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A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l -serine and l -threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5′-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

This paper presented the results of application of super hydrophilicity TiO2 coating on Shinkansen vehicle. Also effect of metalic powder on hydrophilicity of TiO2 coatings was investigated. It was found that durability of protection from dirt adhesion was less than five months in real vehicle tests. The reason of degradation on protection was due to the contamination of metal powders produced by abrasive wear on the coating surface. Accordingly, it was considered that the degradation of hydrophilicity was attributed to the metal powder on the interface between TiO2 and water.

It was presented that the effect of Photocatalysis coating of TiO2 to the body shell of Shinkansen vehicles in this issue. The results indicated that the coating performance was maintained only few months and the application of the present titanium oxide photocatalysis coating was difficult. The reason of loss of performance was contamination of metal powder into coating layer. It was necessary for application of photocatalysis coating to the Shinkansen vehicles that protection on surface from the wear powder of metals which were generated during a running of vehicles.

Purpose: To evaluate antibody-mediated and cytotoxic T cell-mediated pathogenicity that has been implicated as the autoimmune pathophysiological mechanism in Rasmussen's encephalitis. Methods: We examined autoantibodies against the N-methyl-D-aspartate glutamate receptor (NMDA-type GluR) e2 subunit and its epitopes in serum and CSF samples from 20 patients [five histologically proven (definitive) Rasmussen's encephalitis with epilepsia partialis continua (EPC), four definitive Rasmussen's encephalitis without EPC, and 11 clinical Rasmussen's encephalitis with EPC]. We examined 3 H-thymidine uptake into lymphocytes after stimulation by GluRs. Results: All nine definitive patients (five patients with EPC and four without EPC), and 10 of II clinical Rasmussen's encephalitis patients had the autoantibodies. In four patients, the autoantibodies were absent in early stage when epileptic seizures had already become frequent, and appeared subsequently. In two patients, the autoantibodies persisted in the serum after frontal lobe resection or functional hemispherectomy, although epileptic seizures were completely controlled. Autoantibodies to the C2 epitope predominated, while autoantibodies to the extracellular N epitope were rare. The mean 3 H-thymidine uptake ratios (stimulation by GluRe2-containing homogenates/stimulation by PHA) were significantly higher in definitive and clinical Rasmussen encephalitis patients than in controls. The mean 3 H-thymidine uptake ratios (relative to PHA) were significantly higher for GluRe2-containing homogenate than for control homogenate or GluRδ2-containing homogenate. Conclusions: Autoantibodies against GluRe2 may be one of the diagnostic markers for Rasmussen's encephalitis with and without EPC. Patients have activated T cells stimulated by GluRe2 in peripheral blood circulation. We speculate that cellular autoimmunity and the subsequent humoral autoimmunity against GluRe2 may contribute to the pathophysiological processes in Rasmussen's encephalitis.

Classical eyeblink conditioning has been known to depend critically on the cerebellum. Apparently consistent with this, glutamate receptor subunit delta2 null mutant mice, which have serious morphological and functional deficiencies in the cerebellar cortex, are severely impaired in delay paradigm. However, these mutant mice successfully learn in trace paradigm, even in '0-trace paradigm,' in which the unconditioned stimulus starts just after the conditioned stimulus terminates. Our previous studies revealed that the hippocampus and the muscarinic acetylcholine receptors play crucial roles in 0-trace paradigm in glutamate receptor subunit delta2 null mutant mice unlike in wild-type mice, suggesting a large contribution of the forebrain to 0-trace conditioning in this type of mutant mice. In the present study, we investigated the role of N-methyl-D-aspartate receptors in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice. Mice were injected intraperitoneally with the noncompetitive N-methyl-d-aspartate receptor antagonist (+)MK-801 (0.1mg/kg) or saline, and conditioned with 350-ms tone conditioned stimulus followed by 100-ms periorbital shock unconditioned stimulus. Glutamate receptor subunit delta2 null mutant mice that received (+)MK-801 injection exhibited a severe impairment in acquisition of the conditioned response, compared with the saline-injected glutamate receptor subunit delta2 null mutant mice. In contrast, wild-type mice were not impaired in acquisition of 0-trace conditioned response by (+)MK-801 injection. After the injection solution was changed from (+)MK-801 to saline, glutamate receptor subunit delta2 null mutant mice showed a rapid and partial recovery of performance of the conditioned response. On the other hand, when the injection solution was changed from saline to (+)MK-801, glutamate receptor subunit delta2 null mutant mice showed a marked impairment in expression of the pre-acquired conditioned response, whereas impairment of the expression was small in wild-type mice. Injection of (+)MK-801 had no significant effects on spontaneous eyeblink frequency or startle eyeblink frequency to the tone conditioned stimulus in either glutamate receptor subunit delta2 null mutant mice or wild-type mice. These results suggest that N-methyl-D-aspartate receptors play critical roles both in acquisition and expression of the conditioned response in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice.

