Brain-specific Phgdh deletion reveals a pivotal role for L-serine biosynthesis in controlling the level of D-serine, an N-methyl-D-aspartate receptor co-agonist, in adult brain.

In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.

6,7-Difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic acid, a newly designed fluorescence enhancement-type derivatizing reagent for amino compounds.

A novel fluorescence enhancement-type derivatizing reagent for amino compounds, 6,7-difluoro-1,4-dihydro-1-methyl-4-oxo-3-quinolinecarboxylic acid (FMQC), was developed. FMQC reacts with aliphatic primary amino compounds to afford strong fluorescent derivatives having high photo-and thermo-stabilities. The FMQC derivatives of amino compounds showed 12-159 times higher fluorescence quantum efficiencies than those of FMQC in aqueous and polar organic media. Additionally, the absorption and fluorescence emission wavelength of the derivatives are red-shifted from those of FMQC. These differences in the fluorescence properties between FMQC and the fluorescent derivative enabled the simple and highly sensitive determination of amino compounds without removing any excess unreacted FMQC by using a simple spectrofluorometer as well as HPLC.

Fluorescence and chemiluminescence properties of indolylmaleimides: experimental and theoretical studies.

Various indolylmaleimides (IMs) were synthesized, and their fluorescence (FL) and chemiluminescence (CL) were measured. The substitution at the 2-position of the indole ring and the 3- or 4-position of the maleimide moiety caused an obvious change in the FL and CL of the IMs. An almost on-off switching of the FL of the IMs was observed. The intramolecular charge transfer from the indole moiety to the maleimide moiety occurred in 3-(1H-3-indolyl)-2,5-dihydro-1H-2,5-pyrroledione. In the FL of the IMs, CASPT2 calculations showed deprotonation of the NH group of the indole ring and the maleimide moiety at the excited state. The C[double bond, length as m-dash]C bond in the maleimide moiety was needed for strong CL in the IMs without substitution at the 2-position of the indole ring. The relationships between the FL or CL properties and the structures of the IMs were clarified. These results provide significant information on the rational design of IMs as FL and CL probes.

Chemiluminescence enhancement of 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene in the presence of quaternary ammonium ions.

The chemiluminescence intensity of 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene increased in the presence of quaternary ammonium ions, such as acetylcholine chloride, choline chloride or benzyltrimethylammonium chloride. The complex of 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene with acetylcholine chloride, choline chloride or benzyltrimethylammonium chloride was investigated by (1)H-NMR spectroscopy. The structure of the complex formed from 1,2-di[3,4,5-tri(3,4,5-trihydroxybenzoyloxy)benzoyloxy] benzene with choline chloride was described by an ab initio quantum chemical calculation.

Mutant mice and rats lacking D-amino acid oxidase.

D-amino acid oxidase (DAO) catalyzes oxidative deamination of D-amino acids. Since D-amino acids are considered to be rare in eukaryotes, physiological function of this enzyme has been enigmatic for a long time. Mutant mice lacking DAO were found, and their strain was established. The urine of the mutant mice contained large amounts of D-amino acids. D-Amino acids were also present in their organs and blood. The origin of these D-amino acids was pursued. The results indicate that one of the physiological functions of DAO is the metabolism of D-amino acids of internal and external origin. A large amount of D-serine is shown to exist in the brain of mammals. It binds to the coagonist-binding site of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and enhances the neurotransmission. DAO metabolizes this D-serine and, therefore, modulates neurotransmission. Mutant mice displayed phenotypes resulting from the enhanced NMDA receptor function. Recent studies have shown that DAO is associated with schizophrenia. Mutant mice were resistant to the drugs which act on NMDA receptors and elicit schizophrenia-like symptoms. Recently, mutant rats lacking DAO have also been found. They were free from D-serine-induced nephrotoxicity, indicating involvement of DAO in this toxicity. The mutant mice and rats lacking DAO would be useful for the elucidation of the physiological functions of DAO and the etiology of neuronal diseases associated with DAO.

