Study of the K_{L}→π

The rare decay K_{L}→π^{0}νν[over ¯] was studied with the dataset taken at the J-PARC KOTO experiment in 2016, 2017, and 2018. With a single event sensitivity of (7.20±0.05_{stat}±0.66_{syst})×10^{-10}, three candidate events were observed in the signal region. After unveiling them, contaminations from K^{±} and scattered K_{L} decays were studied, and the total number of background events was estimated to be 1.22±0.26. We conclude that the number of observed events is statistically consistent with the background expectation. For this dataset, we set an upper limit of 4.9×10^{-9} on the branching fraction of K_{L}→π^{0}νν[over ¯] at the 90% confidence level.

Search for K_{L}→π

A search for the rare decay K_{L}→π^{0}νν[over ¯] was performed. With the data collected in 2015, corresponding to 2.2×10^{19} protons on target, a single event sensitivity of (1.30±0.01_{stat}±0.14_{syst})×10^{-9} was achieved and no candidate events were observed. We set an upper limit of 3.0×10^{-9} for the branching fraction of K_{L}→π^{0}νν[over ¯] at the 90% confidence level (C.L.), which improved the previous limit by almost an order of magnitude. An upper limit for K_{L}→π^{0}X^{0} was also set as 2.4×10^{-9} at the 90% C.L., where X^{0} is an invisible boson with a mass of 135 MeV/c^{2}.

Depletion of homeodomain-interacting protein kinase 3 impairs insulin secretion and glucose tolerance in mice.

AIMS/HYPOTHESIS: Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes. Here, we focused on a family of serine-threonine kinases known as homeodomain-interacting protein kinases (HIPKs). HIPKs are implicated in the modulation of Wnt signalling, which plays a crucial role in transcriptional activity, and in pancreas development and maintenance. The aim of the present study was to characterise the role of HIPKs in glucose metabolism. METHODS: We used RNA interference to characterise the role of HIPKs in regulating insulin secretion and transcription activity. We conducted RT-PCR and western blot analyses to analyse the expression and abundance of HIPK genes and proteins in the islets of high-fat diet-fed mice. Glucose-induced insulin secretion and beta cell proliferation were measured in islets from Hipk3 ( -/- ) mice, which have impaired glucose tolerance owing to an insulin secretion deficiency. The abundance of pancreatic duodenal homeobox (PDX)-1 and glycogen synthase kinase (GSK)-3ß phosphorylation in Hipk3 ( -/- ) islets was determined by immunohistology and western blot analyses. RESULTS: We found that HIPKs regulate insulin secretion and transcription activity. Hipk3 expression was most significantly increased in the islets of high-fat diet-fed mice. Furthermore, glucose-induced insulin secretion and beta cell proliferation were decreased in the islets of Hipk3 ( -/- ) mice. Levels of PDX1 and GSK-3ß phosphorylation were significantly decreased in Hipk3 ( -/- ) islets. CONCLUSIONS/INTERPRETATION: Depletion of HIPK3 impairs insulin secretion and glucose tolerance. Decreased levels of HIPK3 may play a substantial role in the pathogenesis of type 2 diabetes.

Control of beta cell function and proliferation in mice stimulated by small-molecule glucokinase activator under various conditions.

AIMS/HYPOTHESIS: We investigated changes in the expression of genes involved in beta cell function and proliferation in mouse islets stimulated with glucokinase activator (GKA) in order to elucidate the mechanisms by which GKA stimulates beta cell function and proliferation. METHODS: Islets isolated from mice were used to investigate changes in the expression of genes related to beta cell function and proliferation stimulated by GKA. In addition, Irs2 knockout (Irs2 (-/-)) mice on a high-fat diet or a high-fat diet containing GKA were used to investigate the effects of GKA on beta cell proliferation in vivo. RESULTS: In wild-type mice, Irs2 and Pdx1 expression was increased by GKA. In Irs2 (-/-) mice, GKA administration increased the glucose-stimulated secretion of insulin and Pdx1 expression, but not beta cell proliferation. It was particularly noteworthy that oxidative stress inhibited the upregulation of the Irs2 and Pdx1 genes induced by GKA. Moreover, whereas neither GKA alone nor exendin-4 alone upregulated the expression of Irs2 and Pdx1 in the islets of db/db mice, prior administration of exendin-4 to the mice caused GKA to increase the expression of these genes. CONCLUSIONS/INTERPRETATION: GKA-stimulated IRS2 production affected beta cell proliferation but not beta cell function. Oxidative stress diminished the effects of GKA on the changes in expression of genes involved in beta cell function and proliferation. A combination of GKA and an incretin-related agent might therefore be effective in therapy.

