Investigation on the formation process of metal atomic filament for metal sulfide atomic switches by electrical measurement.

We have studied the formation process of the metal atomic filament for metal sulfide atomic switches by electrical measurement. The switching between ON and OFF states of the atomic switch is controlled by the application of the bias voltage for the atomic switches. The SET (OFF â†’ ON) and RESET (ON â†’ OFF) voltages were investigated for the atomic switch where the Ag2S or Cu2S layer were sandwiched between the Pt and Ag or Cu electrodes. The SET and RESET voltages of the Ag/Cu2S/Pt and Cu/Ag2S/Pt were close to those of the Ag/Ag2S/Pt atomic switch, and different from those of the Cu/Cu2S/Pt atomic switch. These results indicated that the dominant chemical species of the making and breaking part of the metal filament was Ag, and that the source of the metal filament was both the sulfide layer and the metal electrode.

Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin.

AIMS/HYPOTHESIS: The small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function. METHODS: Isolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1(-/-)) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells. RESULTS: BetaRac1(-/-) mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1(-/-) mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion. CONCLUSIONS/INTERPRETATION: In RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.

mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination.

Targeted deletion of metabotropic glutamate receptor-subtype 1 (mGluR1) gene can cause defects in development and function in the cerebellum. We introduced the mGluR1alpha transgene into mGluR1-null mutant [mGluR1 (-/-)] mice with a Purkinje cell (PC)-specific promoter. mGluR1-rescue mice showed normal cerebellar long-term depression and regression of multiple climbing fiber innervation, events significantly impaired in mGluR1 (-/-) mice. The impaired motor coordination was rescued by this transgene, in a dose-dependent manner. We propose that mGluR1 in PCs is a key molecule for normal synapse formation, synaptic plasticity, and motor control in the cerebellum.

Investigation of Ag and Cu Filament Formation Inside the Metal Sulfide Layer of an Atomic Switch Based on Point-Contact Spectroscopy.

The atomic switches have attracted wide attention owing to their applications in nonvolatile electric devices. The atomic switch is operated by the formation and dissipation of a metallic filament inside a metal sulfide film, which is controlled by a solid electrochemical reaction. Although the metallic filament is considered to consist of metal atoms, the chemical species of the metallic filament are difficult to be identified due to challenges in observing the metallic filament inside the solid. In this study, we report the investigation on the metallic filament in the atomic switch with metal sulfide based on point-contact spectroscopy (PCS). By cooling the atomic switch, the switch voltage increased to 1 V, which allowed for the PCS measurement. The PCS revealed that the metallic filament was composed of Ag atoms in the case of the Pt/Ag2S/Ag atomic switch. We applied this technique to the Pt/Cu2S/Ag and Pt/Ag2S/Cu atomic switches to uncover the formation process of the metallic filament. In both atomic switches, the chemical species of the metallic filament were Ag. The metal atoms were supplied from both the metal electrode and the sulfide layer.

Presynaptic inhibition caused by retrograde signal from metabotropic glutamate to cannabinoid receptors.

We report a type of synaptic modulation that involves retrograde signaling from postsynaptic metabotropic glutamate receptors (mGluRs) to presynaptic cannabinoid receptors. Activation of mGluR subtype 1 (mGluR1) expressed in cerebellar Purkinje cells (PCs) reduced neurotransmitter release from excitatory climbing fibers. This required activation of G proteins but not Ca2+ elevation in postsynaptic PCs. This effect was occluded by a cannabinoid agonist and totally abolished by cannabinoid antagonists. Depolarization-induced Ca2+ transients in PCs also caused cannabinoid receptor-mediated presynaptic inhibition. Thus, endocannabinoid production in PCs can be initiated by two distinct stimuli. Activation of mGluR1 by repetitive stimulation of parallel fibers, the other excitatory input to PCs, caused transient cannabinoid receptor-mediated depression of climbing fiber input. Our data highlight a signaling mechanism whereby activation of postsynaptic mGluR retrogradely influences presynaptic functions via endocannabinoid system.

Persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking mGluR1.

