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

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

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.

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.

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

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

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

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.

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.

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

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.

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

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

PubMed

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

2017-10-01

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

Scallop DMT functions as a Ca2+ transporter.

PubMed

Toyohara, Haruhiko; Yamamoto, Sayuri; Hosoi, Masatomi; Takagi, Masaya; Hayashi, Isao; Nakao, Kenji; Kaneko, Shuji

2005-05-09

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca(2+) transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca(2+) from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe(2+) concentration. Scallop DMT distinctly functions as a Ca(2+) transporter unlike other DMTs, however, it also transports Fe(2+) and Cd(2+) similar to them.

Wide Tolerance to Amino Acids Substitutions In The OCTN1 Ergothioneine Transporter

PubMed Central

Frigeni, Marta; Iacobazzi, Francesco; Yin, Xue; Longo, Nicola

2016-01-01

Background Organic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease. Methods Here we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy. Results Substitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased Vmax, with modest changes in Km toward ergothioneine. Conclusions Our results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity. General significance The widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role. PMID:26994919

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.

Glutamine Transporters in Mammalian Cells and Their Functions in Physiology and Cancer

PubMed Central

Bhutia, Yangzom D.; Ganapathy, Vadivel

2016-01-01

The SLC (solute carrier)-type transporters (∼400 in number) in mammalian cells consist of 52 distinct gene families, grouped solely based on the amino acid sequence (primary structure) of the transporter proteins and not on their transport function. Among them are the transporters for amino acids. Fourteen of them, capable of transporting glutamine across the plasma membrane, are found in four families: SLC1, SLC6, SLC7, and SLC38. However, it is generally thought that the members of the SLC38 family are the principal transporters for glutamine. Some of the glutamine transporters are obligatory exchangers whereas some function as active transporters in one direction. While most glutamine transporters mediate the influx of the amino acid into cells, some actually mediate the efflux of the amino acid out of the cells. Glutamine transporters play important roles in a variety of tissues, including the liver, brain, kidney, and placenta, as clearly evident from the biological and biochemical phenotypes resulting from the deletion of specific glutamine transporters in mice. Owing to the obligatory role of glutamine in growth and proliferation of tumor cells, there is increasing attention on glutamine transporters in cancer biology as potential drug targets for cancer treatment. Selective blockers of certain glutamine transporters might be effective in preventing the entry of glutamine and other important amino acids into tumor cells, thus essentially starving these cells to death. This could represent the beginning of a new era in the discovery of novel anticancer drugs with a previously unexplored mode of action. PMID:26724577

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.

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

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.

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.

SH3-like motif-containing C-terminal domain of staphylococcal teichoic acid transporter suggests possible function.

PubMed

Ko, Tzu-Ping; Tseng, Shih-Ting; Lai, Shu-Jung; Chen, Sheng-Chia; Guan, Hong-Hsiang; Shin Yang, Chia; Jung Chen, Chun; Chen, Yeh

2016-09-01

The negatively charged bacterial polysaccharides-wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising a transmembrane subunit TagG and an ATPase subunit TagH. We determined the crystal structure of the C-terminal domain of TagH (TagH-C) to investigate its function. The structure shows an N-terminal SH3-like subdomain wrapped by a C-terminal subdomain with an anti-parallel β-sheet and an outer shell of α-helices. A stretch of positively charged surface across the subdomain interface is flanked by two negatively charged regions, suggesting a potential binding site for negatively charged polymers, such as WTAs or acidic peptide chains. Proteins 2016; 84:1328-1332. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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.

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.

Branched-chain amino acids and brain function.

PubMed

Fernstrom, John D

2005-06-01

Branched-chain amino acids (BCAAs) influence brain function by modifying large, neutral amino acid (LNAA) transport at the blood-brain barrier. Transport is shared by several LNAAs, notably the BCAAs and the aromatic amino acids (ArAAs), and is competitive. Consequently, when plasma BCAA concentrations rise, which can occur in response to food ingestion or BCAA administration, or with the onset of certain metabolic diseases (e.g., uncontrolled diabetes), brain BCAA concentrations rise, and ArAA concentrations decline. Such effects occur acutely and chronically. Such reductions in brain ArAA concentrations have functional consequences: biochemically, they reduce the synthesis and the release of neurotransmitters derived from ArAAs, notably serotonin (from tryptophan) and catecholamines (from tyrosine and phenylalanine). The functional effects of such neurochemical changes include altered hormonal function, blood pressure, and affective state. Although the BCAAs thus have biochemical and functional effects in the brain, few attempts have been made to characterize time-course or dose-response relations for such effects. And, no studies have attempted to identify levels of BCAA intake that might produce adverse effects on the brain. The only "model" of very high BCAA exposure is a very rare genetic disorder, maple syrup urine disease, a feature of which is substantial brain dysfunction but that probably cannot serve as a useful model for excessive BCAA intake by normal individuals. Given the known biochemical and functional effects of the BCAAs, it should be a straightforward exercise to design studies to assess dose-response relations for biochemical and functional effects and, in this context, to explore for adverse effect thresholds.

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

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.

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

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

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

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

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.

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

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.

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.

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

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.

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

δ-Aminolevulinic acid transport in murine mammary adenocarcinoma cells is mediated by beta transporters

PubMed Central

Bermúdez Moretti, M; Correa García, S; Perotti, C; Batlle, A; Casas, A

2002-01-01

δ-aminolevulinic acid, the precursor of porphyrin biosynthesis has been used to induce the endogenous synthesis of the photosensitiser protoporphyrin IX for photodynamic therapy in the treatment of various tumours. The aim of this work was to characterise the δ-aminolevulinic acid transport system in the murine mammary adenocarcinoma cell line LM3 using 14C-δ-aminolevulinic acid, to finally improve δ-aminolevulinic acid incorporation in mammalian cells. Our results showed that δ-aminolevulinic acid is incorporated into these cells by two different mechanisms, passive diffusion which is important at the beginning of the incubation, and active transport. Specificity assays suggested that the transporter involved in δ-aminolevulinic acid incorporation is a BETA transporter, probably GAT-2. British Journal of Cancer (2002) 87, 471–474. doi:10.1038/sj.bjc.6600481 www.bjcancer.com © 2002 Cancer Research UK PMID:12177786

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

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.

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

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.

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.

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

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.

Copper homeostasis in grapevine: functional characterization of the Vitis vinifera copper transporter 1.

PubMed

Martins, Viviana; Bassil, Elias; Hanana, Mohsen; Blumwald, Eduardo; Gerós, Hernâni

2014-07-01

The Vitis vinifera copper transporter 1 is capable of self-interaction and mediates intracellular copper transport. An understanding of copper homeostasis in grapevine (Vitis vinifera L.) is particularly relevant to viticulture in which copper-based fungicides are intensively used. In the present study, the Vitis vinifera copper transporter 1 (VvCTr1), belonging to the Ctr family of copper transporters, was cloned and functionally characterized. Amino acid sequence analysis showed that VvCTr1 monomers are small peptides composed of 148 amino acids with 3 transmembrane domains and several amino acid residues typical of Ctr transporters. Bimolecular fluorescence complementation (BiFC) demonstrated that Ctr monomers are self-interacting and subcellular localization studies revealed that VvCTr1 is mobilized via the trans-Golgi network, through the pre-vacuolar compartment and located to the vacuolar membrane. The heterologous expression of VvCTr1 in a yeast strain lacking all Ctr transporters fully rescued the phenotype, while a deficient complementation was observed in a strain lacking only plasma membrane-bound Ctrs. Given the common subcellular localization of VvCTr1 and AtCOPT5 and the highest amino acid sequence similarity in comparison to the remaining AtCOPT proteins, Arabidopsis copt5 plants were stably transformed with VvCTr1. The impairment in root growth observed in copt5 seedlings in copper-deficient conditions was fully rescued by VvCTr1, further supporting its involvement in intracellular copper transport. Expression studies in V. vinifera showed that VvCTr1 is mostly expressed in the root system, but transcripts were also present in leaves and stems. The functional characterization of VvCTr-mediated copper transport provides the first step towards understanding the physiological and molecular responses of grapevines to copper-based fungicides.

Structure/function relationships in serotonin transporter: new insights from the structure of a bacterial transporter.

PubMed

Rudnick, G

2006-01-01

Serotonin transporter (SERT) serves the important function of taking up serotonin (5-HT) released during serotonergic neurotransmission. It is the target for important therapeutic drugs and psychostimulants. SERT catalyzes the influx of 5-HT together with Na+ and Cl- in a 1:1:1 stoichiometry. In the same catalytic cycle, there is coupled efflux of one K+ ion. SERT is one member of a large family of amino acid and amine transporters that is believed to utilize similar mechanisms of transport. A bacterial member of this family was recently crystallized, revealing the structural basis of these transporters. In light of the new structure, previous results with SERT have been re-interpreted, providing new insight into the substrate binding site, the permeation pathway, and the conformational changes that occur during the transport cycle.

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...

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.

Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

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

Giulliani, S. E.; Frank, A. E.; Collart, F. R.

2008-12-08

We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

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

USGS Publications Warehouse

Bradley, Michael W.; Worland, Scott C.

2015-01-01

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

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

PubMed

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

2018-03-02

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

Transepithelial transport of rosuvastatin and effect of ursolic acid on its transport in Caco-2 monolayers.

PubMed

Hua, Wen Jin; Fang, Hu Jin; Hua, Wei Xiao

2012-09-01

The aim of this study was to determine transepithelial transport characteristics of rosuvastatin and effect of ursolic acid (P-gp potential inhibitor) and ko143 (ABC transporters selective inhibitor) on its transport in Caco-2 monolayers. A reliable Caco-2 cell monolayers model was established. The TEER value was used to inspect integrity of cell model. Apparent permeability coefficients (Papp(BL-AP) and Papp(AP-BL)) were used to analyze transepithelial transport of rosuvastatin. Uptake of rosuvastatin was time- and concentration-dependent in Caco-2 cell. The ko143 but not ursolic acid had effect on the uptake of rosuvastatin in Caco-2 cell monolayer model and affected apparent permeability coefficient and apparent permeability of rosuvastatin. Active transport and passive diffusion absorption existed in transepithelial transport of rosuvastatin in Caco-2 cell model. Ursolic acid had no effect on transport of rosuvastatin in Caco-2 cell monolayer. The result indicated that ursolic acid may not cause effect on intestinal absorption of rosuvastatin.

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

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.

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

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.

Phytomonas: transport of amino acids, hexoses and polyamines.

PubMed

Canepa, Gaspar E; Carrillo, Carolina; Armesto, Arnaldo R; Bouvier, León A; Miranda, Mariana R; Pereira, Claudio A

2007-09-01

Phytomonas cells (Phytomonas Jma) isolated from the latex of Jatropha macrantha were assayed for amino acid, hexose and polyamine transport. Results showed high transport rates for glucose and fructose (193 and 128 pmol min(-1) 10(-7) cells, respectively) and lower, but significant rates, for proline, arginine, cysteine and glutamate (between 1.7 and 5.8 pmol min(-1) 10(-7) cells). Minor transport activities were observed for serine, glycine and aspartate (<1 pmol min(-1) 10(-7) cells). Amino acid transport processes do not seem to be regulated by starvation or during the growth phases. Polyamine transport was also evaluated showing a clear preference for spermidine over putrescine (3.4 and 0.4 pmol min(-1) 10(-7) cells, respectively). This work represents the first report on metabolite transport in phytomonads.

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.

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.

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

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.

Transporter-targeted cholic acid-cytarabine conjugates for improved oral absorption.

PubMed

Zhang, Dong; Li, Dongpo; Shang, Lei; He, Zhonggui; Sun, Jin

2016-09-10

Cytarabine has a poor oral absorption due to its rapid deamination and poor membrane permeability. Bile acid transporters are highly expressed both in enterocytes and hepatocytes and to increase the oral bioavailability and investigate the potential application of cytarabine for liver cancers, a transporter- recognizing prodrug strategy was applied to design and synthesize four conjugates of cytarabine with cholic acid (CA), chenodeoxycholic acid (CDCA), hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA). The anticancer activities against HepG2 cells were evaluated by MTT assay and the role of bile acid transporters during cellular transport was investigated in a competitive inhibition experiment. The in vitro and in vivo metabolic stabilities of these conjugates were studied in rat plasma and liver homogenates. Finally, an oral bioavailability study was conducted in rats. All the cholic acid-cytarabine conjugates (40μM) showed potent antiproliferative activities (up to 70%) against HepG2 cells after incubation for 48h. The addition of bile acids could markedly reduce the antitumor activities of these conjugates. The N(4)-ursodeoxycholic acid conjugate of cytarabine (compound 5) exhibited optimal stability (t1/2=90min) in vitro and a 3.9-fold prolonged half-life of cytarabine in vivo. More importantly, compound 5 increased the oral bioavailability 2-fold compared with cytarabine. The results of the present study suggest that the prodrug strategy based on the bile acid transporters is suitable for improving the oral absorption and the clinical application of cytarabine. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning and functional characterization of a fatty acid transport protein (FATP) from the pheromone gland of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone.

PubMed

Qian, Shuguang; Fujii, Takeshi; Ito, Katsuhiko; Nakano, Ryo; Ishikawa, Yukio

2011-01-01

Sex pheromones of moths are largely classified into two types based on the presence (Type I) or absence (Type II) of a terminal functional group. While Type-I sex pheromones are synthesized from common fatty acids in the pheromone gland (PG), Type-II sex pheromones are derived from hydrocarbons produced presumably in the oenocytes and transported to the PG via the hemolymph. Recently, a fatty acid transport protein (BmFATP) was identified from the PG of the silkworm Bombyx mori, which produces a Type-I sex pheromone (bombykol). BmFATP was shown to facilitate the uptake of extracellular fatty acids into PG cells for the synthesis of bombykol. To elucidate the presence and function of FATP in the PG of moths that produce Type-II sex pheromones, we explored fatp homologues expressed in the PG of a lichen moth, Eilema japonica, which secretes an alkenyl sex pheromone (Type II). A fatp homologue cloned from E. japonica (Ejfatp) was predominantly expressed in the PG, and its expression is upregulated shortly after eclosion. Functional expression of EjFATP in Escherichia coli enhanced the uptake of long chain fatty acids (C₁₈ and C₂₀), but not pheromone precursor hydrocarbons. To the best of our knowledge, this is the first report of the cloning and functional characterization of a FATP in the PG of a moth producing a Type-II sex pheromone. Although EjFATP is not likely to be involved in the uptake of pheromone precursors in E. japonica, the expression pattern of Ejfatp suggests a role for EjFATP in the PG not directly linked to pheromone biosynthesis. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

Genetic polymorphisms in Na+-taurocholate co-transporting polypeptide (NTCP) and ileal apical sodium-dependent bile acid transporter (ASBT) and ethnic comparisons of functional variants of NTCP among Asian populations.

PubMed

Pan, Wei; Song, Im-Sook; Shin, Ho-Jung; Kim, Min-Hye; Choi, Yeong-Lim; Lim, Su-Jeong; Kim, Woo-Young; Lee, Sang-Seop; Shin, Jae-Gook

2011-06-01

Genetic variants of Na(+)-taurocholate co-transporting polypeptide (NTCP; SLC10A1) and ileal apical sodium-dependent bile acid transporter (ASBT; SLC10A2), which greatly contribute to bile acid homeostasis, were extensively explored in the Korean population and functional variants of NTCP were compared among Asian populations. From direct DNA sequencing, six SNPs were identified in the SLC10A1 gene and 14 SNPs in the SLC10A2 gene. Three of seven coding variants were non-synonymous SNPs: two variants from SLC10A1 (A64T, S267F) and one from SLC10A2 (A171S). No linkage was analysed in the SLC10A1 gene because of low frequencies of genetic variants, and the SLC10A2 gene was composed of two separated linkage disequilibrium blocks contrary to the white population. The stably transfected NTCP-A64T variant showed significantly decreased uptakes of taurocholate and rosuvastatin compared with wild-type NTCP. The decreased taurocholate uptake and increased rosuvastatin uptake were shown in the NTCP-S267F variant. The allele frequencies of these functional variants were 1.0% and 3.1%, respectively, in a Korean population. However, NTCP-A64T was not found in Chinese and Vietnamese subjects. The frequency distribution of NTCP-S267F in Koreans was significantly lower than those in Chinese and Vietnamese populations. Our data suggest that NTCP-A64T and -S267F variants cause substrate-dependent functional change in vitro, and show ethnic difference in their allelic frequencies among Asian populations although the clinical relevance of these variants is remained to be evaluated.

