Characterization of the Kynurenine Pathway and Quinolinic Acid Production in Macaque Macrophages
Identifieur interne : 001306 ( Ncbi/Merge ); précédent : 001305; suivant : 001307Characterization of the Kynurenine Pathway and Quinolinic Acid Production in Macaque Macrophages
Auteurs : Chai K. Lim [Australie] ; Margaret M. C. Yap [Australie] ; Stephen J. Kent [Australie] ; Gabriel Gras [France] ; Boubekeur Samah [France] ; Jane C. Batten [Australie] ; Robert De Rose [Australie] ; Benjamin Heng [Australie] ; Bruce J. Brew [Australie] ; Gilles J. Guillemin [Australie]Source :
- International Journal of Tryptophan Research : IJTR [ 1178-6469 ] ; 2013.
Abstract
The kynurenine pathway (KP) and one of its end-products, the excitotoxin quinolinic acid (QUIN), are involved in the pathogenesis of several major neuroinflammatory brain diseases. A relevant animal model to study KP metabolism is now needed to assess whether intervention in this pathway may improve the outcome of such diseases. Humans and macaques share a very similar genetic makeup. In this study, we characterized the KP metabolism in macaque primary macrophages of three different species in comparison to human cells. We found that the KP profiles in simian macrophages were very similar to those in humans when challenged with inflammatory cytokines. Further, we found that macaque macrophages are capable of producing a pathophysiological concentration of QUIN. Our data validate the simian model as a relevant model to study the human cellular KP metabolism in the context of inflammation.
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DOI: 10.4137/IJTR.S11789
PubMed: 23761975
PubMed Central: 3662399
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Lim</name><affiliation wicri:level="3"><nlm:aff id="af1-ijtr-6-2013-007">Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney</wicri:regionArea><placeName><settlement type="city">Sydney</settlement><region type="état">Nouvelle-Galles du Sud</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijtr-6-2013-007">MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University</wicri:regionArea><wicri:noRegion>Macquarie University</wicri:noRegion></affiliation></author><author><name sortKey="Yap, Margaret M C" sort="Yap, Margaret M C" uniqKey="Yap M" first="Margaret M. 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Lim</name><affiliation wicri:level="3"><nlm:aff id="af1-ijtr-6-2013-007">Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney</wicri:regionArea><placeName><settlement type="city">Sydney</settlement><region type="état">Nouvelle-Galles du Sud</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijtr-6-2013-007">MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University</wicri:regionArea><wicri:noRegion>Macquarie University</wicri:noRegion></affiliation></author><author><name sortKey="Yap, Margaret M C" sort="Yap, Margaret M C" uniqKey="Yap M" first="Margaret M. C." last="Yap">Margaret M. C. Yap</name><affiliation wicri:level="3"><nlm:aff id="af3-ijtr-6-2013-007">Applied Neurosciences, St. Vincent’s Center for Applied Medical Research, Sydney, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Applied Neurosciences, St. Vincent’s Center for Applied Medical Research, Sydney</wicri:regionArea><placeName><settlement type="city">Sydney</settlement><region type="état">Nouvelle-Galles du Sud</region></placeName></affiliation></author><author><name sortKey="Kent, Stephen J" sort="Kent, Stephen J" uniqKey="Kent S" first="Stephen J." last="Kent">Stephen J. Kent</name><affiliation wicri:level="4"><nlm:aff id="af4-ijtr-6-2013-007">Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria</wicri:regionArea><orgName type="university">Université de Melbourne</orgName><placeName><settlement type="city">Melbourne</settlement><region type="état">Victoria (État)</region></placeName></affiliation></author><author><name sortKey="Gras, Gabriel" sort="Gras, Gabriel" uniqKey="Gras G" first="Gabriel" last="Gras">Gabriel Gras</name><affiliation wicri:level="1"><nlm:aff id="af5-ijtr-6-2013-007">Laboratoire de Neuro-Immuno-Virologie, Service de Neurovirologie UMR E-01 CEA, France and UMR E1, School of Medicine, University Paris South 11, Le Kremlin-Bicêtre, France.