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Masahiko Watanabe

Publications and source records attributed to Masahiko Watanabe.

At least 19 recordsLinked to original sources
PubMed2026

GIRK Channels Regulate Circadian Rhythms of Excitability in Prokineticin 2 Neurons of the Suprachiasmatic Nucleus and Modulate Behavioral Circadian Rhythms.

The suprachiasmatic nucleus (SCN), the central circadian clock in mammals, generates robust yet adaptable circadian rhythms through electrically mediated coordination among heterogeneous peptidergic neuronal populations with presumed cell type-specific roles. Previous studies have proposed that circadian changes in membrane excitability of individual SCN neurons arise from time-of-day-dependent shifts in the relative balance of subthreshold Na+ and K+ conductances. Although multiple channels have been implicated in these processes, how nocturnally dominant K+ conductances are implemented in a cell type-specific manner remains poorly understood. Prokineticin 2 (Prok2) has been identified as a SCN signaling peptide essential for behavioral circadian regulation; however, the electrophysiological properties of Prok2-expressing neurons and the mechanisms underlying their diurnal rhythmicity remain largely unexplored. Here, using electrophysiological approaches in mice of either sex, we show that Prok2 neurons exhibit diurnal variations in electrical properties, with higher excitability during the day and reduced excitability at night, and that G-protein-coupled inwardly rectifying potassium (GIRK) channel-mediated basal current contributes to nighttime hyperpolarization. Immunofluorescence and single-cell RT-PCR analyses revealed that GIRK1 and GIRK3 are the predominant GIRK subunits expressed in Prok2 neurons. Moreover, Prok2 neuron-specific deletion of GIRK3 using in vivo genome editing resulted in significant nocturnal depolarization and induced abnormalities in behavioral rhythms, including delayed activity onset and circadian period lengthening, with altered SCN network activity. Together, these findings suggest that tonic, G-protein-dependent regulation of GIRK channels provides a night-specific inhibitory mechanism that contributes to intrinsic diurnal neuronal excitability in Prok2 neurons and supports the regulation of behavioral circadian rhythms.

Animals↗
PubMed2006

The effects of hypoxia-ischemia on neutral amino acid transporters in the developing rat brain.

The neutral amino acid transporters SNAT1-3 and ASCT1 play critical roles in the recycling of glutamine, and subsequently glutamate, via the glutamine-glutamate cycle. Hypoxia-ischemia was induced in rat pups using the Rice-Vannucci model. Brains were harvested at 1 h, 24 h and 7 days after ischemia. The expression of NAATs was evaluated using immunoblotting, real-time PCR, and immunohistochemistry. Results were compared with age-matched controls and shams. SNAT1 mRNA decreased at 1 h after injury in both hemispheres when compared with the control animals and correlated with a decrease in protein expression at 24 h in the hippocampus and cortex. SNAT1 protein expression increased globally at 7 days after injury and specifically in the hippocampus. Finally, SNAT2 and 3 demonstrated subtle changes in various brain regions after injury. These data suggest that neutral amino acid transporters remain largely intact after hypoxia-ischemia.

Amino Acid Transport System A↗
PubMed2006

Depolarization-induced suppression of inhibition mediated by endocannabinoids at synapses from fast-spiking interneurons to medium spiny neurons in the striatum.

Endogenous cannabinoids (endocannabinoids) act as retrograde inhibitory messengers in various regions of the brain. We have recently reported that endocannabinoids mediate short-term retrograde suppression of excitatory synaptic transmission from the neocortex to medium spiny (MS) neurons, the major projection neurons from the striatum. However, it remains unclear whether endocannabinoids modulate inhibitory transmission in the striatum. Here we show that depolarization of MS neurons induces transient suppression of inhibition that is mediated by retrograde endocannabinoid signalling. By paired recording from a fast-spiking (FS) interneuron and an MS neuron, we demonstrated that FS-MS inhibitory synapses undergo endocannabinoid-mediated retrograde suppression. We verified that GABAergic inhibitory terminals immunopositive for parvalbumin (PV), a marker for FS interneurons, expressed CB1 receptors. These PV-CB1 double-positive terminals surrounded dopamine D1 receptor-positive and D2 receptor-positive MS neurons; these constitute direct and indirect pathways, respectively. These results suggest that endocannabinoid-mediated retrograde suppression of inhibition influences information flow along both direct and indirect pathways, depending on the activity of MS neurons.