Lipotoxicity, fatty acid uncoupling and mitochondrial carrier function.

PubMed

Rial, Eduardo; Rodríguez-Sánchez, Leonor; Gallardo-Vara, Eunate; Zaragoza, Pilar; Moyano, Eva; González-Barroso, M Mar

2010-01-01

Diseases like obesity, diabetes or generalized lipodystrophy cause a chronic elevation of circulating fatty acids that can become cytotoxic, a condition known as lipotoxicity. Fatty acids cause oxidative stress and alterations in mitochondrial structure and function. The uncoupling of the oxidative phosphorylation is one of the most recognized deleterious fatty acid effects and several metabolite transporters are known to mediate in their action. The fatty acid interaction with the carriers leads to membrane depolarization and/or the conversion of the carrier into a pore. The result is the opening of the permeability transition pore and the initiation of apoptosis. Unlike the other members of the mitochondrial carrier superfamily, the eutherian uncoupling protein UCP1 has evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte and therefore it is activated by the low fatty acid concentrations generated by the noradrenaline-stimulated lipolysis. Copyright © 2010 Elsevier B.V. All rights reserved.

Na+-independent transporters, LAT-2 and b0,+, exchange L-DOPA with neutral and basic amino acids in two clonal renal cell lines.

PubMed

Gomes, P; Soares-da-Silva, P

2002-03-15

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.

Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria

PubMed Central

Romano, Andrea; Trip, Hein; Lonvaud-Funel, Aline; Lolkema, Juke S.

2012-01-01

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol. PMID:22247134

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.

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

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.

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.

OsABCB14 functions in auxin transport and iron homeostasis in rice (Oryza sativa L.).

PubMed

Xu, Yanxia; Zhang, Saina; Guo, Haipeng; Wang, Suikang; Xu, Ligen; Li, Chuanyou; Qian, Qian; Chen, Fan; Geisler, Markus; Qi, Yanhua; Jiang, De An

2014-07-01

Members of the ATP Binding Cassette B/Multidrug-Resistance/P-glyco-protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock-down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock-down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (-Fe). Their Fe concentration is higher and upregulation of Fe deficiency-responsive genes is lower in osabcb14 mutants than in wild-type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

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.

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.

Structure and function of SemiSWEET and SWEET sugar transporters.

PubMed

Feng, Liang; Frommer, Wolf B

2015-08-01

SemiSWEETs and SWEETs have emerged as unique sugar transporters. First discovered in plants with the help of fluorescent biosensors, homologs exist in all kingdoms of life. Bacterial and plant homologs transport hexoses and sucrose, whereas animal SWEETs transport glucose. Prokaryotic SemiSWEETs are small and comprise a parallel homodimer of an approximately 100 amino acid-long triple helix bundle (THB). Duplicated THBs are fused to create eukaryotic SWEETs in a parallel orientation via an inversion linker helix, producing a similar configuration to that of SemiSWEET dimers. Structures of four SemiSWEETs have been resolved in three states: open outside, occluded, and open inside, indicating alternating access. As we discuss here, these atomic structures provide a basis for exploring the evolution of structure-function relations in this new class of transporters. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

2008-01-01

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

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

Light-activated amino acid transport in Halobacterium halobium envelope vesicles

NASA Technical Reports Server (NTRS)

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

1977-01-01

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

Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes*

PubMed Central

Courville, Pascal; Quick, Matthias; Reimer, Richard J.

2010-01-01

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H+-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members. PMID:20424173

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.

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

Amino acid-dependent cMyc expression is essential for NK cell metabolic and functional responses in mice.

PubMed

Loftus, Róisín M; Assmann, Nadine; Kedia-Mehta, Nidhi; O'Brien, Katie L; Garcia, Arianne; Gillespie, Conor; Hukelmann, Jens L; Oefner, Peter J; Lamond, Angus I; Gardiner, Clair M; Dettmer, Katja; Cantrell, Doreen A; Sinclair, Linda V; Finlay, David K

2018-06-14

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system L-amino acid transport is blocked. We identify SLC7A5 as the predominant system L-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

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

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.

Functional significance of the E loop, a novel motif conserved in the lantibiotic immunity ATP-binding cassette transport systems.

PubMed

Okuda, Ken-ichi; Yanagihara, Sae; Sugayama, Tomomichi; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

2010-06-01

Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.

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.

Mendelian randomization analysis associates increased serum urate, due to genetic variation in uric acid transporters, with improved renal function.

PubMed

Hughes, Kim; Flynn, Tanya; de Zoysa, Janak; Dalbeth, Nicola; Merriman, Tony R

2014-02-01

Increased serum urate predicts chronic kidney disease independent of other risk factors. The use of xanthine oxidase inhibitors coincides with improved renal function. Whether this is due to reduced serum urate or reduced production of oxidants by xanthine oxidase or another physiological mechanism remains unresolved. Here we applied Mendelian randomization, a statistical genetics approach allowing disentangling of cause and effect in the presence of potential confounding, to determine whether lowering of serum urate by genetic modulation of renal excretion benefits renal function using data from 7979 patients of the Atherosclerosis Risk in Communities and Framingham Heart studies. Mendelian randomization by the two-stage least squares method was done with serum urate as the exposure, a uric acid transporter genetic risk score as instrumental variable, and estimated glomerular filtration rate and serum creatinine as the outcomes. Increased genetic risk score was associated with significantly improved renal function in men but not in women. Analysis of individual genetic variants showed the effect size associated with serum urate did not correlate with that associated with renal function in the Mendelian randomization model. This is consistent with the possibility that the physiological action of these genetic variants in raising serum urate correlates directly with improved renal function. Further studies are required to understand the mechanism of the potential renal function protection mediated by xanthine oxidase inhibitors.

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.

"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

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

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.

Functional relationship between cationic amino acid transporters and beta-defensins: implications for dry skin diseases and the dry eye.

PubMed

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

2010-04-20

The ocular surface, constantly exposed to environmental pathogens, is particularly vulnerable to infection. Hence an advanced immune defence system is essential to protect the eye from microbial attack. Antimicrobial peptides, such as beta-defensins, are essential components of the innate immune system and are the first line of defence against invaders of the eye. High concentrations of L-arginine and L-lysine are necessary for the expression of beta-defensins. These are supplied by epithelial cells in inflammatory processes. The limiting factor for initiation of beta-defensin production is the transport of L-arginine and L-lysine into the cell. This transport is performed to 80% by only one transporter system in the human, the y(+)-transporter. This group of proteins exclusively transports the cationic amino acids L-arginine, L-lysine and L-ornithine and is also known under the term cationic amino acid transporter proteins (CAT-proteins). Various infections associated with L-arginine deficiency (for example psoriasis, keratoconjuctivitis sicca) are also associated with an increase in beta-defensin production. For the first time, preliminary work has shown the expression of human CATs in ocular surface epithelia and tissues of the lacrimal apparatus indicating their relevance for diseases of the ocular surface. In this review, we summarize current knowledge on the human CATs that appear to be integrated in causal regulation cascades of beta-defensins, thereby offering novel concepts for therapeutic perspectives. Copyright 2010 Elsevier GmbH. All rights reserved.

Na+ Interactions with the Neutral Amino Acid Transporter ASCT1*

PubMed Central

Scopelliti, Amanda J.; Heinzelmann, Germano; Kuyucak, Serdar; Ryan, Renae M.; Vandenberg, Robert J.

2014-01-01

The alanine, serine, cysteine transporters (ASCTs) belong to the solute carrier family 1A (SLC1A), which also includes the excitatory amino acid transporters (EAATs) and the prokaryotic aspartate transporter GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na+ ions and one proton, and the counter-transport of one K+ ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K+ ions and the number of Na+ ions required is not well established. One property common to SLC1A family members is a substrate-activated anion conductance. We have investigated the number and location of Na+ ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na+ binding. Mutations to all three proposed Na+ sites influence the binding of substrate and/or Na+, or the rate of substrate exchange. A G422S mutation near the Na2 site reduced Na+ affinity, without affecting the rate of exchange. D467T and D467A mutations in the Na1 site reduce Na+ and substrate affinity and also the rate of substrate exchange. T124A and D380A mutations in the Na3 site selectively reduce the affinity for Na+ and the rate of substrate exchange without affecting substrate affinity. In many of the mutants that reduce the rate of substrate transport the amplitudes of the substrate-activated anion conductances are not substantially affected indicating altered ion dependence for channel activation compared with substrate exchange. PMID:24808181

Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter.

PubMed

Underwood, William; Somerville, Shauna C

2017-10-03

The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against a number of pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whether PAMP-induced phosphorylation of PEN3 is important for its defense function or focal recruitment has not been addressed. In this study, we evaluated the role of PEN3 phosphorylation in modulating the localization and defense function of the transporter. We report that PEN3 phosphorylation is critical for its function in defense, but dispensable for recruitment to powdery mildew penetration sites. These results indicate that PAMP-induced phosphorylation is likely to regulate the transport activity of PEN3.

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.

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

Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation

PubMed Central

Mendiondo, Guillermina M.; Medhurst, Anne; van Roermund, Carlo W.; Zhang, Xuebin; Devonshire, Jean; Scholefield, Duncan; Fernández, José; Axcell, Barry; Ramsay, Luke; Waterham, Hans R.; Waugh, Robbie; Theodoulou, Frederica L.; Holdsworth, Michael J.

2014-01-01

In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ. PMID:24913629

Functional characterization of Citrus macrophylla BOR1 as a boron transporter.

PubMed

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

2013-11-01

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

Inhibition of l-type amino acid transporter 1 activity as a new therapeutic target for cholangiocarcinoma treatment.

PubMed

Yothaisong, Supak; Dokduang, Hasaya; Anzai, Naohiko; Hayashi, Keitaro; Namwat, Nisana; Yongvanit, Puangrat; Sangkhamanon, Sakkarn; Jutabha, Promsuk; Endou, Hitoshi; Loilome, Watcharin

2017-03-01

Unlike normal cells, cancer cells undergo unlimited growth and multiplication, causing them to require massive amounts of amino acid to support their continuous metabolism. Among the amino acid transporters expressed on the plasma membrane, l-type amino acid transporter-1, a Na + -independent neutral amino acid transporter, is highly expressed in many types of human cancer including cholangiocarcinoma. Our previous study reported that l-type amino acid transporter-1 and its co-functional protein CD98 were highly expressed and implicated in cholangiocarcinoma progression and carcinogenesis. Therefore, this study determined the effect of JPH203, a selective inhibitor of l-type amino acid transporter-1 activity, on cholangiocarcinoma cell inhibition both in vitro and in vivo. JPH203 dramatically suppressed [ 14 C]l-leucine uptake as well as cell growth in cholangiocarcinoma cell lines along with altering the expression of l-type amino acid transporter-1 and CD98 in response to amino acid depletion. We also demonstrated that JPH203 induced both G2/M and G0/G1 cell cycle arrest, as well as reduced the S phase accompanied by altered expression of the proteins in cell cycle progression: cyclin D1, CDK4, and CDK6. There was also cell cycle arrest of the related proteins, P21 and P27, in KKU-055 and KKU-213 cholangiocarcinoma cells. Apoptosis induction, detected by an increase in trypan blue-stained cells along with a cleaved caspase-3/caspase-3 ratio, occurred in JPH203-treated cholangiocarcinoma cells at the highest concentration tested (100 µM). As expected, daily intravenous administration of JPH203 (12.5 and 25 mg/kg) significantly inhibited tumor growth in KKU-213 cholangiocarcinoma cell xenografts in the nude mice model in a dose-dependent manner with no statistically significant change in the animal's body weight and with no differences in the histology and appearance of the internal organs compared with the control group. Our study demonstrates that

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...

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

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.

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

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.

An Active Learning Exercise to Facilitate Understanding of Nephron Function: Anatomy and Physiology of Renal Transporters

ERIC Educational Resources Information Center

Dirks-Naylor, Amie J.

2016-01-01

Renal transport is a central mechanism underlying electrolyte homeostasis, acid base balance and other essential functions of the kidneys in human physiology. Thus, knowledge of the anatomy and physiology of the nephron is essential for the understanding of kidney function in health and disease. However, students find this content difficult to…

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.

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

NASA Astrophysics Data System (ADS)

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

2017-11-01

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

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

Sequencing, bioinformatic characterization and expression pattern of a putative amino acid transporter from the parasitic cestode Echinococcus granulosus.

PubMed

Camicia, Federico; Paredes, Rodolfo; Chalar, Cora; Galanti, Norbel; Kamenetzky, Laura; Gutierrez, Ariana; Rosenzvit, Mara C

2008-03-31

We have sequenced and partially characterized an Echinococcus granulosus cDNA, termed egat1, from a protoscolex signal sequence trap (SST) cDNA library. The isolated 1627 bp long cDNA contains an ORF of 489 amino acids and shows an amino acid identity of 30% with neutral and excitatory amino acid transporters members of the Dicarboxylate/Amino Acid Na+ and/or H+ Cation Symporter family (DAACS) (TC 2.A.23). Additional bioinformatics analysis of EgAT1, confirmed the results obtained by similarity searches and showed the presence of 9 to 10 transmembrane domains, consensus sequences for N-glycosylation between the third and fourth transmembrane domain, a highly similar hydropathy profile with ASCT1 (a known member of DAACS family), high score with SDF (Sodium Dicarboxilate Family) and similar motifs with EDTRANSPORT, a fingerprint of excitatory amino acid transporters. The localization of the putative amino acid transporter was analyzed by in situ hybridization and immunofluorescence in protoscoleces and associated germinal layer. The in situ hybridization labelling indicates the distribution of egat1 mRNA throughout the tegument. EgAT1 protein, which showed in Western blots a molecular mass of approximately 60 kD, is localized in the subtegumental region of the metacestode, particularly around suckers and rostellum of protoscoleces and layers from brood capsules. The sequence and expression analyses of EgAT1 pave the way for functional analysis of amino acids transporters of E. granulosus and its evaluation as new drug targets against cystic echinococcosis.

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

Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity.

PubMed

Wolf, Sabine; Janzen, Annette; Vékony, Nicole; Martiné, Ursula; Strand, Dennis; Closs, Ellen I

2002-06-15

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.

Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity.

PubMed Central

Wolf, Sabine; Janzen, Annette; Vékony, Nicole; Martiné, Ursula; Strand, Dennis; Closs, Ellen I

2002-01-01

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230-236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional. PMID:12049641

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.

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

Transport Coefficients from Large Deviation Functions

NASA Astrophysics Data System (ADS)

Gao, Chloe; Limmer, David

2017-10-01

We describe a method for computing transport coefficients from the direct evaluation of large deviation function. This method is general, relying on only equilibrium fluctuations, and is statistically efficient, employing trajectory based importance sampling. Equilibrium fluctuations of molecular currents are characterized by their large deviation functions, which is a scaled cumulant generating function analogous to the free energy. A diffusion Monte Carlo algorithm is used to evaluate the large deviation functions, from which arbitrary transport coefficients are derivable. We find significant statistical improvement over traditional Green-Kubo based calculations. The systematic and statistical errors of this method are analyzed in the context of specific transport coefficient calculations, including the shear viscosity, interfacial friction coefficient, and thermal conductivity.

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.

Effect of furosemide on ion transport in the turtle bladder: evidence for direct inhibition of active acid-base transport.

PubMed

Ehrenspeck, G; Voner, C

1985-07-25

The diuretic furosemide inhibits acid-base transport in the short-circuited turtle bladder. It inhibits luminal acidification when present in either mucosal or serosal bathing fluids, but decreases alkalinization only from the serosal side of the tissue. The inhibition of both acid-base transport processes is independent of ambient Cl-; and the disulfonic stilbene, SITS, an inhibitor of Cl--HCO3- exchange, fails to prevent the furosemide-elicited inhibition of alkalinization. These results preclude an absolute requirement of a furosemide-sensitive Cl--HCO3- exchange by these transport processes. The drug also interferes with the CO2-induced stimulation of acidification and alkalinization. The inhibition of the residual acidification in acetazolamide-treated, acidotic bladders, however, suggests an action at sites other than cytosolic carbonic anhydrase. Although active Na+ and Cl- reabsorption and tissue oxygen uptake are also decreased by furosemide, the rate of oxygen consumption uncoupled by 2,4-dinitrophenol is not diminished, indicating a primary inhibition of the various ion transport processes, not of metabolism. It is proposed that inhibition of transepithelial acid-base transport by furosemide in the turtle bladder includes inhibition of the acid-base pumps.

Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

PubMed

Quay, S C; Oxender, D L

1977-06-01

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur.

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.

Transport of K+ and other cations across phospholipid membranes by nonesterified fatty acids.

PubMed

Sharpe, M A; Cooper, C E; Wrigglesworth, J M

1994-07-01

The rate of change of internal pH and transmembrane potential has been monitored in liposomes following the external addition of various cation salts. Oleic acid increases the transmembrane movement of H+ following the imposition of a K+ gradient. An initial fast change in internal pH is seen followed by a slower rate of alkalinization. High concentrations of the fatty acid enhance the rate comparable to that seen in the presence of nigericin in contrast to the effect of FCCP (carbonyl cyanide p-(tri-fluoromethoxy)phenyl hydrazone) which saturates at an intermediate value. The ability of nonesterified fatty acids to catalyze the movement of cations across the liposome membrane increases with the degree of unsaturation and decreases with increasing chain length. Li and Na salts cause a similar initial fast pH change but have less effect on the subsequent slower rate. Similarly, the main effect of divalent cation salts is on the initial fast change. The membrane potential can enhance or inhibit cation transport depending on its polarity with respect to the cation gradient. It is concluded that nonesterified fatty acids have the capability to complex with, and transport, a variety of cations across phospholipid bilayers. However, they do not act simply as proton/cation exchangers analogous to nigericin nor as protonophores analogous to FCCP. The full cycle of ionophoric action involves a combination of both functions.

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

PubMed Central

Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

2014-01-01

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation. PMID:24652292

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

PubMed

Porcelli, Vito; Fiermonte, Giuseppe; Longo, Antonella; Palmieri, Ferdinando

2014-05-09

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.

Functional Characterization of 5-Oxoproline Transport via SLC16A1/MCT1*

PubMed Central

Sasaki, Shotaro; Futagi, Yuya; Kobayashi, Masaki; Ogura, Jiro; Iseki, Ken

2015-01-01

Thyrotropin-releasing hormone is a tripeptide that consists of 5-oxoproline, histidine, and proline. The peptide is rapidly metabolized by various enzymes. 5-Oxoproline is produced by enzymatic hydrolysis in a variety of peptides. Previous studies showed that 5-oxoproline could become a possible biomarker for autism spectrum disorders. Here we demonstrate the involvement of SLC16A1 in the transport of 5-oxoproline. An SLC16A1 polymorphism (rs1049434) was recently identified. However, there is no information about the effect of the polymorphism on SLC16A1 function. In this study, the polymorphism caused an observable change in 5-oxoproline and lactate transport via SLC16A1. The Michaelis constant (Km) was increased in an SLC16A1 mutant compared with that in the wild type. In addition, the proton concentration required to produce half-maximal activation of transport activity (K0.5, H+) was increased in the SLC16A1 mutant compared with that in the wild type. Furthermore, we examined the transport of 5-oxoproline in T98G cells as an astrocyte cell model. Despite the fact that 5-oxoproline is an amino acid derivative, Na+-dependent and amino acid transport systems scarcely contributed to 5-oxoproline transport. Based on our findings, we conclude that H+-coupled 5-oxoproline transport is mediated solely by SLC16A1 in the cells. PMID:25371203

Functional characterization of 5-oxoproline transport via SLC16A1/MCT1.

PubMed

Sasaki, Shotaro; Futagi, Yuya; Kobayashi, Masaki; Ogura, Jiro; Iseki, Ken

2015-01-23

Thyrotropin-releasing hormone is a tripeptide that consists of 5-oxoproline, histidine, and proline. The peptide is rapidly metabolized by various enzymes. 5-Oxoproline is produced by enzymatic hydrolysis in a variety of peptides. Previous studies showed that 5-oxoproline could become a possible biomarker for autism spectrum disorders. Here we demonstrate the involvement of SLC16A1 in the transport of 5-oxoproline. An SLC16A1 polymorphism (rs1049434) was recently identified. However, there is no information about the effect of the polymorphism on SLC16A1 function. In this study, the polymorphism caused an observable change in 5-oxoproline and lactate transport via SLC16A1. The Michaelis constant (Km) was increased in an SLC16A1 mutant compared with that in the wild type. In addition, the proton concentration required to produce half-maximal activation of transport activity (K0.5, H (+)) was increased in the SLC16A1 mutant compared with that in the wild type. Furthermore, we examined the transport of 5-oxoproline in T98G cells as an astrocyte cell model. Despite the fact that 5-oxoproline is an amino acid derivative, Na(+)-dependent and amino acid transport systems scarcely contributed to 5-oxoproline transport. Based on our findings, we conclude that H(+)-coupled 5-oxoproline transport is mediated solely by SLC16A1 in the cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

Blood-brain barrier transport of an essential amino acid after cerebral ischemia reperfusion injury.

PubMed

Suzuki, Toyofumi; Miyazaki, Yumiko; Ohmuro, Aya; Watanabe, Masaki; Furuishi, Takayuki; Fukami, Toshiro; Tomono, Kazuo

2013-01-01

Under pathophysiological conditions such as -cerebral ischemia-reperfusion (IR), damage to cerebrovascular endothelial cells causes alterations in the blood-brain barrier (BBB) function that can exacerbate neuronal cell injury and death. Clarifying changes in BBB transport in the early period of IR is important for understanding BBB function during therapy after cerebral ischemia. The present study was aimed at clarifying changes during IR in the BBB transport of L-phenylalanine (Phe) as a substrate of L-type amino acid transporter 1. An IR model was produced in mice by blood recirculation following occlusion of the middle cerebral artery. Permeability of the BBB to [(3)H]Phe was measured after IR injury using the brain perfusion method. Confocal microscopy of the IR injury showed no brain penetration of fluorescent tracer, thus confirming BBB integrity during 45 min of ischemia. Tight junction opening was not observed at 30 min after reperfusion following ischemia for 45 min. At the time of IR, [(3)H]Phe uptake into the brain appeared saturated. The Michaelis constant and maximum transport velocity in the IR group was reduced by 22 % compared with those in controls. These results suggest that the intrinsic transport clearance of Phe is slightly decreased in the early phase of IR.

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.

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.

Acid-functionalized nanoparticles for biomass hydrolysis

NASA Astrophysics Data System (ADS)

Pena Duque, Leidy Eugenia

Cellulosic ethanol is a renewable source of energy. Lignocellulosic biomass is a complex material composed mainly of cellulose, hemicellulose, and lignin. Biomass pretreatment is a required step to make sugar polymers liable to hydrolysis. Mineral acids are commonly used for biomass pretreatment. Using acid catalysts that can be recovered and reused could make the process economically more attractive. The overall goal of this dissertation is the development of a recyclable nanocatalyst for the hydrolysis of biomass sugars. Cobalt iron oxide nanoparticles (CoFe2O4) were synthesized to provide a magnetic core that could be separated from reaction using a magnetic field and modified to carry acid functional groups. X-ray diffraction (XRD) confirmed the crystal structure was that of cobalt spinel ferrite. CoFe2O4 were covered with silica which served as linker for the acid functions. Silica-coated nanoparticles were functionalized with three different acid functions: perfluoropropyl-sulfonic acid, carboxylic acid, and propyl-sulfonic acid. Transmission electron microscope (TEM) images were analyzed to obtain particle size distributions of the nanoparticles. Total carbon, nitrogen, and sulfur were quantified using an elemental analyzer. Fourier transform infra-red spectra confirmed the presence of sulfonic and carboxylic acid functions and ion-exchange titrations accounted for the total amount of catalytic acid sites per nanoparticle mass. These nanoparticles were evaluated for their performance to hydrolyze the beta-1,4 glycosidic bond of the cellobiose molecule. Propyl-sulfonic (PS) and perfluoropropyl-sulfonic (PFS) acid functionalized nanoparticles catalyzed the hydrolysis of cellobiose significantly better than the control. PS and PFS were also evaluated for their capacity to solubilize wheat straw hemicelluloses and performed better than the control. Although PFS nanoparticles were stronger acid catalysts, the acid functions leached out of the nanoparticle during

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

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.

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.

Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis.

PubMed

Kraidlova, Lucie; Schrevens, Sanne; Tournu, Hélène; Van Zeebroeck, Griet; Sychrova, Hana; Van Dijck, Patrick

2016-01-01

Amino acids are key sources of nitrogen for growth of Candida albicans . In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to Sc Gap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae , where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of Sc Sam3, the only S -adenosylmethionine (SAM) transporter in S. cerevisiae , and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of Sc Gap1 and that C. albicans Gap4 is an orthologue of Sc Sam3, an S -adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans .

Na-coupled bicarbonate transporters of the Slc4 family in the nervous system: function, localization, and relevance to neurologic function

PubMed Central

Majumdar, Debeshi; Bevensee, Mark O.

2010-01-01

Many cellular processes including neuronal activity are sensitive to changes in intracellular and/or extracellular pH— both of which are regulated by acid-base transporter activity. HCO3−-dependent transporters are particularly potent regulators of intracellular pH in neurons and astrocytes, and also contribute to the composition of the cerebrospinal fluid (CSF). The molecular physiology of HCO3− transporters has advanced considerably over the past ~14 years as investigators have cloned and characterized the function and localization of many Na-Coupled Bicarbonate Transporters of the Slc4 family (NCBTs). In this review, we provide an updated overview of the function and localization of NCBTs in the nervous system. Multiple NCBTs are expressed in neurons and astrocytes in various brain regions, as well as in epithelial cells of the choroid plexus. Characteristics of human patients with SLC4 gene mutations/deletions and results from recent studies on mice with Slc4 gene disruptions highlight the functional importance of NCBTs in neuronal activity, somatosensory function, and CSF production. Furthermore, energy-deficient states (e.g., hypoxia and ischemia) lead to altered expression and activity of NCBTs. Thus, recent studies expand our understanding of the role of NCBTs in regulating the pH and ionic composition of the nervous system that can modulate neuronal activity. PMID:20884330

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.

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

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.

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.

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

Gene duplication and neo-functionalization in the evolutionary and functional divergence of the metazoan copper transporters Ctr1 and Ctr2.

PubMed

Logeman, Brandon L; Wood, L Kent; Lee, Jaekwon; Thiele, Dennis J

2017-07-07

Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu + across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu + transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu + transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1 -/- mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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,...

[Lipoproteins as a specific circulatory transport system].

PubMed

Titov, V N

1998-01-01

In accordance with the systemic approach, each circulatory transport system is highly specific and transports an elementary substance from cell to cell in the hydrated medium. In the author's opinion, the lipoprotein system has also a functional specificity and carries the elementary substance fatty acid in the blood stream. A great variety of fatty acids, the individuality of their physicochemical properties, great stereochemic differences of saturated and polyenic fatty acids make their transport virtually impossible. The steric individuality of fatty acids can be reduced if the acids are covalently bonded by a matrix as complex lipids. For formation of complex lipids, nature prefers esterification of fatty acids with alcohols which have a varying hydrophoby, such as glycerol, sphingosine, cholesterol, cetyl alcohol. The steric differences of saturated and polyenic fatty acids form a basis for their being structurized in different lipids. Triacyl glycerides are a transport form of saturated, monounsaturated fatty acids and their transforms and give rise to a crystalline phase. Phospholipids and cholesterol esters are a transport form of mainly polyunsaturated fatty acids in the polar phase in the former case and in the crystalline phase in the latter one. The individual apolipoproteins structure complex lipids into individual lipoprotein particles and transport them in the hydrated medium of blood flow. Saturated fatty acids chiefly transport lipoprotein particles formed by apoB-48- and apoB-100-isoproteins. Polyenic acids transport mainly high-density apoA-1-lipoprotein particles, which makes up a main physiological function of the latter. Cholesterol is nothing more than a matrix; it reesterifies polyenic fatty acids from the polar transport form of phospholipids into the unpolar transport form of cholesterol esters. Cholesterol esterification of polyenic fatty acids may structure complex lipid in the unpolar phase and transport it to the cells via apoB-100

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.

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

Decarboxylative functionalization of cinnamic acids.

PubMed

Borah, Arun Jyoti; Yan, Guobing

2015-08-14

Decarboxylative functionalization of α,β-unsaturated carboxylic acids is an emerging area that has been developed significantly in recent years. This critical review focuses on the different decarboxylative functionalization reactions of cinnamic acids leading to the formation of various C-C and C-heteroatom bonds. Apart from metal carboxylates, decarboxylation in cinnamic acids has been achieved efficiently under metal-free conditions, particularly via the use of hypervalent iodine reagents. We believe this review will encourage organic chemists to develop vinylic decarboxylation in a more appealing way with an understanding of new mechanistic insight.

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta.

PubMed

Seward, David J; Koh, Albert S; Boyer, James L; Ballatori, Nazzareno

2003-07-25

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

Unraveling fatty acid transport and activation mechanisms in Yarrowia lipolytica.

PubMed

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

2015-09-01

Fatty acid (FA) transport and activation have been extensively studied in the model yeast species Saccharomyces cerevisiae but have rarely been examined in oleaginous yeasts, such as Yarrowia lipolytica. Because the latter begins to be used in biodiesel production, understanding its FA transport and activation mechanisms is essential. We found that Y. lipolytica has FA transport and activation proteins similar to those of S. cerevisiae (Faa1p, Pxa1p, Pxa2p, Ant1p) but mechanism of FA peroxisomal transport and activation differs greatly with that of S. cerevisiae. While the ScPxa1p/ScPxa2p heterodimer is essential for growth on long-chain FAs, ΔYlpxa1 ΔYlpxa2 is not impaired for growth on FAs. Meanwhile, ScAnt1p and YlAnt1p are both essential for yeast growth on medium-chain FAs, suggesting they function similarly. Interestingly, we found that the ΔYlpxa1 ΔYlpxa2 ΔYlant1 mutant was unable to grow on short-, medium-, or long-chain FAs, suggesting that YlPxa1p, YlPxa2p, and YlAnt1p belong to two different FA degradation pathways. We also found that YlFaa1p is involved in FA storage in lipid bodies and that FA remobilization largely depended on YlFat1p, YlPxa1p and YlPxa2p. This study is the first to comprehensively examine FA intracellular transport and activation in oleaginous yeast. Copyright © 2015. Published by Elsevier B.V.

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

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

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.

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

PubMed

Van Dyke, James U; Beaupre, Steven J

2012-03-01

Viviparity and placentation have evolved from oviparity over 100 times in squamate reptiles (lizards and snakes). The independent origins of placentation have resulted in a variety of placental morphologies in different taxa, ranging from simple apposition of fetal and maternal tissues to endotheliochorial implantation that is homoplasious with mammalian placentation. Because the eggs of oviparous squamates transport gases and water from the environment and calcium from the eggshell, the placentae of viviparous squamates are thought to have initially evolved to accomplish these functions from within the maternal oviduct. Species with complex placentae have also been shown to rely substantially, or even primarily, on placental transport of organic nutrients for embryonic nutrition. However, it is unclear whether species with only simple placentae are also capable of transporting organic nutrients to offspring. Among viviparous squamates, all of the snakes that have been studied thus far have been shown to have simple placentae. However, most studies of snake placentation are limited to a single lineage, the North American Natricinae. We tested the abilities of four species of viviparous snakes - Agkistrodon contortrix (Viperidae), Boa constrictor (Boidae), Nerodia sipedon (Colubridae: Natricinae) and Thamnophis sirtalis (Colubridae: Natricinae) - to transport diet-derived amino acids to offspring during gestation. We fed [(15)N]leucine to pregnant snakes, and compared offspring (15)N content with that of unlabeled controls. Labeled females allocated significantly more (15)N to offspring than did controls, but (15)N allocation did not differ among species. Our results indicate that viviparous snakes are capable of transporting diet-derived amino acids to their offspring during gestation, possibly via placentation.

Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein.

PubMed

Telbisz, Ágnes; Özvegy-Laczka, Csilla; Hegedűs, Tamás; Váradi, András; Sarkadi, Balázs

2013-03-01

The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein. We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators of ABCG2 activity. Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cell membrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter.

A Functional-Phylogenetic Classification System for Transmembrane Solute Transporters

PubMed Central

Saier, Milton H.

