Kazuko Yorita

Abnormal Immune Function of Hemopoietic Cells from Alymphoplasia ( aly ) Mice, a Natural Strain with Mutant NF-κB-Inducing Kinase

Alymphoplasia (aly) mice, a natural strain with a mutant NF-κB-inducing kinase (NIK) gene, manifest a unique phenotype; they lack lymph nodes and Peyer’s patches, have a disturbed spleen architecture, and exhibit defects in both Ab and cellular immune responses. Although a stromal defect caused by impaired lymphotoxin-β receptor signaling accounts for their abnormal lymphoid organogenesis, the exact mechanisms underlying the development of immunodeficiency in aly mice are poorly understood. We therefore investigated the contribution of hemopoietic cells with the aly NIK mutation to the development of immunodeficiency. Transfer of aly/aly bone marrow cells into aly/+ mice resulted in poorly developed B cell follicles and lack of support for the development of germinal centers and isotype switching, indicating that the hemopoietic cells of aly mice contain an autonomous defect. However, follicular dendritic cell clusters were maintained in the spleens of these bone marrow chimeras, suggesting that the lack of follicular dendritic cell clusters in aly mice is probably due to the stromal defect. The aly mice lacked marginal zone B cells in their spleens, and aly/aly B cells showed an impaired proliferative response after in vitro stimulation. IL-2 production by activated T cells was also impaired. By contrast, the dendritic cells of aly mice exhibited grossly normal development and function. Supporting the concept of an autonomous cell defect, Rel protein expression was altered in aly/aly spleens. Thus, the aly NIK mutation affects hemopoietic cell function in an intrinsic fashion and, together with the stromal defect, may contribute to the development of immunodeficiency in aly mice.

Potential pathophysiological role of d-amino acid oxidase in schizophrenia: immunohistochemical and in situ hybridization study of the expression in human and rat brain

Abstractd-Amino acid oxidase (DAO) is a peroxisomal flavoenzyme that catalyzes oxidative deamination of a wide range of d-amino acids. Among the possible substrates of DAO in vivo, d-serine is proposed to be a neuromodulator of the N-methyl-d-aspartate (NMDA) type glutamate receptor. The gene for DAO was reported to be associated with schizophrenia. Since DAO is expected to be one of the key enzymes in the regulation of NMDA neurotransmission, the modulation of the enzyme activity is expected to be therapeutical for neuronal disorders. In search of the pathophysiological role of DAO, we analyzed the distribution of DAO mRNA and protein in the rat and human brain. In rat, the distribution of DAO mRNA was newly detected in choroid plexus (CP) epithelial cells in addition to glial cells of pons, medulla oblongata, and especially Bergmann glia of cerebellum. Moreover, to investigate how DAO expression level is altered in schizophrenia, we performed immunohistochemistry in the human brain. In agreement with the results in the rat brain, the immunoreactivity for DAO was detected in glial cells of rhombencephalon and in CP. Furthermore, higher level of DAO expression was observed in schizophrenic CP epithelial cells than that in non-schizophrenic cases. These results suggest that an increase in DAO expression in parts of the brain is involved in aberrant d-amino acid metabolism. In particular, gene expression of DAO in CP suggests that DAO may regulate d-amino acid concentration by modulating the cerebrospinal fluid and may be regarded as a potential therapeutic target for schizophrenia.

The effect of risperidone on D-amino acid oxidase activity as a hypothesis for a novel mechanism of action in the treatment of schizophrenia:

D-Amino acid oxidase (DAO) has been established to be involved in the oxidation of D-serine, an allosteric activator of the N-methyl-D-aspartate-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The effect of risperidone, a benzisoxazole derivative, atypical antischizophrenic drug, on the activity of human DAO was tested using an in-vitro oxygraph system and rat C6, stable C6 transformant cells overexpressing mouse DAO (designated as C6/DAO) and pig kidney epithelial cells (LLC-PK1). Risperidone has a hyperbolic mixed-type inhibition, designated as ‘partial uncompetitive inhibition effect’, with Ki value of 41 μM on human DAO. Risperidone exhibited a protective effect from D-amino acid induced cell death in both C6/DAO and LLC-PK1 cells with 10% increase in viability. These data indicate the involvement of DAO activity in D-serine metabolism and also suggest a new mechanism of action to risperidone as antischizophrenic drug.

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase, product of a susceptibility gene for schizophrenia

D-Amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (Ki = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.

Conversion of L-Lactate Oxidase to a Long Chain α-Hydroxyacid Oxidase by Site-directed Mutagenesis of Alanine 95 to Glycine

A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long chain α-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-α-hydroxyacids, α-hydroxy-n-butyric acid, α-hydroxy-n-valeric acid, etc., and also the aromatic α-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pKa of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.

Potential cytotoxic effect of hydroxypyruvate produced from d-serine by astroglial d-amino acid oxidase

D-amino acid oxidase (DAO) is a flavoenzyme that exists in the kidney, liver and brain of mammals. This enzyme catalyzes the oxidation of D-amino acids to the corresponding α-keto acid, hydrogen peroxide and ammonia. Recently D-serine, one of the substrates of DAO, has been found in the mammalian brain, and shown to be a co-agonist of the N-methyl-D-aspartate (NMDA) receptor in glutamate neurotransmission. In this study, we investigated the metabolism of extracellular D-serine and the effects of D-serine metabolites to study the pathophysiological role of DAO. Treatment with a high dose of D-serine induced the cell death in dose-dependent manner in DAO-expressing cells. Moreover, overexpression of DAO in astroglial cells induced the enhanced cytotoxicity. The treatment with 1 mM beta-hydroxypyruvate (HPA), uniquely produced from the D-serine metabolism by DAO activity, also induced cell death, comprising apoptosis, in the astroglial cell, but not in the other cells derived from liver and kidney. Taken together, we consider that high dose of extracellular D-serine induced cell death by the production of not only hydrogen peroxide but also HPA as a result of DAO catalytic activity in astroglial cell. Furthermore, this cytotoxicity of HPA is observed uniquely in astroglial cells expressing DAO.

