Nobuaki Okumura

Binding of APC to the Human Homolog of the Drosophila Discs Large Tumor Suppressor Protein

The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein β-catenin. Overexpression of APC blocks cell cycle progression. The APC-β-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function.

Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain.

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.

Effects of intraduodenal injection of Lactobacillus johnsonii La1 on renal sympathetic nerve activity and blood pressure in urethane-anesthetized rats

Previously, it was shown that milk fermented with lactic acid bacteria lowers blood pressure, suggesting that metabolites or components of the bacteria have hypotensive action. To examine whether one of lactobacilli, Lactobacillus johnsonii La1 (LJLa1), a probiotic strain adhesive onto intestinal epithelial cells, or its metabolite has hypotensive action, and if so the mechanism of action, we determined the effects of intraduodenal injection of LJLa1 on blood pressure (BP) and the activity of autonomic nerves in urethane-anesthetized rats. Intraduodenal injection of LJLa1 reduced renal sympathetic nerve activity (RSNA) and BP and enhanced gastric vagal nerve activity (GVNA). Pre-treatment with thioperamide, a histaminergic H3-receptor antagonist, eliminated the effects of LJLa1 on RSNA, GVNA, and BP. Furthermore, bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN), the master circadian oscillator, abolished the suppression of RSNA and BP and the elevation of GVNA caused by LJLa1. These findings suggest that LJLa1 or its metabolites might lower BP by changing autonomic neurotransmission via the central histaminergic nerves and the suprachiasmatic nucleus in rats.

Automated interpretation of low-energy collision-induced dissociation spectra by SeqMS, a software aid for de novo sequencing by tandem mass spectrometry

SeqMS, a software aid for de novo sequencing by tandem mass spectrometry (MS/MS), which was initially developed for the automated interpretation of high‐energy collision‐induced dissociation (CID) MS/MS spectra of peptides, has been applied to the interpretation of low‐energy CID and post‐source decay (PSD) spectra of peptides. Based on peptide backbone fragmented ions and their related ions, which are the dominant ions observed in the latter two techniques, the types of ions and their propensities to be observed have been optimized for efficient interpretation of the spectra. In a typical example, the modified SeqMS allowed the complete sequencing of a 31‐amino acid synthetic peptide, except for the isobaric amino acids (Leu or Ile, and Lys or Gln), based on only the low‐energy CID‐MS/MS spectrum.

Characteristics of glucose transport in neuronal cells and astrocytes from rat brain in primary culture.

Abstract: Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2‐deoxy‐d‐[3H]glucose ([3H]2‐DG) into the cells. Various sugars inhibited 2‐DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Kmvalues for 2‐DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2‐DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2‐DG uptake competitively in both neuronal cells and astrocytes. The uptakes by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis after photoamnity labeling using [3H]cytochalasin B as a probe.

Interaction of Neuronal Nitric-oxide Synthase with α1-Syntrophin in Rat Brain

Neuronal nitric-oxide synthase (nNOS) has a PSD-95/Dlg/ZO-1 (PDZ) domain that can interact with multiple proteins. nNOS has been known to interact with PSD-95 and a related protein, PSD-93, in brain and with α1-syntrophin in skeletal muscle in mammals. In this study, we have purified an nNOS-interacting protein from bovine brain using an affinity column made of Sepharose conjugated with glutathione S-transferase-rat nNOS fusion protein and identified it as α1-syntrophin by microsequencing. Immunostaining of primary cultures of rat embryonic brain neuronal cells with antibodies against these proteins showed that nNOS and α1-syntrophin were colocalized in neuronal cell bodies and neurites. Immunohistochemical analysis indicated that the nNOS- and α1-syntrophin-like immunoreactive substances were highly expressed in the rat hypothalamic suprachiasmatic nucleus (SCN) and paraventricular nucleus. In the SCN, nNOS- and α1-syntrophin-like immunoreactive substances were colocalized in the same neurons as detected by confocal microscopy. These results indicate that nNOS in brain interacts with α1-syntrophin in specific neurons of the SCN and paraventricular nucleus and that this interaction might play a physiological role in functions of these neurons.

