Effects of neutron radiation generated in deep space-like environments on food resources.

The impact of deep space cosmic rays on food resources is as important as the risks of cosmic rays to the human body. This study demonstrates the potential for neutrons as secondary radiation in deep space spacecraft to cause meat activation and oxidative modification of proteins and lipids. We conducted a series of experiments such as the neutron irradiation experiment, the radioactivation analysis and the biochemical analysis. Neutrons with energies from 1 to 5 MeV with doses from 0.01 Gy to 4 Gy were irradiated by the RIKEN accelerated-driven neutron source (RANS). Radioactive nuclei, 24Na, 42K, and 38Cl, were detected in the neutron-irradiated meat. The modification products of the proteins by oxidative nitration, 6-nitrotryptophan (6NO2Trp), and by a lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE), were detected in several proteins with neutron dose dependent. The proteome analysis showed that many oxidative modifications were detected in actin and myosin which are major proteins of myofibrils. This study is of crucial importance not only as risk factors for human space exploration, but also as fundamental effects of radiation on the components of the human body.

Tryptophan nitration of immunoglobulin light chain as a new possible biomarker for atopic dermatitis.

To reduce the incidence and severity of atopic dermatitis, detection and treatment at an early stage are urgently required, but no effective biomarker has been reported. In this study, we attempted to detect a candidate biomarker of early stage atopic dermatitis by focusing on the levels of nitrated residues in the plasma proteins of atopic dermatitis model mice (NC/Nga mice). We found that the immunoglobulin (Ig) light chain was more highly nitrated in the plasma of the animal model than that of control mice. Western blot analysis showed a statistically significant difference between the 6-nitrotryptophan content of the Ig light chain in the NC/Nga mice before onset of atopic dermatitis symptoms and that of the control mice. LC-ESI-MS/MS analysis demonstrated that these light chains contained nitrotryptophan (Trp56) and nitrotyrosine (Tyr66). Immunofluorescence staining revealed a significant increase in manganese superoxide dismutase and inducible nitric oxide synthase production in the skin lesions of the NC/Nga mice. Furthermore, we found protein-bound 6-nitrotryptophan and 3-nitrotyrosine only in the lesioned skin, where their signals partially overlapped with the IgG signal. Our findings suggest that the 6-nitrotryptophan content of Ig light chains could be a new biomarker for detecting early stage atopic dermatitis.

Accumulation of immunoglobulin G against Dermatophagoides farinae tropomyosin in dorsal root ganglia of NC/Nga mice with atopic dermatitis-like symptoms.

Atopic dermatitis (AD), a chronic inflammatory skin disease, manifests as intractable itch, but its underlying mechanisms are poorly understood. This study assessed the relationship between immunoglobulin G (IgG) and dorsal root ganglia (DRG) in NC/Nga mice, a model of AD that manifests AD-like symptoms including itch. Immunohistochemical analysis showed large amounts of IgG in DRG extracts of NC/Nga mice with AD-like dermatitis, with a large fraction of the IgG distributed in satellite glial cells of the DRG. Proteomic analysis showed that this IgG was reactive against tropomyosin of Dermatophagoides farinae. These findings indicate that the accumulation of anti-tropomyosin IgG in DRG of atopic NC/Nga mice may be associated with the pathogenesis of AD-like symptoms, including itch.

Peroxynitrite-mediated oxidation of plasma fibronectin.