Reconsideration of insulin signals induced by improved laboratory animal diets, Japanese and American diets, in IRS-2 deficient mice.

Current Japanese and American diets and Japanese diet immediately after the War were converted to laboratory animal diets. As a result, current laboratory animal diet (CA-1, CLEA) unexpectedly resembled the diet of Japanese after the War. This is considered to result in an under-evaluation of diabetes research using laboratory animals at present. Therefore, changes in insulin signals caused by current Japanese and American diets were examined using IRS-2 deficient mice ( IRS2(-/-) mice) and mechanisms of aggravation of type 2 diabetes due to modern diets were examined. IRS2(-/-) mice at 6 weeks of age were divided into three groups: Japanese diet (Jd) group, American diet (Ad) group and CA-1 diet [regular diet (Rd)] group. Each diet was given to the dams from 7 days before delivery. When the IRS2(-/-) mice reached 6 weeks of age, the glucose tolerance test (GTT), insulin tolerance test (ITT) and organ sampling were performed. The sampled organs and white adipose tissue were used for analysis of RNA, enzyme activity and tissues. In GTT and ITT, the Ad group showed worse glucose tolerance and insulin resistance than the Rd group. Impaired glucose tolerance of the Jd group was the same as that of the Rd group, but insulin resistance was worse than in the Rd group. These results were caused an increase in fat accumulation and adipocytes in the peritoneal cavity by lipogenic enzyme activity in the liver and muscle, and the increase in TNFalpha of hypertrophic adipocyte origin further aggravated insulin resistance and the increase in resistin also aggravated the impaired glucose tolerance, leading to aggravation of type 2 diabetes. The Japanese and American diets given to the IRS2(-/-) mice, which we developed, showed abnormal findings in some IRS2(-/-) mice but inhibited excessive reactions of insulin signals as diets used in ordinary nutritional management.

Crucial role of insulin receptor substrate-2 in compensatory beta-cell hyperplasia in response to high fat diet-induced insulin resistance.

In type 2 diabetes, there is a defect in the regulation of functional beta-cell mass to overcome high-fat (HF) diet-induced insulin resistance. Many signals and pathways have been implicated in beta-cell function, proliferation and apoptosis. The co-ordinated regulation of functional beta-cell mass by insulin signalling and glucose metabolism under HF diet-induced insulin-resistant conditions is discussed in this article. Insulin receptor substrate (IRS)-2 is one of the two major substrates for the insulin signalling. Interestingly, IRS-2 is involved in the regulation of beta-cell proliferation, as has been demonstrated using knockout mice models. On the other hand, in an animal model for human type 2 diabetes with impaired insulin secretion because of insufficiency of glucose metabolism, decreased beta-cell proliferation was observed in mice with beta-cell-specific glucokinase haploinsufficiency (Gck(+/) (-)) fed a HF diet without upregulation of IRS-2 in beta-cells, which was reversed by overexpression of IRS-2 in beta-cells. As to the mechanism underlying the upregulation of IRS-2 in beta-cells, glucose metabolism plays an important role independently of insulin, and phosphorylation of cAMP response element-binding protein triggered by calcium-dependent signalling is the critical pathway. Downstream from insulin signalling via IRS-2 in beta-cells, a reduction in FoxO1 nuclear exclusion contributes to the insufficient proliferative response of beta-cells to insulin resistance. These findings suggest that IRS-2 is critical for beta-cell hyperplasia in response to HF diet-induced insulin resistance.

Fulminant type 1 diabetes mellitus observed in insulin receptor substrate 2 deficient mice.

