Off-line two-dimensional LC-MS/MS determination of tryptophan enantiomers in mammalian urine and alteration of their amounts in d-amino acid oxidase deficient mice.

D-Tryptophan (D-Trp) is one of the minor D-enantiomers of amino acids discovered in microbes and mollusca. In the present study, a highly-selective 2D chiral LC-MS/MS method has been designed and developed focusing on the determination of Trp enantiomers to investigate the presence and regulation of free D-Trp in mammals. The developed system consisted of a reversed-phase separation for the first dimension, an enantioselective separation for the second dimension and also the detection using a triple quadrupole mass spectrometer for the third/fourth dimensions. Using the present method, urinary D-Trp in mammals, including healthy human volunteers and mice, were successfully determined. Although only l-Trp was observed in a mixed urine sample of healthy volunteers, small amounts of D-Trp were detected in the C57BL/6J mice (n = 5, %D=6.18 ± 0.47). In B6DAO- mice lacking the activity of d-amino acid oxidase (DAO), relatively high levels of D-Trp were observed (n = 6, %d=27.43 ± 3.26). The obtained %d values of Trp in the urine of the C57BL/6J mice and B6DAO- mice were confirmed using various enantioselective columns having different separation properties. These results indicate that the urinary D-Trp level is regulated by DAO in mammals, and further investigations, such as tissue distribution and physiological significance of the intrinsic D-Trp, are expected.

Determination of phenylalanine enantiomers in the plasma and urine of mammals and á´ -amino acid oxidase deficient rodents using two-dimensional high-performance liquid chromatography.

A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice.

Gut microbiota-derived D-serine protects against acute kidney injury.

Gut microbiota-derived metabolites play important roles in health and disease. D-amino acids and their L-forms are metabolites of gut microbiota with distinct functions. In this study, we show the pathophysiologic role of D-amino acids in association with gut microbiota in humans and mice with acute kidney injury (AKI). In a mouse kidney ischemia/reperfusion model, the gut microbiota protected against tubular injury. AKI-induced gut dysbiosis contributed to the altered metabolism of D-amino acids. Among the D-amino acids, only D-serine was detectable in the kidney. In injured kidneys, the activity of D-amino acid oxidase was decreased. Conversely, the activity of serine racemase was increased. The oral administration of D-serine mitigated the kidney injury in B6 mice and D-serine-depleted mice. D-serine suppressed hypoxia-induced tubular damage and promoted posthypoxic tubular cell proliferation. Finally, the D-serine levels in circulation were significantly correlated with the decrease in kidney function in AKI patients. These results demonstrate the renoprotective effects of gut-derived D-serine in AKI, shed light on the interactions between the gut microbiota and the kidney in both health and AKI, and highlight D-serine as a potential new therapeutic target and biomarker for AKI.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

Enantioselective determination of citrulline and ornithine in the urine of d-amino acid oxidase deficient mice using a two-dimensional high-performance liquid chromatographic system.

Two-dimensional high-performance liquid chromatographic (2D-HPLC) and 2D-HPLC-mass spectrometric (2D-HPLC-MS) systems have been designed and developed for the determination of the citrulline (Cit) and ornithine (Orn) enantiomers. Several d-amino acids have already been identified as novel physiologically active molecules and biomarkers, and the enantioselective evaluation of the amounts, distributions and metabolisms of non-proteinogenic amino acids gain as well increasing interest. In the present study, highly selective analytical methods were developed using a capillary monolithic ODS column (0.53mm i.d. x 1000mm) for the reversed-phase separation of the target analytes from the matrix compounds in the first dimension, and a narrowbore-Pirkle type enantioselective column, KSAACSP-105S (1.5mm i.d. x 250mm), was used for the enantiomer separation in the second dimension. The amino acids were analyzed after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by the fluorescence detector and MS. The systems were applied to the urine of d-amino acid oxidase (DAO) deficient B6DAO- mice and control C57BL mice to evaluate the presence and metabolism of the Cit and Orn enantiomers in mammals. As a result, all of the 4 target enantiomers (d-Cit, l-Cit, d-Orn, l-Orn) were found in the urine of both strains. The %D value of Cit (d-Cit/Cit×100) increased about 3-fold in the urine of the DAO deficient mice and that of Orn also tended to increase with the DAO deficiency. These results were definitely confirmed by a 2D-HPLC-MS detection system. Further investigations about the biological significance of these d-isomers are currently ongoing.