Enantioselective visualization of D-alanine in rat anterior pituitary gland: localization to ACTH-secreting cells.

The cellular localization of D: -alanine (D: -Ala) in the rat pituitary gland, the tissue containing the highest amount of D: -Ala, has been clarified for the first time by enantioselective visualization of D: -Ala using our own established mouse monoclonal antibody against D: -Ala. D: -Ala immunopositive cells were present predominantly in the anterior lobe, while no intense staining was observed in the intermediate and posterior lobes. The anterior pituitary gland contains five types of cells secreting specific hormones (growth hormone, adrenocorticotropic hormone (ACTH), gonadotropic hormone, prolactin, and thyroid-stimulating hormone), and the double staining results indicated that D: -Ala is localized to the ACTH-secreting cells. The localization of D: -Ala is clearly different from that of D: -aspartic acid (D: -Asp), which is observed in the prolactin cells. Considered together with our previous findings that D: -Ala is localized to the insulin-secreting beta-cells in the pancreas, and both ACTH and insulin are typical regulatory hormones of blood glucose, D: -Ala is suggested to have some functional relationships to blood glucose level regulation in mammals.

Theoretical study of photophysical properties of bisindolylmaleimide derivatives.

The photophysical properties of two bisindolylmaleimide derivatives, 3,4-bis(3-indolyl)-1-H-pyrrole-2,5-dione (arcyriarubin A) and indolo[2,3-a]pyrrolo[3,4-c] carbazole-5,7-(6 H)-dione (arcyriaflavin A), are investigated by using ab initio molecular orbital (MO) and multireference perturbation theory. These compounds are suggested to exist as monovalent anions deprotonated from an indole NH group in aprotic polar solvents. The analysis of MOs shows that the electronic structures of the S(1) and S(2) states are described by the single- or double-electron excitation between the naturally localized MOs on an indole moiety and on the maleimide part. This indicates that the intramolecular charge transfer (ICT) transfer may occur by photoexcitation. The minimum-energy structure of the arcyriarubin A anion is twisted; the dihedral angles between the indole and maleimide rings are 83.4 degrees and 20.2 degrees for the S(1) and S(0) states, respectively. The analysis of the minimum energy path along the coordinate of the twist angle is performed to explore the emission process from the S(1) state. It has been shown that the magnitude of the Stokes shift increases with increasing the twist angle, but the oscillator strength decreases. It has been suggested that the experimentally observed fluorescence arises on the way toward the energy minimum of the S(1) state. The Stokes-shifted emission of arcyriaflavin A is contributed by the S(1)-S(0) electronic relaxation after the excitation in the S(2) state.

Simple and rapid genotyping of D-amino acid oxidase gene recognizing a crucial variant in the ddY strain using microchip electrophoresis.

A rapid genotyping method of D-amino acid oxidase (DAO), an enzyme that catalyzes the oxidative degradation of most of the D-amino acids in mammals, has been established. This method employs a one-step PCR, restriction enzyme digestion and rapid microchip electrophoresis (MCE), and the DAO genotype of the living individual mice was definitely determined within a day by clearly separating the 95 and 107 bp Hpa II digested DNA fragments. For verification of the method, the DAO activity in the kidney of individual mice was also determined, and the obtained values completely matched the estimated genotypes (DAO(+/+), DAO(+/-), and DAO(-/-)). The intrinsic amounts of D-Pro in the serum and kidney of mice with three DAO genotypes were compared for the first time, and demonstrated that the D-amino acid amounts in the DAO(+/+) mice (1.93 +/- 0.66 nmol/mL serum, not detectable in the kidney) and DAO(+/-) mice (1.50 +/- 0.24 nmol/mL serum, not detectable in the kidney) were almost the same. The present method should be a powerful tool to establish various pathologic-model animals under the complete care of their intrinsic DAO activity, which are useful for the screening of D-amino acids having physiological activity and/or diagnostic values.

Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals.

A fully automated 2D-HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column has been developed for the simultaneous enantiomer determination of proline, trans-4-hydroxyproline and cis-4-hydroxyproline in mammals. As a first dimension, a monolithic ODS column of 0.53 mm i.d. showed 3-6 times larger theoretical plate numbers than those of particle-packed ODS columns, and the best enantioseparations were obtained by a Chiralpak QN-2-AX column of 1.5 mm i.d. in the second dimension (separation factors: 1.44-1.83). The R.S.D. values for within-day and dayto-day precisions were less than 5.8%, and the lower limits of quantitation for the D-enantiomers were 1 fmol. The present method was successfully applied to the determination of proline and hydroxyproline enantiomers in the serum and collagen-rich skin tissue. Small amounts of D-proline were found both in the serum (1.57 +/- 0.19 nmol/mL) and in the skin (0.093 +/- 0.015 nmol/mg protein), while the amounts of D-hydroxyproline were smaller than the lower limit of quantitation.

Structural and photophysical properties of novel dual fluorescent compounds, 1-aryl-substituted 6-methoxy-4-quinolones.

Dual fluorescent compounds are useful platforms for the development of ratiometric probes and sensors. We have developed a new series of dual fluorescent compounds, 1-aryl-substituted 6-methoxy-4-quinolones, and investigated their structural and photophysical properties. The X-ray crystallographic analysis and ab initio quantum chemical calculations revealed that the developed compounds exhibited 60-75 degrees twisted structures. The dual fluorescence of the compounds were observed in polar solvents, and the ratiometric fluorescence responses to alterations in the acidity and temperature were obtained.

A genetic variant of the serine racemase gene is associated with schizophrenia.

BACKGROUND: Serine racemase (SRR) is a brain-enriched enzyme that converts L-serine to D-serine, which acts as an endogenous ligand of N-methyl D-aspartate (NMDA) receptors. Dysfunction of SRR may reduce the function of NMDA receptors and susceptibility to schizophrenia. METHODS: We genotyped three single-nucleotide polymorphisms (SNPs) of the 5' region of the SRR gene in 525 patients with schizophrenia and 524 healthy controls. Effects of SNPs on the promoter activity and on serum levels of total and D-serine were examined. RESULTS: We found a significant excess of the IVS1a+465C allele of the SRR gene in schizophrenia, especially in the paranoid subtype (p = .0028). A reporter assay showed that the IVS1a+465C allele had 60% lower promoter activity than did the IVS1a+465G allele. CONCLUSIONS: The IVS1a+465C allele of the SRR gene, which reduces expression of the gene, is a risk factor for schizophrenia, especially the paranoid subtype.

Bisindolylmaleimides with large Stokes shift and long-lasting chemiluminescence properties.

Various bisindolylmaleimides have fluorescence emission maxima wavelengths longer than 500 nm, large Stokes shifts longer than 200 nm, different fluorescence emission wavelengths at an excitation wavelength of 365 nm, and a long-lasting chemiluminescence. The expansion of the pi-conjugation, the pi-bond electronic structure, and oxidation of the C=C bond at the 2,3-position of the maleimide moiety are crucial for producing these fluorescence and chemiluminescence properties.

Comprehensive analysis of branched aliphatic D-amino acids in mammals using an integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns.