Most of the cerebellar Purkinje cells (PCs) of an adult animal are innervated individually by a single climbing fiber (CF) that forms strong excitatory synapses with the PCs. This one-to-one relationship between a PC and a CF is a consequence of a developmentally regulated regression of the innervation of PCs by CFs. We found that, in mice deficient in the type 1 metabotropic glutamate receptor (mGluR1), the regression of supernumerary CFs ceases by the end of the second postnatal week, which is about one week earlier than in normal mice. Consequently, about one third of PCs in the mGluR1 mutant mice are innervated by multiple CFs in adulthood. We conclude that the regression of CFs normally occurs in two developmental phases and that mGluR1 plays a crucial role in the second phase.

Rac1 is required for the formation of three germ layers during gastrulation.

The Rac1, a member of the Rho family proteins, regulates actin organization of cytoskeleton and cell adhesion. We used genetic analysis to elucidate the role of Rac1 in mouse embryonic development. The rac1 deficient embryos showed numerous cell deaths in the space between the embryonic ectoderm and endoderm at the primitive streak stage. Investigation of the primary epiblast culture isolated from rac1 deficient embryos indicated that Rac1 is involved in lamellipodia formation, cell adhesion and cell migration in vivo. These results suggest that Rac1-mediated cell adhesion is essential for the formation of three germ layers during gastrulation.

K-ras is essential for the development of the mouse embryo.

ras genes encode members of the small GTP-binding proteins. Ras protein in highly conserved in various species from yeast to humans and plays a key role in signal transduction. Ras is related to cell proliferation and differentiation. While, in addition, mutations in the ras genes are implicated in a variety of tumors. However, the physiological functions and specific roles of each ras gene, H-ras, K-ras and N-ras, are still not fully understood. To clarify the role of the K-Ras in vivo, we generated K-ras mutant mice by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Besides, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. Our results thus indicate K-Ras to be essential for normal development in mice and residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice.

Targeted deletion of the H-ras gene decreases tumor formation in mouse skin carcinogenesis.

To clarify the role of the H-Ras in vivo, we generated H-ras null mutant mice by gene targeting. In spite of the importance of the Ras in cell proliferation and differentiation, H-ras null mutant mice grew normally and were fertile. The oldest H-ras mutant mice grew to be more than 30 months old. We used the H-ras deficient mice to study the importance of the H-ras and other ras genes in the development of skin tumors induced by initiation with 7, 12-dimethylbenz(a)anthracene (DMBA) followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). We showed that H-ras null mutant mice develop approximately six times less papillomas compared with wild-type littermates after 20 weeks of TPA treatment. While all papillomas examined (17 out of 17) in wild-type mice have mutations of H-ras at codon 61, 13 (62%) out of 21 papillomas in H-ras null mutant mice have mutations of K-ras gene at codon 12, 13, or 61 and another eight (38%) papillomas have no mutations in these codons of K-ras or N-ras genes. This suggests that the activation of H-ras gene is critical in the wild-type mice, but the activation of K-ras gene can replace the H-ras activation in the initiation step of skin tumor development in the H-ras deficient mice. Oncogene (2000).

Regulation of long-term potentiation by H-Ras through NMDA receptor phosphorylation.

The proto-oncogene ras plays a critical role in cell proliferation and differentiation. However, ras genes are abundantly expressed in the adult CNS, although neuronal cells normally do not proliferate. Recently, several lines of evidence implicated the involvement of Ras signaling pathway in synaptic plasticity. To explore the role of the Ras proteins in the CNS, we generated knock-out mice lacking the H-ras gene and then used them to study the roles of Ras in synaptic transmission and plasticity. An investigation of protein phosphorylation and synaptic transmission in H-ras null mutant mice has shown that the NMDA receptor is a final target molecule of the Ras protein pathway in the CNS. In the H-ras null mutant hippocampus, the tyrosine phosphorylation of NR2A (epsilon1) and NR2B (epsilon2) subunits of NMDA receptors is increased, and, correspondingly, NMDA synaptic responses are selectively enhanced. In addition, long-term potentiation is markedly enhanced in mutant mice, most likely because of a selective enhancement of NMDA synaptic responses. Therefore, although Ras proteins have been implicated in cell proliferation and differentiation, the regulation of activity-dependent synaptic plasticity in the adult animals by downregulation of the phosphorylation of the NMDA receptor may be another major and pivotal role for H-Ras protein.

Roles of glutamate receptor delta 2 subunit (GluRdelta 2) and metabotropic glutamate receptor subtype 1 (mGluR1) in climbing fiber synapse elimination during postnatal cerebellar development.