While the phenomena of phase separation in bulk glass have been well examined, those of amorphous thin films have not been fully detailed. From the viewpoint of new applications, the control of phase separation in thin films is very interesting. To investigate phase separation phenomena of amorphous films, Na 2 O–SiO 2 films with various thicknesses were prepared by sputtering and the annealed films examined by SEM. Though a thin film with a thickness in 120 nm did not show phase separation in the vicinity of the surface, it was found that a clear binodal structure was observed in the film after HF etching. The microstructure and behavior of phase separation in the films was different from that found in bulk glass with the same Na 2 O–SiO 2 composition. XPS results revealed that the compositions of Na/Si were inhomogeneous along the thickness, which explained the structural difference of the phase separation.

It has been proposed that the N-methyl-d-aspartate (NMDA)-type glutamate receptor (GluR) plays an important role in synaptic plasticity, learning, and memory. The four GluRepsilon (NR2) subunits, which constitute NMDA receptors with a GluRzeta (NR1) subunit, differ both in their expression patterns in the brain and in their functional properties. In order to specify the distinct participation of each of these subunits, we focused on the GluRepsilon2 subunits, which are expressed mainly in the forebrain. We investigated delay and trace eyeblink conditioning in GluRepsilon2 heterozygous mutant mice whose content of GluRepsilon2 protein was decreased to about half of that in wild-type mice. GluRepsilon2 mutant mice exhibited severe impairment of the attained level of conditioned response (CR) in the delay paradigm, for which the cerebellum is essential and modulation by the forebrain has been suggested. Moreover, GluRepsilon2 mutant mice showed no trend toward CR acquisition in the trace paradigm with a trace interval of 500 ms, in which the forebrain is critically involved in successful learning. On the other hand, the reduction of GluRepsilon2 proteins did not disturb any basic sensory and motor functions which might have explained the observed impairment. These results are different from those obtained with GluRepsilon1 null mutant mice, which attain a normal level of the CR but at a slower rate in the delay paradigm, and showed a severe impairment in the trace paradigm. Therefore, the NMDA receptor GluRepsilon2 plays a more critical role than the GluRepsilon1 subunit in classical eyeblink conditioning.

Layered-type TiO2/WO3 thin films were fabricated by sol–gel and sputtering methods and the effects of those different fabricating methods on the interfacial structures and the photoinduced hydrophilic properties were investigated. A third amorphous phase was observed by cross-sectional transmission electron micrograph at the interface of the sol–gel derived TiO2/WO3 thin film, but was not at that of the sputtered TiO2/WO3 thin film. Under weak ultra-violet light irradiation with intensity of 0.05 mW/cm2, the photoinduced hydrophilic property on the sputtered thin film was superior to that on the sol–gel thin film. These results suggest that the interfacial third phase acts as a barrier against holes and electrons’ transfer and degrades the photoinduced hydrophilic property.

Glutamate receptor (GluR) delta2 selectively expressed in cerebellar Purkinje cells plays a central role in cerebellar long-term depression (LTD), motor learning, and formation of parallel fiber synapses. By yeast two-hybrid screening, we identified members of the Shank family of scaffold proteins as major GluRdelta2-interacting molecules. GluRdelta2 bound directly to the PDZ domain of Shank proteins through an internal motif in the carboxyl-terminal putative cytoplasmic domain. Shank1 and Shank2 proteins as well as GluRdelta2 proteins were localized in the dendritic spines of cultured Purkinje cells. Anti-GluRdelta2 antibodies immunoprecipitated Shank1, Shank2, Homer, and metabotropic GluR1alpha proteins from the synaptosomal membrane fractions of cerebella. Furthermore, Shank2 interacted with GRIP1 in the cerebellum. These results suggest that through Shank1 and Shank2, GluRdelta2 interacts with the metabotropic GluR1alpha, the AMPA-type GluR, and the inositol 1,4,5-trisphosphate receptor (IP3R) that are essential for cerebellar LTD.