</nlm:aff><country xml:lang="fr" wicri:curation="lc">France</country><wicri:regionArea>Laboratoire de Neuro-Immuno-Virologie, Service de Neurovirologie UMR E-01 CEA, France and UMR E1, School of Medicine, University Paris South 11, Le Kremlin-Bicêtre</wicri:regionArea><wicri:noRegion>Le Kremlin-Bicêtre</wicri:noRegion><wicri:noRegion>Le Kremlin-Bicêtre</wicri:noRegion></affiliation></author><author><name sortKey="Samah, Boubekeur" sort="Samah, Boubekeur" uniqKey="Samah B" first="Boubekeur" last="Samah">Boubekeur Samah</name><affiliation wicri:level="1"><nlm:aff id="af5-ijtr-6-2013-007">Laboratoire de Neuro-Immuno-Virologie, Service de Neurovirologie UMR E-01 CEA, France and UMR E1, School of Medicine, University Paris South 11, Le Kremlin-Bicêtre, France.</nlm:aff><country xml:lang="fr" wicri:curation="lc">France</country><wicri:regionArea>Laboratoire de Neuro-Immuno-Virologie, Service de Neurovirologie UMR E-01 CEA, France and UMR E1, School of Medicine, University Paris South 11, Le Kremlin-Bicêtre</wicri:regionArea><wicri:noRegion>Le Kremlin-Bicêtre</wicri:noRegion><wicri:noRegion>Le Kremlin-Bicêtre</wicri:noRegion></affiliation></author><author><name sortKey="Batten, Jane C" sort="Batten, Jane C" uniqKey="Batten J" first="Jane C." last="Batten">Jane C. Batten</name><affiliation wicri:level="4"><nlm:aff id="af4-ijtr-6-2013-007">Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria</wicri:regionArea><orgName type="university">Université de Melbourne</orgName><placeName><settlement type="city">Melbourne</settlement><region type="état">Victoria (État)</region></placeName></affiliation></author><author><name sortKey="De Rose, Robert" sort="De Rose, Robert" uniqKey="De Rose R" first="Robert" last="De Rose">Robert De Rose</name><affiliation wicri:level="4"><nlm:aff id="af4-ijtr-6-2013-007">Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria</wicri:regionArea><orgName type="university">Université de Melbourne</orgName><placeName><settlement type="city">Melbourne</settlement><region type="état">Victoria (État)</region></placeName></affiliation></author><author><name sortKey="Heng, Benjamin" sort="Heng, Benjamin" uniqKey="Heng B" first="Benjamin" last="Heng">Benjamin Heng</name><affiliation wicri:level="1"><nlm:aff id="af2-ijtr-6-2013-007">MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University</wicri:regionArea><wicri:noRegion>Macquarie University</wicri:noRegion></affiliation></author><author><name sortKey="Brew, Bruce J" sort="Brew, Bruce J" uniqKey="Brew B" first="Bruce J." last="Brew">Bruce J. Brew</name><affiliation wicri:level="3"><nlm:aff id="af3-ijtr-6-2013-007">Applied Neurosciences, St. Vincent’s Center for Applied Medical Research, Sydney, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Applied Neurosciences, St. Vincent’s Center for Applied Medical Research, Sydney</wicri:regionArea><placeName><settlement type="city">Sydney</settlement><region type="état">Nouvelle-Galles du Sud</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="af6-ijtr-6-2013-007">Department of Neurology, St. Vincent’s Hospital, Darlinghurst, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Neurology, St. Vincent’s Hospital, Darlinghurst</wicri:regionArea><wicri:noRegion>Darlinghurst</wicri:noRegion></affiliation></author><author><name sortKey="Guillemin, Gilles J" sort="Guillemin, Gilles J" uniqKey="Guillemin G" first="Gilles J." last="Guillemin">Gilles J. Guillemin</name><affiliation wicri:level="3"><nlm:aff id="af1-ijtr-6-2013-007">Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney</wicri:regionArea><placeName><settlement type="city">Sydney</settlement><region type="état">Nouvelle-Galles du Sud</region></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="af2-ijtr-6-2013-007">MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University, Australia.</nlm:aff><country xml:lang="fr" wicri:curation="lc">Australie</country><wicri:regionArea>MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University</wicri:regionArea><wicri:noRegion>Macquarie University</wicri:noRegion></affiliation></author></analytic><series><title level="j">International Journal of Tryptophan Research : IJTR</title><idno type="eISSN">1178-6469</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The kynurenine pathway (KP) and one of its end-products, the excitotoxin quinolinic acid (QUIN), are involved in the pathogenesis of several major neuroinflammatory brain diseases. A relevant animal model to study KP metabolism is now needed to assess whether intervention in this pathway may improve the outcome of such diseases. Humans and macaques share a very similar genetic makeup. In this study, we characterized the KP metabolism in macaque primary macrophages of three different species in comparison to human cells. We found that the KP profiles in simian macrophages were very similar to those in humans when challenged with inflammatory cytokines. Further, we found that macaque macrophages are capable of producing a pathophysiological concentration of QUIN. Our data validate the simian model as a relevant model to study the human cellular KP metabolism in the context of inflammation.