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluRdelta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluRdelta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluRdelta2 mutant PCs were associated with voltage-dependent Ca(2+) signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca(2+) signals involved both proximal and distal dendrites. Morphologically, CFs of GluRdelta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluRdelta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluRdelta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.

Evidence against a role for metabotropic glutamate receptors in mossy fiber LTP: the use of mutant mice and pharmacological antagonists.

We have used a number of approaches to address a possible role of metabotropic glutamate receptors (mGluRs) in mossy fiber long-term potentiation (LTP) in the hippocampus. We have used two types of mutant mice--one lacking the mGluR1 subtype of receptor and one lacking the gamma isoform of protein kinase C. In neither type of mouse did we find any alteration in the magnitude of mossy fiber LTP. We next examined whether mGluRs might modulate the magnitude and/or threshold for the induction of mossy fiber LTP. In these experiments we used tetani that were either just subthreshold or just suprathreshold for generating LTP. The mGluR antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] did not convert a subthreshold tetanus into a suprathreshold tetanus, nor did (+)MCPG have any effect on the small amount of LTP that was generated by a just suprathreshold tetanus. Based on our studies, we have been unable to identify a role for mGluRs in mossy fiber LTP.

[Dopamine receptor knockout mice].

Dopaminergic systems are transmitted by dopamine receptors which couple to GTP binding proteins. Five subtypes of dopamine receptors were so far cloned. To study the functions of each dopamine receptors, mice lacking each of D1R, D2R, D3R and D4R dopamine receptors have been generated. Histological analyses of D1R knockout mice indicated that the expression of dynorphin is reduced in the striatum. In contrast with wild-type mice, D1R knockout mice exhibit a dose dependent decrease in locomotion. D2R knockout mice display a hypoactivity. The expression of enkephalin mRNA in the striatum is increased in the D2R knockout mice. D2R knockout mice showed hyperplastic changes of intermediate lobe of the pituitary and the increased expression of POMC in the pituitary in D2R knockout mice. D3R knockout mice are more active than wild-type mice in a novel environment and they exhibit enhanced behavioral sensitivity to cocaine and amphetamine. D4R knockout mice show a hypoactivity, but they display locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Dopamine synthesis and its conversion to DOPAC are elevated in the dorsal striatum from D4R knockout mice.

Metabotropic glutamate receptor subtype-1 is essential for in vivo growth of melanoma.

Ectopic expression of metabotropic glutamate receptor subtype 1 (mGluR1) in mouse melanocytes induces melanoma formation. Although requirement of mGluR1 for development of melanoma in the initial stage has been demonstrated, its role in melanoma growth in vivo remains unclear. In this study, we developed novel transgenic mice that conditionally express mGluR1 in melanocytes, using a tetracycline regulatory system. Pigmented lesions on the ears and tails of the transgenic mice began to appear 29 weeks after activation of the mGluR1 transgene, and the transgenic mice produced melanomas at a frequency of 100% 52 weeks after transgene activation. Subsequent inactivation of the mGluR1 transgene in melanoma-bearing mice inhibited melanoma growth with reduction of immunoreactivity to phosphorylated ERK1/2, whereas mice with persistent expression of mGluR1 developed larger melanoma burdens. mGluR1 expression is thus required not only for melanoma development but also for melanoma growth in vivo. These findings suggest that growth of melanoma can be inhibited in vivo by eliminating only one of the multiple genetic anomalies involved in tumorigenesis.

Transient paraparesis after laminectomy for thoracic ossification of the posterior longitudinal ligament and ossification of the ligamentum flavum.

STUDY DESIGN: Case report. OBJECTIVES: To report a case with thoracic myelopathy caused by ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), in which postoperative paralysis occurred after laminectomy and was reversed after an additional posterior instrumented fusion. SETTING: A University Hospital in Japan. CASE REPORT: A 71-year-old woman, with a spastic palsy of both lower extremities, had OPLL and OLF at T10-T11, which pinched the spinal cord anteriorly and posteriorly. She underwent a laminectomy at T10-T11, and no further neurological deterioration was seen immediately after surgery. Over the next 18 h, however, myelopathy worsened, showing severe paraparesis. An additional posterior instrumented fusion at T7-L1 was performed without correction of the kyphosis. After fusion, neurological deficits gradually recovered, despite the presence of residual anterior impingement of spinal cord by the OPLL. CONCLUSIONS: The present case provides evidence for the possibility that laminectomy alone produces postoperative paralysis for combined thoracic OPLL and OLF, and we recommend that a posterior instrumented fusion should be added when posterior decompression is performed for this disorder.