This study discusses the ionization mechanisms of sputtered titanium (Ti) in inductively coupled plasma (ICP)-assisted dc magnetron sputtering based on measurements and model analyses. Ionic and atomic densities of sputtered titanium were measured using an optical absorption method under an Ar pressure of 3.5 Pa against rf(13.56MHz) power applied to the inserted coil. A Langmuir probe provided measurements of electron densities against coil rf power, giving input parameters for model analyses. We adopted a model analysis method presented by J. Hopwood and F. Qian [J. Appl. Phys. 78, 758 (1995)]. Variations of ionic and atomic densities and ionization fractions against electron density from model calculation agreed with those obtained by measurements. The results showed clearly that electron impact ionization dominates ionization of sputtered titanium in high er electron density regions compared to Penning ionization through Ar metastable atoms. Penning ionization played an important role at the lower electron density region because of the longer time for radiative decay of Ar metastable atoms.

Spontaneous correlated activity regulates the precision of developing neural circuits. A synchronized elevation of intracellular calcium ion concentration, [Ca(2+)](i), occurred in 5-50 adjacent neurons--known as a "neuronal domain"--in developing neocortex. This coordinated response of neuronal cells is mediated by the diffusion of inositol trisphosphate (IP(3)) via gap-junction channels. In this study, we utilized the N-methyl-D-aspartate (NMDA)-type glutamate receptor epsilon 2 (GluR epsilon 2/NR2B)(-/-) mouse, which does not possess any functional NMDA receptors in the developing neocortex, and showed that NMDA receptors are essential for the generation of "neuronal domains". First, the frequency of spontaneously occurring neuronal domains in brain slices from GluR epsilon 2(-/-) mice was significantly reduced compared to that seen in brain slices from wild-type mice. Secondly, IP(3) injection into a single neuron in a cortical slice from a GluR epsilon 2(-/-) brain resulted in very few neuronal domains being observed, but an injection similarly made into a neuron in a wild-type slice promptly resulted in neuronal domains. Even in the GluR epsilon 2(-/-) brain, the elevation of intracellular [Ca2+](i) was observed frequently in single neurons and microinjection of IP(3) produced an elevation of [Ca2+](i) in the injected cells. These results suggest that the diffusion of IP(3) into the surrounding neurons via gap junctions is almost completely absent in the GluR epsilon 2(-/-) brain. Our results may reflect the critical role of NMDA receptors in the formation of cortical circuitry, probably via the regulation of gap-junction channels between immature cortical neurons.

Mutant mice lacking the glutamate receptor subunit delta2 exhibit changes in the structure and function of the cerebellar cortex. The most prominent functional feature is a deficiency in the long-term depression (LTD) at parallel fiber-Purkinje cell synapses. These mutant mice exhibit severe impairment during delay eyeblink conditioning but learn normally during trace eyeblink conditioning without the cerebellar LTD, even with a 0 trace interval. We investigated the hippocampal contribution to this cerebellar LTD-independent "0 trace interval" learning. The mutant mice whose dorsal hippocampi were aspirated exhibited severe impairment in learning, whereas those that received post-training hippocampal lesions retained the memory. The wild-type mice showed no impairment in either case. These results suggest that the hippocampal component of the eyeblink conditioning task becomes dominant when cerebellar LTD is impaired.

We identified a novel Golgi apparatus-specific protein with the DHHC zinc finger domain and four putative transmembrane regions, designated as GODZ. The amino acid sequences were highly conserved among mouse and human GODZs and homologous proteins in human, mouse, rat, Drosophila melanogaster, and Caenorhabditis elegans, implying a functional significance of the GODZ protein family. Overexpression of mouse GODZ in COS7 cells suppressed the sorting of the glutamate receptor GluRalpha1 from the Golgi apparatus. These results suggest that GODZ plays a role in the membrane protein trafficking.

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.