The objective of this study was to characterise the fulminant type 1 diabetes mellitus (DM) accompanying abrupt hyperglycaemia and ketonuria observed in insulin receptor substrate 2 (IRS2)-deficient mice. IRS2-deficient mice backcrossed onto the original C57BL/6J:Jc1 background (B6J-IRS2(-/-) mice) for more than 10 generations were used. Eight male IRS2-deficient mice with ketonuria and abrupt increase in plasma glucose concentrations over 25 mmol/l were used as the fulminant type 1 diabetic mice (diabetic mice) and 8 male IRS2-deficient mice (8 weeks old) without glycosuria were used as the control mice. Plasma metabolite, immunoreactive insulin (IRI) and C-peptide concentrations, hepatic energy metabolism related enzyme activities and histopathological change in pancreatic islets were investigated. The diabetic mice showed significantly higher plasma glucose and cholesterol concentrations and lower plasma IRI and C-peptide concentrations than the control mice. In livers of the diabetic mice, glycolytic and malate-aspartate shuttle enzyme activities decreased significantly and gluconeogenic, lipogenic and ketone body synthesis enzyme activities increased significantly compared to those in the control mice. The pancreatic islets of the diabetic mice decreased significantly in size and number of beta cells. The diabetic IRS2-deficient mice did not show the islet-related antibodies observed in the diabetic NOD mice in their sera. The characteristics of the diabetic IRS2-deficient mice resembled those of the human nonautoimmune fulminant type 1 DM. IRS2-deficient mice may be a useful animal model for studying the degradation mechanism of pancreatic beta cells in the process of development of fulminant type 1 DM.

Ajuba negatively regulates the Wnt signaling pathway by promoting GSK-3beta-mediated phosphorylation of beta-catenin.

The Wnt signaling pathway is essential for embryonic development and carcinogenesis. Upon Wnt stimulation, beta-catenin is stabilized and associates with T-cell factor or lymphoid enhancing factor, thereby activating transcription of target genes. In the absence of Wnt stimulation, the level of beta-catenin is reduced via glycogen synthase kinase (GSK)-3beta-mediated phosphorylation and subsequent proteasome-dependent degradation. Here, we report the identification of Ajuba as a negative regulator of the Wnt signaling pathway. Ajuba is a member of LIM domain-containing proteins that contribute to cell fate determination and regulate cell proliferation and differentiation. We found that enforced expression of Ajuba destabilized beta-catenin and suppressed target gene expression. Ajuba promoted GSK-3beta-mediated phosphorylation of beta-catenin by reinforcing the association between beta-catenin and GSK-3beta. Furthermore, Wnt stimulation induced both accumulation of beta-catenin and destabilization of Ajuba. Our findings suggest that Ajuba is important for regulation of the Wnt signaling pathway.

Mice conditionally lacking the Wolfram gene in pancreatic islet beta cells exhibit diabetes as a result of enhanced endoplasmic reticulum stress and apoptosis.

AIMS/HYPOTHESIS: Wolfram syndrome is an autosomal recessive disorder characterised by childhood diabetes mellitus, optic atrophy and severe neurodegeneration, resulting in premature death. The aim of this study was to investigate the mechanisms responsible for the phenotype of carbohydrate intolerance and loss of pancreatic beta cells in this disorder. MATERIALS AND METHODS: To study the role of the Wolfram gene (Wfs1) in beta cells, we developed a mouse model with conditional deletion of Wfs1 in beta cells by crossing floxed Wfs1 exon 8 animals with mice expressing Cre recombinase under the control of a rat insulin promoter (RIP2-Cre). Complementary experiments using RNA interference of Wfs1 expression were performed in mouse insulinoma (MIN6) cell lines (WfsKD). RESULTS: Male knockout mice (betaWfs(-/-)) began developing variable and progressive glucose intolerance and concomitant insulin deficiency, compared with littermate controls, by 12 weeks of age. Analysis of islets from betaWfs(-/-) mice revealed a reduction in beta cell mass, enhanced apoptosis, elevation of a marker of endoplasmic reticulum stress (immunoglobulin heavy chain-binding protein [BiP]), and dilated endoplasmic reticulum with decreased secretory granules by electron microscopy. WfsKD cell lines had significantly increased apoptosis and elevated expression of the genes encoding BiP and C/EBP-homologous protein (CHOP), two markers of endoplasmic reticulum stress. CONCLUSIONS/INTERPRETATION: These results indicate that (1) lack of expression of Wfs1 in beta cells was sufficient to result in the diabetes mellitus phenotype; (2) beta cell death occurred by an accelerated process of apoptosis; and (3) lack of Wfs1 was associated with dilated endoplasmic reticulum and increased markers of endoplasmic reticulum stress, which appears to be a significant contributor to the reduction in beta cell survival.

