Takemitsu Furuchi

Kitasato University, Edo, Tōkyō, Japan

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Publications (26)76.21 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have developed and validated a high-performance liquid chromatography method that uses monolithic silica disk-packed spin columns and a monolithic silica column for the simultaneous determination of N(G) -monomethyl-L-arginine, N(G) ,N(G) -dimethyl-L-arginine, and N(G) ,N(G') -dimethyl-L-arginine in human plasma. For solid-phase extraction, our method employs a centrifugal spin column packed with monolithic silica bonded to propyl benzenesulfonic acid as a cation exchanger. After pretreatment, the methylated arginines are converted to fluorescent derivatives with 4-fluoro-7-nitro-2,1,3-benzoxadiazole, and then the derivatives are separated on a monolithic silica column. L-Arginine concentration was also determined in diluted samples. Standard calibration curves revealed that the assay was linear in the concentration range 0.2-1.0 μM for methylated arginines and 40-200 μM for L-arginine. Linear regression of the calibration curve yielded equations with correlation coefficients of 0.999 (r(2) ). The sensitivity was satisfactory, with a limit of detection ranging from 3.75-9.0 fmol for all four compounds. The relative standard deviations were 4.3-4.8% (intra-day) and 3.0-6.8% (inter-day). When this method was applied to samples from six healthy donors, the detected concentrations of N(G) -monomethyl-L-arginine, N(G) ,N(G) -dimethyl- L-arginine, N(G) ,N(G') -dimethyl- L-arginine and L-arginine were 0.05±0.01, 0.41±0.07, 0.59±0.11 and 83.8±30.43μM (n = 6), respectively. This article is protected by copyright. All rights reserved.
    Journal of Separation Science 06/2014; · 2.59 Impact Factor
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    ABSTRACT: D-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are believed to be co-agonists of the N-methyl-D-aspartate (NMDA) receptor. To identify a new class of DAO inhibitor(s) that can be used to elucidate the molecular details of the active site environment of DAO, manifold biologically active compounds of microbial origin and pre-existing drugs were screened for their ability to inhibit DAO activity, and several compounds were identified as candidates. One of these compounds, acyclovir (ACV), a well-known antiviral drug used for the treatment of herpesvirus infections, was characterized and evaluated as a novel DAO inhibitor in vitro. Analysis showed that ACV acts on DAO as a reversible slow-binding inhibitor, and interestingly, the time required to achieve equilibrium between DAO, ACV, and the DAO/ACV complex was highly dependent on temperature. The binding mechanism of ACV to DAO was investigated in detail by several approaches, including kinetic analysis, structural modeling of DAO complexed with ACV, and site-specific mutagenesis of an active site residue postulated to be involved in the binding of ACV. The results confirm that ACV is a novel, active site-directed inhibitor of DAO that can be a valuable tool for investigating the structure-function relationships of DAO, including the molecular details of the active site environment of DAO. In particular, it appears that ACV can serve as an active site probe to study the structural basis of temperature-induced conformational changes of DAO.
    Biochemistry 07/2013; · 3.38 Impact Factor
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    ABSTRACT: D-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are potential co-agonists of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders, such as schizophrenia. Hence, a DAO inhibitor that augments the brain levels of D-serine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure-function relationships of DAO.
    Journal of Medicinal Chemistry 02/2013; · 5.61 Impact Factor
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    ABSTRACT: Recent investigations have shown that a variety of D-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. D-Amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are degradative enzymes stereospecific for D-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of D-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several D- and L-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and D-amino acids in multicellular organisms.
    Molecular and cellular biology 03/2012; 32(10):1967-83. · 6.06 Impact Factor
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    ABSTRACT: D-aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. Unlike DAO, which acts on several neutral and basic D-amino acids, DDO is highly specific for acidic D-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic D-amino acids, and showed a considerable decrease in activity against acidic D-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic D-amino acids and its activity against acidic D-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.