Cerebellar long-term depression (LTD) at the parallel fiber-Purkinje cell synapses has been proposed to be a neural substrate for classical eyeblink conditioning. Mutant mice lacking the glutamate receptor subunit δ2 (GluRδ2), in which the cerebellar LTD is disrupted, exhibited a severe impairment in the delay eyeblink conditioning with a temporal overlap of CS and US. However, they learned normally trace and delay conditioning without CS-US overlap, suggesting a learning mechanism which does not require the cerebellar LTD.In the present study, we tested possible involvement of the hippocampus in this cerebellar LTD-independent learning. We examined effects of scopolamine and hippocampal lesion on the delay conditioning without CS-US overlap. TheGluRδ2 mutant mice that received scopolamine or aspiration of the dorsalhippocampus together with its overlying cortex exhibited a severe impairment in learning, while the control mutant mice that received saline or aspiration of the overlying cortex learned normally. In contrast, wild-type mice that received either treatment learned as normally as the control wild-type mice. These results suggest that the hippocampus is essential in the cerebellar LTD-independent learning in the GluRδ2 mutant mice, indicating a newrole of hippocampus in the paradigm with a short trace interval.

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association of SR to intracellular membranes. Our data highlight an atypical role of FBXO22 in enhancing d-serine synthesis that is unrelated to its classical effects as a component of the ubiquitin-proteasome degradation pathway.

A simple preparation method of bimodal silica was developed by introducing SiO2 sol into large pores of SiO2 gel pellet directly. Cobalt supported on this kind of bimodal silica support, exhibited remarkably high activity in liquid-phase Fischer–Tropsch synthesis, which was attributed to its bimodal structure having not only a higher surface area but also a larger pore size. The support with a large surface area allowed highly dispersed cobalt particle and its large pore size improved the diffusion of reactants and products.

We examined the regulation of the acoustic startle response in mutant mice of the N-methyl-D-aspartate (NMDA)- and delta-subtypes of the glutamate receptor (GluR) channel, which play important roles in neural plasticity in the forebrain and the cerebellum, respectively. Heterozygous mutant mice with reduced GluRepsilon2 subunits of the NMDA receptor showed strongly enhanced startle responses to acoustic stimuli. On the other hand, heterozygous and homozygous mutation of the other NMDA receptor GluRepsilon subunits exerted no, or only small effects on acoustic startle responses. The threshold of the auditory brainstem response of the GluRepsilon2-mutant mice was comparable to that of the wild-type littermates. The primary circuit of the acoustic startle response is a relatively simple oligosynaptic pathway located in the lower brainstem, whilst the expression of GluRepsilon2 is restricted to the forebrain. We thus suggest that the NMDA receptor GluRepsilon2 subunit plays a role in the regulation of the startle reflex. Ablation of the cerebellar Purkinje cell-specific delta2 subunit of the GluR channel exerted little effect on the acoustic startle response but resulted in the enhancement of prepulse inhibition of the reflex. Because inhibition of the acoustic startle response by a weak prepulse is a measure of sensorimotor gating, the process by which an organism filters sensory information, these observations indicate the involvement of the cerebellum in the modulation of sensorimotor gating.

To elucidate the role of the N-methyl-D-aspartate (NMDA) -type glutamate receptor subunit epsilon 1 (GluR epsilon 1) in classical eyeblink conditioning, delay and trace eyeblink conditioning were investigated in GluR epsilon 1-null mutant mice. In delay conditioning and short-trace interval conditioning with a trace interval of 250 ms, GluR epsilon 1 mutant mice attained a normal level of the conditioned response (CR), although acquisition was a little slower than in wild-type mice. In contrast, GluR epsilon 1 mutant mice exhibited severe impairment of the attained level of the CR and disturbed temporal pattern of CR expression in trace conditioning with a longer trace interval of 500 ms. These findings indicate that GluR epsilon 1 is essential for long-trace interval eyeblink conditioning. The impairments of the associative learning with a long temporal separation between the conditioned and unconditioned stimuli observed in the GluR epsilon 1 mutant mice could be attributed to an impairment of hippocampal long-term potentiation in this line of mutant mice.