An expression profile of human pancreatic islet mRNAs by Serial Analysis of Gene Expression (SAGE).

AIMS/HYPOTHESIS: The Human Genome Project seeks to identify all genes with the ultimate goal of evaluation of relative expression levels in physiology and in disease states. The purpose of the current study was the identification of the most abundant transcripts in human pancreatic islets and their relative expression levels using Serial Analysis of Gene Expression. METHODS: By cutting cDNAs into small uniform fragments (tags) and concatemerizing them into larger clones, the identity and relative abundance of genes can be estimated for a cDNA library. Approximately 49,000 SAGE tags were obtained from three human libraries: (i) ficoll gradient-purified islets (ii) islets further individually isolated by hand-picking, and (iii) pancreatic exocrine tissue. RESULTS: The relative abundance of each of the genes identified was approximated by the frequency of the tags. Gene ontology functions showed that all three libraries contained transcripts mostly encoding secreted factors. Comparison of the two islet libraries showed various degrees of contamination from the surrounding exocrine tissue (11 vs 25%). After removal of exocrine transcripts, the relative abundance of 2180 islet transcripts was determined. In addition to the most common genes (e.g. insulin, transthyretin, glucagon), a number of other abundant genes with ill-defined functions such as proSAAS or secretagogin, were also observed. CONCLUSION/INTERPRETATION: This information could serve as a resource for gene discovery, for comparison of transcript abundance between tissues, and for monitoring gene expression in the study of beta-cell dysfunction of diabetes. Since the chromosomal location of the identified genes is known, this SAGE expression data can be used in setting priorities for candidate genes that map to linkage peaks in families affected with diabetes.

Characteristics of wine produced by mushroom fermentation.

Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% beta-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis.

Characteristics of a cheese-like food produced by fermentation of the mushroom Schizophyllum commune.

Lactate bacteria of the Lactobacillus and Streptococcus genera are normally employed in cheese making because these microbes have potent ability to produce lactate dehydrogenase. A milk-clotting enzyme is also necessary to make cheese. Recently, we discovered that some mushroom genera produce both lactate dehydrogenase and a milk-clotting enzyme. Using the mushroom Schizophyllum commune in place of a lactate bacterium, we produced a cheese-like food that contained about 0.58% beta-D-glucan, which has been shown to have preventive effects against cancer. The food also exhibited thrombosis prevention activity, prolonging the thrombin clotting time to 49.6-fold that of the control.

Cloning and characterization of the mouse tob2 gene.

Human Tob2 is a member of the Tob/BTG1 anti-proliferative family of proteins. Here, we report the molecular cloning and characterization of the mouse tob2 gene. The tob2 gene contains an open reading frame of 345 amino acids with an 89% identity to its human counterparts. The coding region of mouse tob2 is not interrupted by introns. The tob2 transcript is 4.2kb long, the size being similar to that of the human tob2 transcript, and detected ubiquitously in various tissues of adult mice. In addition, in situ hybridization shows that tob2 is ubiquitously expressed in embryo, the level of expression being especially high in skeletal muscle. Collectively, Tob2 is suggested to play roles both during embryogenesis and in adults.

Development of mushrooms for thrombosis prevention by protoplast fusion.