A validated two-dimensional HPLC method for the comprehensive analysis of small quantities of branched aliphatic D-amino acids in the presence of large amounts of their L-congeners in mammalian tissues and physiological fluids is described. The quantitative analysis of these aliphatic amino acids (Val, allo-Ile, Ile, and Leu) is important for the diagnosis of various inherent metabolic disorders of amino acids, and the D-enantiomers are expected to be of particular interest from a pharmacological point of view. Target analytes were determined as their fluorescent derivatives, pre-column labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), using an automated two-dimensional column-switching high-performance liquid chromatographic system combining a narrow bore reversed-phase column and an enantioselective column connected with an integrated multi-loop peak fraction storage device. The described two-dimensional analysis concept proved to be successful for the given task in biological samples taken from mammals. Total analysis time for the reversed-phase separation of the four target NBD-amino acids is 60 min, and the integrated enantiomer separation of each of the four analytes is completed in approximately 5 min. In the rat, significant amounts of D-Leu were found in all tissues and physiological fluids tested (trace-1.3 nmol/g tissue), and in the urine, the presence of high amounts of D-allo-Ile (D-isomer of a non-proteinogenic amino acid, 22.2 nmol/ml) was demonstrated. D-allo-Ile was also found in the urine of dog and mouse, which indicates the ubiquitous presence of this unusual D-amino acid and the potential need to clarify its unique metabolism in mammals.

Sensitive high-performance liquid chromatographic assay for D-amino-acid oxidase activity in mammalian tissues using a fluorescent non-natural substrate, 5-fluoro-D-tryptophan.

A sensitive assay for D-amino-acid oxidase (DAO) activity in mammalian tissues has been established. D-Tryptophan (D-Trp) analogs were tested as substrates for DAO, and 5-fluoro-D-tryptophan (D-FTP) was found to be the best substrate. By the enzymatic reaction, D-FTP was converted to 5-fluoroindole-3-acetic acid (FIAA), a highly fluorescent product, and the product was determined by an RP-HPLC system with a fluorescence detector. The detection limit for purified DAO (from hog kidney) was 0.25 microU, and the within-day and day-to-day precisions of the assays were 4.6% (RSD, n=5), and 13.8% (RSD, 5 days), respectively. By the present method, the detailed distribution of DAO activity in the mouse brain was determined using individual animals for the first time, and significant activities were observed in the cerebellum, medulla oblongata and midbrain. Because sensitive DAO assay is frequently required in small tissues or in limited-tissue regions, the present method is useful for various research studies concerning DAO and the related D-amino acids.

Development of a Fully-automated On-line Oxidation Column-switching HPLC System for the Determination of Endogenous Melatonin in Human Clinical Samples.

A fully-automated on-line oxidation column-switching HPLC system has been developed for the determination of endogenous melatonin in human plasma and saliva. This HPLC system consists of four processes. In the first step, melatonin is fractionated using a reversed-phase C4 column (Proteonavi, 75 mm × 1.0 mm i.d.). In the second step, the obtained melatonin fraction is on-line collected, and oxidized to a highly-fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), by mixing with hydrogen peroxide under alkaline conditions. In the third step, the produced 6-MOQMA is concentrated, and the oxidation reagents are removed using an alkaline resistive reversed-phase column, Asahipak ODP (35 mm × 1.0 mm i.d.). In the final fourth step, the 6-MOQMA is determined by a microbore-ODS column packed with ultrafine particles (CAPCELL PAK C18 IF, 250 mm × 1.0 mm i.d.). The limit of detection of melatonin using this system is about 200 amol/injection, and the determination of endogenous melatonin in a small volume of human physiological fluids, such as 100 µL of plasma and 300 µL of saliva, was successfully accomplished.

2-amino-3-phenylpyrazine, a sensitive fluorescence prelabeling reagent for the chromatographic or electrophoretic determination of saccharides.

2-Amino-3-phenylpyrazine is found to be a sensitive fluorescence labeling reagent for saccharides with a reducing end. The labeled monosaccharides show strong fluorescence under various pH conditions, and could be analyzed by both HPLC and HPCE techniques. Laser induced fluorescence detection is also applicable. Following derivatization with 2-amino-3-phenylpyrazine, six monosaccharides are separated by an HPCE system within 23 min in the calibration range of 5 or 10 fmol to 5 pmol (injection amount). The within-day and day-to-day precisions of the monosaccharide determinations are 3.83-4.86% (RSD) and 3.37-4.56% (RSD), respectively. This method was successfully applied to the determination of component monosaccharides in a glycoprotein, bovine serum fetuin.