Amino Acid Sequence↗
PubMed2006

Characterization of a novel low-molecular-mass dual specificity phosphatase-4 (LDP-4) expressed in brain.

Dual-specificity phosphatases (DSPs), which dephosphorylate proteins at Ser/Thr as well as Tyr residues, are thought to be involved in critical signaling events such as control of MAP kinases (MAPKs). We have isolated the cDNA for a novel DSP and termed it low molecular mass DSP-4 (LDP-4). LDP-4 is composed of 211 amino acids with a predicted molecular mass of 23.9-kDa. Northern blot analysis using various mouse tissues showed that the LDP-4 transcript was expressed exclusively in brain. In situ hybridization showed that brain expression of LDP-4 was ubiquitous except for the hippocampus. When expressed in COS-7 cells, FLAG-tagged LDP-4 protein was present within the nucleus and Golgi apparatus. LDP-4 expression did not reduce phosphorylation levels of MAPKs, but rather evoked activation of JNK and p38.

Amino Acid Sequence↗
PubMed2006

Purification and molecular cloning of a DNA ADP-ribosylating protein, CARP-1, from the edible clam Meretrix lamarckii.

The cabbage butterflies Pieris rapae and Pieris brassicae have unique enzymes, named pierisin-1 and -2, respectively, that catalyze the ADP-ribosylation of guanine residues of DNA, which has been linked with induction of apoptosis and mutation in mammalian cell lines. In the present study, we identified ADP-ribosylation activity targeting DNA in six kinds of edible clam. Similar to our observations with pierisin-1 and -2, crude extracts from the clams Meretrix lamarckii, Ruditapes philippinarum, and Corbicula japonica incubated with calf thymus DNA and beta-NAD resulted in production of N(2)-(ADP-ribos-1-yl)-2'-deoxyguanosine. The DNA ADP-ribosylating protein in the hard clam M. lamarckii, designated as CARP-1, was purified by column chromatography, and its cDNA was cloned. The cDNA encodes a 182-aa protein with a calculated molecular mass of 20,332. The protein synthesized in vitro from the cDNA in a reticulocyte lysate exhibited the same ADP-ribosylating activity as that of purified CARP-1. Neither the nucleotide nor the deduced amino acid sequence of CARP-1 showed homology with pierisin-1 or -2. However, a glutamic acid residue (E128) at the putative NAD-binding site, conserved in all ADP-ribosyltransferases, was found in CARP-1, and replacement of aspartic acid for this glutamic acid resulted in loss of almost all ADP-ribosylating activity. CARP-1 in the culture medium showed no cytotoxicity against HeLa and TMK-1 cells; however, introduction of this protein by electroporation induced apoptosis in these cells. The finding of clam ADP-ribosylating protein targeting guanine residues in DNA could offer new insights into the biological significance of ADP-ribosylation of DNA.

ADP Ribose Transferases↗
PubMed2006

From the Cover: Indispensability of the glutamate transporters GLAST and GLT1 to brain development.