D-Amino acid oxidase-induced oxidative stress, 3-bromopyruvate and citrate inhibit angiogenesis, exhibiting potent anticancer effects

Angiogenesis is critical for cancer growth and metastasis. Steps of angiogenesis are energy consuming, while vascular endothelial cells are highly glycolytic. Glioblastoma multiforme (GBM) is a highly vascular tumor and this enhances its aggressiveness. D-amino acid oxidase (DAO) is a promising therapeutic protein that induces oxidative stress upon acting on its substrates. Oxidative stress-energy depletion (OSED) therapy was recently reported (El Sayed et al., Cancer Gene Ther, 19, 1-18, 2012). OSED combines DAO-induced oxidative stress with energy depletion caused by glycolytic inhibitors such as 3-bromopyruvate (3BP), a hexokinase II inhibitor that depleted ATP in cancer cells and induced production of hydrogen peroxide. 3BP disturbs the Warburg effect and antagonizes effects of lactate and pyruvate (El Sayed et al., J Bioenerg Biomembr, 44, 61-79, 2012). Citrate is a natural organic acid capable of inhibiting glycolysis by targeting phosphofructokinase. Here, we report that DAO, 3BP and citrate significantly inhibited angiogenesis, decreased the number of vascular branching points and shortened the length of vascular tubules. OSED delayed the growth of C6/DAO glioma cells. 3BP combined with citrate delayed the growth of C6 glioma cells and decreased significantly the number and size of C6 glioma colonies in soft agar. Human GBM cells (U373MG) were resistant to chemotherapy e.g. cisplatin and cytosine arabinoside, while 3BP was effective in decreasing the viability and disturbing the morphology of U373MG cells.

Modulation of extracellular d-serine content by calcium permeable AMPA receptors in rat medial prefrontal cortex as revealed by in vivo microdialysis.

In mammalian brains, d-serine has been shown to be required for the regulation of glutamate neurotransmission as an endogenous co-agonist for the N-methyl-d-aspartate type glutamate receptor that is essential for the expression of higher-order brain functions. The exact control mechanisms for the extracellular d-serine dynamics, however, await further elucidation. To obtain an insight into this issue, we have characterized the effects of agents acting at the α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPA) type glutamate receptor on the extracellular d-serine contents in the medial prefrontal cortex of freely moving rats by an in vivo microdialysis technique in combination with high-performance liquid chromatography with fluorometric detection. In vivo experiments are needed in terms of a crucial role of d-serine in the neuron-glia communications despite the previous in vitro studies on AMPA receptor-d-serine interactions using the separated preparations of neurons or glial cells. Here, we show that the intra-cortical infusion of (S)-AMPA, an active enantiomer at the AMPA receptor, causes a significant and concentration-dependent reduction in the prefrontal extracellular contents of d-serine, which is reversed by an AMPA/kainate receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, and a calcium permeable AMPA receptor antagonist, 1-naphthyl acetyl spermine. The d-serine reducing effects of (S)-AMPA are augmented by co-infusion of cyclothiazide that prevents AMPA receptor desensitization. Our data support the view that a calcium permeable AMPA receptor subtype may exert a phasic inhibitory control on the extracellular d-serine release in the mammalian prefrontal cortex in vivo.

L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies

Crystals of flavoenzyme L-lactate oxidase from Aerococcus viridans (LOX) have been obtained that diffract to 3.0 A resolution (P2(1)2(1)2(1), a = 118.4 A, b = 138.4 A, c = 194.6 A). Crystallographic studies suggest that the enzyme may exist as an octameric form with non-crystallographic two- and four-fold axes in the center of the octamer. The four-fold axis makes the tetramer tight, and the tetramers lie upon one another by the two-fold axis.

Identification of two promoters for human d-amino acid oxidase gene: implication for the differential promoter regulation mediated by PAX5/PAX2

D-amino acid oxidase (DAO) is a flavoenzyme that metabolizes d-amino acids. Until now, the DAO expression mechanism is still unclear. Our assessment of human DAO (hDAO) promoter activity using luciferase reporter system indicated the proximal upstream region of exon1 (-237/+1) has promoter activity (P1). Interestingly, we identified an alternative promoter in the proximal upstream region of exon2 (+4,126/+4,929) (P2). This alternative promoter has stronger activity than that of P1. Our results also revealed a negative regulatory segment (+1,163/+1,940) in intron1; that would act in concert with P1 and P2. Bioinformatics analyses elucidated the conservation of transcription factor PAX5 family binding sites among species. These sites (-60/-31) and (+4,464/+4,493), locate in P1 and P2 of hDAO, respectively. Gel shift assays demonstrated P1 contains a site (-60/-31) for PAX5 binding while P2 has three sites for both paired box gene 2 (PAX2) and paired box gene 5 (PAX5) binding. The dual roles of PAX5 family in regulating hDAO transcription by modulating promoter activity of P1 and activating promoter activity of P2 were implicated based on the site-directed mutagenesis experiment. Altogether, our data suggested the differential regulation of hDAO expression by two promoters whose activities may be modulated by the binding of PAX2 and PAX5.