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Braidy, N" uniqKey="Braidy N">N Braidy</name></author><author><name sortKey="Grant, R" uniqKey="Grant R">R Grant</name></author><author><name sortKey="Brew, Bj" uniqKey="Brew B">BJ Brew</name></author><author><name sortKey="Adams, S" uniqKey="Adams S">S Adams</name></author><author><name sortKey="Jayasena, T" uniqKey="Jayasena T">T Jayasena</name></author><author><name sortKey="Guillemin, Gj" uniqKey="Guillemin G">GJ Guillemin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Munn, Dh" uniqKey="Munn D">DH Munn</name></author><author><name sortKey="Zhou, M" uniqKey="Zhou M">M Zhou</name></author><author><name sortKey="Attwood, Jt" uniqKey="Attwood J">JT 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Tryptophan Res</journal-id><journal-id journal-id-type="iso-abbrev">Int J Tryptophan Res</journal-id><journal-title-group><journal-title>International Journal of Tryptophan Research : IJTR</journal-title></journal-title-group><issn pub-type="epub">1178-6469</issn><publisher><publisher-name>Libertas Academica</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23761975</article-id><article-id pub-id-type="pmc">3662399</article-id><article-id pub-id-type="doi">10.4137/IJTR.S11789</article-id><article-id pub-id-type="publisher-id">ijtr-6-2013-007</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Research</subject></subj-group></article-categories><title-group><article-title>Characterization of the Kynurenine Pathway and Quinolinic Acid Production in Macaque Macrophages</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Lim</surname><given-names>Chai K.</given-names></name><xref ref-type="aff" rid="af1-ijtr-6-2013-007">1</xref><xref ref-type="aff" rid="af2-ijtr-6-2013-007">2</xref></contrib><contrib contrib-type="author"><name><surname>Yap</surname><given-names>Margaret M.C.</given-names></name><xref ref-type="aff" rid="af3-ijtr-6-2013-007">3</xref></contrib><contrib contrib-type="author"><name><surname>Kent</surname><given-names>Stephen J.</given-names></name><xref ref-type="aff" rid="af4-ijtr-6-2013-007">4</xref></contrib><contrib contrib-type="author"><name><surname>Gras</surname><given-names>Gabriel</given-names></name><xref ref-type="aff" rid="af5-ijtr-6-2013-007">5</xref></contrib><contrib contrib-type="author"><name><surname>Samah</surname><given-names>Boubekeur</given-names></name><xref ref-type="aff" rid="af5-ijtr-6-2013-007">5</xref></contrib><contrib contrib-type="author"><name><surname>Batten</surname><given-names>Jane C.</given-names></name><xref ref-type="aff" rid="af4-ijtr-6-2013-007">4</xref></contrib><contrib contrib-type="author"><name><surname>De Rose</surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="af4-ijtr-6-2013-007">4</xref></contrib><contrib contrib-type="author"><name><surname>Heng</surname><given-names>Benjamin</given-names></name><xref ref-type="aff" rid="af2-ijtr-6-2013-007">2</xref></contrib><contrib contrib-type="author"><name><surname>Brew</surname><given-names>Bruce J.</given-names></name><xref ref-type="aff" rid="af3-ijtr-6-2013-007">3</xref><xref ref-type="aff" rid="af6-ijtr-6-2013-007">6</xref></contrib><contrib contrib-type="author"><name><surname>Guillemin</surname><given-names>Gilles J.</given-names></name><xref ref-type="aff" rid="af1-ijtr-6-2013-007">1</xref><xref ref-type="aff" rid="af2-ijtr-6-2013-007">2</xref><xref ref-type="corresp" rid="c1-ijtr-6-2013-007"></xref></contrib></contrib-group><aff id="af1-ijtr-6-2013-007"><label>1</label>Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia.</aff><aff id="af2-ijtr-6-2013-007"><label>2</label>MND and Neurodegenerative disease Research Group, Australian School of Advance Medicine, Macquarie University, Australia.</aff><aff id="af3-ijtr-6-2013-007"><label>3</label>Applied Neurosciences, St. Vincent’s Center for Applied Medical Research, Sydney, Australia.</aff><aff id="af4-ijtr-6-2013-007"><label>4</label>Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.