    Amino Acids 02/2011; 40(2):467-76. · 3.91 Impact Factor
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    ABSTRACT: D-Aspartate oxidase (DDO) and D-amino acid oxidase (DAO) are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids. While several functionally and structurally important amino acid residues have been identified in the DAO protein, little is known about the structure-function relationships of DDO. In the search for a potent DDO inhibitor as a novel tool for investigating its structure-function relationships, a large number of biologically active compounds of microbial origin were screened for their ability to inhibit the enzymatic activity of mouse DDO. We discovered several compounds that inhibited the activity of mouse DDO, and one of the compounds identified, thiolactomycin (TLM), was then characterized and evaluated as a novel DDO inhibitor. TLM reversibly inhibited the activity of mouse DDO with a mixed type of inhibition more efficiently than meso-tartrate and malonate, known competitive inhibitors of mammalian DDOs. The selectivity of TLM was investigated using various DDOs and DAOs, and it was found that TLM inhibits not only DDO, but also DAO. Further experiments with apoenzymes of DDO and DAO revealed that TLM is most likely to inhibit the activities of DDO and DAO by competition with both the substrate and the coenzyme, FAD. Structural models of mouse DDO/TLM complexes supported this finding. The binding mode of TLM to DDO was validated further by site-directed mutagenesis of an active site residue, Arg-237. Collectively, our findings show that TLM is a novel, active site-directed DDO inhibitor that will be useful for elucidating the molecular details of the active site environment of DDO.
    Biochimie 10/2010; 92(10):1371-8. · 3.14 Impact Factor
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    ABSTRACT: Under physiological conditions, L-aspartyl (L-Asp) and L-asparaginyl residues in proteins are spontaneously isomerized or racemized to D-aspartyl (D-Asp) or D,L-isoaspartyl (D,L-isoAsp) residue. These atypical Asp residues can interfere with protein activity and lead to disruption of cellular function. Protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) is a repair enzyme that initiates the conversion of L-isoAsp (or D-Asp) residues to L-Asp residues. PIMT-Deficient mice exhibit accumulation of L-isoAsp in several tissues and die from progressive epileptic seizures at a mean age of 42 days. However, the biological roles of PIMT are still largely unknown. To further our understanding of the function of this protein, we developed an assay to measure PIMT activity in cell lysates. Additionally, we generated PIMT-knockdown cells by stable transfection of HEK293 cells with PIMT small interfering (si) RNA. Northern blotting and immunoblot analysis revealed that PIMT mRNA and protein levels were significantly decreased in the knockdown cells. In addition, significant levels of proteins that contained isoAsp residues accumulated in these cells, and immunoblot analysis revealed that Raf-1, MEK, and ERK were hyperphosphorylated upon EGF stimulation compared to control cells. These results indicate that the ability to repair atypical Asp residues is important for normal MAP kinase signaling.
    Chemistry & Biodiversity 06/2010; 7(6):1337-48. · 1.81 Impact Factor
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    ABSTRACT: Previously, we cloned cDNAs for four Caenorhabditis elegans genes (F20 Hp, C47Ap, F18Ep, and Y69Ap genes) that were annotated in the database as encoding D-amino acid oxidase (DAO) or D-aspartate oxidase (DDO) proteins. These genes were expressed in Escherichia coli, and the recombinant C47Ap and F18Ep were shown to have functional DDO activities, while Y69Ap had functional DAO activity. In this study, we improved the E. coli culture conditions for the production of recombinant F20 Hp and, following purification of the protein, revealed that it has functional DDO activity. The kinetic properties of recombinant C47Ap (DDO-1), F18Ep (DDO-2), F20 Hp (DDO-3), and Y69Ap (DAO) were also determined and compared with recombinant human DDO and DAO. In contrast to the low catalytic efficiency of human DDO for D-Glu, all three C. elegans DDOs showed higher catalytic efficiencies for D-Glu than D-Asp or N-methyl-D-Asp. The catalytic efficiency of C. elegans DAO for D-Ser was substantially lower than that of human DAO, while the C. elegans DAO was more efficient at deamination of basic D-amino acids (D-Arg and D-His) than human DAO. Collectively, our results indicate that C. elegans contains at least three genes that encode functional DDOs, and one gene encoding a functional DAO, and that these enzymes have different and distinctive properties.