2000-01-01

A comprehensive classification system for transmembrane molecular transporters has been developed and recently approved by the transport panel of the nomenclature committee of the International Union of Biochemistry and Molecular Biology. This system is based on (i) transporter class and subclass (mode of transport and energy coupling mechanism), (ii) protein phylogenetic family and subfamily, and (iii) substrate specificity. Almost all of the more than 250 identified families of transporters include members that function exclusively in transport. Channels (115 families), secondary active transporters (uniporters, symporters, and antiporters) (78 families), primary active transporters (23 families), group translocators (6 families), and transport proteins of ill-defined function or of unknown mechanism (51 families) constitute distinct categories. Transport mode and energy coupling prove to be relatively immutable characteristics and therefore provide primary bases for classification. Phylogenetic grouping reflects structure, function, mechanism, and often substrate specificity and therefore provides a reliable secondary basis for classification. Substrate specificity and polarity of transport prove to be more readily altered during evolutionary history and therefore provide a tertiary basis for classification. With very few exceptions, a phylogenetic family of transporters includes members that function by a single transport mode and energy coupling mechanism, although a variety of substrates may be transported, sometimes with either inwardly or outwardly directed polarity. In this review, I provide cross-referencing of well-characterized constituent transporters according to (i) transport mode, (ii) energy coupling mechanism, (iii) phylogenetic grouping, and (iv) substrates transported. The structural features and distribution of recognized family members throughout the living world are also evaluated. The tabulations should facilitate familial and functional

Novel metabolic and physiological functions of branched chain amino acids: a review.

PubMed

Zhang, Shihai; Zeng, Xiangfang; Ren, Man; Mao, Xiangbing; Qiao, Shiyan

2017-01-01

It is widely known that branched chain amino acids (BCAA) are not only elementary components for building muscle tissue but also participate in increasing protein synthesis in animals and humans. BCAA (isoleucine, leucine and valine) regulate many key signaling pathways, the most classic of which is the activation of the mTOR signaling pathway. This signaling pathway connects many diverse physiological and metabolic roles. Recent years have witnessed many striking developments in determining the novel functions of BCAA including: (1) Insufficient or excessive levels of BCAA in the diet enhances lipolysis. (2) BCAA, especially isoleucine, play a major role in enhancing glucose consumption and utilization by up-regulating intestinal and muscular glucose transporters. (3) Supplementation of leucine in the diet enhances meat quality in finishing pigs. (4) BCAA are beneficial for mammary health, milk quality and embryo growth. (5) BCAA enhance intestinal development, intestinal amino acid transportation and mucin production. (6) BCAA participate in up-regulating innate and adaptive immune responses. In addition, abnormally elevated BCAA levels in the blood (decreased BCAA catabolism) are a good biomarker for the early detection of obesity, diabetes and other metabolic diseases. This review will provide some insights into these novel metabolic and physiological functions of BCAA.

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

Regulation of Ion Transport in the Intestine by Free Fatty Acid Receptor 2 and 3: Possible Involvement of the Diffuse Chemosensory System

PubMed Central

Kuwahara, Atsukazu; Kuwahara, Yuko; Inui, Toshio; Marunaka, Yoshinori

2018-01-01

The diffuse chemosensory system (DCS) is well developed in the apparatuses of endodermal origin like gastrointestinal (GI) tract. The primary function of the GI tract is the extraction of nutrients from the diet. Therefore, the GI tract must possess an efficient surveillance system that continuously monitors the luminal contents for beneficial or harmful compounds. Recent studies have shown that specialized cells in the intestinal lining can sense changes in the luminal content. The chemosensory cells in the GI tract belong to the DCS which consists of enteroendocrine and related cells. These cells initiate various important local and remote reflexes. Although neural and hormonal involvements in ion transport in the GI tract are well documented, involvement of the DCS in the regulation of intestinal ion transport is much less understood. Since activation of luminal chemosensory receptors is a primary signal that elicits changes in intestinal ion transport and motility and failure of the system causes dysfunctions in host homeostasis, as well as functional GI disorders, study of the regulation of GI function by the DCS has become increasingly important. This review discusses the role of the DCS in epithelial ion transport, with particular emphasis on the involvement of free fatty acid receptor 2 (FFA2) and free fatty acid receptor 3 (FFA3). PMID:29510573

Immunohistochemical localization of fatty acid transporters and MCT1 in the sebaceous glands of mouse skin.

PubMed

Zheng, Miao; Lee, Shinhye; Tsuzuki, Satoshi; Inoue, Kazuo; Masuda, Daisaku; Yamashita, Shizuya; Iwanaga, Toshihiko

2016-01-01

The sebaceous glands secrete sebum to protect the epidermis and hairs by the oily products. The glands express several transporters and binding proteins for the production of fatty acids and uptake of their sources. The present immunohistochemical study examined the expression and localization of CD36, MCT1, FATP4, and E-FABP in the sebaceous glands, including the meibomian and preputial glands of mice. CD36 and MCT1 in sebaceous glands were largely co-localized along the plasma membrane of secretory cells, while they were separately expressed in the glandular portion of meibomian and preputial glands. Immunoreactivities for FATP4 and E-FABP appeared diffusely in the cytoplasm of secretory cells. Genetic deletion of CD36 did not affect the immunolocalization of the three other molecules. The sebaceous glands were judged to be useful for analyzing the functions and relation of fatty acid transporters and binding proteins.

Plasma membrane transporters for arginine.

PubMed

Closs, Ellen I; Simon, Alexandra; Vékony, Nicole; Rotmann, Alexander

2004-10-01

The supply of arginine may become rate limiting for enzymatic reactions that use this semiessential amino acid as a substrate (e.g., nitric oxide, agmatine, creatine, and urea synthesis), particularly under conditions of high demand such as growth, sepsis, or wound healing. In addition, arginine acts as a signaling molecule that regulates essential cellular functions such as protein synthesis, apoptosis, and growth. In the past decade, a number of carrier proteins for amino acids have been identified on the molecular level. They belong to different gene families, exhibit overlapping but distinctive substrate specificities, and can further be distinguished by their requirement for the cotransport or countertransport of inorganic ions. A number of these transporters function as exchangers rather than uniporters. Uptake of amino acids by these transporters therefore depends largely on the intracellular substrate composition. Hence, there is a complex crosstalk between transporters for cationic and neutral amino acids as well as for peptides. This article briefly reviews current knowledge regarding mammalian plasma membrane transporters that accept arginine as a substrate.

Acid monolayer functionalized iron oxide nanoparticle catalysts

NASA Astrophysics Data System (ADS)

Ikenberry, Myles

Superparamagnetic iron oxide nanoparticle functionalization is an area of intensely active research, with applications across disciplines such as biomedical science and heterogeneous catalysis. This work demonstrates the functionalization of iron oxide nanoparticles with a quasi-monolayer of 11-sulfoundecanoic acid, 10-phosphono-1-decanesulfonic acid, and 11-aminoundecanoic acid. The carboxylic and phosphonic moieties form bonds to the iron oxide particle core, while the sulfonic acid groups face outward where they are available for catalysis. The particles were characterized by thermogravimetric analysis (TGA), transmission electron microscopy (TEM), potentiometric titration, diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), inductively coupled plasma optical emission spectrometry (ICP-OES), X-ray photoelectron spectrometry (XPS), and dynamic light scattering (DLS). The sulfonic acid functionalized particles were used to catalyze the hydrolysis of sucrose at 80° and starch at 130°, showing a higher activity per acid site than the traditional solid acid catalyst Amberlyst-15, and comparing well against results reported in the literature for sulfonic acid functionalized mesoporous silicas. In sucrose catalysis reactions, the phosphonic-sulfonic nanoparticles (PSNPs) were seen to be incompletely recovered by an external magnetic field, while the carboxylic-sulfonic nanoparticles (CSNPs) showed a trend of increasing activity over the first four recycle runs. Between the two sulfonic ligands, the phosphonates produced a more tightly packed monolayer, which corresponded to a higher sulfonic acid loading, lower agglomeration, lower recoverability through application of an external magnetic field, and higher activity per acid site for the hydrolysis of starch. Functionalizations with 11-aminoundecanoic acid resulted in some amine groups binding to the surfaces of iron oxide nanoparticles. This amine binding is commonly ignored in iron oxide

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

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.

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters*

PubMed Central

Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky

2015-01-01

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393

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.

Ascorbic acid, cognitive function, and Alzheimer’s disease: a current review and future direction

PubMed Central

Bowman, Gene L.

2013-01-01

This narrative review appraises the human and animal studies implicating ascorbic acid (AA) in normal cognitive function and Alzheimer’s disease. A research framework for how nutrition affects brain aging is proposed with emphasis on AA intake, status, metabolism, and transport into brain tissue. A final synopsis highlights areas for future research regarding AA nourishment and healthy brain aging. PMID:22419527

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.

Specific analogues uncouple transport, signalling, oligo-ubiquitination and endocytosis in the yeast Gap1 amino acid transceptor

PubMed Central

Van Zeebroeck, Griet; Rubio-Texeira, Marta; Schothorst, Joep; Thevelein, Johan M

2014-01-01

The Saccharomyces cerevisiae amino acid transceptor Gap1 functions as receptor for signalling to the PKA pathway and concomitantly undergoes substrate-induced oligo-ubiquitination and endocytosis. We have identified specific amino acids and analogues that uncouple to certain extent signalling, transport, oligo-ubiquitination and endocytosis. l-lysine, l-histidine and l-tryptophan are transported by Gap1 but do not trigger signalling. Unlike l-histidine, l-lysine triggers Gap1 oligo-ubiquitination without substantial induction of endocytosis. Two transported, non-metabolizable signalling agonists, β-alanine and d-histidine, are strong and weak inducers of Gap1 endocytosis, respectively, but both causing Gap1 oligo-ubiquitination. The non-signalling agonist, non-transported competitive inhibitor of Gap1 transport, l-Asp-γ-l-Phe, induces oligo-ubiquitination but no discernible endocytosis. The Km of l-citrulline transport is much lower than the threshold concentration for signalling and endocytosis. These results show that molecules can be transported without triggering signalling or substantial endocytosis, and that oligo-ubiquitination and endocytosis do not require signalling nor metabolism. Oligo-ubiquitination is required, but apparently not sufficient to trigger endocytosis. In addition, we demonstrate intracellular cross-induction of endocytosis of transport-defective Gap1Y395C by ubiquitination- and endocytosis-deficient Gap1K9R,K16R. Our results support the concept that different substrates bind to partially overlapping binding sites in the same general substrate-binding pocket of Gap1, triggering divergent conformations, resulting in different conformation-induced downstream processes. PMID:24852066

Acid-functionalized polyolefin materials and their use in acid-promoted chemical reactions

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

Oyola, Yatsandra; Tian, Chengcheng; Bauer, John Christopher

An acid-functionalized polyolefin material that can be used as an acid catalyst in a wide range of acid-promoted chemical reactions, wherein the acid-functionalized polyolefin material includes a polyolefin backbone on which acid groups are appended. Also described is a method for the preparation of the acid catalyst in which a precursor polyolefin is subjected to ionizing radiation (e.g., electron beam irradiation) of sufficient power and the irradiated precursor polyolefin reacted with at least one vinyl monomer having an acid group thereon. Further described is a method for conducting an acid-promoted chemical reaction, wherein an acid-reactive organic precursor is contacted inmore » liquid form with a solid heterogeneous acid catalyst comprising a polyolefin backbone of at least 1 micron in one dimension and having carboxylic acid groups and either sulfonic acid or phosphoric acid groups appended thereto.« less

Air swallowing, belching, acid and non-acid reflux in patients with functional dyspepsia.

PubMed

Conchillo, J M; Selimah, M; Bredenoord, A J; Samsom, M; Smout, A J P M

2007-04-15

Frequent belching is a common symptom in patients with functional dyspepsia with a reported incidence up to 80%. We hypothesized that patients with functional dyspepsia possibly have a higher frequency of belching than healthy subjects secondary to frequent air swallowing. To assess air swallowing, belching, acid and non-acid reflux patterns of patients with functional dyspepsia. Combined 24-h oesophageal impedance and pH monitoring was performed in 10 functional dyspepsia patients and 10 controls. Analysis of the impedance-pH signals included incidence of air swallows, belching, acid and non-acid reflux. The incidence of air swallows in functional dyspepsia patients were significantly higher compared with controls (153 +/- 15 vs. 79 +/- 10, P < 0.001), while the incidence of liquid-only swallows were not significantly increased. The proportions of gas-containing reflux episodes (belches) and non-acid reflux episodes in functional dyspepsia patients were significantly higher when compared with controls (66.4 vs. 44.4%, P = 0.04 and 70.1 vs. 45.9%, P = 0.009, respectively). Patients with functional dyspepsia swallow air more frequently than controls and this is associated with an increased incidence of non-acid gaseous gastro-oesophageal reflux.

Amino Acid Transport Associated to Cluster of Differentiation 98 Heavy Chain (CD98hc) Is at the Cross-road of Oxidative Stress and Amino Acid Availability.

PubMed

de la Ballina, Laura R; Cano-Crespo, Sara; González-Muñoz, Elena; Bial, Susanna; Estrach, Soline; Cailleteau, Laurence; Tissot, Floriane; Daniel, Hannelore; Zorzano, Antonio; Ginsberg, Mark H; Palacín, Manuel; Féral, Chloé C

2016-04-29

CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with β-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

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

PubMed Central

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

2008-01-01

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

Pharmacology of Glutamate Transport in the CNS: Substrates and Inhibitors of Excitatory Amino Acid Transporters (EAATs) and the Glutamate/Cystine Exchanger System x c -

NASA Astrophysics Data System (ADS)

Bridges, Richard J.; Patel, Sarjubhai A.

As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamate participates not only in standard fast synaptic communication, but also contributes to higher order signal processing, as well as neuropathology. Given this variety of functional roles, interest has been growing as to how the extracellular concentrations of l-glutamate surrounding neurons are regulated by cellular transporter proteins. This review focuses on two prominent systems, each of which appears capable of influencing both the signaling and pathological actions of l-glutamate within the CNS: the sodium-dependent excitatory amino acid transporters (EAATs) and the glutamate/cystine exchanger, system x c - (Sx c -). While the family of EAAT subtypes limit access to glutamate receptors by rapidly and efficiently sequestering l-glutamate in neurons and glia, Sxc - provides a route for the export of glutamate from cells into the extracellular environment. The primary intent of this work is to provide an overview of the inhibitors and substrates that have been developed to delineate the pharmacological specificity of these transport systems, as well as be exploited as probes with which to selectively investigate function. Particular attention is paid to the development of small molecule templates that mimic the structural properties of the endogenous substrates, l-glutamate, l-aspartate and l-cystine and how strategic control of functional group position and/or the introduction of lipophilic R-groups can impact multiple aspects of the transport process, including: subtype selectivity, inhibitory potency, and substrate activity.

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.

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

Identification of a Disulfide Bridge in Sodium-Coupled Neutral Amino Acid Transporter 2(SNAT2) by Chemical Modification.

PubMed

Chen, Chen; Wang, Jiahong; Cai, Ruiping; Yuan, Yanmeng; Guo, Zhanyun; Grewer, Christof; Zhang, Zhou

2016-01-01

Sodium-coupled neutral amino acid transporter 2 (SNAT2) belongs to solute carrier 38 (SLC38) family of transporters, which is ubiquitously expressed in mammalian tissues and mediates transport of small, neutral amino acids, exemplified by alanine(Ala, A). Yet structural data on SNAT2, including the relevance of intrinsic cysteine residues on structure and function, is scarce, in spite of its essential roles in many tissues. To better define the potential of intrinsic cysteines to form disulfide bonds in SNAT2, mutagenesis experiments and thiol-specific chemical modifications by N-ethylmaleimide (NEM) and methoxy-polyethylene glycol maleimide (mPEG-Mal, MW 5000) were performed, with or without the reducing regent dithiothreitol (DTT) treatment. Seven single mutant transporters with various cysteine (Cys, C) to alanine (Ala, A) substitutions, and a C245,279A double mutant were introduced to SNAT2 with a hemagglutinin (HA) tag at the C-terminus. The results showed that the cells expressing C245A or C279A were labeled by one equivalent of mPEG-Mal in the presence of DTT, while wild-type or all the other single Cys to Ala mutants were modified by two equivalents of mPEG-Mal. Furthermore, the molecular weight of C245,279A was not changed in the presence or absence of DTT treatment. The results suggest a disulfide bond between Cys245 and Cys279 in SNAT2 which has no effect on cell surface trafficking, as well as transporter function. The proposed disulfide bond may be important to delineate proximity in the extracellular domain of SNAT2 and related proteins.