Novel stable fluorophore, 6-methoxy-4-quinolone, with strong fluorescence in wide pH range of aqueous media, and its application as a fluorescent labeling reagent.

6-Methoxy-4-quinolone (6-MOQ, 1), an oxidation product derived from 5-methoxyindole-3-acetic acid, is a novel fluorophore, which has several useful characteristics for biomedical analysis. Compound 1 has strong fluorescence with a large Stokes' shift in aqueous media, and the maximum fluorescence excitation and emission wavelengths are 243 nm and 374 nm, respectively. The molar absorptivity at the maximum excitation wavelength and fluorescence quantum yield in aqueous 10% (v/v) methanol are 32 600 L mol(-1) cm(-1) and 0.38, respectively. The fluorescence intensity of 1 is scarcely affected by changing the medium pH, showing strong fluorescence from pH 2.0 to 11.0. In addition, 1 is highly stable against light and heat, and no degradation was observed at 60 degrees C for 3 days with exposure to daylight. As a fluorescent labeling reagent, [(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]amine (6-MOQ-NH2, 2) was synthesized, and determination of carboxylic acids was demonstrated; 50 pmol of standard propionic acid and isobutyric acid were derivatized, and the obtained S/N ratios for 10 fmol (injection amount) of these two acids were 206 and 164, respectively.

Reversible fluorescence labeling of amino groups of protein using dansylaminomethylmaleic anhydride.

The reversible fluorescence labeling of insulin, catalase and lysozyme has been demonstrated. As a derivatizing reagent, dansylaminomethylmaleic acid (DAM) has been used after investigating the precolumn and precapillary derivatization conditions. This reagent (DAM) reacts with the amino groups of proteins via its anhydride in the presence of a suitable dehydrating reagent, which then could be liberated under mild acidic conditions and the native proteins are regenerated. After the derivatization of insulin, catalase and lysozyme with DAM, no peaks of these native proteins were observed while several peaks of the derivatized proteins due to the multiple labeling were observed. However, after the regeneration, increasing amounts of the native proteins were observed as the regeneration period increased. For the lysozyme, the bacteriolytic activity of the enzyme decreased after the derivatization, and only 0.9% of the activity remained. The activity increases by the regeneration, and 95.6% of the bacteriolytic activity of the native enzyme was observed after a 48-h regeneration at pH 2.5 and 40 degrees C.

D-Amino acids in mammals and their diagnostic value.

Substantial amounts of D-amino acids are present in mammalian tissues; their function, origin and relationship between pathophysiological processes have been of great interest over the last two decades. In the present article, analytical methods including chromatographic, electrophoretic and enzymatic methods to determine D-amino acids in mammalian tissues are reviewed, and the distribution of these D-amino acids in mammals is discussed. An overview of the function, origin and relationship between the amino acids and pathophysiological processes is also given.

A sensitive internal standard method for the determination of melatonin in mammals using precolumn oxidation reversed-phase high-performance liquid chromatography.

A sensitive and accurate internal standard method to determine melatonin in mammalian tissues and physiological fluids has been described. This method includes the oxidation of melatonin to a highly fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), and the determination of 6-MOQMA by a reversed-phase HPLC system. For the accurate and reliable determination, several melatonin analogs were designed and utilized as the internal standards, and ethyl and isopropyl analogs (having the corresponding alkyl group via the amide bond of melatonin instead of the methyl group) were found to be promising. Using these internal standards, highly accurate and sensitive determination could be accomplished using rat pineal gland samples, and the clear circadian rhythms are demonstrated. This method was also successfully applied to the determination of melatonin in a small amount (20 microL) of human saliva.