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ABSTRACT: Calcineurin is a Ca(2+) /calmodulin-dependent protein phosphatase consisting of two subunits - catalytic subunit A (CnA) and regulatory subunit B (CnB) - and plays a critical role in transducing Ca(2+) signals into cellular responses. In this study, we investigated the expression of calcineurin in the mouse developing tooth. In-situ hybridization detected mRNAs for the CnAα and CnAβ isoforms of CnA and for the CnB1 isoform of CnB in the upper molar tooth germ at embryonic day 15. Immunohistochemistry with antibodies specific for CnAα, CnAβ, and CnB1 showed strong immunoreactivity of these proteins in secretory-stage ameloblasts and in odontoblasts during dentin formation. CnAβ and CnB1 were strongly immunoreactive in ruffle-ended ameloblasts at the enamel-maturation stage. In ameloblasts and odontoblasts, we also noted different subcellular distributions of CnAα and CnAβ. From these data, temporal profiles of calcineurin expression appear to correlate with active mineralization in tooth development. Furthermore, the distinct subcellular distribution of the two CnA subunits may reflect their distinct substrates or responsive sites within single cells, thus contributing to the diversity of calcineurin-dependent cellular responses during active tooth mineralization.
European Journal Of Oral Sciences 10/2012; 120(5):386-94. · 1.88 Impact Factor
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Seiji Negoro,
Naoki Shibata,
Yusuke Tanaka,
Kengo Yasuhira,
Hiroshi Shibata,
Haruka Hashimoto,
Young-Ho Lee, Shohei Oshima,
Ryuji Santa,
Kozo Mochiji,
Yuji Goto,
Takahisa Ikegami,
Keisuke Nagai,
Dai-Ichiro Kato,
Masahiro Takeo,
Yoshiki Higuchi
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ABSTRACT: We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.
Journal of Biological Chemistry 12/2011; 287(7):5079-90. · 4.77 Impact Factor
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ABSTRACT: In secondary ion mass spectrometry (SIMS) of organic substances, the dissociation of the sample molecules is crucial. We have developed SIMS equipment capable of bombardment, where the primary ions are argon cluster ions with kinetic energy per atom controllable down to 1 eV. We previously reported the detection of intact ions of insulin and cytochrome C using this equipment. In this paper, we present a detailed characterization of the emission of secondary ions from insulin, focusing on the difference in secondary ion yield between intact ions and fragment ions by varying the incident angle of the cluster ions. The emission intensity of the intact ions was changed drastically due to the exposed dosage and incident angle of the cluster ions in contrast to the fragment ions. We discuss these results based on the manner in which the argon-cluster ions collide with the organic solid.
Rapid Communications in Mass Spectrometry 04/2011; 25(8):1070-4. · 2.79 Impact Factor
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ABSTRACT: Production of nitric oxide (NO) in the hypothalamic paraventricular nucleus (PVN) was examined by microdialysis in rats subjected to immobilization (IMO) stress. A dialysis probe was implanted in the posterior magnocellular subdivision of the PVN and nitrite (NO(2)(-)), an oxidized product of NO, was measured continuously. NO(2)(-) concentration in dialysate was enhanced to 156% after 30 min of IMO compared with the NO(2)(-) level before IMO. Intraperitoneal administration of N(G)-monomethyl-l-arginine (10 mg/kg), a NO synthase inhibitor, before IMO completely inhibited the increase of NO production that IMO was to induce. Depletion of catecholamines innervating the PVN by an injection of 6-hydroxydopamine into the lateral ventricle before the microdialysis had no suppressive effect on the increase of NO production by IMO. In contrast, NO(2)(-) levels in the PVN were lowered by continuous perfusion of the solution containing the ionotropic glutamate receptor antagonists 2-amino-5-phosphonovaleric acid (500 microm) and 6-cyano-7-nitroquinoxaline-2, 3 dione (50 microm) through the dialysis probe, and the IMO-induced increase of NO production was attenuated by the treatment. These results suggest that catecholaminergic drive to the hypothalamus is not necessary for the IMO-induced increase of NO production and that ionotropic glutamate receptors play a role in the basal and IMO-induced NO production.
Endocrinology 09/2004; 145(8):3603-7. · 4.46 Impact Factor
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ABSTRACT: The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy.
Neuroscience Letters 08/2004; 365(2):97-101. · 2.11 Impact Factor
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ABSTRACT: We examined the expression profile of catechol O-methyltransferase (COMT) mRNA and its protein in the neonatal rat hypothalamus by in situ hybridization and immunohistochemistry to clarify the sites of dopamine degradation. Strong COMT mRNA expression was observed in the suprachiasmatic nucleus (SCN) throughout its rostrocaudal extent at postnatal day 1 (P1) and P2, and the mRNA levels decreased gradually until P16. COMT mRNA was predominantly localized to the ventral and medial parts of the SCN. Intense COMT immunoreactivity was demonstrated in the ventral SCN and was detected in neuronal perikarya and processes at P1. Ependymal and microglial cells also exhibited strong COMT immunoreactivity. These results indicate that COMT may directly be involved in dopaminergic signaling in the neonatal SCN.
Neuroreport 07/2004; 15(8):1239-43. · 1.66 Impact Factor
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ABSTRACT: Oro-facial sensorimotor function conducted by the brainstem is vital to newborn mammals, and N-methyl-D-aspartate (NMDA) receptors play an important role in the regulation. Here we examined the expression of NMDA receptor subunits in the mouse hypoglossal nucleus from embryonic day 13 (E13) through postnatal day 21 (P21). Compared with other brainstem regions, early onset of GluRepsilon1 (NR2A) mRNA expression was conspicuous to the embryonic hypoglossal nucleus. The expression peaked at P1-P7, when other brainstem regions just started to express it. At P1, GluRepsilon1 subunit was localized to asymmetrical synapses on motoneuron dendrites, particularly, on the postsynaptic junction membrane. In developing motoneurons, expressions of GluRepsilon2 (NR2B), GluRepsilon4 (NR2D), and GluRzeta1 (NR1) mRNAs were accompanied. Until P21, however, all of these subunits were down-regulated with particular reduction for GluRepsilon2 and GluRepsilon4 mRNAs. Similar patterns of temporal expressions were observed in motoneurons of other brainstem motor nuclei. Taking that high levels of GluRepsilon1, GluRepsilon2, and GluRzeta1 subunits are also found in the adult hippocampus and cerebral cortex, it can be assumed that NMDA receptors in developing motoneurons are highly potent and potentially involved in structural and functional development of the brainstem motor system.
Neuroscience Research 08/2002; 43(3):239-50. · 2.25 Impact Factor