Previous in vitro studies have shown that the neurotransmitter glutamate is important in brain development. Paradoxically, loss-of-function mouse models of glutamatergic signaling that are generated by genetic deletion of glutamate receptors or glutamate release show normal brain assembly. We examined the direct consequences on brain development of extracellular glutamate buildup due to the depletion of the glutamate transporters GLAST and GLT1. GLAST/GLT1 double knockout mice show multiple brain defects, including cortical, hippocampal, and olfactory bulb disorganization with perinatal mortality. Here, we report abnormal formation of the neocortex in GLAST/GLT1 mutants. Several essential aspects of neuronal development, such as stem cell proliferation, radial migration, neuronal differentiation, and survival of SP neurons, were impaired. These results provide direct in vivo evidence that GLAST and GLT1 are necessary for brain development through regulation of extracellular glutamate concentration and show that an important mechanism is likely to be maintenance of glutamate-mediated synaptic transmission.

Animals↗
PubMed2006

Effects of RNA interference of Atg4B on the limited proteolysis of LC3 in PC12 cells and expression of Atg4B in various rat tissues.

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.

Animals↗
PubMed2006

Dehydration-induced expression of LEA proteins in an anhydrobiotic chironomid.

Late embryogenesis abundant (LEA) proteins are associated with desiccation tolerance in anhydrobiotic organisms. The larvae of an African chironomid, Polypedilum vanderplanki, are able to withstand almost complete desiccation during which they enter a state of suspended animation. Here, we developed an EST database from desiccating larvae and isolated three cDNAs encoding proteins (PvLEA1, PvLEA2, and PvLEA3) with highly significant matches to Group 3 LEA proteins. Both mRNA and protein levels of all three examples were increased by dehydration stress imposed by either desiccation or hypersalinity, and one protein, PvLEA2, is likely to be post-translationally processed into smaller molecules. This first description of LEA protein genes in arthropods suggests that this protein family is widespread throughout invertebrate phyla, and that animals, plants, and microorganisms possess similar mechanisms for combating dehydration stress.

Animals↗
PubMed2006

Functional uncoupling between Ca2+ release and afterhyperpolarization in mutant hippocampal neurons lacking junctophilins.

Junctional membrane complexes (JMCs) composed of the plasma membrane and endoplasmic/sarcoplasmic reticulum seem to be a structural platform for channel crosstalk. Junctophilins (JPs) contribute to JMC formation by spanning the sarcoplasmic reticulum membrane and binding with the plasma membrane in muscle cells. In this article, we report that mutant JP double-knockout (JP-DKO) mice lacking neural JP subtypes exhibited an irregular hindlimb reflex and impaired memory. Electrophysiological experiments indicated that the activation of small-conductance Ca(2+)-activated K(+) channels responsible for afterhyperpolarization in hippocampal neurons requires endoplasmic reticulum Ca(2+) release through ryanodine receptors, triggered by NMDA receptor-mediated Ca(2+) influx. We propose that in JP-DKO neurons lacking afterhyperpolarization, the functional communications between NMDA receptors, ryanodine receptors, and small-conductance Ca(2+)-activated K(+) channels are disconnected because of JMC disassembly. Moreover, JP-DKO neurons showed an impaired long-term potentiation and hyperactivation of Ca(2+)/calmodulin-dependent protein kinase II. Therefore, JPs seem to have an essential role in neural excitability fundamental to plasticity and integrated functions.

Animals↗
PubMed2006

Disturbance of cerebellar synaptic maturation in mutant mice lacking BSRPs, a novel brain-specific receptor-like protein family.

By DNA cloning, we have identified the BSRP (brain-specific receptor-like proteins) family of three members in mammalian genomes. BSRPs were predominantly expressed in the soma and dendrites of neurons and localized in the endoplasmic reticulum (ER). Expression levels of BSRPs seemed to fluctuate greatly during postnatal cerebellar maturation. Triple-knockout mice lacking BSRP members exhibited motor discoordination, and Purkinje cells (PCs) were often innervated by multiple climbing fibers with different neuronal origins in the mutant cerebellum. Moreover, the phosphorylation levels of protein kinase Calpha (PKCalpha) were significantly downregulated in the mutant cerebellum. Because cerebellar maturation and plasticity require metabotropic glutamate receptor signaling and resulting PKC activation, BSRPs are likely involved in ER functions supporting PKCalpha activation in PCs.