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.

Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells.

PubMed

Mabashi-Asazuma, Hideaki; Shi, Xianzong; Geisler, Christoph; Kuo, Chu-Wei; Khoo, Kay-Hooi; Jarvis, Donald L

2013-02-01

Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N-acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N-acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

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.

Ursodeoxycholic acid attenuates colonic epithelial secretory function

PubMed Central

Kelly, Orlaith B; Mroz, Magdalena S; Ward, Joseph B J; Colliva, Carolina; Scharl, Michael; Pellicciari, Roberto; Gilmer, John F; Fallon, Padraic G; Hofmann, Alan F; Roda, Aldo; Murray, Frank E; Keely, Stephen J

2013-01-01

Dihydroxy bile acids, such as chenodeoxycholic acid (CDCA), are well known to promote colonic fluid and electrolyte secretion, thereby causing diarrhoea associated with bile acid malabsorption. However, CDCA is rapidly metabolised by colonic bacteria to ursodeoxycholic acid (UDCA), the effects of which on epithelial transport are poorly characterised. Here, we investigated the role of UDCA in the regulation of colonic epithelial secretion. Cl− secretion was measured across voltage-clamped monolayers of T84 cells and muscle-stripped sections of mouse or human colon. Cell surface biotinylation was used to assess abundance/surface expression of transport proteins. Acute (15 min) treatment of T84 cells with bilateral UDCA attenuated Cl− secretory responses to the Ca2+ and cAMP-dependent secretagogues carbachol (CCh) and forskolin (FSK) to 14.0 ± 3.8 and 40.2 ± 7.4% of controls, respectively (n= 18, P < 0.001). Investigation of the molecular targets involved revealed that UDCA acts by inhibiting Na+/K+-ATPase activity and basolateral K+ channel currents, without altering their cell surface expression. In contrast, intraperitoneal administration of UDCA (25 mg kg−1) to mice enhanced agonist-induced colonic secretory responses, an effect we hypothesised to be due to bacterial metabolism of UDCA to lithocholic acid (LCA). Accordingly, LCA (50–200 μm) enhanced agonist-induced secretory responses in vitro and a metabolically stable UDCA analogue, 6α-methyl-UDCA, exerted anti-secretory actions in vitro and in vivo. In conclusion, UDCA exerts direct anti-secretory actions on colonic epithelial cells and metabolically stable derivatives of the bile acid may offer a new approach for treating intestinal diseases associated with diarrhoea. PMID:23507881

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.

Multiple functions of neuronal plasma membrane neurotransmitter transporters.

PubMed

Raiteri, Luca; Raiteri, Maurizio

2015-11-01

Removal from receptors of neurotransmitters just released into synapses is one of the major steps in neurotransmission. Transporters situated on the plasma membrane of nerve endings and glial cells perform the process of neurotransmitter (re)uptake. Because the density of transporters in the membranes can fluctuate, transporters can determine the transmitter concentrations at receptors, thus modulating indirectly the excitability of neighboring neurons. Evidence is accumulating that neurotransmitter transporters can exhibit multiple functions. Being bidirectional, neurotransmitter transporters can mediate transmitter release by working in reverse, most often under pathological conditions that cause ionic gradient dysregulations. Some transporters reverse to release transmitters, like dopamine or serotonin, when activated by 'indirectly acting' substrates, like the amphetamines. Some transporters exhibit as one major function the ability to capture transmitters into nerve terminals that perform insufficient synthesis. Transporter activation can generate conductances that regulate directly neuronal excitability. Synaptic and non-synaptic transporters play different roles. Cytosolic Na(+) elevations accompanying transport can interact with plasmalemmal or/and mitochondrial Na(+)/Ca(2+) exchangers thus generating calcium signals. Finally, neurotransmitter transporters can behave as receptors mediating releasing stimuli able to cause transmitter efflux through multiple mechanisms. Neurotransmitter transporters are therefore likely to play hitherto unknown roles in multiple therapeutic treatments. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

KCl stimulation increases norepinephrine transporter function in PC12 cells.

PubMed

Mandela, Prashant; Ordway, Gregory A

2006-09-01

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

Ion Transport Function of SLC4A11 in Corneal Endothelium

PubMed Central

Jalimarada, Supriya S.; Ogando, Diego G.; Vithana, Eranga N.; Bonanno, Joseph A.

2013-01-01

Purpose. Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na+ borate and Na+:OH− transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]4−), bicarbonate (HCO3−), or hydroxyl (OH−) anions coupled to Na+. Methods. SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na+ ([Na+]i) in response to Ringer solutions with/without B(OH)4− or HCO3− after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11. Results. SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)4− (2.5–10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05–0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)4−. However, the rate of B(OH)4−-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)4− did not induce significant changes in resting [Na+i] or the amplitude and rate of acidification caused by Na+ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO2/HCO3−-rich Ringer, Na+-free induced acidification, or the rate of Na+ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na+ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH4+ prepulse was decreased significantly (27%) by SLC4A11 silencing. Conclusions. In corneal endothelium, SLC4A11 displays robust Na+-coupled OH− transport, but does not transport B(OH)4− or HCO3−. PMID:23745003

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.

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

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.

Human Erythrocyte Glucose Transporter: Normal Asymmetric Orientation and Function in Liposomes

NASA Astrophysics Data System (ADS)

Chen, Chia-Chen; Kurokawa, Tomonori; Shaw, Shyh-Yu; Tillotson, Loyal G.; Kalled, Susan; Isselbacher, Kurt J.

1986-04-01

The transport function and orientation of the reconstituted human erythrocyte glucose transporter was studied with liposomes made with bovine brain lipid or Escherichia coli lipid. Reconstitution was achieved by a simple octyl glucoside dilution method. The reconstituted transporters with either lipid showed identical counterflow transport activity, the same response to various inhibitors, and characteristic cytochalasin B (CB) labeling. Functional location and purification of the glucose transporter was performed by using gel-permeation high-performance liquid chromatography with octyl glucoside-containing buffer. The reconstituted transport activity was associated only with band 4.5 protein (preactin) and not with band 3 protein. Both CB binding and transport function of the reconstituted transporters were resistant to trypsin but susceptible to chymotrypsin digestion. However, both trypsin and chymotrypsin treatment of unsealed ghosts completely eliminated the CB labeling and transport function of the glucose transporter. In our reconstitution system the glucose transporters maintained a normal asymmetrical (rightside-out) orientation and good transport function. A specific monoclonal antibody against the glucose transporter inhibited CB labeling of the transporters on unsealed ghosts. This was not found with the reconstituted system; however, after freeze-thawing there was a significant inhibition of CB binding by the antibody. These findings suggest that the CB-binding site of the reconstituted transporter is on the inner side of the proteoliposomes.

Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

PubMed

Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

2017-05-01

The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple functions of the crustacean gill: osmotic/ionic regulation, acid-base balance, ammonia excretion, and bioaccumulation of toxic metals

PubMed Central

Henry, Raymond P.; Lucu, Čedomil; Onken, Horst; Weihrauch, Dirk

2012-01-01

The crustacean gill is a multi-functional organ, and it is the site of a number of physiological processes, including ion transport, which is the basis for hemolymph osmoregulation; acid-base balance; and ammonia excretion. The gill is also the site by which many toxic metals are taken up by aquatic crustaceans, and thus it plays an important role in the toxicology of these species. This review provides a comprehensive overview of the ecology, physiology, biochemistry, and molecular biology of the mechanisms of osmotic and ionic regulation performed by the gill. The current concepts of the mechanisms of ion transport, the structural, biochemical, and molecular bases of systemic physiology, and the history of their development are discussed. The relationship between branchial ion transport and hemolymph acid-base regulation is also treated. In addition, the mechanisms of ammonia transport and excretion across the gill are discussed. And finally, the toxicology of heavy metal accumulation via the gill is reviewed in detail. PMID:23162474

Structural basis of intramitochondrial phosphatidic acid transport mediated by Ups1-Mdm35 complex.

PubMed

Yu, Fang; He, Fangyuan; Yao, Hongyan; Wang, Chengyuan; Wang, Jianchuan; Li, Jianxu; Qi, Xiaofeng; Xue, Hongwei; Ding, Jianping; Zhang, Peng

2015-07-01

Ups1 forms a complex with Mdm35 and is critical for the transport of phosphatidic acid (PA) from the mitochondrial outer membrane to the inner membrane. We report the crystal structure of the Ups1-Mdm35-PA complex and the functional characterization of Ups1-Mdm35 in PA binding and transfer. Ups1 features a barrel-like structure consisting of an antiparallel β-sheet and three α-helices. Mdm35 adopts a three-helical clamp-like structure to wrap around Ups1 to form a stable complex. The β-sheet and α-helices of Ups1 form a long tunnel-like pocket to accommodate the substrate PA, and a short helix α2 acts as a lid to cover the pocket. The hydrophobic residues lining the pocket and helix α2 are critical for PA binding and transfer. In addition, a hydrophilic patch on the surface of Ups1 near the PA phosphate-binding site also plays an important role in the function of Ups1-Mdm35. Our study reveals the molecular basis of the function of Ups1-Mdm35 and sheds new light on the mechanism of intramitochondrial phospholipid transport by the MSF1/PRELI family proteins. © 2015 The Authors.

Monoterpene glycoside ESK246 from Pittosporum targets LAT3 amino acid transport and prostate cancer cell growth.

PubMed

Wang, Qian; Grkovic, Tanja; Font, Josep; Bonham, Sarah; Pouwer, Rebecca H; Bailey, Charles G; Moran, Anne M; Ryan, Renae M; Rasko, John E J; Jormakka, Mika; Quinn, Ronald J; Holst, Jeff

2014-06-20

The L-type amino acid transporter (LAT) family consists of four members (LAT1-4) that mediate uptake of neutral amino acids including leucine. Leucine is not only important as a building block for proteins, but plays a critical role in mTORC1 signaling leading to protein translation. As such, LAT family members are commonly upregulated in cancer in order to fuel increased protein translation and cell growth. To identify potential LAT-specific inhibitors, we established a function-based high-throughput screen using a prefractionated natural product library. We identified and purified two novel monoterpene glycosides, ESK242 and ESK246, sourced from a Queensland collection of the plant Pittosporum venulosum. Using Xenopus laevis oocytes expressing individual LAT family members, we demonstrated that ESK246 preferentially inhibits leucine transport via LAT3, while ESK242 inhibits both LAT1 and LAT3. We further show in LNCaP prostate cancer cells that ESK246 is a potent (IC50 = 8.12 μM) inhibitor of leucine uptake, leading to reduced mTORC1 signaling, cell cycle protein expression and cell proliferation. Our study suggests that ESK246 is a LAT3 inhibitor that can be used to study LAT3 function and upon which new antiprostate cancer therapies may be based.

Monoterpene Glycoside ESK246 from Pittosporum Targets LAT3 Amino Acid Transport and Prostate Cancer Cell Growth

PubMed Central

2014-01-01

The l-type amino acid transporter (LAT) family consists of four members (LAT1–4) that mediate uptake of neutral amino acids including leucine. Leucine is not only important as a building block for proteins, but plays a critical role in mTORC1 signaling leading to protein translation. As such, LAT family members are commonly upregulated in cancer in order to fuel increased protein translation and cell growth. To identify potential LAT-specific inhibitors, we established a function-based high-throughput screen using a prefractionated natural product library. We identified and purified two novel monoterpene glycosides, ESK242 and ESK246, sourced from a Queensland collection of the plant Pittosporum venulosum. Using Xenopus laevis oocytes expressing individual LAT family members, we demonstrated that ESK246 preferentially inhibits leucine transport via LAT3, while ESK242 inhibits both LAT1 and LAT3. We further show in LNCaP prostate cancer cells that ESK246 is a potent (IC50 = 8.12 μM) inhibitor of leucine uptake, leading to reduced mTORC1 signaling, cell cycle protein expression and cell proliferation. Our study suggests that ESK246 is a LAT3 inhibitor that can be used to study LAT3 function and upon which new antiprostate cancer therapies may be based. PMID:24762008

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.

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

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.

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

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.

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.

Functional Analysis of Arabidopsis Sucrose Transporters

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

John M. Ward

2009-03-31

Sucrose is the main photosynthetic product that is transported in the vasculature of plants. The long-distance transport of carbohydrates is required to support the growth and development of net-importing (sink) tissues such as fruit, seeds and roots. This project is focused on understanding the transport mechanism sucrose transporters (SUTs). These are proton-coupled sucrose uptake transporters (membrane proteins) that are required for transport of sucrose in the vasculature and uptake into sink tissues. The accomplishments of this project included: 1) the first analysis of substrate specificity for any SUT. This was accomplished using electrophysiology to analyze AtSUC2, a sucrose transporter frommore » companion cells in Arabidopsis. 2) the first analysis of the transport activity for a monocot SUT. The transport kinetics and substrate specificity of HvSUT1 from barley were studied. 3) the first analysis of a sucrose transporter from sugarcane. and 4) the first analysis of transport activity of a sugar alcohol transporter homolog from plants, AtPLT5. During this period four primary research papers, funded directly by the project, were published in refereed journals. The characterization of several sucrose transporters was essential for the current effort in the analysis of structure/function for this gene family. In particular, the demonstration of strong differences in substrate specificity between type I and II SUTs was important to identify targets for site-directed mutagenesis.« less

A structure-function analysis of ion transport in crustacean gills and excretory organs.

PubMed

Freire, Carolina A; Onken, Horst; McNamara, John C

2008-11-01

Osmotic and ionic regulation in the Crustacea is mostly accomplished by the multifunctional gills, together with the excretory organs. In addition to their role in gas exchange, the gills constitute organs of active, transepithelial, ion transport, an activity of major importance that underlies many essential physiological functions like osmoregulation, calcium homeostasis, ammonium excretion and extracellular pH regulation. This review focuses on structure-function relationships in crustacean gills and excretory effectors, from the organ to molecular levels of organization. We address the diversity of structural architectures encountered in different crustacean gill types, and in constituent cell types, before examining the physiological mechanisms of Na(+), Cl(-), Ca(2+) and NH(4)(+) transport, and of acid-base equivalents, based on findings obtained over the last two decades employing advanced techniques. The antennal and maxillary glands constitute the principal crustacean excretory organs, which have received less attention in functional studies. We examine the diversity present in antennal and maxillary gland architecture, highlighting the structural similarities between both organ types, and we analyze the functions ascribed to each glandular segment. Emphasis is given to volume and osmoregulatory functions, capacity to produce dilute urine in freshwater crustaceans, and the effect of acclimation salinity on urine volume and composition. The microanatomy and diversity of function ascribed to gills and excretory organs are appraised from an evolutionary perspective, and suggestions made as to future avenues of investigation that may elucidate evolutionary and adaptive trends underpinning the invasion and exploitation of novel habitats.

The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.

PubMed

Shang, Peng; Valapala, Mallika; Grebe, Rhonda; Hose, Stacey; Ghosh, Sayan; Bhutto, Imran A; Handa, James T; Lutty, Gerard A; Lu, Lixia; Wan, Jun; Qian, Jiang; Sergeev, Yuri; Puertollano, Rosa; Zigler, J Samuel; Xu, Guo-Tong; Sinha, Debasish

2017-04-01

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H + -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding βA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter.

PubMed

Cater, Rosemary J; Ryan, Renae M; Vandenberg, Robert J

2016-03-01

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl(-)) channels. The EAATs couple the transport of glutamate to the co-transport of three Na(+) ions and one H(+) ion into the cell, and the counter-transport of one K(+) ion out of the cell. The EAAT Cl(-) channel is activated by the binding of glutamate and Na(+), but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, GltPh, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl(-) permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl(-) permeation and the mechanisms that underlie their split personality.

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.

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

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J

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

PubMed

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

2018-02-01

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

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

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.

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.