</aff><aff id="af5-ijtr-6-2013-007"><label>5</label>Laboratoire de Neuro-Immuno-Virologie, Service de Neurovirologie UMR E-01 CEA, France and UMR E1, School of Medicine, University Paris South 11, Le Kremlin-Bicêtre, France.</aff><aff id="af6-ijtr-6-2013-007"><label>6</label>Department of Neurology, St. Vincent’s Hospital, Darlinghurst, Australia.</aff><author-notes><corresp id="c1-ijtr-6-2013-007">Corresponding author email: <email>gilles.guillemin@mq.edu.au</email></corresp></author-notes><pub-date pub-type="collection"><year>2013</year></pub-date><pub-date pub-type="epub"><day>15</day><month>5</month><year>2013</year></pub-date><volume>6</volume><fpage>7</fpage><lpage>19</lpage><permissions><copyright-statement>© 2013 the author(s), publisher and licensee Libertas Academica Ltd.</copyright-statement><copyright-year>2013</copyright-year><license license-type="open-access"><license-p>This is an open access article published under the Creative Commons CC-BY-NC 3.0 license.</license-p></license></permissions><abstract><p>The kynurenine pathway (KP) and one of its end-products, the excitotoxin quinolinic acid (QUIN), are involved in the pathogenesis of several major neuroinflammatory brain diseases. A relevant animal model to study KP metabolism is now needed to assess whether intervention in this pathway may improve the outcome of such diseases. Humans and macaques share a very similar genetic makeup. In this study, we characterized the KP metabolism in macaque primary macrophages of three different species in comparison to human cells. We found that the KP profiles in simian macrophages were very similar to those in humans when challenged with inflammatory cytokines. Further, we found that macaque macrophages are capable of producing a pathophysiological concentration of QUIN. Our data validate the simian model as a relevant model to study the human cellular KP metabolism in the context of inflammation.</p></abstract><kwd-group><kwd>simian macrophages</kwd><kwd>kynurenine pathway</kwd><kwd>indoleamine 2</kwd><kwd>3-dioxygenase</kwd><kwd>quinolinic acid</kwd></kwd-group></article-meta></front><floats-group><fig id="f1-ijtr-6-2013-007" position="float"><label>Figure 1</label><caption><p>Simplified version of the kynurenine pathway.</p></caption><graphic xlink:href="ijtr-6-2013-007f1"></graphic></fig><fig id="f2-ijtr-6-2013-007" position="float"><label>Figure 2</label><caption><p>Macrophages cultures of human (<bold>A</bold>) and pigtail macaque (<bold>B</bold>) at 7 day in vitro. Pictures were taken with phase contrast microscope, at 400× magnification. Bar scale = 10 μm. Immunocytochemical stains for macrophage identification with surface marker, CD-68 on isolated (<bold>C</bold>) human, (<bold>D</bold>) pigtail macaque, (<bold>E</bold>) cynomolgus macaque and (<bold>F</bold>) rhesus macaque macrophage cultures.</p></caption><graphic xlink:href="ijtr-6-2013-007f2"></graphic></fig><fig id="f3-ijtr-6-2013-007" position="float"><label>Figure 3</label><caption><p>Expression of the kynurenine pathway enzymes using RT-PCR in macrophages stimulated with IFN-γ for 24 h. (<bold>A</bold>) Ethidium bromide-stained gels showing RT-PCR for IDO-1, TDO-2, KAT-2, KYNU, KMO, 3-HAO, QPRT, ACMSD and GAPDH expression (top to bottom). The first column corresponds to human macrophages, the second to pigtail macaque macrophages, the third to cynomolgus macaque macrophages and fourth to rhesus macaque macrophages. (<bold>B</bold>) The histograms indicate the ratio of the expression of the eight kynurenine pathway enzyme mRNA relative to the GAPDH mRNA expression. Each column of the histogram also corresponds to the columns in (<bold>A</bold>).</p><p><bold>Note:</bold> Individual samples were analyzed in triplicate, and the SEM for all data was determined to be ±7%.</p></caption><graphic xlink:href="ijtr-6-2013-007f3"></graphic></fig><fig id="f4-ijtr-6-2013-007" position="float"><label>Figure 4</label><caption><p>Immunodetection of various KP enzymes and QUIN in (<bold>A</bold>) human, (<bold>B</bold>) pigtail macaque, (<bold>C</bold>) cynomolgus macaque and (<bold>D</bold>) rhesus macaque macrophages. (<bold>E</bold>) Control preparations of cultures of human macrophages: without IFN-γ stimulation, without antibodies (unlabelled), irrelevant primary antibodies and secondary antibodies only.