Fibronectin is a large dimeric glycoprotein present in both human plasma and in basement membranes. The latter are specialized extracellular matrices underlying endothelial cells in the artery wall. Peroxynitrous acid (ONOOH) a potent oxidizing and nitrating agent, is formed in vivo from superoxide and nitric oxide radicals by stimulated macrophages and other cells. Considerable evidence supports ONOOH involvement in human atherosclerotic lesion development and rupture, possibly via extracellular matrix damage. Here we demonstrate that Tyr and Trp residues on human plasma fibronectin are highly sensitive to ONOOH with this resulting in the formation of 3-nitrotyrosine, 6-nitrotryptophan and dityrosine as well as protein aggregation and fragmentation. This occurs with equimolar or greater levels of oxidant, and in a dose-dependent manner. Modification of Tyr was quantitatively more significant than Trp (9.1% versus 1.5% conversion with 500µM ONOOH) after accounting for parent amino acid abundance, but only accounts for a small percentage of the total oxidant added. LC-MS studies identified 28 nitration sites (24 Tyr, 4 Trp) with many of these present within domains critical to protein function, including the cell-binding and anastellin domains. Human coronary artery endothelial cells showed decreased adherence and cell-spreading on ONOOH-modified fibronectin compared to control, consistent with cellular dysfunction induced by the modified matrix. Studies on human atherosclerotic lesions have provided evidence for co-localization of 3-nitrotyrosine and fibronectin. ONOOH-mediated fibronectin modification and compromised cell-matrix interactions, may contribute to endothelial cell dysfunction, a weakening of the fibrous cap of atherosclerotic lesions, and an increased propensity to rupture.

Electrophysiological properties of brain-natriuretic peptide- and gastrin-releasing peptide-responsive dorsal horn neurons in spinal itch transmission.

Spinal itch transmission has been reported to be mediated by at least two neuronal populations in spinal dorsal horn, neurons expressing brain-natriuretic peptide (BNP) receptor (Npra) and gastrin-releasing peptide (GRP) receptor (GRPR). Although Npra-expressing neurons were shown to be upstream of GRPR- expressing neurons in spinal itch transmission, the roles of BNP and GRP in the spinal neurotransmission of histamine-dependent and -independent itch remains unclear. Using in vivo electrophysiology and behavior analysis, this study examined the responses of chloroquine (histamine-independent pruritogen)-responsive and histamine-responsive dorsal horn neurons to spinal applications of BNP and GRP. Electrophysiologically, 9.5% of chloroquine-responsive neurons responded to BNP, 33.3% to GRP, and 4.8% to both, indicating that almost half of chloroquine-responsive neurons were unresponsive to both BNP and GRP. In contrast, histamine-responsive neurons did not respond to spinal BNP application, whereas 30% responded to spinal GRP application, indicating that 70% of histamine-responsive neurons were unresponsive to both BNP and GRP. Behavioral analyses showed differences in the time-course and frequency of scratching responses evoked by intrathecal BNP and GRP. These findings provide evidence that most BNP-Npra and GRP-GRPR signaling involve different pathways of spinal itch transmission, and that multiple neurotransmitters, in addition to BNP and GRP, are involved in spinal itch transmission. The electrophysiological results also suggest that spinal BNP contributes little to histaminergic itch directly.

Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

Sumoylated α-skeletal muscle actin in the skeletal muscle of adult rats.

Skeletal muscles are composed of two major muscle fiber types: slow-twitch oxidative fibers and fast-twitch glycolytic fibers. The proteins in these muscle fibers are known to differ in their expression, relative abundance, and post-translational modifications. In this study, we report a previously unreported post-translational modification of α-skeletal muscle actin in the skeletal muscles of adult male F344 rats in vivo. Using two-dimensional electrophoresis (2D-PAGE), we first examined the differences in the protein expression profiles between the soleus and plantaris muscles. We found higher intensity protein spots at approximately 60 kDa and pH 9 on 2D-PAGE for the soleus muscle compared with the plantaris muscle. These spots were identified as α-skeletal muscle actin by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry and western blot analyses. In addition, we found that the 60 kDa α-skeletal muscle actin is modified by small ubiquitin-like modifier (SUMO) 1, using 2D-PAGE and western blot analyses. Furthermore, we found that α-skeletal muscle actin with larger molecular weight was localized in the nuclear and cytosol of the skeletal muscle, but not in the myofibrillar fraction by the combination of subcellular fractionation and western blot analyses. These results suggest that α-skeletal muscle actin is modified by SUMO-1 in the skeletal muscles, localized in nuclear and cytosolic fractions, and the extent of this modification is much higher in the slow muscles than in the fast muscles. This is the first study to show the presence of SUMOylated actin in animal tissues.

Importance of tryptophan nitration of carbonic anhydrase III for the morbidity of atopic dermatitis.