In mice lacking glutamate receptor subunit delta 2 (GluR delta 2(-/-_ mice), cerebellar long-term depression (LTD) at the parallel fibre-Purkinje cell synapses is disrupted. Unlike the cerebellar LTD-deficient mice previously used for eyeblink conditioning, however, the abnormalities of the GluR delta 2(-/-) mice are restricted to the cerebellar cortex. In delay eyeblink conditionings (interstimulus interval of 252 and 852 ms), in which the conditioned stimulus (CS) overlaps temporally with a coterminating unconditioned stimulus (US), GluR delta 2(-/-) mice are severely impaired in learning, strongly supporting the hypothesis that cerebellar cortical LTD is essential for delay conditioning. In the trace paradigm, in which a stimulus-free trace interval of 500 ms intervened between the CS and US, GluR delta 2(-/-) mice learned as successfully as wild-type mice, indicating that cerebellar LTD is not necessary for trace conditioning. Thus, the present study has revealed a cerebellar LTD-independent learning in eyeblink conditioning.

NMDA receptors, an ionotropic subtype of glutamate receptors (GluRs) forming high Ca2+-permeable cation channels, are composed by assembly of the GluRζ subunit (NR1) with any one of four GluRε subunits (GluRε1–4; NR2A-D). In the present study, we investigated neuronal functions in mice lacking the GluRε1 subunit. GluRε1 mutant mice exhibited a malfunction of NMDA receptors, as evidenced by alterations of [3H]MK-801 binding as well as45Ca2+uptake through the NMDA receptors. A postmortem brain analysis revealed that both dopamine and serotonin metabolism were increased in the frontal cortex and striatum of GluRε1 mutant mice. The NMDA-stimulated [3H]dopamine release from the striatum was increased, whereas [3H]GABA release was markedly diminished in GluRε1 mutant mice. When (+)bicuculline, a GABAAreceptor antagonist, was added to the superfusion buffer, NMDA-stimulated [3H]dopamine release was significantly increased in wild-type, but not in the mutant mice. GluRε1 mutant mice exhibited an increased spontaneous locomotor activity in a novel environment and an impairment of latent learning in a water-finding task. Hyperlocomotion in GluRε1 mutant mice was attenuated by treatment with haloperidol and risperidone, both of which are clinically used antipsychotic drugs, at doses that had no effect in wild-type mice. These findings provide evidence that NMDA receptors are involved in the regulation of behavior through the modulation of dopaminergic and serotonergic neuronal systems. In addition, our findings suggest that GluRε1 mutant mice are useful as an animal model of psychosis that is associated with NMDA receptor malfunction and hyperfunction of dopaminergic and serotonergic neuronal systems.

Telencephalin (TLCN) is a cell adhesion molecule selectively expressed in the telencephalon of the mammalian brain. The mutant mice lacking TLCN had no detectable abnormalities in their neural development and synaptic structures. Ablation of TLCN increased the hippocampal long-term potentiation and its saturation level. The TLCN mutation selectively enhanced the performance of the radial maze and water-finding tasks, learning tasks with appetitive reinforcers, but not the contextual fear conditioning and Morris water maze tasks with aversive stimuli for conditioning. Furthermore, the TLCN mutant mice showed an increase of prepulse inhibition of the acoustic startle response. These results suggest that TLCN is a determinant of the dynamic range of synaptic plasticity and plays roles in reward-motivated learning and memory and sensorimotor gating.

In the present work, the ductile fracture of structural steels and the effect of plastic pre-strain on ductility have been investigated by tensile test. The fracture process consisting of void nucleation, growth and coalescence was observed by scanning electron microscopy (SEM). The nucleation strain of microvoid was measured experimentally. The critical interfacial strength of particle/matrix interface was calculated by dislocation model and continuum analysis, and the void growth was evaluated by Rice-Tracey model. It was found that longitudinal void coalescence does not affect fracture even though large cracks exist, but particle position affects void nucleation. Thomason's plastic limit-load model was used to predict void growth strain. Through the analysis of ductile fracture process, the influence of plastic pre-strain on ductility was discussed.

Influence of a silica layer on oxidation of free carbon in Si-Ti-C-O fiber-bonded ceramics has been investigated. The rate of oxidation of the Si-Ti-C-O fibers has been measured by thermogravimetric analysis at temperatures ranging between 773 and 1573K. The relative mass of the fibers increased parabolically and the kinetics of the oxidation followed the parabolic rate characteristic of diffusion controlled processes. The thickness of the silica layer, calculated according to a simple equation, well agreed well with the experimentally observed one. Pores created by the oxidation of free carbon were observed inside the fibers after etching the surface of a heat-treated Si-Ti-C-O fiber-bonded ceramics at 773K. Using the parabolic rate constant and the volume expansion coefficient of the fiber, the time required to close the pores by the formation of silica layer was calculated. The thickness of the free carbon layer and the oxidation rate of the fibers were the key parameters for the analysis of the oxidation behavior of the materials.

