Akito Tomomura

Differential expression and function of apoptosis-associated tyrosine kinase (AATYK) in the developing mouse brain

Apoptosis-associated tyrosine kinase (AATYK) is a non-receptor type tyrosine kinase that is predominantly expressed in adult mouse brain. Although it is also expressed in developing brains, its expression pattern and physiological functions are unclear. In the present study, we analyzed expression profiles of AATYK in developing mouse brains and its functional role and subcellular localization in cultured cerebellar granule cells. Expression of AATYK mRNA and protein increased during postnatal brain development. Immunohistochemical analysis indicated that the protein was differentially expressed in postmitotic neurons within various brain areas including the olfactory bulb, cerebral cortex, hippocampus, thalamus, colliculus, cerebellum, and brain stem. Developmental increases in its expression were also observed in cultured cerebellar granule cells. AATYK protein was largely fractionated into the microsomal fraction and was immunocytochemically distributed in an ER-like meshwork of the granule cell soma, suggesting a possible association with the ER membrane. AATYK protein was also present in neurites. In immature granule cells, overexpression of wild-type AATYK promoted neurite outgrowth, whereas that of tyrosine kinase-defective mutant significantly inhibited it. These results suggest that, in addition to its role in cell death in mature neurons, AATYK has a unique role in promoting neurite extension through its tyrosine kinase activity in developing neurons.

Role of the conserved Ser–Tyr–Lys triad of the SDR family in sepiapterin reductase

Sepiapterin reductase (EC 1.1.1.153; SPR) is an enzyme involved in the biosynthesis of tetrahydrobiopterin; and SPR has been identified as a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family based on its catalytic properties for exogenous carbonyl compounds and molecular structure. To examine possible differences in the catalytic sites of SPR for exogenous carbonyl compounds and the native pteridine substrates, we investigated by site-directed mutagenesis the role of the highly conserved Ser-Tyr-Lys triad (Ser and YXXXK motif) in SPR, which was shown to be the catalytic site of SDR-family enzymes. From the analysis of catalytic constants for single- and double-point mutants against the triad, Ser and YXXXK motif, in the SPR molecule, participate in the reduction of the carbonyl group of both pteridine and exogenous carbonyl compounds. The Ser and the Tyr of the triad may co-act in proton transfer and stabilization for the carbonyl group of substrates, as was demonstrated for those in the SDR family. But either the Tyr or the Ser of SPR can function alone for proton transfer to a certain extent and show low activity for both substrates.

Determination of amino acid sequence responsible for suppression of bone resorption by serum calcium-decreasing factor (caldecrin).

We previously reported on the serum calcium‐decreasing activity of recombinant protein factor referred to as caldecrin [Tomomura et al. (1995) J. Biol. Chem. 270, 30315–30321]. To address the mechanism of this serum calcium‐decreasing activity, we investigated the effect of rat caldecrin on osteoclastic bone‐resorbing activity. Wild‐type caldecrin suppressed resorption pit formation by osteoclast on a dentine slice in a dose‐dependent manner. The suppressive effect on the bone resorption was not affected by treatment of caldecrin with phenylmethyl sulfonyl fluoride or by use of protease‐deficient mutant caldecrins. Recombinant procaldecrin (−13–239), and its fragments (−13–125), (1–111), (1–46), (47–111), and (126–239) were expressed as His‐tagged thioredoxin fusion proteins and investigated for their ability to suppress bone resorption. The proform (−13–239) and fragment (−13–125) did not affect the suppressive activity, whereas fragments (1–111) and (126–239) did suppress the bone resorption. The bone‐resorbing activity was also suppressed by fragment (47–111), not by fragment (1–46). Overlapping fragments (47–62), (47–79), (47–98), (56–111), (71–111), and (85–111) were compared for their suppressive activity. The fragments (47–62) and (85–111) did not affect the activity, but the other fragments suppressed the bone resorption. A synthetic peptide having the (71–79) sequence suppressed the bone resorption. These results suggest that amino acid sequence corresponding to rat caldecrin (aa 71–79) is responsible for the suppression of bone resorption by caldecrin.

Rat brain expresses serum calcium-decreasing factor (caldecrin)

We previously cloned the serum calcium-decreasing factor referred to as caldecrin from pancreas (J. Biol. Chem. 270 (1995) 30315). Caldecrin has been shown to be a chymotrypsin-type serine protease and to inhibit parathyroid hormone or parathyroid hormone-related peptide-induced bone resorption. In the present study, caldecrin was detected in adult rat brain by Western blotting and reverse transcription-polymerase chain reaction analysis. The caldecrin gene was constitutively expressed during postnatal days 1-28 in the brain. By in situ hybridization, the caldecrin mRNA was detected in the whole brain, including the olfactory bulb, cerebrum, hippocampus, thalamus, and cerebellum. These results suggest that caldecrin may play a role in the calcium homeostasis of the central nervous system.