    Chemistry & Biodiversity 06/2010; 7(6):1424-34. · 1.81 Impact Factor
  • ChemInform 01/2010; 41(36).
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    ABSTRACT: We have characterized release of D-aspartate (D-Asp), a regulator of hormone synthesis and secretion, via a volume-sensitive organic anion channel (VSOC) in PC12 cells by studying its response to apoptotic stimuli. PC12 cells have been demonstrated to endogenously synthesize D-Asp. Apoptotic inducers, including staurosporin (STS), tumor necrosis factor (TNF)-alpha, H(2)O(2), and C2-ceramide, activate the release of D-Asp through a hypotonic stimulus-triggered mechanism. Putative blockers of the anion channel, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-sulphonic acid (DIDS), significantly inhibited stress-induced D-Asp release under hypotonic conditions following the application of apoptotic inducers. Hypotonic conditions are essential for activation by apoptotic inducers. Phorbol 12-mirystate 13-acetate and the Ca(2+) ionophore A23187 increased D-Asp efflux via the VSOC, implying the involvement of intracellular Ca(2+) in the activation of the D-Asp efflux. However, hypotonic stress and STS had no effect on the concentration of intracellular Ca(2+) in PC12 cells. Furthermore, an unknown EGTA-sensitive factor(s), other than Ca(2+), and peroxynitrite may play pivotal roles in STS-enhanced D-Asp release.
    Archives of Biochemistry and Biophysics 09/2009; 490(2):118-28. · 3.37 Impact Factor
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    ABSTRACT: Protein L-isoaspartyl/D-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N]=3), and the quantitation limit of the activity was approximately 18 microg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.
    Analytical Biochemistry 11/2008; 384(2):207-12. · 2.58 Impact Factor
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    ABSTRACT: l-Aspartyl (l-Asp) and l-asparaginyl residues in proteins isomerize or racemize to d,l-isoaspartyl (d,l-isoAsp) or d-aspartyl (d-Asp) residues during protein aging. These atypical aspartyl residues can interfere with the biological function of the protein and lead to cellular dysfunction. Protein l-isoaspartyl (d-aspartyl) methyltransferase (PIMT) is a repair enzyme that facilitates conversion of l-isoAsp and d-Asp to l-Asp. PIMT deficient mice exhibit accumulation of l-isoAsp in several tissues and die, on average, 12 days after birth from progressive epileptic seizures with grand mal and myoclonus features. However, little is known about the molecular mechanisms by which accumulation of the aberrant residues leads to cellular abnormalities. In this study, we established PIMT-knockdown cells using a short interfering RNA expression system and characterized the resultant molecular abnormalities in intracellular signaling pathways. PIMT-knockdown cells showed significant accumulation of proteins with isomerized residues, compared to control cells. In the PIMT-knockdown cells, Raf-1, MEK, and ERK, members of the MAPK cascade, were hyperphosphorylated after EGF stimulation compared to control cells. These results suggest that PIMT repair of abnormal proteins is necessary to maintain normal MAPK signaling.