With thrombosis a major cause of death in Japan and the Western world, thrombin-inhibitory agents that constrain the formation of fibrin are sought. We screened for basidiomycetes showing anti-thrombin activity and isolated Laetiporus sulphureus. However, it was difficult to cultivate and its form was not satisfactory. We therefore used protoplast fusion between L. sulphureus and the commonly cultivated basidiomycete Hypsizygus marmoreaus to obtain cultivable basidiomycetes that produced an anti-thrombin substance. For the protoplast fusion of L. sulphureus and H. marmoreaus, the protoplast concentration, alternating electric field intensity, dielectrophoresis duration, and field pulse intensity used were of 1 x 10(7) protoplasts/ml, 100 V/cm.1 MHz, 60 s, and 8 kV/cm, respectively. The number of regenerated colonies obtained was 4961, from which 43 strains were selected for electrophoretic analysis. Four of the fusants were found to have a band from each parent in isozyme patterns obtained using their crude extract. The fruiting bodies of the fusants were very similar to those of H. marmoreaus. Crude extract from each of the fusants and from L. sulphureus showed anti-coagulative activity in terms of the thrombin clotting time. We thus obtained improved basidiomycetes that produce an anti-thrombin substance, are easily cultivated, and whose form resembles H. marmoreaus, a commonly used culinary mushroom.

Tob2, a novel anti-proliferative Tob/BTG1 family member, associates with a component of the CCR4 transcriptional regulatory complex capable of binding cyclin-dependent kinases.

Human cDNAs encoding a novel member of Tob/BTG1 anti-proliferative family proteins were cloned. The putative protein product termed Tob2 consisted of 344 amino acids with high similarity to the Tob protein. The tob2 mRNA was 4.1 kb long and was ubiquitously expressed in human adult tissues, as was revealed by Northern blot hybridization. However, further in situ hybridization analysis showed a characteristic expression of the tob2 mRNA in oocytes, suggesting a unique role of Tob2 in oogenesis. Like the Tob protein, Tob2 inhibited cell cycle progression from the G0/G1 to S phases. Intriguingly, the amino-terminal half of Tob2 as well as that of Tob was associated with a human homologue of yeast Caf1, a component of the CCR4 transcription factor complex. Moreover, Caf1 was associated with cyclin dependent kinases. These data suggested that both Tob and Tob2 were involved in cell cycle regulation through their interaction with Caf1. Finally, the tob2 gene was mapped to human chromosome 22q13.1-q13.31.

Beta-catenin is a major tyrosine-phosphorylated protein during mouse oocyte maturation and preimplantation development.

During mouse preimplantation development, the components of the E-cadherin-catenin complex are derived from both maternal and zygotic gene activity and the adhesion complex is increasingly accumulated and stored in a nonfunctional form, ready to be used for compaction and the formation of the trophectoderm cell layer (Ohsugi et al., Dev. Dyn. 206:391-402, 1996). Here, we show that beta-catenin is a major tyrosine-phosphorylated protein in oocytes and early cleavage-stage embryos and that the relative amount of phosphorylated beta-catenin is greatly reduced during the morula-blastocyst transition. Peptide-specific antibodies indicate that beta-catenin undergoes conformational changes and/or that the carboxy-terminal region of beta-catenin is blocked during preimplantation development. Moreover, the availability of a carboxy-terminal epitope seems to depend on the tyrosine phosphorylation state of beta-catenin and becomes unmasked when oocytes are treated with the tyrosine kinase inhibitor genistein. Our results suggest that tyrosine phosphorylation of beta-catenin represents a molecular mechanism to keep the accumulating E-cadherin adhesion complex in a nonfunctional form. Dev Dyn 1999;216:168-176.

Identification of a novel Sry-related gene and its germ cell-specific expression.

Sox family proteins are characterized by a unique DNA-binding domain, a HMG box which shows at least 50% sequence similarity with mouse Sry, the sex-determining factor. At present almost 30 Sox genes have been identified. Members of this family have been shown to be conserved during evolution and to play key roles during animal development. Some are involved in human diseases, including sex reversal. Here we report the isolation of a novel member of the Sox gene family, Sox30, which may constitute a distinct subgroup of this family. Using a bacterially expressed DNA-binding domain of Sox30, we show that it is able to specifically recognize the ACAAT motif. Furthermore, Sox30 is capable of activating transcription from a synthetic promoter containing the ACAAT motif. The specific expression of Sox30 in normal testes, but not in maturing germ cell-deficient testes, suggests the involvement of Sox30 in differentiation of male germ cells. Mapping analyses revealed that the Sox30 gene is located on human chromosome 5 (5q33) and on mouse chromosome 11.