Changes in D-aspartic acid and D-glutamic acid levels in the tissues and physiological fluids of mice with various D-aspartate oxidase activities.

D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

Cellular origin and regulation of D- and L-serine in in vitro and in vivo models of cerebral ischemia.

D-Serine is known to be essential for the activation of the N-methyl-D-aspartate (NMDA) receptor in the excitation of glutamatergic neurons, which have critical roles in long-term potentiation and memory formation. D-Serine is also thought to be involved in NMDA receptor-mediated neurotoxicity. The deletion of serine racemase (SRR), which synthesizes D-serine from L-serine, was recently reported to improve ischemic damage in mouse middle cerebral artery occlusion model. However, the cell type in which this phenomenon originates and the regulatory mechanism for D-/L-serine remain elusive. The D-/L-serine content in ischemic brain increased until 20 hours after recanalization and then leveled off gradually. The results of in vitro experiments using cultured cells suggested that D-serine is derived from neurons, while L-serine seems to be released from astroglia. Immunohistochemistry studies of brain tissue after cerebral ischemia showed that SRR is expressed in neurons, and 3-phosphoglycerate dehydrogenase (3-PGDH), which synthesizes L-serine from 3-phosphoglycerate, is located in astrocytes, supporting the results of the in vitro experiments. A western blot analysis showed that neither SRR nor 3-PGDH was upregulated after cerebral ischemia. Therefore, the increase in D-/L-serine was not related to an increase in SRR or 3-PGDH, but to an increase in the substrates of SRR and 3-PGDH.

Ischemic acute kidney injury perturbs homeostasis of serine enantiomers in the body fluid in mice: early detection of renal dysfunction using the ratio of serine enantiomers.

The imbalance of blood and urine amino acids in renal failure has been studied mostly without chiral separation. Although a few reports have shown the presence of D-serine, an enantiomer of L-serine, in the serum of patients with severe renal failure, it has remained uncertain how serine enantiomers are deranged in the development of renal failure. In the present study, we have monitored serine enantiomers using a two-dimensional HPLC system in the serum and urine of mice after renal ischemia-reperfusion injury (IRI), known as a mouse model of acute kidney injury. In the serum, the level of D-serine gradually increased after renal IRI in parallel with that of creatinine, whereas the L-serine level decreased sharply in the early phase after IRI. The increase of D-serine was suppressed in part by genetic inactivation of a D-serine-degrading enzyme, D-amino acid oxidase (DAO), but not by disruption of its synthetic enzyme, serine racemase, in mice. Renal DAO activity was detected exclusively in proximal tubules, and IRI reduced the number of DAO-positive tubules. On the other hand, in the urine, D-serine was excreted at a rate nearly triple that of L-serine in mice with sham operations, indicating that little D-serine was reabsorbed while most L-serine was reabsorbed in physiological conditions. IRI significantly reduced the ratio of urinary D-/L-serine from 2.82 ± 0.18 to 1.10 ± 0.26 in the early phase and kept the ratio lower than 0.5 thereafter. The urinary D-/L-serine ratio can detect renal ischemia earlier than kidney injury molecule-1 (KIM-1) or neutrophil gelatinase-associated lipocalin (NGAL) in the urine, and more sensitively than creatinine, cystatin C, or the ratio of D-/L-serine in the serum. Our findings provide a novel understanding of the imbalance of amino acids in renal failure and offer a potential new biomarker for an early detection of acute kidney injury.

Retinal NMDA receptor function and expression are altered in a mouse lacking D-amino acid oxidase.

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.

Two-dimensional high-performance liquid chromatographic determination of day-night variation of D-alanine in mammals and factors controlling the circadian changes.

