Search

Phencyclidine (PCP) causes mental symptoms that closely resemble schizophrenia through the inhibition of the glutamatergic system. The kynurenine (KYN) pathway (KP) generates metabolites that modulate glutamatergic systems such as kynurenic acid (KA), quinolinic acid (QA), and xanthurenic acid (XA). Kynurenine 3-monooxygenase (KMO) metabolizes KYN to 3-hydroxykynurenine (3-HK), an upstream metabolite of QA and XA. Clinical studies have reported lower KMO mRNA and higher KA levels in the postmortem brains of patients with schizophrenia and exacerbation of symptoms in schizophrenia by PCP. However, the association between KMO deficiency and PCP remains elusive. Here, we demonstrated that a non-effective dose of PCP induced impairment of prepulse inhibition (PPI) in KMO KO mice. KA levels were increased in the prefrontal cortex (PFC) and hippocampus (HIP) of KMO KO mice, but 3-HK levels were decreased. In wild-type C57BL/6 N mice, the PPI impairment induced by PCP is exacerbated by KA, while attenuated by 3-HK, QA and XA. Taken together, KMO KO mice were vulnerable to the PPI impairment induced by PCP through an increase in KA and a decrease in 3-HK, suggesting that an increase in the ratio of KA to 3-HK (QA and XA) may play an important role in the pathophysiology of schizophrenia.

Tryptophan (TRP) metabolism via the indoleamine 2,3-dioxygenase (IDO) subset of the kynurenine (KYN) pathway is one of the most important mechanisms of immune escape in cancer. TRP is converted into several biologically active KYN metabolites. However, the role of KYN metabolic products and related enzymes has not been clarified in patients with hematological malignant tumors. Here, we examined the serum concentrations of TRP, KYN, and the KYN metabolites kynurenic acid, anthranilic acid, and 3-hydroxyanthranilic acid in 157 patients stratified into five different hematological malignant tumors. KYN was the most abundant product of the TRP metabolic pathway among all five diagnostic categories. Serum KYN was increased in myelodysplastic syndrome (MDS) patients. The KYN/TRP ratio was significantly higher in MDS patients than in acute myeloid leukemia patients. In conclusion, IDO activity is increased in MDS patients, and IDO inhibitors might represent a new therapeutic approach for MDS treatment.

Indoleamine 2,3-dioxygenase 1 (IDO1) is the first rate-limiting enzyme that metabolizes tryptophan to the kynurenine pathway. Its activity is highly inducible by pro-inflammatory cytokines and correlates with the severity of major depressive disorder (MDD). MicroRNAs (miRNAs) are involved in gene regulation and the development of neuropsychiatric disorders including MDD. However, the role of miRNAs in targeting IDO1 in the pathophysiology of MDD is still unknown. In the present study, we investigated the role of novel miRNAs in the regulation of IDO1 activity and its effect on lipopolysaccharide (LPS)-induced depression-like behavior in mice. LPS upregulated miR-874-3p concomitantly with increase of IDO1 expression in the prefrontal cortex (PFC), increase of immobility in the forced swimming test as depression-like behavior and decrease of locomotor activity as sickness behavior without motor dysfunction. The miR-874-3p increased in both neuron and microglia after LPS. Its mimic significantly suppressed LPS-induced IDO1 expression in the PFC. Infusion of IDO1 inhibitor (1-methyl-l-tryptophan) and miR-874-3p into PFC prevented an increase of immobility in the forced swimming test, but did not decrease of locomotor activity induced by LPS. These results suggest that miR-874-3p may play an important role in preventing the LPS-induced depression-like behavior through inhibition of IDO1 expression. This may also serve as a novel potential target molecule for the treatment of MDD.