    Biochemical and Biophysical Research Communications 07/2008; 371(1):22-7. · 2.41 Impact Factor
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    ABSTRACT: The role of Ser-308 of murine D-aspartate oxidase (mDASPO), particularly its side chain hydroxyl group, was investigated through the use of site-specific mutational analysis of Ser-308. Recombinant mDASPO carrying a substitution of Gly, Ala, or Tyr for Ser-308 was generated, and fused to either His (His-mDASPO), or glutathione S-transferase, His, and S (GHS-mDASPO) at its N-terminus. Wild-type His-mDASPO or GHS-mDASPO or their mutant derivatives were expressed in Escherichia coli and purified by affinity chromatography. All purified recombinant proteins had functional DASPO activity. The Gly-308 and Ala-308 mutants had significantly higher catalytic efficiency towards D-Asp and N-methyl-D-Asp, and a higher affinity for flavin adenine dinucleotide (FAD) compared to the wild-type enzyme. The Tyr-308 mutant had lower catalytic efficiency and binding capacity. These results suggest that the side chain hydroxyl group of a critical residue of mDASPO, Ser-308, down-regulates enzymatic activity, substrate binding, and FAD binding. This study provides information on the active site of DASPO that will considerably enhance our understanding of the biological significance of this enzyme.
    Amino Acids 07/2008; 35(1):75-82. · 3.91 Impact Factor
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    ABSTRACT: The understanding of D-amino acid metabolism in higher plants lags far behind that in mammals, for which the biological functions of these unique amino acids have already been elucidated. In this article, we report on the biochemical behavior of D-amino acids (particularly D-Asp) and relevant metabolic enzymes in Arabidopsis thaliana. During germination and growth of the plant, a transient increase in D-Asp levels was observed, suggesting that D-Asp is synthesized in the plant. Administration of D-Asp suppressed growth, although the inhibitory mechanism responsible for this remains to be clarified. Exogenous D-Asp was efficiently incorporated and metabolized, and was converted to other D-amino acids (D-Glu and D-Ala). We then studied the related metabolic enzymes, and consequently cloned and characterized A. thaliana D-amino acid aminotransferase, which is presumably involved in the metabolism of D-Asp in the plant by catalyzing transamination between D-amino acids. This is the first report of cDNA cloning and functional characterization of a D-amino acid aminotransferase in eukaryotes. The results presented here provide important information for understanding the significance of D-amino acids in the metabolism of higher plants.
    FEBS Journal 04/2008; 275(6):1188-200. · 4.25 Impact Factor
  • Takemitsu Furuchi, Hiroshi Homma
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    ABSTRACT: Proteins are subject to various types of spontaneous modifications that can disrupt their structures with sometimes adverse affects on biological activity. The formation of L-isoaspartyl (or D-aspartyl) residues, through either the deamidation of asparagine or dehydration of aspartate, is one of the most frequent types of deterioration occurring under physiological conditions. Protein L-isoaspartate/D-aspartate o-methyltransferase (PIMT) is a conserved and ubiquitous enzyme that participates in the repair of various isomerized proteins. PIMT catalyzes the transfer of the methyl group of S-adenosyl-L-methionine onto the alpha-carboxyl group of an L-isoaspartyl (or the beta-carboxyl group of an D-aspartyl) residue, which initiates the conversion of this residue to an L-aspartyl residue. PIMT-deficient mice have been shown to die at a mean age of 42 days from progressive epileptic seizures with grand mal and myoclonus. Although PIMT-deficiency clearly leads to the accumulation of isomerized proteins, it is currently unclear how this causes progressive epilepsy in PIMT-deficient mice. As a first step towards understanding this, we developed a new assay to measure PIMT activity in cell lysates. Additionally, we isolated PIMT knockdown cells from HEK293 cells that were stably transfected with a PIMT small interfering RNA expression vector. PIMT activities were significantly decreased in the PIMT knockdown cells, and analysis of the transfectants revealed that MEK and ERK were hyperactivated after cell stimulation with epidermal growth factor (EGF). These results indicate that the ability to repair L-isoaspartyl-(or D-aspartyl-) containing proteins is important for the maintenance of normal MEK-ERK signaling.
    Yakugaku zasshi journal of the Pharmaceutical Society of Japan 01/2008; 127(12):1927-36. · 0.46 Impact Factor
  • Takemitsu Furuchi, Hiroshi Homma
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    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 01/2008; 39(15).
  • M Katane, T Furuchi, M Sekine, H Homma
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    ABSTRACT: The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT-PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.