D-Alanine (D-Ala) is one of the naturally occurring D-amino acids in mammals, and its amount is known to have characteristic circadian changes. It is a candidate for a novel physiologically active substance and/or a biomarker, and the regulation mechanisms of the intrinsic amounts of D-Ala are expected to be clarified. In the present study, the effects of the possible factors controlling the D-Ala amounts, e.g., diet, D-amino acid oxidase (DAO) and intestinal bacteria, on the day-night changes in the intrinsic D-Ala amounts have been investigated using a highly sensitive and selective two-dimensional high-performance liquid chromatographic system combining a reversed-phase column and an enantioselective column. The circadian rhythm was not changed under fasting conditions. In the mice lacking D-amino acid oxidase activity (ddY/DAO(-) mice), clear day-night changes were still observed, suggesting that the factors controlling the D-Ala rhythm were not their food and DAO activity. On the other hand, in the germ-free mice, quite low amounts of D-Ala were detected compared with those in the control mice, indicating that the main origin of D-Ala in the mice is intestinal bacteria. Because the D-Ala amounts in the digesta containing intestinal bacteria did not show the day-night changes, the controlling factor of the circadian changes of the D-Ala amount was suggested to be the intestinal absorption.

Alteration of intrinsic amounts of D-serine in the mice lacking serine racemase and D-amino acid oxidase.

For elucidation of the regulation mechanisms of intrinsic amounts of D-serine (D-Ser) which modulates the neuro-transmission of N-methyl-D-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and D-amino acid oxidase (DAO) were established, and the amounts of D-Ser in the tissues and physiological fluids were determined. D-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in D-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR(-/-)) mice compared with those of control mice, although the amounts of D-Ser in these tissues were low. The amounts of D-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous D-Ser into the brain tissues, we have determined the D-Ser of SRR(-/-) mice after oral administration of D-Ser for the first time, and a drastic increase in D-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic D-Ser amounts was investigated. In the frontal brain, most of the intrinsic D-Ser was biosynthesized by SRR. On the other hand, half of the D-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic D-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.

Functional roles of endogenous D-serine in pain-induced ultrasonic vocalization.

The N-methyl-D-aspartate receptor (NMDAR) is crucial for pain-related behaviors. D-Serine is synthesized from L-serine by serine racemase (SR) and modulates NMDAR functions by acting as an agonist at the glycine-binding site. We analyzed noxious stimulus-induced ultrasonic vocalization and locomotor activity in the open-field test using SR knockout (SR-KO) mice to examine the role of endogenous D-serine in mammalian behaviors. SR-KO mice emitted less ultrasonic vocalization after noxious stimulation (VAS) than wild-type (WT) mice. The locomotor activity of WT mice decreased with repeated daily exposures to the open field, whereas that of SR-KO mice remained unchanged. VAS was significantly enhanced during arthritis in WT mice, whereas it was not enhanced during arthritis in SR-KO mice. These results indicate that mice lacking the ability to produce D-serine endogenously in the brain differ from normal mice with respect to the chronic pain-induced behavioral changes.

D-Amino acids in the brain and mutant rodents lacking D-amino-acid oxidase activity.

D-Amino acids are stereoisomers of L-amino acids. They are often called unnatural amino acids, but several D-amino acids have been found in mammalian brains. Among them, D-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. D-Amino-acid oxidase (DAO), which degrades neutral and basic D-amino acids, is mainly present in the hindbrain. DAO catabolizes D-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of D-serine and other D-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of D-serine. D-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that D-amino acids and DAO have pivotal functions in the central nervous system.

Functional roles of endogenous D-serine in the chronic pain-induced plasticity of NMDAR-mediated synaptic transmission in the central amygdala of mice.

The amygdala is implicated in chronic pain-induced emotional changes. Chronic pain induces plastic changes of the N-methyl-d-aspartate receptor (NMDAR) functions in the brain including the amygdala. d-Serine is synthesized endogenously by serine racemase and modulates NMDAR-mediated synaptic transmission as a coagonist of glycine binding site. To clarify the functional roles of endogenous d-serine in chronic pain-induced plasticity of NMDAR mediated synaptic transmission, we investigated the NMDAR-mediated excitatory synaptic current (EPSC) of neurons in the latero-capsular division of the central amygdala (CeLC) using brain slices from serine racemase knockout (SR-KO) mice with chronic pain induced by monoarthritis. The decay time of NMDAR-mediated EPSC was significantly elongated by monoarthritis in wild type (WT) mice, but not in SR-KO mice. The d-serine application-induced increase of NMDAR-mediated EPSC was significantly facilitated by monoarthritis in WT mice, but not in SR-KO mice. These results suggest that endogenous d-serine facilitates chronic pain-induced plastic changes of NMDAR mediated synaptic transmission in CeLC.