The nitration of proteins results from the vigorous production of reactive nitrogen species in inflammatory disease. We previously reported the proteomic analysis of nitrated tryptophan residues in in vitro model cells for inflammatory diseases using a 6-nitrotryptophan-specific antibody. In this paper, we applied this method to the analysis of a disease model animal and identified the 6-nitrotryptophan-containing proteins in the skin of atopic dermatitis model mice (AD-NC/Nga mice). We found three nitrotryptophan-containing proteins, namely, carbonic anhydrase III (CAIII), α-enolase (α-ENO), and cytoskeletal keratin type II (KTII), and identified the positions of the nitrotryptophan residues in their amino acid sequences: Trp47 and Trp123 in CAIII, Trp365 in α-ENO, and Trp221 in KTII. Among these, the nitration of CAIII was increased not only in the lesional skin of AD-NC/Nga mice but also in the mice that did not present any symptoms. The in vitro nitration of purified CAIII by peroxynitrite reduced its CO2 hydratase activity in a dose-dependent manner. In addition, we found that CAIII was induced during the differentiation of normal human epidermal keratinocytes. Furthermore, we found the presence of CAIII and the formation of 6-nitrotryptophan-containing proteins in both the lesional and the nonlesional sections of the skin of patients with atopic dermatitis through immunohistochemical staining. This study provides the first demonstration of the formation of 6-nitrotryptophan in human tissues and disease.

Proteomic analysis of endogenous nitrotryptophan-containing proteins in rat hippocampus and cerebellum.

Nitration of tryptophan residues is a novel post-translational modification. In the present study, we examined whether NO2Trp (nitrotryptophan)-containing proteins are produced in the hippocampus and cerebellum of the adult rat under physiological conditions in vivo. Using Western blot analysis with anti-6-NO2Trp-specific antibody, we found many similar immunoreactive spots in the protein extracts from both regions. These spots were subsequently subjected to trypsin digestion and LC-ESI-MS/MS (LC-electrospray ionization-tandem MS) analysis. We identified several cytoskeletal proteins and glycolytic enzymes as NO2Trp-containing proteins and determined the position of nitrated tryptophan residues with significant ion score levels (P<0.05) in several proteins in both regions. We also observed that the total amount of NO2Trp-containing proteins in the cerebellum was significantly greater than that in the hippocampus (P<0.05). Moreover, IP (immunoprecipitation) assays using anti-aldolase C antibody showed that the relative intensity of immunostaining for NO2Trp over aldolase C was much higher in cerebellum than in hippocampus. The amounts of nNOS (neuronal nitric oxide synthase) and eNOS (endothelial nitric oxide synthase) were much greater in cerebellum than in hippocampus. This is the first evidence of several specific sites of nitrated tryptophan in proteins under physiological conditions in vivo.

Indolepyruvate ferredoxin oxidoreductase: An oxygen-sensitive iron-sulfur enzyme from the hyperthermophilic archaeon Thermococcus profundus.

Thermococcus profundus is a strictly anaerobic sulfur-dependent archaeon that grows optimally at 80°C by peptide fermentation. Indolepyruvate ferredoxin oxidoreductase (IOR), an enzyme involved in the peptide fermentation pathway, was purified to homogeneity from the archaeon under strictly anaerobic conditions. The maximal activity was obtained above the boiling temperature of water (105°C), with a half-life of 62min at 100°C and 20min at 105°C. IOR was oxygen-sensitive with a half-life of 7h at 25°C under aerobic conditions. The specific activity of T. profundus IOR was found to be dependent on the number of [4Fe-4S] clusters in the enzyme.

Nitration of tryptophan in ribosomal proteins is a novel post-translational modification of differentiated and naïve PC12 cells.