Tryptophan (TRP) is metabolized via the kynurenine (KYN) pathway, which is related to the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the metabolism of KYN to 3-hydroxykynurenine. In rodents, KMO deficiency induces a depression-like behavior and increases the levels of kynurenic acid (KA), a KYN metabolite formed by kynurenine aminotransferases (KATs). KA antagonizes α7 nicotinic acetylcholine receptor (α7nAChR). Here, we investigated the involvement of KA in depression-like behavior in KMO knockout (KO) mice. KYN, KA, and anthranilic acid but not TRP or 3-hydroxyanthranilic acid were elevated in the prefrontal cortex of KMO KO mice. The mRNA levels of KAT1 and α7nAChR but not KAT2-4, α4nAChR, or β2nAChR were elevated in the prefrontal cortex of KMO KO mice. Nicotine blocked increase in locomotor activity, decrease in social interaction time, and prolonged immobility in a forced swimming test, but it did not decrease sucrose preference in the KMO KO mice. Methyllycaconitine (an α7nAChR antagonist) antagonized the effect of nicotine on decreased social interaction time and prolonged immobility in the forced swimming test, but not increased locomotor activity. Galantamine (an α7nAChR allosteric agonist) blocked the increased locomotor activity and prolonged immobility in the forced swimming test, but not the decreased social interaction time in the KMO KO mice. In conclusion, elevation of KA levels contributes to depression-like behaviors in KMO KO mice by α7nAChR antagonism. The ameliorating effects of nicotine and galantamine on depression-like behaviors in KMO KO mice are associated with the activation of α7nAChR.

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of kynurenine (KYN) to kynurenic acid (KYNA). Although the isozymes KAT1–4 have been identified, KYNA is mainly produced by KAT2 in brain tissues. KNYA is an antagonist of N-methyl-D-aspartate and α-7-nicotinic acetylcholine receptors, and accumulation of KYNA in the brain has been associated with the pathology of schizophrenia. Therefore, KAT2 could be exploited as a therapeutic target for the management of schizophrenia. Although currently available KAT2 inhibitors irreversibly bind to pyridoxal 5′-phosphate (PLP), inhibition via this mechanism may cause adverse side effects because of the presence of other PLP-dependent enzymes. Therefore, we identified novel selective KAT2 inhibitors by screening approximately 13,000 molecules. Among these, glycyrrhizic acid (GL) and its analogues, glycyrrhetinic acid (GA) and carbenoxolone (CBX), were identified as KAT2 inhibitors. These compounds were highly selective for KAT2 and competed with its substrate KYN, but had no effects on the other 3 KAT isozymes. Furthermore, we demonstrated that in complex structures that were predicted in docking calculations, GL, GA and CBX were located on the same surface as the aromatic ring of KYN. These results indicate that GL and its analogues are highly selective and competitive inhibitors of KAT2.

The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader , M. B. ; Costantino , N. ; Elkins , C. A. ; Chen , C. Y. ; Patel , I. ; Makusky , A. J. ; Choy , J. S. ; Court , D. L. ; Markey , S. P. ; Kowalak , J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011 , 10 , M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography-tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.

Indoleamine 2,3-dioxygenase (IDO) induction can deplete l -Trp in target cells, and this effect is partially responsible for the antimicrobial, antiviral, and antiproliferative activities of several cytokines. Although a lot of studies have indicated the biological importance of IDO, the post-translational modification of IDO remains unknown. In this study, we analyzed IDO protein by liquid chromatography and tandem mass spectrometry (LC-MS/MS) to find post-translational modifications. LC-MS/MS analysis revealed that: (1) immunoprecipitated-IDO from a human monocyte cell line has been processed to cleave the N-terminal methionine, and the resulting N-terminal alanine is acetylated; (2) peroxynitrite, an NO-derived reactive species, which can modify proteins via formation of 3-nitrotyrosine, induced nitration and inactivation of recombinant IDO, specifically nitrating Tyr15, 345, and 353 residues. We successfully revealed these two types of post-translational modifications of IDO, and further findings of the post-translational modification may shed light on the mechanisms of IDO induction.

Indoleamine 2,3-dioxygenase (IDO) is up-regulated by interferon-γ (IFN-γ)-dependent and/or -independent mechanisms. IFN-γ-dependent mechanism is regulated through signal transducer and activator of transcription 1α (STAT1α) and interferon regulatory factor 1 (IRF-1). On the other hand, IFN-γ-independent mechanism of IDO induction has not been identified. In this study, we explored whether IDO induction by LPS requires the STAT1α and IRF-1 signaling pathways. IDO was induced by LPS or IFN-γ in THP-1 cells and a synergistic IDO induction occurred in THP-1 cultured with a combination of tumor necrosis factor-α, interleukin-6 and interleukin-1β. An electrophoretic mobility shift assay using STAT1α and IRF-1 consensus oligonucleotide probes showed no STAT1α or IRF-1 binding activities in LPS-stimulated THP-1 cells. Further, the LPS-induced IDO activity was inhibited by both p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) inhibitors. These results suggest that IDO induction by LPS is not regulated through the STAT1α or IRF-1 binding activities that are induced by IFN-γ, and may be related to the activity of the p38 MAPK pathway and/or NF-κB.