Anti-herpes simplex virus activity of moronic acid purified from Rhus javanica in vitro and in vivo.

Rhus javanica, a medicinal herb, has been shown to exhibit oral therapeutic anti-herpes simplex virus (HSV) activity in mice. We purified two major anti-HSV compounds, moronic acid and betulonic acid, from the herbal extract by extraction with ethyl acetate at pH 10 followed by chromatographic separations and examined their anti-HSV activity in vitro and in vivo. Moronic acid was quantitatively a major anti-HSV compound in the ethyl acetate-soluble fraction. The effective concentrations for 50% plaque reduction of moronic acid and betulonic acid for wild-type HSV type 1 (HSV-1) were 3.9 and 2.6 microgram/ml, respectively. The therapeutic index of moronic acid (10.3-16.3) was larger than that of betulonic acid (6.2). Susceptibility of acyclovir-phosphonoacetic acid-resistant HSV-1, thymidine kinase-deficient HSV-1, and wild-type HSV type 2 to moronic acid was similar to that of the wild-type HSV-1. When this compound was administered orally to mice infected cutaneously with HSV-1 three times daily, it significantly retarded the development of skin lesions and/or prolonged the mean survival times of infected mice without toxicity compared with the control. Moronic acid suppressed virus yields in the brain more efficiently than those in the skin. This was consistent with the prolongation of mean survival times. Thus, moronic acid was purified as a major anti-HSV compound from the herbal extract of Rhus javanica. Mode of the anti-HSV activity was different from that of ACV. Moronic acid showed oral therapeutic efficacy in HSV-infected mice and possessed novel anti-HSV activity that was consistent with that of the extract.

Active-oxygen scavenging activity of traditional nourishing-tonic herbal medicines and active constituents of Rhodiola sacra.

The active-oxygen scavenging activity of 70 traditional herbal medicines used in China and Japan as nourishing tonics were evaluated by electron spin resonance (ESR) technique, in order to evaluate their effectiveness for anti-aging and to search for new active-oxygen scavengers from natural resources. Most of the 70 herbal medicines showed scavenging activity with various intensities. Areca catechu (methanol extract), Dendrobium plicatile (methanol extract), Juglans regia (water extract), Paeonia lactiflora (methanol extract), Psychotria serpens (water and methanol extracts), Rhodiola sacra (water and methanol extracts) and Uncaria rhynchophylla (water extract) especially showed strong scavenging activity against superoxide anion radical (*O2-), while J. regia (water and methanol extracts), Morus alba (water extract) and Schisandra chinensis (water extract) revealed strong scavenging activity against hydroxyl radical (HO*). In addition, the active-oxygen scavenging activities of 19 compounds isolated from R. sacra were also examined, and hydroquinone (1), caffeic acid (3), protocatechuic acid (6), gallic acid (7), (-)-epigallocatechin 3-O-gallate (8), 3-O-galloylepigallocatechin-(4beta-->8)-epigallocatechin+ ++ 3-O-gallate (10), heterodendrin (17) and gallic acid 4-O-beta-D-glucopyranoside (19) were found to show mild or strong inhibitory activity against superoxide anion radical (*O2-), while 4-hydroxybenzoic acid (2), 3, 4-hydroxycinnamic acid (4), 6-8 and 19 inhibited hydroxyl radical (OH*). These active-oxygen scavengers may contribute, to different extents, to their anti-aging action.

Aspartate dehydrogenase in vitamin B12-producing Klebsiella pneumoniae IFO 13541.

We found a new reaction of aspartic acid dehydrogenation, catalyzed by NADP(+)-dependent aspartate dehydrogenase, in vitamin B12-producing Klebsiella pneumoniae IFO 13541. The enzyme, which was purified from a crude extract of K.pneumoniae IFO 13541, catalyzes the oxidative deamination of aspartic acid to form oxaloacetic acid. This enzyme had a molecular mass of about 124 kDa consisting of two identical subunits. L-Aspartic acid was a substrate, although D-aspartic acid and L-glutamic acid were inactive. The enzyme showed maximal activity at about pH 7.0-8.0 for the oxidative deamination of L-aspartic acid, and it required NADP+ as a coenzyme, while NAD+ was inactive.