Neuron growth factor (NGF) signaling in PC12 cell, which is derived from pheochromocytoma of rat adrenal medulla, induces expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production. Subsequently, NO causes differentiation of PC12 cell to neuronal cell with morphological changes, such as neurite extension. In this study, we showed that 6-nitrotryptophan-containing proteins were produced in PC12 cell (naïve PC12 cell) and/or NGF-induced PC12 cell (differentiated PC12 cell). Western blot analysis of the protein extract of naïve PC12 cell and differentiated PC12 cell using anti 6-nitrotryptophan antibody showed several immunoreactive bands, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. The peptides from five ribosomal proteins, namely, 60S ribosomal protein L7 (Trp154), 60S acidic ribosomal protein P1 (Trp43), 40S ribosomal protein S2 (Trp60), 40S ribosomal protein S6 (Trp45), and 40S ribosomal protein S19 (Trp52), were identified as nitrotryptophan residue-containing proteins with significant ion score levels (p<0.05). Among these, tryptophan nitration was observed only in differentiated PC12 cell for S19 protein, and only in naïve PC12 cell for L7 protein. Tryptophan nitration of the other ribosomal proteins P1, S2, and S6 was observed in both naive and differentiated PC12 cells. The positive signal of nitrotryptophan-containing proteins in the Western blotting around 16 kDa (Band 1), which includes 40S ribosomal protein S19, was suppressed by treatment with NOS inhibitor, L-NAME. The tryptophan nitration of 40S ribosomal protein was not observed by LC-ESI-MS-MS analysis of this sample. This is the first study to identify several specific sites of nitrated tryptophan on proteins not only in viable culture cells but also in a physiological process: cell differentiation.

Mass spectrometric identification of tryptophan nitration sites on proteins in peroxynitrite-treated lysates from PC12 cells.

One of the important sites of peroxynitrite action that affects cellular function is known to be nitration of tyrosine residues. However, tryptophan residues could be another target of peroxynitrite-dependent modification of protein function, as we have shown previously using a model protein (F. Yamakura et al., J. Biochem. 138:57-69; 2005). Here, we report the identification of several proteins that allowed us to determine the position of nitrotryptophan in their amino acid sequences in a more complex system. We modified lysates from PC12 cells with and without nerve growth factor (NGF) by treatment with peroxynitrite (0.98 or 4.9 mM). Western blot analyses using anti-6-nitrotryptophan antibody showed several immunoreactive bands and spots, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. We identified several tryptic peptides including nitrotryptophan residues, which were derived from L-lactate dehydrogenase A, malate dehydrogenase 1, M2 pyruvate kinase, and heat-shock protein 90 α, in peroxynitrite-treated lysates from PC12 cells, and l-lactate dehydrogenase A, malate dehydrogenase 1, transaldorase, and lactoylglutathione lyase, in peroxynitrite-treated lysates from NGF/PC12 cells. The molar ratio of 3-nitrotyrosine to 6-nitrotryptophan in protease-digested PC12 cell lysates treated with peroxynitrite was determined to be 5.8 to 1 by using an HPLC-CoulArray system. This is the first report to identify several specific sites of nitrated tryptophan on proteins in a complex system treated with peroxynitrite and to compare the susceptibility of nitration between tryptophan and tyrosine residues of the proteins.

Effect of endurance exercise training on oxidative stress in spontaneously hypertensive rats (SHR) after emergence of hypertension.

The purpose of this study is to elucidate the effect of wheel training on oxidative stress maker levels in spontaneous hypertensive rats (SHR). 4-hydroxynonenal and 3-nitrotyrosine levels in the aorta of SHRs were allowed to run for 10 weeks from the age of 15 weeks were measured and compared with those of nonexercised SHRs. The 4-hydroxynonenal and 3-nitrotyrosine levels in the exercised group were significantly lower than those in the nonexercised group. The exercised group showed a significant increase of manganese-containing superoxide dismutase. Endurance exercise showed a possible suppressing effect on the arteriosclerosis development by reducing oxidative stress, even after emergence of hypertension.

Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr.

Glycine 165, which is located near the active site metal, is mostly conserved in aligned amino acid sequences of manganese-containing superoxide dismutase (Mn-SOD) proteins, but is substituted to threonine in most iron-containing SODs (Fe-SODs). Because threonine 165 is located between Trp128 and Trp130, and Trp128 is one of the metal-surrounding aromatic amino acids, the conversion of this amino acid may affect the metal-specific activity of Escherichia coli Mn-SOD. In order to clarify this possibility, we prepared a mutant of E. coli Mn-SOD with the replacement of Gly165 by Thr. The ratio of the specific activities of Mn- to Fe-reconstituted enzyme increased from 0.006 in the wild-type to 0.044 in the mutant SOD; therefore, the metal-specific SOD was converted to a metal-tolerant SOD. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs indicated the loss of Mn-SOD character. It was concluded that Gly at position 165 plays a catalytic role in maintaining the integrity of the metal specificity of Mn-SOD.