Increase in Tetrahydrobiopterin Release from PC 12 Cells under Hypotonic Culture Conditions is Inhibited by HgCl2

Abstract Tetrahydrobiopterin (BH4) was released from PC 12 cells to the extracellular fluid. We found that the BH4 outward flow from the cells placed in Hanks medium was increased when NaCl concentration of the medium was decreased. Increase in the BH4 out flow was not observed when NaCl in the Hanks medium was substituted with sodium glutamate (0.14 M), choline chloride (0.14 M) or sucrosc (0.25 M). HgCl2, an inhibitor of water channel, aquaporin, prevented the increase in BH4 out flow caused by the hypotonic medium. The results suggested the presence of a site in BH4 transport which was stimulated by osmotic pressure and/or water influx.

Morphological Changes of Myoepithelial Cells in the Rat Submandibular Gland Following the Application of Surgical Stimuli

Myoepithelial cells (MECs) exist on the basal surface of acini in major exocrine glands, include myofilaments and various constructive proteins, and share characteristics with smooth muscle and epithelial cells. MECs project several ramified processes to invest acini, and possibly contract to compress acini to support the secretion by the glandular cells. However, the functional roles of MECs in salivary secretion are still unclear. We investigated morphological changes in immunostained MECs using the anti-α-smooth muscle actin (αSMA) antibody in operated or non-operated contralateral (NC) submandibular glands after partial or total resection. Furthermore, we investigated and discuss other salivary glands of rats. MECs in the parotid, sublingual and submandibular gland of adult rats exhibited different shapes and localizations. After surgery, in both operated and NC glands, the number of MECs and αSMA-immunopositive areas increased significantly. Three-dimensional analysis using a confocal laser-scanning microscope revealed that substantial and significant enhancement became evident in the number, length, and thickness of MEC-processes covering acini of the operated and NC submandibular glands. The preset findings indicate that MECs alter the morphology of their processes in operated and NC glands after surgery of the partial or total resection. It is suggested that MECs promote salivary secretion using elongated, thickened, and more ramified processes.

Rhinacanthin C Inhibits Osteoclast Differentiation and Bone Resorption: Roles of TRAF6/TAK1/MAPKs/NF-κB/NFATc1 Signaling

Rhinacanthin C is a naphthoquinone ester with anti-inflammatory activity, found in Rhinacanthus nasutus (L) Kurz (Acanthaceae). We found that rhinacanthin C inhibited osteoclast differentiation stimulated by the receptor activator of nuclear factor-κB ligand (RANKL) in mouse bone marrow macrophage cultures, although the precise molecular mechanisms underlying this phenomenon are unclear. In this study, we investigated the inhibitory mechanisms of rhinacanthin C in osteoclastogenesis. Rhinacanthin C suppressed RANKL-induced nuclear factor of activated T cells c1 (NFATc1) expression. Phosphorylation of ERK, JNK, and NF-κB, but not p38, was inhibited by rhinacanthin C, which also inhibited RANKL-stimulated TRAF6-TAK1 complex formation. Thus, the anti-osteoclastogenic effect of rhinacanthin C is mediated by a cascade of inhibition of RANKL-induced TRAF6-TAK1 association followed by activation of MAPKs/NF-κB; this leads to suppression of c-Fos and NFATc1, which regulate transcription of genes associated with osteoclast differentiation. In vivo, rhinacanthin C also reduced RANKL-induced osteoclast formation and bone resorption in mouse calvaria. Rhinacanthin C also suppressed LPS-stimulated osteoclastogenesis and bone resorption in vitro and in vivo. Rhinacanthin C may provide a novel therapy for abnormal bone lysis that occurs during inflammatory bone resorption.

Serum calcium-decreasing factor, caldecrin, protects against amyloid-β peptide toxicity by proteolytic cleavage of the peptide

Recent studies have implicated Rac1, a Rho family GTPases, participates in Alzheimer’s disease. In this study, we examined the neuroprotective effects of NSC23766, a specific Rac1 inhibitor, on neurotoxicity induced by amyloid betapeptide (Abeta). Abeta42 treatment increased Rac1 activity in primary hippocampal neurons. NSC23766 pretreatment reduced both Rac1 activity and neurotoxicity induced by Abeta. These results indicate that Rac1 activation is involved in Abetainduced neurotoxicity. Activation of glycogen synthase kinase 3beta (GSK3beta) is also reported to be involved in Abeta-induced neurotoxicity; however, the signaling pathway of GSK3beta remains to be elucidated. Interestingly, Abeta42 treatment increased GSK3beta activity and NSC23766 pretreatment reduced this activity. Our results suggest that Rac1, which acts as an upstream factor of GSK3beta, is involved in Abeta-induced neurotoxicity.

Determination by Site-directed Mutagenesis of Sites in Sepiapterin Reductase Phosphorylated by Ca2+/Calmodulin-dependent Protein Kinase II

Summary Phosphorylation sites of sepiapterin reductase (SPR) phosphorylated by Ca2+-dependent protein kinase II (CaM KII) were studied. By immunoreaction against phosphorylated amino acids, we found that Ser residues of SPR were phosphorylated. We constructed several point mutants of SPR by site-directed mutagenesis and expressed then in E. coli. In assays with anti-phospho Ser antibody, we determined that each of the three Ser residues, S46, S 196, and S214, of SPR was phosphorylated by CaM KIl. Each of these serine residues in SPR was found in a CaM KII phosphorylation site sequelce (Arg-X-X-Ser/Thr).