Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake☆
Identifieur interne : 000272 ( Pmc/Checkpoint ); précédent : 000271; suivant : 000273Functional expression of TcoAT1 reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake☆
Auteurs : Jane C. Munday [Royaume-Uni] ; Karla E. Rojas L Pez [Royaume-Uni] ; Anthonius A. Eze [Royaume-Uni] ; Vincent Delespaux [Belgique] ; Jan Van Den Abbeele [Belgique] ; Tim Rowan [Royaume-Uni] ; Michael P. Barrett [Royaume-Uni] ; Liam J. Morrison [Royaume-Uni] ; Harry P. De Koning [Royaume-Uni]Source :
- International Journal for Parasitology, Drugs and Drug Resistance [ 2211-3207 ] ; 2013.
Abstract
► Diminazene transporter in
Url:
DOI: 10.1016/j.ijpddr.2013.01.004
PubMed: 24533295
PubMed Central: 3862423
Affiliations:
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PMC:3862423Le document en format XML
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Barrett</name><affiliation wicri:level="4"><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. 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De Koning</name><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">24533295</idno><idno type="pmc">3862423</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3862423</idno><idno type="RBID">PMC:3862423</idno><idno type="doi">10.1016/j.ijpddr.2013.01.004</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">000379</idno><idno type="wicri:Area/Pmc/Curation">000379</idno><idno type="wicri:Area/Pmc/Checkpoint">000272</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Functional expression of <italic>TcoAT1</italic> reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake<sup><xref ref-type="fn" rid="d32e303">☆</xref></sup></title><author><name sortKey="Munday, Jane C" sort="Munday, Jane C" uniqKey="Munday J" first="Jane C." last="Munday">Jane C. 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Rojas L Pez</name><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="Eze, Anthonius A" sort="Eze, Anthonius A" uniqKey="Eze A" first="Anthonius A." last="Eze">Anthonius A. Eze</name><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="Delespaux, Vincent" sort="Delespaux, Vincent" uniqKey="Delespaux V" first="Vincent" last="Delespaux">Vincent Delespaux</name><affiliation wicri:level="1"><nlm:aff id="aff3">Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp</wicri:regionArea><wicri:noRegion>B-2000 Antwerp</wicri:noRegion></affiliation></author><author><name sortKey="Van Den Abbeele, Jan" sort="Van Den Abbeele, Jan" uniqKey="Van Den Abbeele J" first="Jan" last="Van Den Abbeele">Jan Van Den Abbeele</name><affiliation wicri:level="1"><nlm:aff id="aff3">Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp</wicri:regionArea><wicri:noRegion>B-2000 Antwerp</wicri:noRegion></affiliation></author><author><name sortKey="Rowan, Tim" sort="Rowan, Tim" uniqKey="Rowan T" first="Tim" last="Rowan">Tim Rowan</name><affiliation wicri:level="3"><nlm:aff id="aff4">GALVmed, Pentlands Science Park, Bush Loan, Edinburgh, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>GALVmed, Pentlands Science Park, Bush Loan, Edinburgh</wicri:regionArea><placeName><settlement type="city">Édimbourg</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="Barrett, Michael P" sort="Barrett, Michael P" uniqKey="Barrett M" first="Michael P." last="Barrett">Michael P. Barrett</name><affiliation wicri:level="4"><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. Morrison</name><affiliation wicri:level="4"><nlm:aff id="aff1">Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation><affiliation wicri:level="4"><nlm:aff id="aff5">Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Roslin Institute, University of Edinburgh, Easter Bush, Midlothian</wicri:regionArea><orgName type="university">Université d'Édimbourg</orgName><placeName><settlement type="city">Édimbourg</settlement><region type="country">Écosse</region></placeName></affiliation></author><author><name sortKey="De Koning, Harry P" sort="De Koning, Harry P" uniqKey="De Koning H" first="Harry P." last="De Koning">Harry P. De Koning</name><affiliation wicri:level="4"><nlm:aff id="aff2">Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow</wicri:regionArea><orgName type="university">Université de Glasgow</orgName><placeName><settlement type="city">Glasgow</settlement><region type="country">Écosse</region></placeName></affiliation></author></analytic><series><title level="j">International Journal for Parasitology, Drugs and Drug Resistance</title><idno type="eISSN">2211-3207</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Graphical abstract</title><p><fig id="f0040" position="anchor"><graphic xlink:href="fx2"></graphic></fig></p></sec><sec><title>Highlights</title><p>► Diminazene transporter in <italic>Trypanosoma congolense</italic> has been proposed to be TcoAT1. ► Here, <italic>TcoAT1</italic> was cloned and functionally expressed in <italic>Trypanosoma brucei</italic>. ► TcoAT1 did not mediate the uptake of diminazene, only of purine nucleosides. ► Expression of TcoAT1 did not alter drug sensitivity in trypanosomes. ► We conclude that TcoAT1 is a transporter for purine nucleosides, not for diminazene.