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

Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1

PubMed Central

Gui, Chunshan; Hagenbuch, Bruno

2009-01-01

The liver-specific organic anion transporting polypeptides OATP1B1 and OATP1B3 are highly homologous and share numerous substrates. However, at low concentrations OATP1B1 shows substrate selectivity for estrone-3-sulfate. In this study, we investigated the molecular mechanism for this substrate selectivity of OATP1B1 by constructing OATP1B1/1B3 chimeric transporters and by site-directed mutagenesis. Functional studies of chimeras showed that transmembrane domain 10 is critical for the function of OATP1B1. We further identified four amino acid residues, namely L545, F546, L550, and S554 in TM10, whose simultaneous mutation caused almost complete loss of OATP1B1-mediated estrone-3-sulfate transport. Comparison of the kinetics of estrone-3-sulfate transport confirmed a biphasic pattern for OATP1B1, but showed a monophasic pattern for the quadruple mutant L545S/F546L/L550T/S554T. This mutant also showed reduced transport for other OATP1B1 substrates such as bromosulfophthalein and [d-penicillamine2,5]enkephalin. Helical wheel analysis and molecular modeling suggest that L545 is facing the substrate translocation pathway, whereas F546, L550, and S554 are located inside the protein. These results indicate that L545 might contribute to OATP1B1 function by interacting with substrates, whereas F546, L550, and S554 seem important for protein structure. In conclusion, our results show that TM10 is critical for the function of OATP1B1. PMID:19760661

Structural and functional interaction of fatty acids with human liver fatty acid-binding protein (L-FABP) T94A variant.

PubMed

Huang, Huan; McIntosh, Avery L; Martin, Gregory G; Landrock, Kerstin K; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2014-05-01

The human liver fatty acid-binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride levels. How this amino acid substitution elicits these effects is not known. This issue was addressed using human recombinant wild-type (WT) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC and CC). The T94A substitution did not alter or only slightly altered L-FABP binding affinities for saturated, monounsaturated or polyunsaturated long chain fatty acids, nor did it change the affinity for intermediates of triglyceride synthesis. Nevertheless, the T94A substitution markedly altered the secondary structural response of L-FABP induced by binding long chain fatty acids or intermediates of triglyceride synthesis. Finally, the T94A substitution markedly decreased the levels of induction of peroxisome proliferator-activated receptor α-regulated proteins such as L-FABP, fatty acid transport protein 5 and peroxisome proliferator-activated receptor α itself meditated by the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in cultured primary human hepatocytes. Thus, although the T94A substitution did not alter the affinity of human L-FABP for long chain fatty acids, it significantly altered human L-FABP structure and stability, as well as the conformational and functional response to these ligands. © 2014 FEBS.

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

STRUCTURAL AND FUNCTIONAL INTERACTION OF FATTY ACIDS WITH HUMAN LIVER FATTY ACID BINDING PROTEIN (L-FABP) T94A VARIANT

PubMed Central

Huang, Huan; McIntosh, Avery L.; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Gupta, Shipra; Atshaves, Barbara P.; Kier, Ann B.; Schroeder, Friedhelm

2014-01-01

The human liver fatty acid binding protein (L-FABP) T94A variant, the most common in the FABP family, has been associated with elevated liver triglyceride (TG) levels. How this amino acid substitution elicits these effects is not known. This issue was addressed with human recombinant wild-type (WT, T94T) and T94A variant L-FABP proteins as well as cultured primary human hepatocytes expressing the respective proteins (genotyped as TT, TC, and CC). T94A substitution did not or only slightly alter L-FABP binding affinities for saturated, monounsaturated, or polyunsaturated long chain fatty acids (LCFA), nor did it change the affinity for intermediates in TG synthesis. Nevertheless, T94A substitution markedly altered the secondary structural response of L-FABP induced by binding LCFA or intermediates of TG synthesis. Finally, T94A substitution markedly diminished polyunsaturated fatty acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), induction of peroxisome proliferator-activated receptor alpha (PPARα) - regulated proteins such as L-FABP, fatty acid transport protein 5 (FATP5), and PPARα itself in cultured primary human hepatocytes. Thus, while T94A substitution did not alter the affinity of human L-FABP for LCFAs, it significantly altered human L-FABP structure and stability as well as conformational and functional response to these ligands. PMID:24628888

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

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids.

PubMed

Tan, Kemin; Chang, Changsoo; Cuff, Marianne; Osipiuk, Jerzy; Landorf, Elizabeth; Mack, Jamey C; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R

2013-10-01

Lignin comprises 15-25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP-binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute-binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins. Copyright © 2013 Wiley Periodicals, Inc.

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids

PubMed Central

Tan, Kemin; Chang, Changsoo; Cuff, Marianne; Osipiuk, Jerzy; Landorf, Elizabeth; Mack, Jamey C.; Zerbs, Sarah; Joachimiak, Andrzej; Collart, Frank R.

2013-01-01

Lignin comprises 15.25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP.binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute.binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins. PMID:23606130

Expression, Purification, and Structural Insights for the Human Uric Acid Transporter, GLUT9, Using the Xenopus laevis Oocytes System

PubMed Central

Clémençon, Benjamin; Lüscher, Benjamin P.; Fine, Michael; Baumann, Marc U.; Surbek, Daniel V.; Bonny, Olivier; Hediger, Matthias A.

2014-01-01

The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies. PMID:25286413

Study of electron transport in the functionalized nanotubes and their impact on the electron transfer in the active site of horseradish peroxidase

NASA Astrophysics Data System (ADS)

Feizabadi, Mina; Ajloo, Davood; Soleymanpour, Ahmad; Faridnouri, Hassan

2018-05-01

Electrochemical characterization of functionalized carbon nanotubes (f-CNT) including carboxyl (CNT-COOH), amine (CNT-NH2) and hydroxyl (CNT-OH) functional groups were studied using differential pulse voltammetry (DPV). The current-voltage (I-V) curves were obtained from each system and the effect of f-CNT on redox interaction of horseradish peroxidase (HRP) immobilized on the electrode surface was investigated. The non-equilibrium Green's function (NEGF) combined with density functional theory (DFT) were used to study the transport properties of f-CNT. Additionally, the effect of the number of functional groups on transport properties of CNT, I-V characteristics, electronic transmission coefficients and spatial distribution of f-CNTs have been calculated and analyzed. The results showed that the carboxyl derivative has larger transmission coefficients and current value than other f-CNTs. Then, the effect of functional groups on the electron transport in heme group of HRP is discussed. Finally, the effect of a covalent bond between active site amino acids and amine functional group of CNT was investigated and discussed.

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

Polymers functionalized with bronsted acid groups

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

Van Humbeck, Jeffrey; Long, Jeffrey R.; McDonald, Thomas M.

Porous aromatic framework polymers functionalized with Bronsted acid moieties are prepared by polymerization of a three-dimensional organic aryl or heteroaryl monomer and its copolymerization with a second aryl or heteroaryl monomer functionalized with one or more Bronsted acid moiety. The polymers are characterized by a stable three-dimensional structure, which, in exemplary embodiments, includes interpenetrating subunits within one or more domain of the bulk polymer structure. The polymers are of use in methods of adsorbing ammonia and amines and in devices and systems configured for this purpose.

High-Resolution NMR Reveals Secondary Structure and Folding of Amino Acid Transporter from Outer Chloroplast Membrane

PubMed Central

Zook, James D.; Molugu, Trivikram R.; Jacobsen, Neil E.; Lin, Guangxin; Soll, Jürgen; Cherry, Brian R.; Brown, Michael F.; Fromme, Petra

2013-01-01

Solving high-resolution structures for membrane proteins continues to be a daunting challenge in the structural biology community. In this study we report our high-resolution NMR results for a transmembrane protein, outer envelope protein of molar mass 16 kDa (OEP16), an amino acid transporter from the outer membrane of chloroplasts. Three-dimensional, high-resolution NMR experiments on the 13C, 15N, 2H-triply-labeled protein were used to assign protein backbone resonances and to obtain secondary structure information. The results yield over 95% assignment of N, HN, CO, Cα, and Cβ chemical shifts, which is essential for obtaining a high resolution structure from NMR data. Chemical shift analysis from the assignment data reveals experimental evidence for the first time on the location of the secondary structure elements on a per residue basis. In addition T 1Z and T2 relaxation experiments were performed in order to better understand the protein dynamics. Arginine titration experiments yield an insight into the amino acid residues responsible for protein transporter function. The results provide the necessary basis for high-resolution structural determination of this important plant membrane protein. PMID:24205117

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.

Biological response to purification and acid functionalization of carbon nanotubes

NASA Astrophysics Data System (ADS)

Figarol, Agathe; Pourchez, Jérémie; Boudard, Delphine; Forest, Valérie; Tulliani, Jean-Marc; Lecompte, Jean-Pierre; Cottier, Michèle; Bernache-Assollant, Didier; Grosseau, Philippe

2014-07-01

Acid functionalization has been considered as an easy way to enhance the dispersion and biodegradation of carbon nanotubes (CNT). However, inconsistencies between toxicity studies of acid functionalized CNT remain unexplained. This could be due to a joint effect of the main physicochemical modifications resulting from an acid functionalization: addition of surface acid groups and purification from catalytic metallic impurities. In this study, the impact on CNT biotoxicity of these two physiochemical features was assessed separately. The in vitro biological response of RAW 264.7 macrophages was evaluated after exposure to 15-240 µg mL-1 of two types of multi-walled CNT. For each type of CNT (small: 20 nm diameter, and big: 90 nm diameter), three different surface chemical properties were studied (total of six CNT samples): pristine, acid functionalized and desorbed. Desorbed CNT were purified by the acid functionalization but presented a very low amount of surface acid groups due to a thermal treatment under vacuum. A Janus effect of acid functionalization with two opposite impacts is highlighted. The CNT purification decreased the overall toxicity, while the surface acid groups intensified it when present at a specific threshold. These acid groups especially amplified the pro-inflammatory response. The threshold mechanism which seemed to regulate the impact of acid groups should be further studied to determine its value and potential link to the other physicochemical state of the CNT. The results suggest that, for a safer-design approach, the benefit-risk balance of an acid functionalization has to be considered, depending on the CNT primary state of purification. Further research should be conducted in this direction.

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.

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

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.

Dietary fatty acids and membrane protein function.

PubMed

Murphy, M G

1990-02-01

In recent years, there has been growing public awareness of the potential health benefits of dietary fatty acids, and of the distinction between the effects of the omega6 and omega3 polyunsaturated fatty acids that are concentrated in vegetable and fish oils, respectively. A part of the biologic effectiveness of the two families of polyunsaturated fatty acids resides in their relative roles as precursors of the eicosanoids. However, we are also beginning to appreciate that as the major components of the hydrophobic core of the membrane bilayer, they can interact with and directly influence the functioning of select integral membrane proteins. Among the most important of these are the enzymes, receptors, and ion channels that are situated in the plasma membrane of the cell, since they carry out the communication and homeostatic processes that are necessary for normal cell function. This review examines current information regarding the effects of diet-induced changes in plasma membrane fatty acid composition on several specific enzymes (adenylate cyclase, 5'-nucleotidase, Na(+)/K(+)-ATPase) and cell-surface receptors (opiate, adrenergic, insulin). Dietary manipulation studies have demonstrated a sensitivity of each to a fatty acid environment that is variably dependent on the nature of the fatty acid(s) and/or source of the membrane. The molecular mechanisms appear to involve fatty acid-dependent effects on protein conformation, on the "fluidity" and/or thickness of the membrane, or on protein synthesis. Together, the results of these studies reinforce the concept that dietary fats have the potential to regulate physiologic function and to further our understanding of how this occurs at a membrane level.

Transport of 5-aminolevulinic acid by the dipeptide permease in Salmonella typhimurium.

PubMed

Elliott, T

1993-01-01

In a previous search for mutants of Salmonella typhimurium that are defective in heme synthesis, one class that is apparently defective in 5-aminolevulinic acid (ALA) uptake (alu) was found. Here, I describe the characterization of these mutations. The mutations all map to a single locus near 77.5 min on the genetic map, which is transcribed counterclockwise. Nutritional tests, genetic and physical mapping, and partial DNA sequence analysis revealed that alu mutants are defective in a periplasmic binding protein-dependent permease that also transports dipeptides, encoded by the dpp operon. The uptake of labeled ALA is defective in dpp mutants and is markedly increased in a strain that has elevated transcription of the dpp locus. Unlabeled L-leucyl-glycine competes with labeled ALA for uptake. In a strain carrying both a dpp-lac operon fusion and a functional copy of the dpp locus, the expression of beta-galactosidase is not induced by ALA, nor, in a hemL mutant, does expression of dpp change substantially during starvation for ALA. The dipeptide permease displays a relaxed substrate specificity that allows transport of the important nonpeptide nutrient ALA, whose structure is closely related to that of glycyl-glycine.

Monitoring the function of membrane transport proteins in detergent-solubilized form

PubMed Central

Quick, Matthias; Javitch, Jonathan A.

2007-01-01

Transport proteins constitute ≈10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment. PMID:17360689

Functional characterization of a ClC transporter by solid-supported membrane electrophysiology

PubMed Central

Garcia-Celma, Juan; Szydelko, Adrian

2013-01-01

EcClC, a prokaryotic member of the ClC family of chloride channels and transporters, works as coupled H+/Cl− exchanger. With a known structure and the possibility of investigating its behavior with different biochemical and biophysical techniques, the protein has become an important model system for the family. Although many aspects of its function have been previously characterized, it was difficult to measure transport on the same sample under different environmental conditions. To overcome this experimental limitation, we have studied EcClC by solid-supported membrane electrophysiology. The large transport-related transient currents and a simple way of relating transport rates to the measured signal have allowed a thorough investigation of ion selectivity, inhibition, and the dependence of transport on changes in ion concentration and pH. Our results confirm that the protein transports larger anions with about similar rates, whereas the smaller fluoride is not a substrate. We also show that 4,4′-diisothiocyano-2,2’-stilbenedisulfonic acid (DIDS), a known inhibitor of other anion transport protein, irreversibly inhibits EcClC from the intracellular side. The chloride dependence shows an apparent saturation at millimolar concentrations that resembles a similar behavior in eukaryotic ClC channels. Our experiments have also allowed us to quantify the pH dependence of transport. EcClC shows a strong activation at low pH with an apparent pKa of 4.6. The pronounced pH dependence is lost by the mutation of a conserved glutamate facing the extracellular solution that was previously shown to be an acceptor for transported protons, whereas it is largely retained by the mutation of an equivalent residue at the intracellular side. Our results have provided a quantitative basis for the transport behavior of EcClC, and they will serve as a reference for future investigations of novel electrogenic transporters with still-uncharacterized properties. PMID:23478993

Vitamin C Function in the Brain: Vital Role of the Ascorbate Transporter (SVCT2)

PubMed Central

Harrison, Fiona E.; May, James M.

2009-01-01

Ascorbate (vitamin C) is a vital antioxidant molecule in the brain. However, it also has a number of other important functions, participating as a co-factor in several enzyme reactions including catecholamine synthesis, collagen production and regulation of HIF-1α. Ascorbate is transported into the brain and neurons via the Sodium-dependent Vitamin C Transporter-2 (SVCT2), which causes accumulation of ascorbate within cells against a concentration gradient. Dehydroascorbic acid, the oxidized form of ascorbate, is transported via glucose transporters of the GLUT family. Once in cells, it is rapidly reduced to ascorbate. The highest concentrations of ascorbate in the body are found in the brain and neuroendocrine tissues such as adrenal, although the brain is the most difficult organ to deplete of ascorbate. Combined with regional asymmetry in ascorbate distribution within different brain areas, these facts suggest an important role for ascorbate in the brain. Ascorbate is proposed as a neuromodulator of glutamatergic, dopaminergic, cholinergic and GABAergic transmission and related behaviors. Neurodegenerative diseases typically involve high levels of oxidative stress and thus ascorbate has been posited to have potential therapeutic roles against ischemic stroke, Alzheimer's disease, Parkinson's disease and Huntingdon's disease. PMID:19162177

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

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

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.