</p></caption><graphic xlink:href="ijtr-6-2013-007f4"></graphic></fig><fig id="f5-ijtr-6-2013-007" position="float"><label>Figure 5</label><caption><p>Histogram showing levels of tryptophan (<bold>A</bold>) kynurenine (<bold>B</bold>) and the level of KP activation reflected by K/T ratio (<bold>C</bold>) in human and simian macrophages using HPLC.</p><p><bold>Notes:</bold> Controls (Cont) were untreated macrophages cultures whereas interferon-γ (IFN- γ) stimulated macrophages were treated for 24, 48 and 72 h. Only IFN-γ stimulated macrophages cultures for 24, 48 and 72 h results were presented in (C) to illustrate KP activation in time-dependent manner to IFN-γ stimulation. Statistical differences comparing (i) the untreated and its counterpart IFN-γ-treated conditions are denoted by <sup>*</sup>; (ii) the changes in concentration to 24 h time point are denoted by # and; (iii) the change in concentration detected at the 48 or 72 h compared to 24 h time point are denoted by $. One-symbol, two-symbol and three-symbols are indicated as <italic>P</italic> < 0.05; <italic>P</italic> < 0.01 and <italic>P</italic> < 0.0001, respectively (Dunnett’s test).</p></caption><graphic xlink:href="ijtr-6-2013-007f5"></graphic></fig><fig id="f6-ijtr-6-2013-007" position="float"><label>Figure 6</label><caption><p>Histogram showing de novo synthesis of quinolinic acid (QUIN; <bold>A</bold>) and picolinic acid (PIC; <bold>B</bold>) in human and simian macrophages using GCMS.</p><p><bold>Notes:</bold> Control (Cont) were untreated macrophages cultures whereas interferon-γ (IFN-γ) stimulated macrophages were treated for 24, 48 and 72 h. Statistical differences comparing the untreated and its counterpart IFN-γ-treated conditions are denoted by <sup>*</sup> whereas the change in concentration detected at the 48 or 72 h compared to 24 h time point are denoted by $. One-symbol, two-symbol and three-symbol are indicated as <italic>P</italic> < 0.05; <italic>P</italic> < 0.01 and <italic>P</italic> < 0.0001, respectively (Dunnett’s test).</p></caption><graphic xlink:href="ijtr-6-2013-007f6"></graphic></fig><table-wrap id="t1-ijtr-6-2013-007" position="float"><label>Table 1</label><caption><p>Details of primary and secondary antibodies used in immunocytochemistry.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Primary antibodies</th><th align="left" valign="top" rowspan="1" colspan="1">Clonality</th><th align="left" valign="top" rowspan="1" colspan="1">Isotype</th><th align="left" valign="top" rowspan="1" colspan="1">Dilution</th><th align="left" valign="top" rowspan="1" colspan="1">Company (Cat. #)</th><th align="left" valign="top" rowspan="1" colspan="1">Secondary antibodies</th><th align="left" valign="top" rowspan="1" colspan="1">Dilution</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">IDO-1</td><td align="left" valign="top" rowspan="1" colspan="1">Monoclonal</td><td align="left" valign="top" rowspan="1" colspan="1">IgG</td><td align="left" valign="top" rowspan="1" colspan="1">1:200</td><td align="left" valign="top" rowspan="1" colspan="1">Gift from O. Takikawa</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 594</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">TDO-2</td><td align="left" valign="top" rowspan="1" colspan="1">Polyclonal</td><td align="left" valign="top" rowspan="1" colspan="1">–</td><td align="left" valign="top" rowspan="1" colspan="1">1:100</td><td align="left" valign="top" rowspan="1" colspan="1">Gift from C. Miller</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 488</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">KAT-2/AADAT</td><td align="left" valign="top" rowspan="1" colspan="1">Polyclonal</td><td align="left" valign="top" rowspan="1" colspan="1">–</td><td align="left" valign="top" rowspan="1" colspan="1">1:100</td><td align="left" valign="top" rowspan="1" colspan="1">Abnova (H00051166-A01)</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 488</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">KMO</td><td align="left" valign="top" rowspan="1" colspan="1">Polyclonal</td><td align="left" valign="top" rowspan="1" colspan="1">IgG</td><td align="left" valign="top" rowspan="1" colspan="1">1:100</td><td align="left" valign="top" rowspan="1" colspan="1">Abcam (ab93195)</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 594</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">KYNU</td><td align="left" valign="top" rowspan="1" colspan="1">Polyclonal</td><td align="left" valign="top" rowspan="1" colspan="1">–</td><td align="left" valign="top" rowspan="1" colspan="1">1:100</td><td align="left" valign="top" rowspan="1" colspan="1">Abnova (H00008942-A01)</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 488</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">QPRT</td><td align="left" valign="top" rowspan="1" colspan="1">Monoclonal clone 5D11</td><td align="left" valign="top" rowspan="1" colspan="1">IgG<sub>1</sub></td><td align="left" valign="top" rowspan="1" colspan="1">1:200</td><td align="left" valign="top" rowspan="1" colspan="1">Abnova (H00023475-M01)</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 594</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">ACMSD</td><td align="left" valign="top" rowspan="1" colspan="1">Polyclonal</td><td align="left" valign="top" rowspan="1" colspan="1">–</td><td align="left" valign="top" rowspan="1" colspan="1">1:100</td><td align="left" valign="top" rowspan="1" colspan="1">Abnova (H00130013-A01)</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 488</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">QUIN</td><td align="left" valign="top" rowspan="1" colspan="1">Monoclonal</td><td align="left" valign="top" rowspan="1" colspan="1">IgG</td><td align="left" valign="top" rowspan="1" colspan="1">1:20</td><td align="left" valign="top" rowspan="1" colspan="1">Millipore</td><td align="left" valign="top" rowspan="1" colspan="1">Alexa fluor 594</td><td align="left" valign="top" rowspan="1" colspan="1">1:400</td></tr></tbody></table><table-wrap-foot><fn id="tfn1-ijtr-6-2013-007"><p><bold>Note:</bold> All secondary antibodies used in this study were purchased from Life Technologies.</p></fn></table-wrap-foot></table-wrap><table-wrap id="t2-ijtr-6-2013-007" position="float"><label>Table 2</label><caption><p>Comparison of homology for various KP enzymes between rhesus macaque and human.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">KP enzymes</th><th align="left" valign="top" rowspan="1" colspan="1">Spieces</th><th align="left" valign="top" rowspan="1" colspan="1">Accession</th><th align="left" valign="top" rowspan="1" colspan="1">Size (basepair)</th><th align="left" valign="top" rowspan="1" colspan="1">Homology (%)</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">IDO-1</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM001077483</td><td align="left" valign="top" rowspan="1" colspan="1">1392</td><td align="left" valign="top" rowspan="1" colspan="1">95</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM-002164</td><td align="left" valign="top" rowspan="1" colspan="1">1944</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">TDO-2</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XR_002804253</td><td align="left" valign="top" rowspan="1" colspan="1">1551</td><td align="left" valign="top" rowspan="1" colspan="1">95</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_005651.3</td><td align="left" valign="top" rowspan="1" colspan="1">1703</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">KAT-2</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XM_001082463.2</td><td align="left" valign="top" rowspan="1" colspan="1">1519</td><td align="left" valign="top" rowspan="1" colspan="1">95</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_001008661.2</td><td align="left" valign="top" rowspan="1" colspan="1">2165</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">KYNU</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XM_002798863.1</td><td align="left" valign="top" rowspan="1" colspan="1">1463</td><td align="left" valign="top" rowspan="1" colspan="1">95</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_001199241.1</td><td align="left" valign="top" rowspan="1" colspan="1">1774</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">KMO</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XM_001093900.2</td><td align="left" valign="top" rowspan="1" colspan="1">2361</td><td align="left" valign="top" rowspan="1" colspan="1">90</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_003679.4</td><td align="left" valign="top" rowspan="1" colspan="1">5266</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">3-HAO</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XM_001111024.2</td><td align="left" valign="top" rowspan="1" colspan="1">1281</td><td align="left" valign="top" rowspan="1" colspan="1">96</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_012205.2</td><td align="left" valign="top" rowspan="1" colspan="1">1301</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">QPRT</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XM_001097266.2<sup>*</sup></td><td