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Al Salabi, M I" uniqKey="Al Salabi M">M.I. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Parasitol Drugs Drug Resist</journal-id><journal-id journal-id-type="iso-abbrev">Int J Parasitol Drugs Drug Resist</journal-id><journal-title-group><journal-title>International Journal for Parasitology, Drugs and Drug Resistance</journal-title></journal-title-group><issn pub-type="epub">2211-3207</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">24533295</article-id><article-id pub-id-type="pmc">3862423</article-id><article-id pub-id-type="publisher-id">S2211-3207(13)00005-5</article-id><article-id pub-id-type="doi">10.1016/j.ijpddr.2013.01.004</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Functional expression of <italic>TcoAT1</italic> reveals it to be a P1-type nucleoside transporter with no capacity for diminazene uptake<sup><xref ref-type="fn" rid="d32e303">☆</xref></sup></article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Munday</surname><given-names>Jane C.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Rojas López</surname><given-names>Karla E.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Eze</surname><given-names>Anthonius A.</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Delespaux</surname><given-names>Vincent</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Van Den Abbeele</surname><given-names>Jan</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Rowan</surname><given-names>Tim</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Barrett</surname><given-names>Michael P.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Morrison</surname><given-names>Liam J.</given-names></name><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref><xref rid="aff5" ref-type="aff">e</xref></contrib><contrib contrib-type="author"><name><surname>de Koning</surname><given-names>Harry P.</given-names></name><email>Harry.de-Koning@glasgow.ac.uk</email><xref rid="aff2" ref-type="aff">b</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, United Kingdom</aff><aff id="aff2"><label>b</label>Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom</aff><aff id="aff3"><label>c</label>Institute of Tropical Medicine, Nationalestraat 155, B-2000 Antwerp, Belgium</aff><aff id="aff4"><label>d</label>GALVmed, Pentlands Science Park, Bush Loan, Edinburgh, United Kingdom</aff><aff id="aff5"><label>e</label>Roslin Institute, University of Edinburgh, Easter Bush, Midlothian, United Kingdom</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Address: Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, United Kingdom. Tel.: +44 141 3303753; fax: +44 141 3306900. <email>Harry.de-Koning@glasgow.ac.uk</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>10</day><month>2</month><year>2013</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was
based on .</pmc-comment>
<pub-date pub-type="epub"><day>10</day><month>2</month><year>2013</year></pub-date><pub-date pub-type="collection"><month>12</month><year>2013</year></pub-date><volume>3</volume><fpage>69</fpage><lpage>76</lpage><history><date date-type="received"><day>14</day><month>12</month><year>2012</year></date><date date-type="rev-recd"><day>19</day><month>1</month><year>2013</year></date><date date-type="accepted"><day>23</day><month>1</month><year>2013</year></date></history><permissions><copyright-statement>© 2013 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Australian Society for Parasitology</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract abstract-type="graphical"><sec><title>Graphical abstract</title><p><fig id="f0040" position="anchor"><graphic xlink:href="fx2"></graphic></fig></p></sec><sec><title>Highlights</title><p>► Diminazene transporter in <italic>Trypanosoma congolense</italic> has been proposed to be TcoAT1. ► Here, <italic>TcoAT1</italic> was cloned and functionally expressed in <italic>Trypanosoma brucei</italic>. ► TcoAT1 did not mediate the uptake of diminazene, only of purine nucleosides. ► Expression of TcoAT1 did not alter drug sensitivity in trypanosomes. ► We conclude that TcoAT1 is a transporter for purine nucleosides, not for diminazene.