Kumiko Sakai

ANALYTICAL CHEMISTRY AND BIOCHEMISTRY OF D-AMINO ACIDS

The methodologies for the analysis of D-amino acids in biological materials have been reviewed, including the use of enzymes, gas and liquid chromatography with chiral stationary phases and diastereomer derivatization with chiral reagents followed by GC or HPLC separation. The distribution of D-amino acids in the body, their origin, metabolism and possible roles in human diseases are discussed.

Emergence of D-aspartic acid in the differentiating neurons of the rat central nervous system.

The rat embryonic brain was probed with anti-d-aspartic acid (d-Asp) antiserum at different stages of development. At gestational day (E) 12, weak immunoreactivity (IR) of d-Asp was apparent at the hindbrain, midbrain and caudal forebrain, whereas it became more intense and extended over the whole brain at E20. However, IR markedly decreased after parturition. In the region of the immature forebrain at an early stage of development (E12), IR was mainly a characteristic of the cytoplasm of the neuronal cells, while in the more mature hindbrain it was localized in the axonal zone. In the more differentiated forebrain at a later stage of development (E18), the IR became restricted to zones which mainly consisted of axons and processes. Consequently, in the rat central nervous system, d-Asp first emerges during embryonic development as a feature of the cytoplasm and thereafter spreads into the axonal regions of neuronal cells, before disappearing almost completely after parturition.

The Transcription Factor CCAAT-binding Factor CBF/NF-Y Regulates the Proximal Promoter Activity in the Human α1(XI) Collagen Gene (COL11A1)

We have characterized the proximal promoter region of the human COL11A1 gene. Transient transfection assays indicate that the segment from –199 to +1 is necessary for the activation of basal transcription. Electrophoretic mobility shift assays (EMSAs) demonstrated that the ATTGG sequence, within the –147 to –121 fragment, is critical to bind nuclear proteins in the proximal COL11A1 promoter. We demonstrated that the CCAAT binding factor (CBF/NF-Y) bound to this region using an interference assay with consensus oligonucleotides and a supershift assay with specific antibodies in an EMSA. In a chromatin immunoprecipitation assay and EMSA using DNA-affinity-purified proteins, CBF/NF-Y proteins directly bound this region in vitro and in vivo. We also showed that four tandem copies of the CBF/NF-Y-binding fragment produced higher transcriptional activity than one or two copies, whereas the absence of a CBF/NF-Y-binding fragment suppressed the COL11A1 promoter activity. Furthermore, overexpression of a dominant-negative CBF-B/NF-YA subunit significantly inhibited promoter activity in both transient and stable cells. These results indicate that the CBF/NF-Y proteins regulate the transcription of COL11A1 by directly binding to the ATTGG sequence in the proximal promoter region.

Calcium disodium edetate enhances type a monoamine oxidase activity in monkey brain

The effects of metal chelators on monoamine oxidase (MAO) isozymes, MAO-A and MAO-B, in monkey brain mitochondria were investigated in vitro. MAO-A activity increased to about 40% with 0.1 µM calcium disodium edetate (CaNa2EDTA) using serotonin as a substrate, and this activation was proportional to the concentration of CaNa2EDTA. On the other hand, MAO-A activities were decreased gradually with an increasing concentration of o-phenanthroline and diethyldithiocarbamic acid, but these metal chelators had no effect on MAO-B activity in monkey brain. The activation of MAO-A activity by CaNa2EDTA was reversible. CaNa2EDTA did not activate both MAO-A and MAO-B activities in rat brain mitochondria. Zn and Fe ions were found in the mitochondria of monkey brain. Zn ions potently inhibited MAO-A activity, but Fe ions did not inhibit either MAO-A or MAO-B activity in monkey brain mitochondria. These results indicate that the activating action of CaNa2EDTA on MAO-A was the result of the chelating of Zn ions contained in mitochondria by CaNa2EDTA. These results also indicate the possibility that Zn ions may regulate physiologically the level of serotonin and norepinephrine content in brain by inhibiting a MAO-A activity.