There are three stages in ductile fracture process: voids initiation, void growth and void coalescence. In the final stage, ductile fracture occurs due to the void coalescence. Thomason suggested that voids begin to coalesce when a plastic limit-load condition is fulfilled for localized plastic failure of the intervoid matrix. On the basis of this critical void-coalescence condition, we developed a three-dimensional model for ductile fracture considering two types of voids with different sizes, and applied this model to a SA440 steel with and without pre-straining. With the critical strength of particle/matrix interface and the theoretical void growth strain obtained from the model, the reason for the decrease in fracture strain caused by plastic pre-strain was discussed.

Ductile fracture processes in structural steels under high strain rates have been investigated by tensile test. Under high strain rate loading, the ductility of the steels decreases. The reasons for this decrease is discussed from the analysis of ductile fracture process, as especially of void nucleation strain and void growth strain up to fracture. A three-dimensional void coalescence model for the high strain rate loading was obtained by modifying Thomason's static three-dimensional void coalescence model. The model was used to predict the void growth strain and evaluate the effect of strain rate on void coalescence. The influence of plastic pre-strain on ductility under high strain rate loading was also investigated.

The NMDA-type glutamate receptor (GluR) channel, composed of the GluRepsilon and GluRzeta subunits, plays a key role in synaptic plasticity in the CNS. The mutant mice lacking the GluRepsilon1 subunit exhibited a reduction in hippocampal long-term potentiation (LTP), but a stronger tetanic stimulation restored the impairment and the saturation level of LTP was unaltered. These results suggest an increase of threshold for LTP induction in the GluRepsilon1 mutant mice. After a series of backcrosses we established a GluRepsilon1 mutant mouse line with a 99.99% pure C57BL/6 genetic background. The performance of the mutant mice in tone- and context-dependent fear conditioning tests was comparable with that of the wild-type mice. However, a significant difference in the extent of contextual learning became apparent when the chamber exposure time before footshock was shortened. Furthermore, there was a significant difference in freezing responses immediately after footshock on the conditioning day between the wild-type and mutant mice, and the difference was not restored by longer chamber exposure in contrast to the contextual learning on the next day of the conditioning. These results suggest that the GluRepsilon1 subunit of the NMDA receptor channel is a determinant of thresholds for both hippocampal LTP and contextual learning and plays differential roles in two forms of contextual fear memories.

The synaptic localization of the N-methyl-D-aspartate (NMDA) type glutamate receptor (GluR) channel is a prerequisite for synaptic plasticity in the brain. We generated mutant mice carrying the carboxy-terminal truncated GluR epsilon2 subunit of the NMDA receptor channel. The mutant mice died neonatally and failed to form barrelette structures in the brainstem. The mutation greatly decreased the NMDA receptor-mediated component of hippocampal excitatory postsynaptic potentials and punctate immunofluorescent labelings of GluR epsilon2 protein in the neuropil regions, while GluR epsilon2 protein expression was comparable. Immunostaining of cultured cerebral neurons showed the reduced punctate staining of the truncated GluR epsilon2 protein at synapses. These results suggest that the carboxy-terminal region of the GluRepsilon2 subunit is important for efficient clustering and synaptic localization of the NMDA receptor channel.

KCNE1 encodes a modulator of KCNH2 and KCNQ1 delayed rectifier K(+) current channels. KCNE1 mutations might cause long QT syndrome (LQTS) by impairing KCNE1 subunit's modulatory actions on these channels. There are major and minor polymorphismic KCNE1 variants whose 38(th) amino acids are glycine and serine [KCNE1(38G) and KCNE1(38S) subunits], respectively. Despite its frequent occurrence, the influence of this polymorphism on the K(+) channels' function is unclear. Patch-clamp recordings were obtained from human embryonic kidney -293T cells. KCNH2 channel current density in KCNE1(38S)-transfected cells was smaller than that in KCNE1(38G)-transfected cells by 34%. The voltage-sensitivity of the KCNQ1 channel current in KCNE1(38S)-transfected cells was lowered compared to that in KCNE1(38G)-transfected cells, with a +13mV shift in the half-maximal activation voltage. KCNH2 channel current density or KCNQ1 channel voltage-sensitivity was not different between KCNE1(38G)-transfected cells and cells transfected with both KCNE1(38G) and KCNE1(38S). Moreover, the KCNH2 channel current in KCNE1(38S)-transfected cells was more susceptible to E4031, a QT prolonging drug and a condition with hypokalemia, than that in KCNE1(38G)-transfected cells. Homozygous inheritance of KCNE1(38S) might cause a mild reduction of the delayed rectifier K(+) currents and might thereby increase an arrhythmogenic potential particularly in the presence of QT prolonging factors. By contrast, heterozygous inheritance of KCNE1(38G) and KCNE1(38S) might not affect the K(+) currents significantly. (Circ J 2014; 78: 610-618).