</p></sec></abstract><abstract><p>It has long been established that the <italic>Trypanosoma brucei</italic> TbAT1/P2 aminopurine transporter is involved in the uptake of diamidine and arsenical drugs including pentamidine, diminazene aceturate and melarsoprol. Accordingly, it was proposed that the closest <italic>Trypanosoma congolense</italic> paralogue, <italic>TcoAT1</italic>, might perform the same function in this parasite, and an apparent correlation between a Single Nucleotide Polymorphism (SNP) in that gene and diminazene tolerance was reported for the strains examined. Here, we report the functional cloning and expression of <italic>TcoAT1</italic> and show that in fact it is the syntenic homologue of another <italic>T. brucei</italic> gene of the same Equilibrative Nucleoside Transporter (ENT) family: TbNT10. The <italic>T. congolense</italic> genome does not seem to contain a syntenic equivalent to <italic>TbAT1</italic>. Two <italic>TcoAT1</italic> alleles, differentiated by three independent SNPs, were expressed in the <italic>T. brucei</italic> clone B48, a <italic>TbAT1</italic>-null strain that further lacks the High Affinity Pentamidine Transporter (HAPT1); <italic>TbAT1</italic> was also expressed as a control. The <italic>TbAT1</italic> and <italic>TcoAT1</italic> transporters were functional and increased sensitivity to cytotoxic nucleoside analogues. However, only <italic>TbAT1</italic> increased sensitivity to diamidines and to cymelarsan. Uptake of [<sup>3</sup>H]-diminazene was detectable only in the B48 cells expressing <italic>TbAT1</italic> but not <italic>TcoAT1</italic>, whereas uptake of [<sup>3</sup>H]-inosine was increased by both <italic>TcoAT1</italic> alleles but not by <italic>TbAT1</italic>. Uptake of [<sup>3</sup>H]-adenosine was increased by all three ENT genes. We conclude that TcoAT1 is a P1-type purine nucleoside transporter and the syntenic equivalent to the previously characterised TbNT10; it does not mediate diminazene uptake and is therefore unlikely to play a role in diminazene resistance in <italic>T. congolense</italic>.</p></abstract><kwd-group><title>Keywords</title><kwd><italic>Trypanosoma congolense</italic></kwd><kwd>Diminazene aceturate</kwd><kwd>Drug resistance</kwd><kwd>TcoAT1</kwd></kwd-group></article-meta></front><floats-group><fig id="f0005"><label>Fig. 1</label><caption><p>Phylogenetic tree of ENT genes in trypanosomatid species <italic>T. b. brucei</italic> (red), <italic>T. congolense</italic> (blue), <italic>T. vivax</italic> (green) and <italic>T. cruzi</italic> (black). Separate clusters for nucleobase transporters, P1-type nucleoside transporters and P2-type transporters are indicated with boxes of dashed lines. The positions of <italic>TbAT1</italic> (Tb927.5.286b), <italic>TbNT10</italic> (Tb09.160.5480) and <italic>TcoAT1</italic> (TcIL3000.9.2500) are indicated with arrows. The tree was constructed using the Maximum Likelihood Tree function of MEGA5, with 500 bootstrap replications and, otherwise, the default parameters (<xref rid="b0240" ref-type="bibr">Tamura et al., 2011</xref>). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The scale bar represents 0.5 amino acid substitutions per position. The tree is rooted with a divergent <italic>T. cruzi</italic> sequence (TcCLB.506445.10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="f0010"><label>Fig. 2</label><caption><p>Sensitivity of various <italic>T. b. brucei</italic> B48-derived cell lines to adenosine analogues. A, tubercidin; B, 5′-deoxyadenosine; C, cordycepin; D, phenylarsine oxide (arsenical control). The strains were all derived from B48 (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>), by transfection with the empty vector (EV) pHD1336 or the same vector containing <italic>TbAT1</italic> or <italic>TcoAT1</italic> ‘sensitive variant’ (<italic>TcoAT1</italic>S). Bars show the average and SEM of 5 independent determinations, using the Alamar blue assay. <sup>∗</sup><italic>P</italic> < 0.05; <sup>∗∗</sup>P < 0.01; <sup>∗∗∗</sup>P < 0.001, Student <italic>T</italic>-test, relative to B48 + EV.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="f0015"><label>Fig. 3</label><caption><p>Sensitivity of <italic>T. b. brucei</italic> bloodstream forms of various clonal lines to diminazene (A), pentamidine (B), cymelarsan (C) and phenylarsine oxide (D), as determined using standard Alamar blue-based assay. All clonal strains were derived from the B48 line, which is highly resistant to diamidines and melaminophenyl arsenicals (<xref rid="b0025" ref-type="bibr">Bridges et al., 2007</xref>). The B48 strain was transformed with either empty vector pHD1336 (EV), <italic>TbAT1</italic>, <italic>TcoAT1</italic> ‘sensitive variant’ (<italic>TcoAT1</italic>S) or <italic>TcoAT1</italic> ‘resistant variant’ (<italic>TcoAT1</italic>R) and for each two independent clones were tested with identical results; only one is shown. Bar graphs represent the average and standard error of 4–5 independent experiments. <sup>∗∗</sup><italic>P</italic> < 0.01 by unpaired Student’s <italic>T</italic>-test, relative to B48 + EV.