Murine acquired immunodeficiency syndrome (MAIDS) induced by LP-BM5 murine leukemia virus is used as a model of human immunodeficiency virus (HIV)-related neurologic dysfunction. Mice infected with LP-BM5 have mnemonic abnormalities (i.e., spontaneous alternation behavior in the Y-maze and performance in the Morris water maze) and biochemical alternations (i.e., cytokines, platelet-activating factor, quinolinate, glutamate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) that produce neurologic symptoms similar to those observed in HIV-related neurologic dysfunction. To identify proteins associated with dysmnesia in the MAIDS model, we examined the expression of neuronal proteins in LP-BM5-infected mice using two-dimensional polyacrylamide gel electrophoresis (2-DE). Neuronal protein expression in LP-BM5-infected mice was compared with that in non-infected mice using the Image Master 2D. We detected approximately 800 protein spots, of which 35 were distinguishable between non-infected and LP-BM5-infected mice. Most of these spots were downregulated in LP-BM5-infected mice. Three of the spots were identified as 14-3-3 protein zeta/delta, synapsin 2 and protein disulfide isomerase using a capillary nanoliquid chromatography tandem mass spectrometric system. We verified the expression levels of these proteins by Western blot. Analysis of these 35 spots could provide insight into mechanisms of dysmnesia in the MAIDS model of HIV-related neuronal dysfunction.

Indoleamine 2,3-dioxygenase (IDO) is induced by interferon (IFN)-gamma-mediated effects of the signal transducer and activator of transcription 1alpha (STAT1alpha) and interferon regulatory factor (IRF)-1. The induction of IDO can also be mediated through an IFN-gamma-independent mechanism, although the mechanism of induction has not been identified. In this study, we explored whether lipopolysaccharide (LPS) or several proinflammatory cytokines can induce IDO via an IFN-gamma-independent mechanism, and whether IDO induction by LPS requires the STAT1alpha and IRF-1 signaling pathways. IDO was induced by LPS or IFN-gamma in peripheral blood mononuclear cells and THP-1 cells, and a synergistic IDO induction occurred when THP-1 cells were cultured in the presence of a combination of tumor necrosis factor-alpha, interleukin-6 or interleukin-1beta. An electrophoretic mobility shift assay using STAT1alpha and IRF-1 consensus oligonucleotide probes showed no STAT1alpha or IRF-1 binding activities in LPS-stimulated THP-1 cells. Further, the LPS-induced IDO activity was inhibited by both p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) inhibitors. These findings suggest that the induction of IDO by LPS in THP-1 cells is not regulated by IFN-gamma via recruitment of STAT1alpha or IRF-1 to the intracellular signaling pathway, and may be related to the activity of the p38 MAPK pathway and NF-kappaB.

IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.

l-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-γ)-induced antimicrobial effects. In the present study, the effects ofToxoplasmagondiiinfection on IDO were investigated by using wild-type and IFN-γ-gene-deficient (knockout) (IFN-γ KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lungl-tryptophan concentrations were dramatically decreased followingT.gondiiinfection. In contrast, these metabolic changes did not occur inT.gondii-infected IFN-γ KO mice or in uninfected IFN-γ KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-γ KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression ofT.gondiisurface antigen gene 2 (SAG2) induced in lungs and brain byT.gondiiinfection was significantly enhanced in IFN-γ KO mice compared to wild-type mice on day 7 postinfection. Treatment withN-nitro-l-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasmal-kynurenine afterT. gondiiinfection. This in vivo study provides evidence thatl-tryptophan depletion caused byT. gondiiis directly mediated by IFN-γ in the lungs, where iNOS is not induced by IFN-γ. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.