D-Serine, an endogenous coagonist of the N-methyl-D-aspartate receptor (NMDAR), is widely distributed in the central nervous system and is synthesized from L-serine by serine racemase (SR). NMDAR plays an important role in pain processing including central sensitization that eventually causes hyperalgesia. To elucidate the roles of D-serine and SR in pain transmission, we evaluated the behavioral changes and spinal nociceptive processing induced by formalin using SR knock-out (KO) mice. We found that SR is mainly distributed in lamina II of the dorsal horn of the spinal cord in wild-type (WT) mice. Although the formalin injected subcutaneously induced the biphasic pain response of licking in SR-KO and WT mice, the time spent on licking was significantly longer in the SR-KO mice during the second phase of the formalin test. The number of neurons immunopositive for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK), which are molecular pain markers, in laminae I-II of the ipsilateral dorsal horn was significantly larger in the SR-KO mice. Immunohistochemical staining revealed that the distribution of SR changed from being broad to being concentrated in cell bodies after the formalin injection. On the other hand, the expression level of the cytosolic SR in the ipsilateral dorsal horn significantly decreased. Oral administration of 10 mM D-serine in drinking water for one week cancelled the difference in pain behaviors between WT and SR-KO mice in phase 2 of the formalin test. These findings demonstrate that the SR-KO mice showed increased sensitivity to inflammatory pain and the WT mice showed translocation of SR and decreased SR expression levels after the formalin injection, which suggest a novel antinociceptive mechanism via SR indicating an important role of D-serine in pain transmission.

Although phenotypically polarized macrophages are now generally classified into two major subtypes termed proinflammatory M1 and anti-inflammatory M2 macrophages, a contributory role of lung M2 macrophages in the pathophysiological features of acute lung injury is not fully understood. Herein, we show in an endotoxemic murine model that M2 macrophages serve as key anti-inflammatory cells that play a regulatory role in the severity of lung injury. To study whether M2 macrophages can modify inflammation, we depleted M2 macrophages from lungs of CD206-diphtheria toxin (DT) receptor transgenic (Tg) mice during challenge with lipopolysaccharide. The i.p. administration of DT depleted CD206-positive cells in bronchoalveolar lavage fluid. The use of M2 macrophage markers Ym1 and arginase-1 identified pulmonary CD206-positive cells as M2 macrophages. A striking increase in neutrophils in bronchoalveolar lavage fluid cell contents was found in DT-treated CD206-DT receptor Tg mice. In CD206-DT receptor Tg mice given DT, endotoxin challenge exaggerated lung inflammation, including up-regulation of proinflammatory cytokines and increased histological lung damage, but the endotoxemia-induced increase in NF-κB activity was significantly reduced, suggesting that M2 phenotype-dependent counteraction of inflammatory insult cannot be attributed to the inhibition of the NF-κB pathway. Our results indicate a critical role of CD206-positive pulmonary macrophages in triggering inflammatory cascade during endotoxemic lung inflammation.