</p></caption><graphic xlink:href="gr3"></graphic></fig><fig id="f0020"><label>Fig. 4</label><caption><p>Diminazene uptake in transformed <italic>T. b. brucei</italic> B48. B48 cells transformed with either vector without insert (circles), <italic>TbAT1</italic> (squares) or <italic>TcoAT1</italic>S (up triangles) were incubated with 0.1 μM [<sup>3</sup>H]-diminazene with (open symbols) or without (filled symbols) 1 mM unlabeled diminazene aceturate for up to 5 min as indicated. Symbols represent the average and SEM of three independent experiments, each performed in triplicate. Lines were calculated by linear regression; only uptake of 0.1 μM [<sup>3</sup>H]-diminazene by B48 expressing <italic>TbAT1</italic> was significantly different from zero (<italic>P</italic> < 0.0001, <italic>F</italic>-test; <italic>r</italic><sup>2</sup> > 0.99). Diminazene uptake by cells expressing <italic>TbAT1</italic> was significantly higher than in control cells (B48 with empty vector). <sup>∗∗</sup><italic>P</italic> < 0.01; <sup>∗∗∗</sup><italic>P</italic> < 0.001 (relative to EV control; Student’s <italic>T</italic>-test).</p></caption><graphic xlink:href="gr4"></graphic></fig><fig id="f0025"><label>Fig. 5</label><caption><p>Uptake of (A) inosine and (B) adenosine in transformed <italic>T. b. brucei</italic> B48. B48 cells transformed with either vector without insert (circles), <italic>TbAT1</italic> (squares), <italic>TcoAT1</italic>S (up triangles) or <italic>TcoAT1</italic>R (down triangles) were incubated with 0.1 μM [<sup>3</sup>H]-inosine (A) or 0.1 lM [<sup>3</sup>H]-adenosine (B) in the presence (open symbols and dotted lines) or absence (filled symbols and continuous lines) of 1 mM unlabeled permeant for up to 2 min as indicated. In the presence of 1 mM permeant uptake was inhibited by >99%, showing that uptake was saturable and therefore most likely transporter-mediated. Inosine uptake by cells expressing the <italic>TcoAT1</italic> alleles was significantly higher than in control cells (B48 with empty vector). Data shown are the average and SEM of three independent experiments, each performed in triplicate. <sup>∗</sup><italic>P</italic> < 0.05; <sup>∗∗</sup><italic>P</italic> < 0.01; <sup>∗∗∗</sup><italic>P</italic> < 0.001 (Student’s <italic>T</italic>-test). <italic>Note:</italic> in frame B each of the experimental data points were significantly different from the empty vector control at 20 s, by <italic>P</italic> < 0.01 or better.</p></caption><graphic xlink:href="gr5"></graphic></fig></floats-group></pmc><affiliations><list><country><li>Belgique</li><li>Royaume-Uni</li></country><region><li>Écosse</li></region><settlement><li>Glasgow</li><li>Édimbourg</li></settlement><orgName><li>Université d'Édimbourg</li><li>Université de Glasgow</li></orgName></list><tree><country name="Royaume-Uni"><region name="Écosse"><name sortKey="Munday, Jane C" sort="Munday, Jane C" uniqKey="Munday J" first="Jane C." last="Munday">Jane C. Munday</name></region><name sortKey="Barrett, Michael P" sort="Barrett, Michael P" uniqKey="Barrett M" first="Michael P." last="Barrett">Michael P. Barrett</name><name sortKey="Barrett, Michael P" sort="Barrett, Michael P" uniqKey="Barrett M" first="Michael P." last="Barrett">Michael P. Barrett</name><name sortKey="De Koning, Harry P" sort="De Koning, Harry P" uniqKey="De Koning H" first="Harry P." last="De Koning">Harry P. De Koning</name><name sortKey="Eze, Anthonius A" sort="Eze, Anthonius A" uniqKey="Eze A" first="Anthonius A." last="Eze">Anthonius A. Eze</name><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. Morrison</name><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. Morrison</name><name sortKey="Morrison, Liam J" sort="Morrison, Liam J" uniqKey="Morrison L" first="Liam J." last="Morrison">Liam J. Morrison</name><name sortKey="Munday, Jane C" sort="Munday, Jane C" uniqKey="Munday J" first="Jane C." last="Munday">Jane C. Munday</name><name sortKey="Rojas L Pez, Karla E" sort="Rojas L Pez, Karla E" uniqKey="Rojas L Pez K" first="Karla E." last="Rojas L Pez">Karla E. Rojas L Pez</name><name sortKey="Rowan, Tim" sort="Rowan, Tim" uniqKey="Rowan T" first="Tim" last="Rowan">Tim Rowan</name></country><country name="Belgique"><noRegion><name sortKey="Delespaux, Vincent" sort="Delespaux, Vincent" uniqKey="Delespaux V" first="Vincent" last="Delespaux">Vincent Delespaux</name></noRegion><name sortKey="Van Den Abbeele, Jan" sort="Van Den Abbeele, Jan" uniqKey="Van Den Abbeele J" first="Jan" last="Van Den Abbeele">Jan Van Den Abbeele</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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