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli.

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.

Identification and sequence analysis of Escherichia coli purE and purK genes encoding 5'-phosphoribosyl-5-amino-4-imidazole carboxylase for de novo purine biosynthesis.

It has been shown that the Escherichia coli purE locus specifying 5'-phosphoribosyl-5-amino-4-imidazole carboxylase in de novo purine nucleotide synthesis is divided into two cistrons. We cloned and determined a 2,449-nucleotide sequence including the purE locus. This sequence contains two overlapped open reading frames, ORF-18 and ORF-39, encoding proteins with molecular weights of 18,000 and 39,000, respectively. The purE mutations of CSH57A and DCSP22 were complemented by plasmids carrying ORF-18, while that of NK6051 was complemented by plasmids carrying ORF-39. Thus, the purE locus consists of two distinct genes, designated purE and purK for ORF-18 and ORF-39, respectively. These genes constitute a single operon. A highly conserved 16-nucleotide sequence, termed the PUR box, was found in the upstream region of purE by comparing the sequences of the purF and purMN operons. We also found three entire and one partial repetitive extragenic palindromic (REP) sequences in the downstream region of purK. Roles of the PUR box and REP sequences are discussed in relation to the genesis of the purEK operon.

Reduced hippocampal long-term potentiation and context-specific deficit in associative learning in mGluR1 mutant mice.

We generated a novel strain of mutant mouse with a deletion in the gene encoding metabotropic glutamate receptor 1 (mGluR1). Gross anatomy of the hippocampus, excitatory synaptic transmission, long-term depression, and short-term potentiation in the hippocampal CA1 region are all apparently normal in the mutant mice. In contrast, long-term potentiation (LTP) is substantially reduced, and a moderate level of impairment is observed in context-specific associative learning. We propose that mGluR1 is not "in line" in LTP production, but rather modulates the plasticity process, and hence affects context-specific associative learning.

Impaired synapse elimination during cerebellar development in PKC gamma mutant mice.

PKC gamma is highly expressed in Purkinje cells (PCs) but not in other types of neurons in the cerebellum. The expression of PKC gamma changes markedly during cerebellar development, being very low at birth and reaching a peak around the third postnatal week. This temporal pattern of PKC gamma expression coincides with the developmental transition from multiple to single climbing fiber innervation onto each PC. In adult mutant mice deficient in PKC gamma, we found that 41% of PCs are still innervated by multiple climbing fibers, while other aspects of the cerebellum including the morphology and excitatory synaptic transmission of PCs appear normal. Thus, elimination of multiple climbing fiber innervation appears to be specifically impaired in the mutant cerebellum. We suggest that the developmental role of PKC gamma may be to act as a downstream element in the signal cascade necessary for the elimination of surplus climbing fiber synapses.

Dopamine D2 receptor plays a critical role in cell proliferation and proopiomelanocortin expression in the pituitary.

BACKGROUND: It has been suggested that the dopaminergic system is involved in the inhibitory control of secretion of pituitary hormones and in the regulation of motor function in the striatum. To elucidate the specific role of the dopamine D2 receptor (D2R) in the pituitary and brain, we generated D2R deficient (D2R mutant) mice using the gene targeting method. RESULTS: Electrophysiological studies as well as ligand binding assays show no functional D2R in mutant mice. The D2R mutant mice display a hypoactivity and a slow, creeping movement. The expression of enkephalin mRNA in the striatum is increased in the mutant mice, but not that of dynorphin and substance P. D2R mutant mice have significantly darker coat colour than their wild-type littermates and show an elevation of plasma alpha-melanocyte stimulating hormone (alpha-MSH) levels. We found corresponding hyperplastic changes of intermediate lobe of the pituitary and the increased expression of pro opiomelanocortin (POMC) in the pituitary in D2R mutant mice. CONCLUSIONS: D2R plays a critical role in the inhibitory regulation of endocrine cell proliferation and the transcription of POMC mRNA, and consequently in the regulation of alpha-MSH in plasma. D2R might be involved in the regulation of enkephalin expression in the striatum, and hence might affect the movement and the behaviour.