Animals↗
PubMed2006

Randomized trial of the efficacy of adjuvant chemotherapy for colon cancer with combination therapy incorporating the oral pyrimidine 1-hexylcarbamoyl-5-fluorouracil.

BACKGROUND AND AIMS: The purpose of the present trial was to clarify the efficacy of postoperative adjuvant chemotherapy including an oral fluoropyrimidine anticancer drug, the 1-hexylcarbamoyl-5-fluorouracil (HCFU), for the treatment of colon cancer. METHOD: Patients with clinical stage Dukes' B and C colon cancer, who had been treated surgically, were assigned to a chemotherapy group treated with mitomycin C, 5-fluorouracil (5-FU), and HCFU and to a control group that received no postoperative adjuvant chemotherapy. RESULTS: Of the 1,001 patients registered for the study, 17 (1.7%) were ineligible. The incidence of toxicity was significantly higher in the chemotherapy group than in the control group. However, there were few severe side effects and no deaths related to the treatment. Overall survival showed no significant difference between the groups. The disease-free survival or the recurrence-free intervals was significantly higher in the chemotherapy group than in the control group. The incidence of hepatic recurrence was significantly (P=0.003) lower in the chemotherapy group than in the control group. CONCLUSION: The results of this study demonstrated the efficacy of adjuvant chemotherapy for colon cancer, i.e., combined chemotherapy that included the 5-FU oral anticancer drug HCFU.

Aged↗
PubMed2006

Gene expression and localization of diacylglycerol kinase isozymes in the rat spinal cord and dorsal root ganglia.

The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKzeta and DGKiota in the DRG. By immunohistochemical analysis, DGKzeta immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKiota immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKzeta in the nucleus and DGKiota in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKzeta between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.

Animals↗
PubMed2006

ATP-binding cassette transporter G2 mediates the efflux of phototoxins on the luminal membrane of retinal capillary endothelial cells.

PURPOSE: The purpose of this study was to clarify the localization and function of the ATP-binding cassette transporter G2 (ABCG2; BCRP/MXR/ABCP) in retinal capillary endothelial cells, which form the inner blood-retinal barrier, as an efflux transport system. METHODS: The expression was determined by reverse transcriptase polymerase chain reaction and Western blotting. The localization was identified by immunostaining. The transport function of ABCG2 was measured by flow cytometry. RESULTS: Western blotting indicated that ABCG2 was expressed as a glycosylated disulfide-linked complex in the mouse retina and in peripheral tissues, including liver, kidney, and small intestine. Double immunolabeling of ABCG2 and glucose transporter 1 suggested that ABCG2 was localized on the luminal membrane of mouse retinal capillary endothelial cells. ABCG2 mRNA and protein were found to be expressed in a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB, and rat retina. Treatment with Ko143, an ABCG2 inhibitor, restored the accumulation of pheophorbide a and protoporphyrin IX in TR-iBRB cells. CONCLUSION: ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where ABCG2 acts as the efflux transporter for photosensitive toxins such as pheophorbide a and protoporphyrin IX. ABCG2 could play an important role at the inner blood-retinal barrier in restricting the distribution of phototoxins and xenobiotics in retinal tissue.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed2006

Complementary stripes of phospholipase Cbeta3 and Cbeta4 expression by Purkinje cell subsets in the mouse cerebellum.