D-amino acid oxidase controls motoneuron degeneration through D-serine.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. D-serine, an endogenous coagonist of N-methyl-D-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the D-amino acid oxidase (DAO) gene, encoding a D-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and D-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the D-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through D-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing D-serine or controlling DAO activity in ALS should be tested in future studies.

D-Amino acid metabolism in mammals: biosynthesis, degradation and analytical aspects of the metabolic study.

It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of D-amino acids are present in mammals. The most abundant D-amino acids are D-serine and D-aspartate. D-Serine, which is synthesized by serine racemase and is degraded by D-amino-acid oxidase, is present in the brain and modulates neurotransmission. D-Aspartate, which is synthesized by aspartate racemase and degraded by D-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. D-Serine and D-aspartate bind to the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these D-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and D-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on D-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and D-amino-acid oxidases.

D-serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor.

D-serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.

Simultaneous two-dimensional HPLC determination of free D-serine and D-alanine in the brain and periphery of mutant rats lacking D-amino-acid oxidase.

A fully automated two-dimensional HPLC system combining a microbore-ODS column and a narrowbore-enantioselective column was designed and validated, and the amounts of D-serine (D-Ser) and D-alanine (D-Ala) in various tissues and physiological fluids of Long-Evans agouti/SENDAI (LEA/Sen) rats lacking D-amino-acid oxidase (DAO) were determined. Intra- and inter-day precision was less than 4.3% and accuracy ranged between 99.9 and 104%. LEA/Sen rats were reported to lack DAO in their kidneys and expected to be a novel mutant animal lacking DAO, however, the amounts of D-amino acids in the LEA/Sen rats have not been investigated. In the present study, the intrinsic amounts of D-Ser and D-Ala, which are neuromodulators of the N-methyl-D-aspartate (NMDA) receptors, were determined in seven brain tissues, four peripheral tissues, plasma and urine of the LEA/Sen rats and compared to those of the control (Wistar and SD) rats having normal DAO activity. The levels of D-Ser in the tissues and physiological fluids of the LEA/Sen rats were significantly higher than those of the Wistar and SD rats except for the frontal brain regions. Concerning D-Ala, the amounts in the tissues and physiological fluids of the LEA/Sen rats were drastically increased compared to those of the Wistar and SD rats. These results indicate that the intrinsic amounts of D-Ser and D-Ala in the tissues of rats are regulated by DAO, and that LEA/Sen rats would be useful for the study of NMDA receptor-related diseases in which DAO is implicated.

Behavioral characterization of a mutant mouse strain lacking D-amino acid oxidase activity.

D-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO(-)) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO-/- mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO-/- mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO-/- mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO-/- mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia.

Protective effects of metallothionein I and II against metal- and ultraviolet radiation-induced damage in cultured lens epithelial cells.

PURPOSE: Few quantitative analyses of the induction of metallothioneins (MTs) have been conducted, and there are no reports on the contribution of MTs to the protective mechanism against ultraviolet (UV) radiation in the lens. In this study, we quantitatively analyzed the induction of MTs and analyzed the resulting protective effects against both metal- and UV radiation-induced damage in the cultured lens epithelial cell line, alphaTN4-1. METHODS: The induction profiles of MTs by ZnCl(2) treatment in alphaTN4-1 cells were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The cells in which MTs were induced were either treated with high concentrations of ZnCl(2) or CdCl(2), or irradiated with UV-C, UV-B, or UV-A radiation, followed by analysis of cell viability. The (3)H-thymidine incorporation rate was used as an indicator of cell viability. RESULTS: mRNA expression of MT-I and MT-II, the main MT isoform classes, was induced by ZnCl(2) treatment in a dose-dependent manner. MT induction increased the protective effects against both metaland UV-A radiation-induced cell damage. CONCLUSIONS: Our results suggest that MTs play an important role in the protection against damage induced by both toxic metals and UV-A radiation in lens epithelial cells.