Apolipoproteins in the cerebrospinal fluid (CSF) play important roles in lipid metabolism in the central nervous system. Although it has been demonstrated that apo E is synthesized in the neuron, the synthesis of apo A-I has only been determined in fish and chicken. It was demonstrated that apo A-I concentrations in the CSF were increased in poliovirus-infected macaques, however, the origin of the CSF apo A-I was not determined. The present immunohistochemical study provided evidence that apo A-I was localized within the nerve cell body of the rat spinal cord. In situ hybridization also showed that apo A-I mRNA was predominantly expressed in the neurons. As a further experiment, we compared apo A-I levels in the spinal cord from control rats and rats with experimental allergic encephalomyelitis (EAE), which was induced by sensitization with myelin basic protein. Although no significant changes in serum apo A-I levels were observed, apo A-I levels in the spinal cord were significantly elevated in EAE rats. Furthermore, apo A-I in the spinal cord of rats with EAE was not seen in the nerve cell body, but at the interstitium, particularly in lesions where inflammation had occurred. The current study clearly demonstrated that apo A-I is synthesized in the neurons of the rat spinal cord and the synthesis was suppressed in EAE rats.

Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in the L-tryptophan-kynurenine pathway, which converts an essential amino acid, L-tryptophan, to N-formylkynurenine. It has been speculated that IFN-γ is a dominant IDO inducer in vivo. The present study used IFN-γ or TNF-α gene-disrupted mice and IFN-γ antibody-treated mice to demonstrate that lipopolysaccharide (LPS)-induced systemic IDO is largely dependent on TNF-α rather than IFN-γ. IFN-γ-independent IDO induction was also demonstrated in vitro with LPS-stimulated monocytic THP-1 cells. These findings clearly indicate that there is an IFN-γ-independent mechanism of IDO induction in addition to the IFN-γ-dependent mechanism.

Background —It has been reported that tumor necrosis factor-α (TNF-α) is expressed in the heart with viral myocarditis and that its expression aggravates the condition. The pathophysiological effects of TNF-α on viral myocarditis, however, have not been fully elucidated. Methods and Results —To investigate the role of TNF-α in the progression of viral myocarditis, we used TNF-α gene–deficient mice (TNF-α −/− ) and induced acute myocarditis by infection with encephalomyocarditis virus (EMCV). The survival rate of TNF-α −/− mice after EMCV infection was significantly lower than that of TNF-α +/+ mice (0% versus 67% on day 14). Injection of recombinant human TNF-α (0.2 to 4.0 μg/mouse IV) improved the survival of TNF-α −/− mice in a dose-dependent manner, indicating that TNF-α is essential for protection against viral myocarditis. The levels of viral titer and viral genomic RNA of EMCV in the myocardium were significantly higher in TNF-α −/− than in TNF-α +/+ mice. Histopathological examination showed that the inflammatory changes of the myocardium were less marked in TNF-α −/− than in TNF-α +/+ mice. Immunohistochemical analysis revealed that the levels of immunoreactivity of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the myocardium were decreased in TNF-α −/− mice compared with TNF-α +/+ mice. Conclusions —These observations suggested that TNF-α is necessary for adhesion molecule expression and to recruit leukocytes to inflammatory sites, and thus, the lack of this cytokine resulted in failure of elimination of infectious agents. We concluded that TNF-α plays a protective role in the acute stage of viral myocarditis.

The effect of cytokines on apolipoprotein E (apo E) production and secretion was investigated in a human hepatoma cell line HepG2. Incubation of HepG2 cells with interleukin 1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF-alpha) for 48 h resulted in a significant dose-related decrease of apo E concentration in the culture medium, while intracellular apo E content increased without change in mRNA level. In contrast, IL-1 beta and TNF-alpha decreased both intracellular and medium apo A-I. Elution profiles of cholesterol and apolipoproteins revealed that apo E was present in apo E-rich high density lipoprotein (HDL) fraction and apo A-I was in apo E-rich HDL and small HDL fractions. IL-1 beta and TNF-alpha decreased both apo E and apo A-I in these fractions. The present results suggest that IL-1 beta and TNF-alpha suppress hepatic apo A-I expression and secretion but not expression of apo E, which could contribute to the abnormal lipid metabolism in certain cytokine-mediated inflammatory diseases.