align="left" valign="top" rowspan="1" colspan="1">888<sup>*</sup></td><td align="left" valign="top" rowspan="1" colspan="1">96</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_014298.3</td><td align="left" valign="top" rowspan="1" colspan="1">1575</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">ACMSD</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">XM_001097996.2</td><td align="left" valign="top" rowspan="1" colspan="1">1369</td><td align="left" valign="top" rowspan="1" colspan="1">95</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_138326.2</td><td align="left" valign="top" rowspan="1" colspan="1">1278</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">GAPDH</td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Macaca Mulatta</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_001195426.1</td><td align="left" valign="top" rowspan="1" colspan="1">1106</td><td align="left" valign="top" rowspan="1" colspan="1">97</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1"></td><td align="left" valign="top" rowspan="1" colspan="1"><italic>Homo Sapien</italic></td><td align="left" valign="top" rowspan="1" colspan="1">NM_002046.4</td><td align="left" valign="top" rowspan="1" colspan="1">1401</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr></tbody></table></table-wrap><table-wrap id="t3A-ijtr-6-2013-007" position="float"><label>Table 3A</label><caption><p>Primers sequences used to target KP enzymes for both simian and human cDNA.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Gene</th><th align="left" valign="top" rowspan="1" colspan="1">Forward</th><th align="left" valign="top" rowspan="1" colspan="1">Reverse</th><th align="left" valign="top" rowspan="1" colspan="1">Band size</th><th align="left" valign="top" rowspan="1" colspan="1">Temp. (°C)</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">1. IDO-1</td><td align="left" valign="top" rowspan="1" colspan="1">ggcccaaagaagtttgcag</td><td align="left" valign="top" rowspan="1" colspan="1">ggaagttcctgtgagctggt</td><td align="left" valign="top" rowspan="1" colspan="1">160</td><td align="left" valign="top" rowspan="1" colspan="1">60.0</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">2. TDO-2</td><td align="left" valign="top" rowspan="1" colspan="1">cagacagtgccttttcacca</td><td align="left" valign="top" rowspan="1" colspan="1">gtgcatccgagaaacaacct</td><td align="left" valign="top" rowspan="1" colspan="1">367</td><td align="left" valign="top" rowspan="1" colspan="1">60.8</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">3. KAT-2</td><td align="left" valign="top" rowspan="1" colspan="1">tgcatcagcgatgaggttta</td><td align="left" valign="top" rowspan="1" colspan="1">ttcttcagcagcatccagtg</td><td align="left" valign="top" rowspan="1" colspan="1">621</td><td align="left" valign="top" rowspan="1" colspan="1">59.7</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">4. KYNU</td><td align="left" valign="top" rowspan="1" colspan="1">ggaagcgtccttggattaca</td><td align="left" valign="top" rowspan="1" colspan="1">atcgtatgggcatgcttttc</td><td align="left" valign="top" rowspan="1" colspan="1">589</td><td align="left" valign="top" rowspan="1" colspan="1">60.8</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">5. KMO</td><td align="left" valign="top" rowspan="1" colspan="1">ccaagcttcaatctgcacac</td><td align="left" valign="top" rowspan="1" colspan="1">caaacggagttgattgctca</td><td align="left" valign="top" rowspan="1" colspan="1">535</td><td align="left" valign="top" rowspan="1" colspan="1">57.3</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">6. 3-HAO</td><td align="left" valign="top" rowspan="1" colspan="1">aagaagcccctgtggtgac</td><td align="left" valign="top" rowspan="1" colspan="1">cacaaacaggatgcccagta</td><td align="left" valign="top" rowspan="1" colspan="1">506</td><td align="left" valign="top" rowspan="1" colspan="1">59.7</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">7. QPRT</td><td align="left" valign="top" rowspan="1" colspan="1">caactgccaagtctcctggt</td><td align="left" valign="top" rowspan="1" colspan="1">ctccaccttcagagcgaagt</td><td align="left" valign="top" rowspan="1" colspan="1">397</td><td align="left" valign="top" rowspan="1" colspan="1">58.0</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">8. ACMSD</td><td align="left" valign="top" rowspan="1" colspan="1">agctaccccaggaggtttgt</td><td