Functional specialization in nucleotide sugar transporters occurred through differentiation of the gene cluster EamA (DUF6) before the radiation of Viridiplantae

PubMed Central

2011-01-01

Background The drug/metabolite transporter superfamily comprises a diversity of protein domain families with multiple functions including transport of nucleotide sugars. Drug/metabolite transporter domains are contained in both solute carrier families 30, 35 and 39 proteins as well as in acyl-malonyl condensing enzyme proteins. In this paper, we present an evolutionary analysis of nucleotide sugar transporters in relation to the entire superfamily of drug/metabolite transporters that considers crucial intra-protein duplication events that have shaped the transporters. We use a method that combines the strengths of hidden Markov models and maximum likelihood to find relationships between drug/metabolite transporter families, and branches within families. Results We present evidence that the triose-phosphate transporters, domain unknown function 914, uracil-diphosphate glucose-N-acetylglucosamine, and nucleotide sugar transporter families have evolved from a domain duplication event before the radiation of Viridiplantae in the EamA family (previously called domain unknown function 6). We identify previously unknown branches in the solute carrier 30, 35 and 39 protein families that emerged simultaneously as key physiological developments after the radiation of Viridiplantae, including the "35C/E" branch of EamA, which formed in the lineage of T. adhaerens (Animalia). We identify a second cluster of DMTs, called the domain unknown function 1632 cluster, which has non-cytosolic N- and C-termini, and thus appears to have been formed from a different domain duplication event. We identify a previously uncharacterized motif, G-X(6)-G, which is overrepresented in the fifth transmembrane helix of C-terminal domains. We present evidence that the family called fatty acid elongases are homologous to transporters, not enzymes as had previously been thought. Conclusions The nucleotide sugar transporters families were formed through differentiation of the gene cluster EamA (domain

Tyrosine Phosphorylation of the Human Serotonin Transporter: A Role in the Transporter Stability and Function

PubMed Central

Annamalai, Balasubramaniam; Mannangatti, Padmanabhan; Arapulisamy, Obulakshmi; Shippenberg, Toni S.; Jayanthi, Lankupalle D.

2012-01-01

The serotonin (5-HT) transporter (SERT) regulates serotoninergic neurotransmission by clearing 5-HT released into the synaptic space. Phosphorylation of SERT on serine and threonine mediates SERT regulation. Whether tyrosine phosphorylation regulates SERT is unknown. Here, we tested the hypothesis that tyrosine-phosphorylation of SERT regulates 5-HT transport. In support of this, alkali-resistant 32P-labeled SERT was found in rat platelets, and Src-tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4,d]pyrimidine (PP2) decreased platelet SERT function and expression. In human placental trophoblast cells expressing SERT, PP2 reduced transporter function, expression, and stability. Although siRNA silencing of Src expression decreased SERT function and expression, coexpression of Src resulted in PP2-sensitive increases in SERT function and expression. PP2 treatment markedly decreased SERT protein stability. Compared with WT-SERT, SERT tyrosine mutants Y47F and Y142F exhibited reduced 5-HT transport despite their higher total and cell surface expression levels. Moreover, Src-coexpression increased total and cell surface expression of Y47F and Y142F SERT mutants without affecting their 5-HT transport capacity. It is noteworthy that Y47F and Y142F mutants exhibited higher protein stability compared with WT-SERT. However, similar to WT-SERT, PP2 treatment decreased the stability of Y47F and Y142F mutants. Furthermore, compared with WT-SERT, Y47F and Y142F mutants exhibited lower basal tyrosine phosphorylation and no further enhancement of tyrosine phosphorylation in response to Src coexpression. These results provide the first evidence that SERT tyrosine phosphorylation supports transporter protein stability and 5HT transport. PMID:21992875

Ion transport controlled by nanoparticle-functionalized membranes.

PubMed

Barry, Edward; McBride, Sean P; Jaeger, Heinrich M; Lin, Xiao-Min

2014-12-17

From proton exchange membranes in fuel cells to ion channels in biological membranes, the well-specified control of ionic interactions in confined geometries profoundly influences the transport and selectivity of porous materials. Here we outline a versatile new approach to control a membrane's electrostatic interactions with ions by depositing ligand-coated nanoparticles around the pore entrances. Leveraging the flexibility and control by which ligated nanoparticles can be synthesized, we demonstrate how ligand terminal groups such as methyl, carboxyl and amine can be used to tune the membrane charge density and control ion transport. Further functionality, exploiting the ligands as binding sites, is demonstrated for sulfonate groups resulting in an enhancement of the membrane charge density. We then extend these results to smaller dimensions by systematically varying the underlying pore diameter. As a whole, these results outline a previously unexplored method for the nanoparticle functionalization of membranes using ligated nanoparticles to control ion transport.

Ion transport controlled by nanoparticle-functionalized membranes

NASA Astrophysics Data System (ADS)

Barry, Edward; McBride, Sean P.; Jaeger, Heinrich M.; Lin, Xiao-Min

2014-12-01

From proton exchange membranes in fuel cells to ion channels in biological membranes, the well-specified control of ionic interactions in confined geometries profoundly influences the transport and selectivity of porous materials. Here we outline a versatile new approach to control a membrane’s electrostatic interactions with ions by depositing ligand-coated nanoparticles around the pore entrances. Leveraging the flexibility and control by which ligated nanoparticles can be synthesized, we demonstrate how ligand terminal groups such as methyl, carboxyl and amine can be used to tune the membrane charge density and control ion transport. Further functionality, exploiting the ligands as binding sites, is demonstrated for sulfonate groups resulting in an enhancement of the membrane charge density. We then extend these results to smaller dimensions by systematically varying the underlying pore diameter. As a whole, these results outline a previously unexplored method for the nanoparticle functionalization of membranes using ligated nanoparticles to control ion transport.

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.

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.

Membrane Transporters: Structure, Function and Targets for Drug Design

NASA Astrophysics Data System (ADS)

Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

Cation-Coupled Bicarbonate Transporters

PubMed Central

Aalkjaer, Christian; Boedtkjer, Ebbe; Choi, Inyeong; Lee, Soojung

2016-01-01

Cation-coupled HCO3− transport was initially identified in the mid-1970s when pioneering studies showed that acid extrusion from cells is stimulated by CO2/HCO3− and associated with Na+ and Cl− movement. The first Na+-coupled bicarbonate transporter (NCBT) was expression-cloned in the late 1990s. There are currently five mammalian NCBTs in the SLC4-family: the electrogenic Na,HCO3-cotransporters NBCe1 and NBCe2 (SLC4A4 and SLC4A5 gene products); the electroneutral Na,HCO3-cotransporter NBCn1 (SLC4A7 gene product); the Na+-driven Cl,HCO3-exchanger NDCBE (SLC4A8 gene product); and NBCn2/NCBE (SLC4A10 gene product), which has been characterized as an electroneutral Na,HCO3-cotransporter or a Na+-driven Cl,HCO3-exchanger. Despite the similarity in amino acid sequence and predicted structure among the NCBTs of the SLC4-family, they exhibit distinct differences in ion dependency, transport function, pharmacological properties, and interactions with other proteins. In epithelia, NCBTs are involved in transcellular movement of acid-base equivalents and intracellular pH control. In nonepithelial tissues, NCBTs contribute to intracellular pH regulation; and hence, they are crucial for diverse tissue functions including neuronal discharge, sensory neuron development, performance of the heart, and vascular tone regulation. The function and expression levels of the NCBTs are generally sensitive to intracellular and systemic pH. Animal models have revealed pathophysiological roles of the transporters in disease states including metabolic acidosis, hypertension, visual defects, and epileptic seizures. Studies are being conducted to understand the physiological consequences of genetic polymorphisms in the SLC4-members, which are associated with cancer, hypertension, and drug addiction. Here, we describe the current knowledge regarding the function, structure, and regulation of the mammalian cation-coupled HCO3− transporters of the SLC4-family. PMID:25428855

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

PubMed

Ligaba, Ayalew; Dreyer, Ingo; Margaryan, Armine; Schneider, David J; Kochian, Leon; Piñeros, Miguel

2013-12-01

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

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.

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.

Lysosome Transport as a Function of Lysosome Diameter

PubMed Central

Bandyopadhyay, Debjyoti; Cyphersmith, Austin; Zapata, Jairo A.; Kim, Y. Joseph; Payne, Christine K.

2014-01-01

Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular transport of the same organelle as a function of diameter. Lysosome transport was measured using live cell fluorescence microscopy and single particle tracking. We find, as expected, the diffusive component of intracellular transport is decreased proportional to the increased lysosome diameter. Active transport of the enlarged lysosomes is not affected by the increased lysosome diameter. PMID:24497985

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.

The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

PubMed Central

King, Steven C

2004-01-01

Background In establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity. A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters. Results Using the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration. The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations). The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (kcat/Km) for [14C]GABA (4-aminobutyrate) versus [3H]NA (nipecotic acid). Conclusions The TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates. A change in the TSR phenotype, called a Δ(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (ΔGb) that translocation catalysts rely upon to decrease activation energy (). TSR analysis is therefore a

Serum Amino Acid Profiling in Citrin-Deficient Children Exhibiting Normal Liver Function During the Apparently Healthy Period.

PubMed

Miyazaki, Teruo; Nagasaka, Hironori; Komatsu, Haruki; Inui, Ayano; Morioka, Ichiro; Tsukahara, Hirokazu; Kaji, Shunsaku; Hirayama, Satoshi; Miida, Takashi; Kondou, Hiroki; Ihara, Kenji; Yagi, Mariko; Kizaki, Zenro; Bessho, Kazuhiko; Kodama, Takahiro; Iijima, Kazumoto; Yorifuji, Tohru; Matsuzaki, Yasushi; Honda, Akira

2018-04-14

Citrin (mitochondrial aspartate-glutamate transporter) deficiency causes the failures in both carbohydrate-energy metabolism and the urea cycle, and the alterations in the serum levels of several amino acids in the stages of newborn (NICCD) and adult (CTLN2). However, the clinical manifestations are resolved between the NICCD and CTLN2, but the reasons are still unclear. This study evaluated the serum amino acid profile in citrin-deficient children during the healthy stage. Using HPLC-MS/MS analysis, serum amino acids were evaluated among 20 citrin-deficient children aged 5-13 years exhibiting normal liver function and 35 age-matched healthy controls. The alterations in serum amino acids characterized in the NICCD and CTLN2 stages were not observed in the citrin-deficient children. Amino acids involved in the urea cycle, including arginine, ornithine, citrulline, and aspartate, were comparable in the citrin-deficient children to the respective control levels, but serum urea was twofold higher, suggestive of a functional urea cycle. The blood sugar level was normal, but glucogenic amino acids and glutamine were significantly decreased in the citrin-deficient children compared to those in the controls. In addition, significant increases of ketogenic amino acids, branched-chain amino acids (BCAAs), a valine intermediate 3-hydroxyisobutyrate, and β-alanine were also found in the citrin-deficient children. The profile of serum amino acids in the citrin-deficient children during the healthy stage showed different characteristics from the NICCD and CTLN2 stages, suggesting that the failures in both urea cycle function and energy metabolism might be compensated by amino acid metabolism. In the citrin-deficient children during the healthy stage, the characteristics of serum amino acids, including decrease of glucogenic amino acids, and increase of ketogenic amino acids, BCAAs, valine intermediate, and β-alanine, were found by comparison to the age-matched healthy control

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

Pathways of Polyunsaturated Fatty Acid Utilization: Implications for Brain Function in Neuropsychiatric Health and Disease

PubMed Central

Liu, Joanne J.; Green, Pnina; Mann, J. John; Rapoport, Stanley I.; Sublette, M. Elizabeth

2014-01-01

Essential polyunsaturated fatty acids (PUFAs) have profound effects on brain development and function. Abnormalities of PUFA status have been implicated in neuropsychiatric diseases such as major depression, bipolar disorder, schizophrenia, Alzheimer’s disease, and attention deficit hyperactivity disorder. Pathophysiologic mechanisms could involve not only suboptimal PUFA intake, but also metabolic and genetic abnormalities, defective hepatic metabolism, and problems with diffusion and transport. This article provides an overview of physiologic factors regulating PUFA utilization, highlighting their relevance to neuropsychiatric disease. PMID:25498862

Calixarene-Mediated Liquid-Membrane Transport of Choline Conjugates.

PubMed

Adhikari, Birendra Babu; Fujii, Ayu; Schramm, Michael P

2014-05-01

A series of supramolecular calixarenes efficiently transport distinct molecular species through a liquid membrane when attached to a receptor-complementary choline handle. Calix-[6]arene hexacarboxylic acid was highly effective at transporting different target molecules against a pH gradient. Both carboxylic- and phosphonic-acid-functionalized calix[4]arenes effect transport without requiring a pH or ion gradient. NMR binding studies, two-phase solvent extraction, and three-phase transport experiments reveal the necessary and subtle parameters to effect the transport of molecules attached to a choline "handle". On the other hand, rescorin[4]arene cavitands, which have similar guest recognition profiles, did not transport guest molecules. These developments reveal new approaches towards attempting synthetic-receptor-mediated selective small-molecule transport in vesicular and cellular systems.

Preference of Conjugated Bile Acids over Unconjugated Bile Acids as Substrates for OATP1B1 and OATP1B3

PubMed Central

Suga, Takahiro; Sato, Toshihiro; Maekawa, Masamitsu; Goto, Junichi; Mano, Nariyasu

2017-01-01

Bile acids, the metabolites of cholesterol, are signaling molecules that play critical role in many physiological functions. They undergo enterohepatic circulation through various transporters expressed in intestine and liver. Human organic anion-transporting polypeptides (OATP) 1B1 and OATP1B3 contribute to hepatic uptake of bile acids such as taurocholic acid. However, the transport properties of individual bile acids are not well understood. Therefore, we selected HEK293 cells overexpressing OATP1B1 and OATP1B3 to evaluate the transport of five major human bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid) together withtheir glycine and taurine conjugates via OATP1B1 and OATP1B3. The bile acids were quantified by liquid chromatography-tandem mass spectrometry. The present study revealed that cholic acid, chenodeoxyxcholic acid, and deoxycholic acid were transported by OATP1B1 and OATP1B3, while ursodeoxycholic acid and lithocholic acid were not significantly transported by OATPs. However, all the conjugated bile acids were taken up rapidly by OATP1B1 and OATP1B3. Kinetic analyses revealed the involvement of saturable OATP1B1- and OATP1B3-mediated transport of bile acids. The apparent Km values for OATP1B1 and OATP1B3 of the conjugated bile acids were similar (0.74–14.7 μM for OATP1B1 and 0.47–15.3 μM for OATP1B3). They exhibited higher affinity than cholic acid (47.1 μM for OATP1B1 and 42.2 μM for OATP1B3). Our results suggest that conjugated bile acids (glycine and taurine) are preferred to unconjugated bile acids as substrates for OATP1B1 and OATP1B3. PMID:28060902

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

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.

The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for 3,4-methylenedioxymethamphetamine (MDMA) translocation and the induction of substrate efflux.

PubMed

Sealover, Natalie R; Felts, Bruce; Kuntz, Charles P; Jarrard, Rachel E; Hockerman, Gregory H; Lamb, Patrick W; Barker, Eric L; Henry, L Keith

2016-11-15

The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser. Copyright © 2016 Elsevier Inc. All rights reserved.

Transport Behavior of Functionalized Multi-Wall Carbon Nanotubes in Water-Saturated Quartz Sand as a Function of Tube Length

PubMed Central

Wang, Yonggang; Kim, Jae-Hong; Baek, Jong-Beom; Miller, Gary W.; Pennell, Kurt D.

2012-01-01

A series of one-dimensional column experiments was conducted to examine the effects of tube length on the transport and deposition of 4-ethoxybenzoic acid functionalized multi-wall carbon nanotubes (MWCNTs) in water-saturated porous media. Aqueous MWCNTs suspensions were prepared to yield three distributions of tube lengths; 0.02–1.3 μm (short), 0.2–7.5 μm (medium), and 0.2–21.4 μm (long). Results of the column studies showed that MWCNT retention increased with increasing tube length. Nevertheless, more than 76% of the MWCNT mass delivered to the columns was detected in effluent samples under all experimental conditions, indicating that the functionalized MWCNTs were readily transported through 40–50 mesh Ottawa sand. Examination of MWCNT length distributions in the effluent samples revealed that nanotubes with lengths greater than 8 μm were preferentially deposited. In addition, measured retention profiles exhibited the greatest MWCNT deposition near the column inlet, which was most pronounced for the long MWCNTs, and decreased sharply with travel distance. Scanning electron microscope (SEM) images showed that MWCNTs were deposited on sand surfaces over the entire column length, while larger MWCNT bundles were retained at grain intersections and near the column inlet. A mathematical model based on clean bed filtration theory (CBFT) was unable to accurately simulate the measured retention profile data, even after varying the weighting function and incorporating a nonuniform attachment rate coefficient expression. Modification of the mathematical model to account for physical straining greatly improved predictions of MWCNT retention, yielding straining rate coefficients that were four orders-of-magnitude greater than corresponding attachment rate coefficients. Taken in concert, these experimental and modeling results demonstrate the potential importance of, and need to consider, particle straining and tube length distribution when describing MWCNT

Transport behavior of functionalized multi-wall carbon nanotubes in water-saturated quartz sand as a function of tube length.

