alanine, serine, cysteine-preferring transporter 2 (ASCT2), is a potential therapeutic strategy. The ASCT2 inhibitor, benzylserine (BenSer), is also able to block LAT1 activity, thus inhibiting both leucine and glutamine uptake. We therefore aimed to investigate the effects of BenSer in breast cancer cell lines to determine whether combined LAT1 and ASCT2 inhibition could inhibit cell growth and proliferation. BenSer treatment significantly inhibited both leucine and glutamine uptake in MCF-7, HCC1806 and MDA-MB-231 breast cancer cells, causing decreased cell viability and cell cycle progression. These effects were not primarily leucine-mediated, as BenSer was more cytostatic than the LAT family inhibitor, BCH. Oocyte uptake assays with ectopically expressed amino acid transporters identified four additional targets of BenSer, and gas chromatography-mass spectrometry (GCMS) analysis of intracellular amino acid concentrations revealed that this BenSer-mediated inhibition of amino acid uptake was sufficient to disrupt multiple pathways of amino acid metabolism, causing reduced lactate production and activation of an amino acid response (AAR) through activating transcription factor 4 (ATF4). Together these data showed that BenSer blockade inhibited breast cancer cell growth and viability through disruption of intracellular amino acid homeostasis and inhibition of downstream metabolic and growth pathways.

Health Impacts of Active Transportation in Europe.

PubMed

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.

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

"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.)

Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486.

PubMed

Kalandadze, Avtandil; Wu, Ying; Robinson, Michael B

2002-11-29

Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an

Growing Mouse Oocytes Transiently Activate Folate Transport via Folate Receptors As They Approach Full Size.

PubMed

Meredith, Megan; MacNeil, Allison H; Trasler, Jacquetta M; Baltz, Jay M

2016-06-01

The folate cycle is central to cellular one-carbon metabolism, where folates are carriers of one-carbon units that are critical for synthesis of purines, thymidylate, and S-adenosylmethionine, the universal methyl donor that forms the cellular methyl pool. Although folates are well-known to be important for early embryo and fetal development, their role in oogenesis has not been clearly established. Here, folate transport proteins were detected in developing neonatal ovaries and growing oocytes by immunohistochemistry, Western blot, and immunofluorescence. The folate receptors FOLR1 and FOLR2 as well as reduced folate carrier 1 (RFC1, SLC19A1 protein) each appeared to be present in follicular cells including granulosa cells. In growing oocytes, however, only FOLR2 immunoreactivity appeared abundant. Localization of apparent FOLR2 immunofluorescence near the plasma membrane increased with oocyte growth and peaked in oocytes as they neared full size. We assessed folate transport using the model folate leucovorin (folinic acid). Unexpectedly, there was a transient burst of folate transport activity for a brief period during oocyte growth as they neared full size, while folate transport was otherwise undetectable for the rest of oogenesis and in fully grown germinal vesicle stage oocytes. This folate transport was inhibited by dynasore, an inhibitor of endocytosis, but insensitive to the anion transport inhibitor stilbene 4-acetamido-40-isothiocyanato-stilbene-2,20-disulfonic acid, consistent with folate receptor-mediated transport but not with RFC1-mediated transport. Thus, near the end of their growth, growing oocytes may take up folates that could support the final stage of oogenesis or be stored to provide the endogenous folates needed in early embryogenesis. © 2016 by the Society for the Study of Reproduction, Inc.

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

Chloride transport in functionally active phagosomes isolated from Human neutrophils.

PubMed

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

2012-12-15

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 properties

Functional activity of L-carnitine transporters in human airway epithelial cells.

PubMed

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

2016-02-01

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2005-07-01

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

Transport of beta-casein-derived peptides by the oligopeptide transport system is a crucial step in the proteolytic pathway of Lactococcus lactis.

PubMed

Kunji, E R; Hagting, A; De Vries, C J; Juillard, V; Haandrikman, A J; Poolman, B; Konings, W N

1995-01-27

In the proteolytic pathway of Lactococcus lactis, milk proteins (caseins) are hydrolyzed extracellularly to oligopeptides by the proteinase (PrtP). The fate of these peptides, i.e. extracellular hydrolysis followed by amino acid uptake or transport followed by intracellular hydrolysis, has been addressed. Mutants have been constructed that lack a functional di-tripeptide transport system (DtpT) and/or oligopeptide transport system (Opp) but do express the P1-type proteinase (specific for hydrolysis of beta- and to a lesser extent kappa-casein). The wild type strain and the DtpT- mutant accumulate all beta-casein-derived amino acids in the presence of beta-casein as protein substrate and glucose as a source of metabolic energy. The amino acids are not accumulated significantly inside the cells by the Opp- and DtpT- Opp- mutants. When cells are incubated with a mixture of amino acids mimicking the composition of beta-casein, the amino acids are taken up to the same extent in all four strains. Analysis of the extracellular peptide fraction, formed by the action of PrtP on beta-casein, indicates that distinct peptides disappear only when the cells express an active Opp system. These and other experiments indicate that (i) oligopeptide transport is essential for the accumulation of all beta-casein-derived amino acids, (ii) the activity of the Opp system is sufficiently high to support high growth rates on beta-casein provided leucine and histidine are present as free amino acids, and (iii) extracellular peptidase activity is not present in L. lactis.

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