Transverse boundaries divide the cerebellar cortex into four transverse zones, and within each zone the cortex is further subdivided into a symmetrical array of parasagittal stripes. Several molecules believed to mediate long-term depression at the parallel fiber-Purkinje cell synapse are known to be expressed in stripes. We have therefore explored the distributions of phospholipase Cbeta3 and phospholipase Cbeta4, key components in the transduction of type 1 metabotropic glutamate receptor-mediated responses. The data reveal that both phospholipase Cbeta isotypes are expressed strongly in the mouse cerebellum in subsets of Purkinje cells. The two distributions are distinct and largely nonoverlapping. The pattern of phospholipase Cbeta3 expression is unique, revealing stripes in three of the four transverse zones and a uniform distribution in the fourth. In contrast, phospholipase Cbeta4 appears to be confined largely to the Purkinje cells that are phospholipase Cbeta3-negative. PLCbeta3 is restricted to the zebrin II-immunopositive Purkinje cell subset. Not all zebrin II-immunoreactive Purkinje cells express PLCbeta3: in lobules IX and X it is restricted to that zebrin II-immunopositive subset that also expresses the small heat shock protein HSP25. PLCbeta4 expression is restricted to, and coextensive with, the zebrin II-immunonegative Purkinje cell subset. These nonoverlapping expression patterns suggest that long-term depression may be manifested differently between cerebellar modules.

Animals↗
PubMed2006

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity.

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

Amygdala↗
PubMed2006

Localization of diacylglycerol lipase-alpha around postsynaptic spine suggests close proximity between production site of an endocannabinoid, 2-arachidonoyl-glycerol, and presynaptic cannabinoid CB1 receptor.

2-arachidonoyl-glycerol (2-AG) is an endocannabinoid that is released from postsynaptic neurons, acts retrogradely on presynaptic cannabinoid receptor CB1, and induces short- and long-term suppression of transmitter release. To understand the mechanisms of the 2-AG-mediated retrograde modulation, we investigated subcellular localization of a major 2-AG biosynthetic enzyme, diacylglycerol lipase-alpha (DAGLalpha), by using immunofluorescence and immunoelectron microscopy in the mouse brain. In the cerebellum, DAGLalpha was predominantly expressed in Purkinje cells. DAGLalpha was detected on the dendritic surface and occasionally on the somatic surface, with a distal-to-proximal gradient from spiny branchlets toward somata. DAGLalpha was highly concentrated at the base of spine neck and also accumulated with much lower density on somatodendritic membrane around the spine neck. However, DAGLalpha was excluded from the main body of spine neck and head. In hippocampal pyramidal cells, DAGLalpha was also accumulated in spines. In contrast to the distribution in Purkinje cells, DAGLalpha was distributed in the spine head, neck, or both, whereas somatodendritic membrane was labeled very weakly. These results indicate that DAGLalpha is essentially targeted to postsynaptic spines in cerebellar and hippocampal neurons, but its fine distribution within and around spines is differently regulated between the two neurons. The preferential spine targeting should enable efficient 2-AG production on excitatory synaptic activity and its swift retrograde modulation onto nearby presynaptic terminals expressing CB1. Furthermore, different fine localization within and around spines suggests that the distance between postsynaptic 2-AG production site and presynaptic CB1 is differentially controlled depending on neuron types.

Animals↗
PubMed2006

Midterm results of Metasul metal-on-metal total hip arthroplasty.

We assessed 106 total hip arthroplasties performed with a new metal-on-metal hip system; the patients were monitored for at least 5 years. The average Harris Hip Score of the patients was 39.5 points before surgery and 87.8 points at final follow-up evaluation. Radiographically, the acetabular component was stable in 103 hips and possibly unstable in 3 hips. The femoral component was bone ingrown in 97 hips and stable and fibrous in 9. After surgery, 6 hips dislocated. The polyethylene liner dissociated in one patient. No patient exhibited clear signs of loosening, migration, or osteolysis. Distal femoral cortical hypertrophy was seen in 35.8% of the cases. Survival at the mean follow-up point (6.4 years) was 99.1%. There was no significant difference in serum chromium concentration between metal-on-metal bearings and polyethylene-on-metal bearings (control subjects). This study found that metal-on-metal total hip arthroplasty produces excellent midterm results.

Acetabulum↗