align="left" valign="top" rowspan="1" colspan="1">gcaaatggctatggtggtct</td><td align="left" valign="top" rowspan="1" colspan="1">297</td><td align="left" valign="top" rowspan="1" colspan="1">58.0</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">9. GAPDH</td><td align="left" valign="top" rowspan="1" colspan="1">acccagaagactgtggatgg</td><td align="left" valign="top" rowspan="1" colspan="1">cttggcaggtttttccagac</td><td align="left" valign="top" rowspan="1" colspan="1">213</td><td align="left" valign="top" rowspan="1" colspan="1">60.0</td></tr></tbody></table></table-wrap><table-wrap id="t3B-ijtr-6-2013-007" position="float"><label>Table 3B</label><caption><p>Summary of PCR run sequences.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" valign="top" rowspan="1" colspan="1">Cycle repeats on step (2): 35</th><th align="left" valign="top" rowspan="1" colspan="1">Temp. (°C)</th><th align="left" valign="top" rowspan="1" colspan="1">Run time (min.)</th><th align="left" valign="top" rowspan="1" colspan="1">Steps</th></tr></thead><tbody><tr><td align="left" valign="top" rowspan="1" colspan="1">Denaturation</td><td align="left" valign="top" rowspan="1" colspan="1">94</td><td align="left" valign="top" rowspan="1" colspan="1">4.5</td><td align="left" valign="top" rowspan="1" colspan="1">1</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Denaturation</td><td align="left" valign="top" rowspan="1" colspan="1">94</td><td align="left" valign="top" rowspan="1" colspan="1">1</td><td align="left" valign="top" rowspan="1" colspan="1">2</td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Hybridization</td><td align="left" valign="top" rowspan="1" colspan="1">Specify in <xref ref-type="table" rid="t3A-ijtr-6-2013-007">Table 3A</xref></td><td align="left" valign="top" rowspan="1" colspan="1">1</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Extension</td><td align="left" valign="top" rowspan="1" colspan="1">72</td><td align="left" valign="top" rowspan="1" colspan="1">1.5</td><td align="left" valign="top" rowspan="1" colspan="1"></td></tr><tr><td align="left" valign="top" rowspan="1" colspan="1">Extension</td><td align="left" valign="top" rowspan="1" colspan="1">72</td><td align="left" valign="top" rowspan="1" colspan="1">10</td><td align="left" valign="top" rowspan="1" colspan="1">3</td></tr></tbody></table></table-wrap></floats-group></pmc><affiliations><list><country><li>Australie</li><li>France</li></country><region><li>Nouvelle-Galles du Sud</li><li>Victoria (État)</li></region><settlement><li>Melbourne</li><li>Sydney</li></settlement><orgName><li>Université de Melbourne</li></orgName></list><tree><country name="Australie"><region name="Nouvelle-Galles du Sud"><name sortKey="Lim, Chai K" sort="Lim, Chai K" uniqKey="Lim C" first="Chai K." last="Lim">Chai K. Lim</name></region><name sortKey="Batten, Jane C" sort="Batten, Jane C" uniqKey="Batten J" first="Jane C." last="Batten">Jane C. Batten</name><name sortKey="Brew, Bruce J" sort="Brew, Bruce J" uniqKey="Brew B" first="Bruce J." last="Brew">Bruce J. Brew</name><name sortKey="Brew, Bruce J" sort="Brew, Bruce J" uniqKey="Brew B" first="Bruce J." last="Brew">Bruce J. Brew</name><name sortKey="De Rose, Robert" sort="De Rose, Robert" uniqKey="De Rose R" first="Robert" last="De Rose">Robert De Rose</name><name sortKey="Guillemin, Gilles J" sort="Guillemin, Gilles J" uniqKey="Guillemin G" first="Gilles J." last="Guillemin">Gilles J. Guillemin</name><name sortKey="Guillemin, Gilles J" sort="Guillemin, Gilles J" uniqKey="Guillemin G" first="Gilles J." last="Guillemin">Gilles J. Guillemin</name><name sortKey="Heng, Benjamin" sort="Heng, Benjamin" uniqKey="Heng B" first="Benjamin" last="Heng">Benjamin Heng</name><name sortKey="Kent, Stephen J" sort="Kent, Stephen J" uniqKey="Kent S" first="Stephen J." last="Kent">Stephen J. Kent</name><name sortKey="Lim, Chai K" sort="Lim, Chai K" uniqKey="Lim C" first="Chai K." last="Lim">Chai K. Lim</name><name sortKey="Yap, Margaret M C" sort="Yap, Margaret M C" uniqKey="Yap M" first="Margaret M. C." last="Yap">Margaret M. C. Yap</name></country><country name="France"><noRegion><name sortKey="Gras, Gabriel" sort="Gras, Gabriel" uniqKey="Gras G" first="Gabriel" last="Gras">Gabriel Gras</name></noRegion><name sortKey="Samah, Boubekeur" sort="Samah, Boubekeur" uniqKey="Samah B" first="Boubekeur" last="Samah">Boubekeur Samah</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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