L-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) induction and reactive nitrogen intermediates produced by inducible nitric oxide synthase (iNOS) induction are important factors for IFN-gamma-induced anti-toxoplasma activities. In the present study, the effects of acute Toxoplasma gondii (T. gondii) infection on IDO and iNOS were investigated using wild-type (WT) and IFN-gamma gene-deficient (IFN-gamma KO) mice. In the WT C57BL/6J mice, enzyme activities and mRNA levels of IDO in both lung and brain were markedly increased, and lung L-tryptophan concentrations were dramatically decreased following infection. In contrast, these metabolic changes did not occur in infected IFN-gamma KO mice. The level of iNOS induction in the infected IFN-gamma KO mice was high in lung and low in brain compared to that in infected WT mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) in lung and brain induced by infection was significantly enhanced in the IFN-gamma KO mice compared to that in WT mice. Treatment with N-nitro-L-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain, but not in lung following infection. This in vivo study provides evidence that L-tryptophan depletion caused by T. gondii is directly mediated by IFN-gamma in the lung, where iNOS is not induced by IFN-gamma. This study suggests that there is an anti-toxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of main anti-parasite effector mechanisms of iNOS and/or IDO may vary among tissues.

Hepatic ACMSD [EC4.1.1.45] plays a key role in regulating NAD biosynthesis from tryptophan. We previously reported that ingestion of polyunsaturated fatty acids by rats leads to a decrease in their hepatic ACMSD activity. We purified ACMSD and cloned cDNA encoding rat ACMSD. Therefore, in this study, we examined whether dietary linoleic acid altered ACMSD gene expression and its protein level. Moreover we measured the tryptophan catabolite quinolinic acid level in rats. In the rats fed with linoleic acid, ACMSD mRNA and its protein levels in the liver were strongly suppressed and serum quinolinic acid was significantly increased as compared with the rats fed on a fat-free diet. These results suggest that the transcription level of ACMSD is modulated by linoleic acids or their metabolites and probably there is an inverse relationship between ACMSD activity and the production of quinolinic acid converted from tryptophan.

Hepatic ACMSD [EC4.1.1.45] plays a key role in regulating NAD biosynthesis from tryptophan. We previously reported that ingestion of polyunsaturated fatty acids by rats leads to a decrease in their hepatic ACMSD activity. We purified ACMSD and cloned cDNA encoding rat ACMSD. Therefore, in this study, we examined whether dietary linoleic acid altered ACMSD gene expression and its protein level. Moreover we measured the tryptophan catabolite quinolinic acid level in rats.

L-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) induction and reactive nitrogen intermediates produced by inducible nitric oxide synthase (iNOS) induction are important factors for IFN-gamma-induced anti-toxoplasma activities. In the present study, the effects of acute Toxoplasma gondii (T. gondii) infection on IDO and iNOS were investigated using wild-type (WT) and IFN-gamma gene-deficient (IFN-gamma KO) mice. In the WT C57BL/6J mice, enzyme activities and mRNA levels of IDO in both lung and brain were markedly increased, and lung L-tryptophan concentrations were dramatically decreased following infection. In contrast, these metabolic changes did not occur in infected IFN-gamma KO mice. The level of iNOS induction in the infected IFN-gamma KO mice was high in lung and low in brain compared to that in infected WT mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) in lung and brain induced by infection was significantly enhanced in the IFN-gamma KO mice compared to that in WT mice. Treatment with N-nitro-L-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain, but not in lung following infection. This in vivo study provides evidence that L-tryptophan depletion caused by T. gondii is directly mediated by IFN-gamma in the lung, where iNOS is not induced by IFN-gamma. This study suggests that there is an anti-toxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of main anti-parasite effector mechanisms of iNOS and/or IDO may vary among tissues.

