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Diazotization of kynurenine by acidified nitrite secreted from indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells.
Hara, Toshiaki; Yamakura, Fumiyuki; Takikawa, Osamu; Hiramatsu, Rie; Kawabe, Tsutomu; Isobe, Ken-Ichi; Nagase, Fumihiko.
Afiliación
  • Hara T; Department of Medical Technology, Nagoya University School of Health Sciences, Nagoya, Aichi, Japan.
J Immunol Methods ; 332(1-2): 162-9, 2008 Mar 20.
Article en En | MEDLINE | ID: mdl-18158157
ABSTRACT
Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway regulates T-cell responses in some dendritic cells (DC) such as plasmacytoid DC. A Kyn assay using HPLC showed that samples were frequently deproteinized with trichloroacetic acid (TCA). In the present study, bone marrow-derived myeloid DC (BMDC) were differentiated from mouse bone marrow cells with GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with NO production in BMDC cultured for 24 h. The concentrations of Kyn in the culture supernatants were not increased by stimulation with CpG but rather decreased by based on the Kyn assay after deproteinization with TCA. The level of Kyn exogenously added into the cell-free culture supernatant of BMDC stimulated with CpG was severely decreased by deproteinization with TCA but not methanol, and the decrease was prevented when BMDC was stimulated with CpG in the presence of a NOS inhibitor. Under acidic conditions, Kyn reacted with nitrite produced by BMDC, and generated a new compound that was not detected by Ehrlich reagent reacting with the aromatic amino residue of Kyn. An analysis by mass spectrometry showed the new compound to be a diazotization form of Kyn. In conclusion, the deproteinization of samples by acidic treatment should be avoided for the Kyn assay when NO is produced.
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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Compuestos Azo / Células Dendríticas / Indolamina-Pirrol 2,3,-Dioxigenasa / Quinurenina / Nitritos Tipo de estudio: Diagnostic_studies Límite: Animals Idioma: En Revista: J Immunol Methods Año: 2008 Tipo del documento: Article País de afiliación: Japón Pais de publicación: HOLANDA / HOLLAND / NETHERLANDS / NL / PAISES BAJOS / THE NETHERLANDS
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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Compuestos Azo / Células Dendríticas / Indolamina-Pirrol 2,3,-Dioxigenasa / Quinurenina / Nitritos Tipo de estudio: Diagnostic_studies Límite: Animals Idioma: En Revista: J Immunol Methods Año: 2008 Tipo del documento: Article País de afiliación: Japón Pais de publicación: HOLANDA / HOLLAND / NETHERLANDS / NL / PAISES BAJOS / THE NETHERLANDS