Role of l-carnosine in the control of blood glucose, blood pressure, thermogenesis, and lipolysis by autonomic nerves in rats: involvement of the circadian clock and histamine

Abstractl-Carnosine (β-alanyl-l-histidine; CAR) is synthesized in mammalian skeletal muscle. Although the physiological roles of CAR have not yet been clarified, there is evidence that the release of CAR from skeletal muscle during physical exercise affects autonomic neurotransmission and physiological functions. In particular, CAR affects the activity of sympathetic and parasympathetic nerves innervating the adrenal glands, liver, kidney, pancreas, stomach, and white and brown adipose tissues, thereby causing changes in blood pressure, blood glucose, appetite, lipolysis, and thermogenesis. CAR-mediated changes in neurotransmission and physiological functions were eliminated by histamine H1 or H3 receptor antagonists (diphenhydramine or thioperamide) and bilateral lesions of the hypothalamic suprachiasmatic nucleus (SCN), a master circadian clock. Moreover, a carnosine-degrading enzyme (carnosinase 2) was shown to be localized to histamine neurons in the hypothalamic tuberomammillary nucleus (TMN). Thus, CAR released from skeletal muscle during exercise may be transported into TMN-histamine neurons and hydrolyzed. The resulting l-histidine may subsequently be converted into histamine, which could be responsible for the effects of CAR on neurotransmission and physiological function. Thus, CAR appears to influence hypoglycemic, hypotensive, and lipolytic activity through regulation of autonomic nerves and with the involvement of the SCN and histamine. These findings are reviewed and discussed in the context of other recent reports, including those on carnosine synthetases, carnosinases, and carnosine transport.

Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2.

l-Carnosine is a bioactive dipeptide (β-alanyl-l-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of l-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn2+, and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn2+ at a resolution of 1.7Å and that with Mn2+ at 2.3Å. CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn2+ and Zn2+ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.

Localization of basic FGF-like immunoreactivity in the hypothalamo-hypophyseal neuroendocrine axis

We examined the localization of basic fibroblast growth factor (basic FGF) in the adult rat brain by immunohistochemical and Western blotting analysis using a specific antibody against a synthetic basic FGF fragment (N-terminal 12 residues). The antibody did not cross-react with acidic FGF. Basic FGF-like immunoreactivity was located exclusively in the neuronal elements and had very heterogeneous distribution. Immunoreactive cell bodies were observed in the paraventricular, supraoptic and circular nuclei of the hypothalamus. Numerous immunoreactive neuronal processes originating from these basic FGF-positive cells extended lateroventrally and then caudally to the internal layer of the median eminence. In addition, the neurohypophysis contained a significant number of basic FGF-like immunoreactive fibers. Western-blotting analysis revealed that the hypothalamus and the hypophysis contained a main band of basic FGF immunoreactive with an apparent molecular weight of 17 kDa. These results show that the hypothalamo-hypophyseal neuroendocrine pathway contains basic FGF.

Evidence for phosphorylation of rat liver glucose-regulated protein 58, GRP58/ERp57/ER-60, induced by fasting and leptin

Glucose‐regulated protein 58 (GRP58)‐like immunoreactivity in rat liver obtained in the evening or after fasting underwent an electrophoretic band‐shift, which disappeared after phosphatase‐treatment. Since mass spectrometric analysis raised a possibility that Ser150 of GRP58 is phosphorylated, an antibody against the phosphoserine150 GRP58 was generated. Immunoreactivity to this antibody was increased in the evening and after fasting. Since GRP58 was shown to interact with signal transducer and activator of transduction 3 (STAT3), a leptin‐related protein, the effect of leptin was examined. Immunoreactivity to the anti‐phosphoGRP58 antibody was markedly elevated after the leptin injection, indicating that Ser150 of GRP58 is phosphorylated after fasting and leptin‐treatment.