L-Tryptophan degradation by indoleamine 2,3-dioxygenase (IDO) might have an important role in gamma interferon (IFN-gamma)-induced antimicrobial effects. In the present study, the effects of Toxoplasma gondii infection on IDO were investigated by using wild-type and IFN-gamma-gene-deficient (knockout) (IFN-gamma KO) mice. In wild-type C57BL/6J mice, enzyme activities and mRNA levels for IDO in both lungs and brain were markedly increased and lung L-tryptophan concentrations were dramatically decreased following T. gondii infection. In contrast, these metabolic changes did not occur in T. gondii-infected IFN-gamma KO mice or in uninfected IFN-gamma KO mice. The levels of inducible nitric oxide synthase (iNOS) induction in infected IFN-gamma KO mice were high in lungs and low in brain compared to those in infected wild-type mice. The extent of increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) induced in lungs and brain by T. gondii infection was significantly enhanced in IFN-gamma KO mice compared to wild-type mice on day 7 postinfection. Treatment with N-nitro-L-arginine methyl ester, an iNOS inhibitor, increased the levels of SAG2 mRNA in brain but not in lungs and of plasma L-kynurenine after T. gondii infection. This in vivo study provides evidence that L-tryptophan depletion caused by T. gondii is directly mediated by IFN-gamma in the lungs, where iNOS is not induced by IFN-gamma. This study suggests that there is an antitoxoplasma mechanism of cross-regulation between iNOS and IDO and that the expression of the main antiparasite effector mechanisms for iNOS and/or IDO may vary among tissues.

Background: The purpose of this study was to evaluate the performance of two homogeneous assays for LDL-cholesterol (LDL-C), a polyethylene/cyclodextrin (PC) assay and a detergent (D) assay, which are based on different principles, in cholestatic serum.Methods: We compared serum LDL-C concentrations determined by the two assays for healthy normolipidemic subjects (n = 42) and cholestatic patients (n = 51). LDL-C concentrations obtained with the homogeneous assays were also compared with those obtained by HPLC for patients’ sera. In the interference study, conjugated bile acids were added to normal serum, and their effects on the two assays were examined. The effects of lipoprotein-X (LP-X), intermediate-density lipoprotein (IDL), and apolipoprotein (apo) E-rich HDL on the LDL-C assays were also investigated by adding these lipoproteins to normal serum.Results: The LDL-C concentrations obtained with the D assay were higher than those obtained with the PC assay in the serum with high LP-X. The bias for LDL-C vs LP-X in cholestatic serum correlated with LP-X concentration (r = 0.582; P Conclusions: Although both homogeneous LDL-C assays are suitable for most cases, the present study showed that each homogeneous assay has a different limitation for cholestatic serum with gross alterations in lipoproteins.

Marked increases in metabolites of thel-tryptophan-kynurenine pathway, l-kynurenine and quinolinic acid (Quin), were observed in serum and cerebrospinal fluid (CSF) of both the rat and human with renal insufficiency. The mechanisms responsible for their accumulation after renal insufficiency were investigated. In patients with chronic renal insufficiency, elevated levels of serum l-kynurenine and Quin were reduced by hemodialysis. In renal-insufficient rats, Quin andl-kynurenine levels in serum, brain, and CSF were also increased parallel to the severity of renal insufficiency. Urinary excretion of Quin (3.5-fold) and l-kynurenine (2.8-fold) was also increased. Liver l-tryptophan 2,3-dioxygenase activity (TDO), a rate-limiting enzyme of the kynurenine pathway, was increased in proportion to blood urea nitrogen and creatinine levels. Kynurenine 3-hydroxylase and quinolinic acid phosphoribosyltransferase were unchanged, but the activities of kynureninase, 3-hydroxyanthranilate dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase (ACMSDase) were significantly decreased. Systemic administrations of pyrazinamide (ACMSDase inhibitor) increased serum Quin concentrations in control rats, demonstrating that changes in body ACMSDase activities in response to renal insufficiency are important factors for the determination of serum Quin concentrations. We hypothesize the following ideas: that increased serum l-kynurenine concentrations are mainly due to the increased TDO and decreased kynureninase activities in the liver and increased serum Quin concentrations are due to the decreased ACMSDase activities in the body after renal insufficiency. The accumulation of CSFl-kynurenine is caused by the entry of increased seruml-kynurenine, and the accumulation of CSF Quin is secondary to Quin from plasma and/or Quin precursor into the brain.

Anthranilic acid (AA) has been attracted considerable attention as one of the L-tryptophan-kynurenine pathway metabolites in the central nervous system. In this study, the concentration of L-kynurenine (L-KYN) and AA in serum and CSF, and its related enzyme activities were determined in several species. In rabbits, CSF AA concentrations were lower and serum AA concentrations were slightly higher than those in other species. However, the concentrations of L-KYN were substantially higher in rabbits in both serum and CSF compared with other species. Tissue enzyme activities varied among species. In rabbits, lung IDO activities were higher, but liver kynurenine 3-hydroxylase activities were lower than those of the other species tested. Furthermore, brain kynurenine 3-hydroxylase activities were higher in gerbils than those in other species. These results clearly demonstrated that kynurenine pathway enzyme activities and metabolite concentrations vary with species.