    Amino Acids 02/2007; 32(1):69-78. · 3.91 Impact Factor
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    ABSTRACT: Four cDNA clones that were annotated in the database as encoding d-amino acid oxidase (DAAO) or d-aspartate oxidase (DASPO) were isolated by RT-PCR from Caenorhabditis elegans RNA. The proteins (Y69Ap, C47Ap, F18Ep, and F20Hp) encoded by the cloned cDNAs were expressed in Escherichia coli as recombinant proteins with an N-terminal His-tag. All proteins except F20Hp were recovered in the soluble fractions. The recombinant Y69Ap has functional DAAO activity, as it can deaminate neutral and basic d-amino acids, whereas the recombinants C47Ap and F18Ep have functional DASPO activities, as they can deaminate acidic d-amino acids. Additional experiments using purified recombinant proteins revealed that Y69Ap deaminates d-Arg more efficiently than d-Ala and d-Met, and that C47Ap and F18Ep show distinct kinetic properties against d-Asp, d-Glu, and N-methyl-d-Asp. This is the first time that cDNA cloning of invertebrate DAAO and DASPO genes has been reported. In addition, our study reveals for the first time that C. elegans has at least two genes encoding functional DASPOs and one gene encoding DAAO, although it had previously been thought that organisms only bear one copy each of these genes. The two C. elegans DASPOs differ in their substrate specificities and possibly also in their subcellular localization.
    FEBS Journal 02/2007; 274(1):137-49. · 4.25 Impact Factor
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    ABSTRACT: In our previous reports [Z. Long, H. Homma, J.-A. Lee, T. Fukushima, T. Santa, T. Iwatsubo, R. Yamada, K. Imai, FEBS Lett. 434 (1998) 231-235; Z. Long, M. Sekine, M. Adachi, T. Furuchi, K. Imai, N. Nimura, H. Homma, Arch. Biochem. Biophys. 404 (2002) 92-97], we demonstrated for the first time that D-aspartate (D-Asp) is actually synthesized in cultured mammalian cells such as PC12, MPT1, and GH3 cells. After its synthesis, this unique amino acid is spontaneously and continuously released into the extracellular space during cell culture. In the current study, we characterized two different types of D-Asp efflux in PC12 cells. One is a spontaneous and continuous form of release of cytoplasmic origin that does not involve exocytotic efflux of vesicular origin. Endogenous D-Asp is predominantly localized to the cytoplasm of cells, and this form of D-Asp release presents a striking contrast to exocytotic, quantal discharge of vesicular dopamine. The other form of efflux is also of cytoplasmic origin and occurs through volume-sensitive organic anion channels that are opened upon hyposmotic stimuli. Interestingly, this latter form of efflux is potentiated by acetylcholine stimulation.
    Archives of Biochemistry and Biophysics 03/2006; 446(2):131-9. · 3.37 Impact Factor
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    Takemitsu Furuchi, Hiroshi Homma
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    ABSTRACT: D-Aspartate (D-Asp) is an endogenous amino acid present in nervous and endocrine tissues in mammals. A high concentration of D-Asp is observed in embryos, which disappears in nervous tissues after delivery, but increases temporarily in endocrine glands, particularly in the pituitary, pineal and adrenal glands at the specific stages. In the pineal gland, D-Asp that is apparently derived from other tissues suppresses melatonin secretion from parenchymal cells. Additionally, D-Asp levels increase in the testis just before birth and during maturation. The amino acid is presumed to be synthesized by the pituitary gland and testis. In the testis, D-Asp produced inside the seminiferous tubules acts on Leydig cells following release to enhance testosterone synthesis by activating the expression of Steroidogenic Acute Regulatory protein. Mammalian cells appear to contain all the molecular components required to regulate D-Asp homeostasis, as they can synthesize, release, take up, and degrade the amino acid. These findings collectively indicate that D-Asp is a novel type of messenger in the mammalian body.
    Biological & Pharmaceutical Bulletin 10/2005; 28(9):1566-70. · 1.85 Impact Factor