PubMed

Wang, Yonggang; Kim, Jae-Hong; Baek, Jong-Beom; Miller, Gary W; Pennell, Kurt D

2012-09-15

A series of one-dimensional column experiments was conducted to examine the effects of tube length on the transport and deposition of 4-ethoxybenzoic acid functionalized multi-wall carbon nanotubes (MWCNTs) in water-saturated porous media. Aqueous MWCNTs suspensions were prepared to yield three distributions of tube lengths; 0.02-1.3 μm (short), 0.2-7.5 μm (medium), and 0.2-21.4 μm (long). Results of the column studies showed that MWCNT retention increased with increasing tube length. Nevertheless, more than 76% of the MWCNT mass delivered to the columns was detected in effluent samples under all experimental conditions, indicating that the functionalized MWCNTs were readily transported through 40-50 mesh Ottawa sand. Examination of MWCNT length distributions in the effluent samples revealed that nanotubes with lengths greater than 8 μm were preferentially deposited. In addition, measured retention profiles exhibited the greatest MWCNT deposition near the column inlet, which was most pronounced for the long MWCNTs, and decreased sharply with travel distance. Scanning electron microscope (SEM) images showed that MWCNTs were deposited on sand surfaces over the entire column length, while larger MWCNT bundles were retained at grain intersections and near the column inlet. A mathematical model based on clean bed filtration theory (CBFT) was unable to accurately simulate the measured retention profile data, even after varying the weighting function and incorporating a nonuniform attachment rate coefficient expression. Modification of the mathematical model to account for physical straining greatly improved predictions of MWCNT retention, yielding straining rate coefficients that were four orders-of-magnitude greater than corresponding attachment rate coefficients. Taken in concert, these experimental and modeling results demonstrate the potential importance of, and need to consider, particle straining and tube length distribution when describing MWCNT transport in

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.

Proxy functions for turbulent transport optimization of stellarators

NASA Astrophysics Data System (ADS)

Rorvig, Mordechai; Hegna, Chris; Mynick, Harry; Xanthopoulos, Pavlos

2012-10-01

The design freedom of toroidal confinement shaping suggests the possibility of optimizing the magnetic geometry for turbulent transport, particularly in stellarators. The framework for implementing such an optimization was recently established [1] using a proxy function as a measure of the ITG induced turbulent transport associated with a given geometry. Working in the framework of local 3-D equilibrium [2], we investigate the theory and implications of such proxy functions by analyzing the linear instability dependence on curvature and local shear, and the associated quasilinear transport estimates. Simple analytic models suggest the beneficial effect of local shear enters through polarization effects, which can be controlled by field torsion in small net current regimes. We test the proxy functions with local, electrostatic gyrokinetics calculations [3] of ITG modes for experimentally motivated local 3-D equilibria.[4pt] [1] H. E. Mynick, N. Pomphrey, and P. Xanthopoulos, Phys. Rev. Lett. 105, 095004 (2010).[0pt] [2] C. C. Hegna, Physics of Plasmas 7, 3921 (2000).[0pt] [3] F. Jenko, W. Dorland, M. Kotschenreuther, and B. N. Rogers, Physical Review Letters 7, 1904 (2000).

Transendothelial Transport and Its Role in Therapeutics

PubMed Central

Upadhyay, Ravi Kant

2014-01-01

Present review paper highlights role of BBB in endothelial transport of various substances into the brain. More specifically, permeability functions of BBB in transendothelial transport of various substances such as metabolic fuels, ethanol, amino acids, proteins, peptides, lipids, vitamins, neurotransmitters, monocarbxylic acids, gases, water, and minerals in the peripheral circulation and into the brain have been widely explained. In addition, roles of various receptors, ATP powered pumps, channels, and transporters in transport of vital molecules in maintenance of homeostasis and normal body functions have been described in detail. Major role of integral membrane proteins, carriers, or transporters in drug transport is highlighted. Both diffusion and carrier mediated transport mechanisms which facilitate molecular trafficking through transcellular route to maintain influx and outflux of important nutrients and metabolic substances are elucidated. Present review paper aims to emphasize role of important transport systems with their recent advancements in CNS protection mainly for providing a rapid clinical aid to patients. This review also suggests requirement of new well-designed therapeutic strategies mainly potential techniques, appropriate drug formulations, and new transport systems for quick, easy, and safe delivery of drugs across blood brain barrier to save the life of tumor and virus infected patients. PMID:27355037

Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

PubMed

Pan, Yijun; Short, Jennifer L; Choy, Kwok H C; Zeng, Annie X; Marriott, Philip J; Owada, Yuji; Scanlon, Martin J; Porter, Christopher J H; Nicolazzo, Joseph A

2016-11-16

Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5 +/+ and FABP5 -/- mice using a battery of memory paradigms. FABP5 -/- mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring 14 C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5 +/+ and FABP5 -/- mice. In line with a crucial role of FABP5 in the brain uptake of DHA, 14 C-DHA uptake into brain endothelial cells and brain capillaries of FABP5 -/- mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5 +/+ mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5 -/- mice are associated with reduced CNS access of DHA. Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5 -/- mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5

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

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.

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

Transcriptional Dysregulation of γ-Aminobutyric Acid Transporter in Parvalbumin-Containing Inhibitory Neurons in the Prefrontal Cortex in Schizophrenia

PubMed Central

Bitanihirwe, Byron K. Y.; Woo, Tsung-Ung W.

2015-01-01

Parvalbumin (PV)-containing neurons are functionally compromised in schizophrenia. Using double in situ hybridization in postmortem human prefrontal cortex, we found that the messenger RNA (mRNA) for the γ-aminobutyric acid transporter GAT-1 was undetectable in 22-41% of PV neurons in layers 3-4 in schizophrenia. In the remaining PV neurons with detectable GAT-1 mRNA, transcript expression was decreased by 26% in layer 3. Hence, the dysfunction of PV neurons involves the molecular dysregulation of presynaptic GABA reuptake. PMID:25312391

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone.

PubMed

Armas, Pablo; Nasif, Sofía; Calcaterra, Nora B

2008-02-15

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression. Copyright 2007 Wiley-Liss, Inc.

Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter β-subunit.

PubMed

Christian, Whitney V; Hinkle, Patricia M

2017-05-25

Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostβ). Although previous investigations have demonstrated the importance of the TMD of Ostβ for its activity, further studies were conducted to assess the contributions of other regions of the Ostβ subunit. Transport activity was retained when Ostβ was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whereas shorter fragments were inactive. To probe the broader functions of Ostβ segments, chimeric proteins were constructed in which N-terminal, TMD or C-terminal regions of Ostβ were fused to corresponding regions of receptor activity-modifying protein (RAMP1), a single TMD protein required by several seven-TMD G-protein-coupled receptors including the calcitonin receptor-like receptor (CLR). Ostβ/RAMP1 chimeras were expressed with Ostα and CLR. As expected, replacing the Ostβ TMD abolished transport activity; however, replacing either the entire N-terminal or entire C-terminal domain of Ostβ with RAMP1 sequences did not prevent plasma membrane localization or the ability to support [ 3 H]taurocholate uptake. Co-immunoprecipitation experiments revealed that the C-terminus of Ostβ is a previously unrecognized site of interaction with Ostα. All chimeras containing N-terminal RAMP1 segments allowed co-expressed CLR to respond to agonists with strong increases in cyclic AMP. These results provide new insights into the structure and function of the heteromeric Ost transporter complex. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

β-Subunit of the Ostα-Ostβ Organic Solute Transporter Is Required Not Only for Heterodimerization and Trafficking but Also for Function*

PubMed Central

Christian, Whitney V.; Li, Na; Hinkle, Patricia M.; Ballatori, Nazzareno

2012-01-01

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostβ required for heterodimerization with Ostα, trafficking of the Ostα-Ostβ complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostβ transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [3H]taurocholate transport activity. Deletion of the single TM domain of Ostβ abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostβ to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostβ resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostβ abolished [3H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct Nexo/Ccyt topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostβ is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostβ critical for these processes. PMID:22535958

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

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.

ETMB-RBF: discrimination of metal-binding sites in electron transporters based on RBF networks with PSSM profiles and significant amino acid pairs.

PubMed

Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

2013-01-01

Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation-reduction reactions. In these oxidation-reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins.

ETMB-RBF: Discrimination of Metal-Binding Sites in Electron Transporters Based on RBF Networks with PSSM Profiles and Significant Amino Acid Pairs

PubMed Central

Ou, Yu-Yen; Chen, Shu-An; Wu, Sheng-Cheng

2013-01-01

Background Cellular respiration is the process by which cells obtain energy from glucose and is a very important biological process in living cell. As cells do cellular respiration, they need a pathway to store and transport electrons, the electron transport chain. The function of the electron transport chain is to produce a trans-membrane proton electrochemical gradient as a result of oxidation–reduction reactions. In these oxidation–reduction reactions in electron transport chains, metal ions play very important role as electron donor and acceptor. For example, Fe ions are in complex I and complex II, and Cu ions are in complex IV. Therefore, to identify metal-binding sites in electron transporters is an important issue in helping biologists better understand the workings of the electron transport chain. Methods We propose a method based on Position Specific Scoring Matrix (PSSM) profiles and significant amino acid pairs to identify metal-binding residues in electron transport proteins. Results We have selected a non-redundant set of 55 metal-binding electron transport proteins as our dataset. The proposed method can predict metal-binding sites in electron transport proteins with an average 10-fold cross-validation accuracy of 93.2% and 93.1% for metal-binding cysteine and histidine, respectively. Compared with the general metal-binding predictor from A. Passerini et al., the proposed method can improve over 9% of sensitivity, and 14% specificity on the independent dataset in identifying metal-binding cysteines. The proposed method can also improve almost 76% sensitivity with same specificity in metal-binding histidine, and MCC is also improved from 0.28 to 0.88. Conclusions We have developed a novel approach based on PSSM profiles and significant amino acid pairs for identifying metal-binding sites from electron transport proteins. The proposed approach achieved a significant improvement with independent test set of metal-binding electron transport proteins

Calixarene-Mediated Liquid-Membrane Transport of Choline Conjugates

PubMed Central

Adhikari, Birendra Babu; Fujii, Ayu

2015-01-01

A series of supramolecular calixarenes efficiently transport distinct molecular species through a liquid membrane when attached to a receptor-complementary choline handle. Calix-[6]arene hexacarboxylic acid was highly effective at transporting different target molecules against a pH gradient. Both carboxylic- and phosphonic-acid-functionalized calix[4]arenes effect transport without requiring a pH or ion gradient. NMR binding studies, two-phase solvent extraction, and three-phase transport experiments reveal the necessary and subtle parameters to effect the transport of molecules attached to a choline “handle”. On the other hand, rescorin[4]arene cavitands, which have similar guest recognition profiles, did not transport guest molecules. These developments reveal new approaches towards attempting synthetic-receptor-mediated selective small-molecule transport in vesicular and cellular systems. PMID:26161034

[Significance of considering hemoglobin derivatives and acid-base balance in the evaluation of the blood oxygen-transport system].

PubMed

Matiushichev, V B; Shamratova, V G; Krapivko, Iu K

2009-12-01

Factor analysis was used to study the pattern of relationships of a number of hematological parameters in clinically healthy young subjects and in patients with moderate anemia. The level of total hemoglobin and the concentration of red blood cells were ascertained to control blood oxygen-transporting function in not full measure and these might be referred to as basic characteristics only conventionally. To clarify the picture, these criteria should be supplemented by the information on other parameters. It is concluded that the introduction of the ratio of a number of hemoglobin derivatives, blood oxygen regimen and acid-base balance can substantially increase the validity of clinical opinions as to this blood function.

Functional Properties and Genomics of Glucose Transporters

PubMed Central

Zhao, Feng-Qi; Keating, Aileen F

2007-01-01

Glucose is the major energy source for mammalian cells as well as an important substrate for protein and lipid synthesis. Mammalian cells take up glucose from extracellular fluid into the cell through two families of structurallyrelated glucose transporters. The facilitative glucose transporter family (solute carriers SLC2A, protein symbol GLUT) mediates a bidirectional and energy-independent process of glucose transport in most tissues and cells, while the NaM+/glucose cotransporter family (solute carriers SLC5A, protein symbol SGLT) mediates an active, Na+-linked transport process against an electrochemical gradient. The GLUT family consists of thirteen members (GLUT1-12 and HMIT). Phylogenetically, the members of the GLUT family are split into three classes based on protein similarities. Up to now, at least six members of the SGLT family have been cloned (SGLT1-6). In this review, we report both the genomic structure and function of each transporter as well as intra-species comparative genomic analysis of some of these transporters. The affinity for glucose and transport kinetics of each transporter differs and ranges from 0.2 to 17mM. The ability of each protein to transport alternative substrates also differs and includes substrates such as fructose and galactose. In addition, the tissue distribution pattern varies between species. There are different regulation mechanisms of these transporters. Characterization of transcriptional control of some of the gene promoters has been investigated and alternative promoter usage to generate different protein isoforms has been demonstrated. We also introduce some pathophysiological roles of these transporters in human. PMID:18660845

Intestinal-fatty acid binding protein and lipid transport in human intestinal epithelial cells

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

Montoudis, Alain; Delvin, Edgard; Canadian Institute of Health Research, Group of the Functional Development and Physiopathology of the Digestive Tract, and Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, Universite de Sherbrooke, Sherbrooke, Que., Canada J1H 5N4

2006-01-06

Intestinal-fatty acid binding protein (I-FABP) is a 14-15 kDa cytoplasmic molecule highly expressed in the enterocyte. Although different functions have been proposed for various FABP family members, the specific function of I-FABP in human intestine remains unclear. Here, we studied the role of I-FABP in molecularly modified normal human intestinal epithelial cells (HIEC-6). cDNA transfection resulted in 90-fold I-FABP overexpression compared to cells treated with empty pQCXIP vector. The high-resolution immunogold technique revealed labeling mainly in the cytosol and confirmed the marked phenotype abundance of I-FABP in cDNA transfected cells. I-FABP overexpression was not associated with alterations in cell proliferationmore » and viability. Studies using these transfected cells cultured with [{sup 14}C]oleic acid did not reveal higher efficiency in de novo synthesis or secretion of triglycerides, phospholipids, and cholesteryl esters compared to cells treated with empty pQCXIP vector only. Similarly, the incubation with [{sup 35}S]methionine did not disclose a superiority in the biogenesis of apolipoproteins (apo) A-I, A-IV, B-48, and B-100. Finally, cells transfected with I-FABP did not exhibit an increased production of chylomicrons, VLDL, LDL, and HDL. Our observations establish that I-FABP overexpression in normal HIEC-6 is not related to cell proliferation, lipid esterification, apo synthesis, and lipoprotein assembly, and, therefore, exclude its role in intestinal fat transport.« less

Regional expression and dietary regulation of rat small intestinal peptide and amino acid transporter mRNAs.

PubMed

Erickson, R H; Gum, J R; Lindstrom, M M; McKean, D; Kim, Y S

1995-11-02

RT-PCR was used to obtain rat small intestinal cDNAs for two peptide transporters, showing conclusively for the first time that both are present in normal intestinal mucosa. Sequencing of these cDNAs showed them to be highly homologous and similar to two different types of peptide transport proteins from either colorectal carcinoma cells (Caco-2) or human and rabbit intestine. An even distribution profile of steady state levels of mRNA for both peptide transporters was observed along the longitudinal axis of small intestine. Both were upregulated in the distal regions of intestine by a high protein diet. Also, high levels of the rat high affinity glutamate transporter EAAC1 were observed in the distal intestine. These results suggest that the distal regions of small intestine play an important role in the absorption of some amino acids and peptides. Furthermore this area appears to be a primary site where dietary-induced changes in peptide and amino acid transport occurs.

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

PubMed

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

2017-10-25

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

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

PubMed

Danylovych, H V

2016-01-01

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system.

PubMed

Souffriau, Ben; den Abt, Tom; Thevelein, Johan M