Quantitative and non-destructive analyses of fatty acid esters and cholesterol in brain tissues by Fourier transform infrared spectroscopy

Abstract Brain tissues obtained from rats were examined with Fourier transform infrared spectroscopy using a beam condenser apparatus to determine quantitatively and non-destructively the amount of fatty acid esters and cholesterol in the small specimens. For the analysis of fatty acid esters we calculated the ratio between the peak intensities of the fatty ester C  O (1730 cm −1 ) or methylene CH (2850 cm −1 ) stretches and amide II band (1550 cm −1 ), and compared it with the amount of fatty acid esters per mg protein determined chemically by a routine method. For analysis of cholesterol we measured the second derivative spectra between 1300 and 1400 cm −1 and the intensity of a band at 1366 cm −1 in the second derivative spectrum was compared with that of the amide II band in the normal absorption spectrum. This infrared analysis of cholesterol was compared and showed good correlations with the chemically defined data. These results suggest that the infrared technique can provide non-destructively quantitative data for the content of fatty acid esters and cholesterol in biological tissues.

Zinc benzoate, a contaminating environmental compound derived from polystyrene resin inhibits A-type monoamine oxidase

The contaminants in deionized and distilled water (DDI water) boiled with polystyrene resin inhibited A-type monoamine oxidase (MAO, MAO-A preferentially deaminates serotonin and norepinephrine and regulates these amines concentration) activity in monkey brain mitochondria. To identify these contaminants, we attempted measurements by HPLC, FT-IR and NMR. The compound inhibiting MAO-A activity was zinc benzoate. Although it potently inhibited MAO-A activity, zinc benzoate did not effect MAO-B in monkey brain mitochondria. It also reversibly and competitively inhibited MAO-A activity in a dose-dependent manner. Zinc benzoate, however, did not inhibit either MAO-A or -B activities in rat brain mitochondria. These results indicate that zinc benzoate, which inhibits MAO-A activity, is easily incorporated in DDI water by boiling polystyrene and also may be a contaminating environmental chemical compound that alters the levels of serotonin and norepinephrine in the central nervous system.

Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task.

We have analyzed oligosaccharide chains in brain microsomes of rats fed an n‐3 polyunsaturated fatty acid‐deficient (safflower oil group; S group) or ‐rich (perilla oil group; P group) diet before and after brightness‐discrimination learning tasks. The amount of concanavalin A‐binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex‐type oligosaccharides became dominant after the learning task and especially a tetra‐antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S‐group than the P group. When polysialylated glycans were analyzed on serotonin‐conjugated HPLC column, the glycans in the S‐group microsomes before the learning task contained larger amount of higher affinity‐polysialylated glycans to serotonin column than those in the P‐group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P‐group. Removal of mannoside from microsomes by α‐mannosidase‐treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1‐anilinonaphthalene‐8‐sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho‐glycans were changed by dietary n‐3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin. J. Neurosci. Res. 63:185–195, 2001.

Styrene inhibits monoamine oxidase A, but not monoamine oxidase B in monkey brain mitochondria.

The effects of styrene on mitochondrial monoamine oxidase (MAO) activity in rat and monkey brains were compared in vitro. After preincubation at 25 degrees C for 20 min with 1 mM styrene monomer MAO-A activity in monkey brain was inhibited potently using 5-HT (for MAO-A substrate), but MAO-B activity in monkey brain and platelets were slightly inhibited using beta-PEA (for MAO-B substrate). Styrene monomer also competitively inhibited MAO-A activity in a dose-dependent manner. MAO-A in monkey brain was inhibited by styrene in ascending order of potency: styrene trimer>styrene dimer>styrene monomer. In contrast styrene monomer slightly inhibited both MAO-A and MAO-B activities in rat brain mitochondria. In the present study styrene monomer potently inhibits MAO-A activity, but not MAO-B activity, in monkey brain mitochondria in vitro. These results indicate the inhibiting action of styrene differs depending on animal species and MAO isoforms.

Changes of oligosaccharides and fatty acids in monkey hippocampus by synaptic potentiation

We measured the release of free fatty acids and structural changes of glycoprotein glycans induced by tetraethylammonium (TEA) salt in hippocampal slices of cynomolgus monkey brain. The release of free fatty acids in the hippocampal slices occurred after synaptic potentiation by TEA in a different manner from rat hippocampus. Arachidonic acid release in monkey hippocampus occurred much faster than that in rat. Several types of glycans of monkey hippocampal glycoproteins were determined depending on the duration time after TEA treatment. 5-Mannose was increased within 2 min, while polysialoglycans were increased after 5 min or later. Comparative study of glycans of monkey and rat hippocampal slices revealed the presence of relatively larger amount of sialo- and multi-anntenary glycans in rat than in monkey. These results indicate that the depolarizing stimulation of monkey hippocampal slices induced the change of glycoprotein glycan structures and release of free fatty acids in a different manner from rat hippocampus.