A new assignment technique of 2D-NMR spectra by spin-lock sequence to a tripeptide containing tryptophan in water.

We developed a new assignment technique of tryptophan residues using pulsed field gradient TOCSY-ROESY (PFG-TORO) and pulsed field gradient TOCSY-ROESY-TOCSY (PFG-TOROTO) techniques in water. Connectivity from betaH to zeta2H (H-7) via epsilon1H (H-1) and delta1H (H-2) in the TORO spectrum and from betaH to zeta3H (H-5) and eta2H (H-6) via epsilon1H (H-1) and delta1H (H-2) in the TOROTO spectrum could be able to assign each of the protons of the indole rings.

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei.

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

Post-translational modifications of superoxide dismutase.

Post-translational modifications of proteins control many biological processes through the activation, inactivation, or gain-of-function of the proteins. Recent developments in mass spectrometry have enabled detailed structural analyses of covalent modifications of proteins and also have shed light on the post-translational modification of superoxide dismutase. In this review, we introduce some covalent modifications of superoxide dismutase, nitration, phosphorylation, glutathionylaion, and glycation. Nitration has been the most extensively analyzed modification both in vitro and in vivo. Reaction of human Cu,Zn superoxide dismutase (SOD) with reactive nitrogen species resulted in nitration of a single tryptophan residue to 6-nitrotryptophan, which could be a new biomarker of a formation of reactive nitrogen species. On the other hand, tyrosine 34 of human MnSOD was exclusively nitrated to 3-nitrotyrosine and almost completely inactivated by the reaction with peroxynitrite. The nitrated MnSOD has been found in many diseases caused by ischemia/reperfusion, inflammation, and others and may have a pivotal role in the pathology of the diseases. Most of the post-translational modifications have given rise to a reduced activity of SOD. Since phosphorylation and nitration of SOD have been shown to have a possible reversible process, these modifications may be related to a redox signaling process in cells. Finally we briefly introduce a metal insertion system of SOD, focusing particularly on the iron misincorporation of nSOD, as a part of post-translational modifications.

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia.

We reported previously that Ascaris suum cytochrome b5, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b5 functions as an electron carrier between NADH-mediated cytochrome b5 reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b5, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of +7.2 mV and +19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b5 were comparable to that for mammalian hemoglobin and cytochrome b5; association constants were 0.585 x 10(3) M(-1) and 2.32 x 10(3) M(-1), respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.

Antimicrobial action of histone H2B in Escherichia coli: evidence for membrane translocation and DNA-binding of a histone H2B fragment after proteolytic cleavage by outer membrane proteinase T.

Previous studies have led to the isolation of histone H2B with antibacterial properties from an extract of the skin of the Schlegel's green tree frog Rhacophorus schlegelii and it is now demonstrated that the intact peptide is released into norepinephrine-stimulated skin secretions. In order to investigate the mechanism of action of this peptide, a maltose-binding protein (MBP)-fused histone H2B (MBP-H2B) conjugate was prepared and subjected to antimicrobial assay. The fusion protein showed bacteriostatic activity against Escherichia coli strain JCM5491 with a minimum inhibitory concentration of 11 microM. The lysate prepared from JCM5491 cells was capable of fragmenting MBP-H2B within the histone H2B region, but the lysate from the outer membrane proteinase T (OmpT) gene-deleted BL21(DE3) cells was not. FITC-labeled MBP-H2B (FITC-MBP-H2B) penetrated into the bacterial cell membrane of JCM5491 and ompT-transformed BL21(DE3) cells, but not into ompT-deleted BL21(DE3) cells. Gel retardation assay using MBP-H2B-deletion mutants indicated that MBP-H2B bound to DNA at a site within the N-terminal region of histone H2B. Consequently, it is proposed that the antimicrobial action of histone H2B involves, at least in part, penetration of an OmpT-produced N-terminal histone H2B fragment into the bacterial cell membrane with subsequent inhibition of cell functions.

Diazotization of kynurenine by acidified nitrite secreted from indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells.

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.