Accumulation of L-kynurenine and 3-hydroxyanthranilic acid (3HAA) occurs in the monocyte-derived cells following immune stimulation, and may derive from L-tryptophan following induction of indoleamine-2,3-dioxygenase. In the present study, we evaluate the possibility that 3HAA acts as an endogenous inducer of monocyte/macrophage apoptosis. Supplementation with 200 μM of 3HAA, but not other L-tryptophan metabolites tested, significantly increased the number of apoptotic cells in both THP-1 and U937 cells. Catalase, superoxide dismutase and manganese ions markedly enhanced apoptosis in the presence of 3HAA in these cells. The present results suggest that 3HAA induces the macrophage/monocyte apoptosis under certain conditions, which may be relevant to patho-physiology of inflammatory conditions.

Anthranilic acid (AA) has attracted considerable attention as one of the l -tryptophan–kynurenine pathway metabolites in the central nervous system. In this study, a highly sensitive and accurate method for the quantification of AA has been developed using reversed-phase high-performance liquid chromatography with electrochemical detection. Serum and cerebrospinal fluid (CSF) AA concentrations in different animal species were measured. CSF AA concentrations in rabbits were 1.1 ± 0.1 nmol/liter, which were 5.7–33.0 times lower than those in other species studied. Serum AA concentrations, however, were slightly higher in rabbits than in other species. In contrast, the concentrations of l -kynurenine (L-KYN) in both serum and CSF were substantially higher in rabbits than in other species. Tissue kynurenine pathway enzymes, indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase, kynurenine 3-hydroxylase, and kynureninase were determined in rabbits, rats, gerbils, and mice. These enzymes varied among species, especially lung IDO activities in rabbits were 146–516 times higher than those found in other species, but rabbit liver kynurenine 3-hydroxylase activities were lower by one order of magnitude than those of the other species tested. Furthermore, brain kynurenine 3-hydroxylasae activities were 12.3–23.2 times higher in gerbils than those in the other species tested. In addition, AA concentrations in serum following intravenous administration of L-KYN (5 mg/kg) were also measured in rabbits. AA levels peaked sharply within 5 min after administration and decreased in a time-dependent manner. At 5 min after administration, CSF L-KYN and AA concentrations were also increased by 1.76- and 2.56-fold, respectively, compared with basal levels. Increased AA concentrations in CSF following L-KYN administration may reflect the entry of AA into the CSF after conversion to AA in systemic tissue and/or the local synthesis of AA from L-KYN in the CNS.

Apolipoproteins in cerebrospinal fluid (CSF) might have important functional roles in the pathophysiology of brain and lipid metabolism in the vascular component. The present study examined apolipoprotein A-I (apo-A-I) and apolipoprotein E (apo-E) levels in CSF and serum from poliovirus-infected macaques. Poliovirus-infected macaques developed motor deficits and were classified into three groups: (1) muscle weakness in one or both legs; (2) partial paralysis in one or both legs; (3) complete paralysis in one or both legs. No motor deficits were evident in the control or sham-treated macaques. Apo-A-I concentrations in CSF were markedly elevated in poliovirus-infected macaques with weakness, partial or complete paralysis, in comparison with either control or sham-treated animals, and were proportional to the severity of motor impairment. Apo-E concentrations in CSF were also significantly elevated in poliovirus-infected macaques with complete paralysis. The magnitude of increase in CSF apo-A-I or apo-E concentrations was also closely associated with the degree of histologic neurological damage and inflammation (lesion scores). However, no changes in serum apo-A-I and apo-E concentrations were observed in the poliovirus-infected macaques compared with control macaques. Furthermore there were no significant correlations apo-A-I or apo-E concentrations between serum and CSF. We hypothesize that the elevation of apo-A-I and apo-E concentrations after poliovirus infection is caused by immune stimulation within the central nervous system (CNS). Measures of CSF apo-A-I and apo-E levels might serve as a useful marker for the severity and/or the range of CNS injury.