KCNQ1 encodes the α subunit of the voltage-gated channel that mediates the cardiac slow delayed rectifier K(+) current (IKs). Here, we report a KCNQ1 allele encoding an A590T mutation [KCNQ1(A590T)] found in a 39-year-old female with a mild QT prolongation. A590 is located in the C-terminal α helical region of KCNQ1 that mediates subunit tetramerization, membrane trafficking, and interaction with Yotiao. This interaction is known to be required for the proper modulation of IKs by cAMP. Since previous studies reported that mutations in the vicinity of A590 impair IKs channel surface expression and function, we examined whether and how the A590T mutation affects the IKs channel. Electrophysiological measurements in HEK-293T cells showed that the A590T mutation caused a reduction in IKs density and a right-shift of the current-voltage relation of channel activation. Immunocytochemical and immunoblot analyses showed the reduced cell surface expression of KCNQ1(A590T) subunit and its rescue by coexpression of the wild-type KCNQ1 [KCNQ1(WT)] subunit. Moreover, KCNQ1(A590T) subunit interacted with Yotiao and had a cAMP-responsiveness comparable to that of KCNQ1(WT) subunit. These findings indicate that the A590 of KCNQ1 subunit plays important roles in the maintenance of channel surface expression and function via a novel mechanism independent of interaction with Yotiao.

d-Serine and glycine are coagonists of NMDA receptors (NMDARs), but their relative contributions for several NMDAR-dependent processes are unclear. We now report that the alanine–serine–cysteine transporter-1 (Asc-1) mediates release of bothd-serine and glycine from neurons, and, in turn, this modulates NMDAR synaptic activity. Asc-1 antiporter activity is enhanced byd-isoleucine (d-Ile), which releasesd-serine and glycine from Asc-1-transfected cells, primary neuronal cultures, and hippocampal slices.d-Ile has no effect on astrocytes, which do not express Asc-1. We show thatd-Ile enhances the long-term potentiation (LTP) in rat and mouse hippocampal CA1 by stimulating Asc-1-mediated endogenousd-serine release.d-Ile effects on synaptic plasticity are abolished by enzymatically depletingd-serine or by using serine racemase knock-out (SR-KO) mice, confirming its specificity and supporting the notion that LTP depends mostly ond-serine release. Conversely, our data also disclose a role of glycine in activating synaptic NMDARs. Although acute enzymatic depletion ofd-serine also drastically decreases the isolated NMDAR synaptic potentials, these responses are still enhanced byd-Ile. Furthermore, NMDAR synaptic potentials are preserved in SR-KO mice and are also enhanced byd-Ile, indicating that glycine overlaps withd-serine binding at synaptic NMDARs. Altogether, our results disclose a novel role of Asc-1 in regulating NMDAR-dependent synaptic activity by mediating concurrent non-vesicular release ofd-serine and glycine. Our data also highlight an important role of neuron-derivedd-serine and glycine, indicating that astrocyticd-serine is not solely responsible for activating synaptic NMDARs.

Astrocytes play a critical role in neurovascular coupling by providing a physical linkage from synapses to arterioles and releasing vaso-active gliotransmitters. We identified a gliotransmitter pathway by which astrocytes influence arteriole lumen diameter. Astrocytes synthesize and release NMDA receptor coagonist, D -serine, in response to neurotransmitter input. Mouse cortical slice astrocyte activation by metabotropic glutamate receptors or photolysis of caged Ca 2+ produced dilation of penetrating arterioles in a manner attenuated by scavenging D -serine with D -amino acid oxidase, deleting the enzyme responsible for D -serine synthesis (serine racemase) or blocking NMDA receptor glycine coagonist sites with 5,7-dichlorokynurenic acid. We also found that dilatory responses were dramatically reduced by inhibition or elimination of endothelial nitric oxide synthase and that the vasodilatory effect of endothelial nitric oxide synthase is likely mediated by suppressing levels of the vasoconstrictor arachidonic acid metabolite, 20-hydroxy arachidonic acid. Our results provide evidence that D -serine coactivation of NMDA receptors and endothelial nitric oxide synthase is involved in astrocyte-mediated neurovascular coupling.

Twin-roll-cast AMX magnesium alloys have recently been developed as a lightweight flame retardant alloy for use in automotive and railway vehicles. However, magnesium alloys containing Ca show inferior plastic deformation and poor formability. In this study, we have developed an extraordinarily high strength and elongation rare-earth-free magnesium alloy by multi-pass rolling of a Mg-10Al-0.2Mn-1Ca (wt.%) twin-roll-cast alloy. The as-rolled sample exhibited a yield stress of 380 MPa, tensile strength of 400 MPa, and elongation to failure of 8 % as a result of grain refinement. The recrystallized region of the rolled material accounted for 70 %, and the mean grain size was 1.4 μm. In addition, excellent rollability was found over a wide range of rolling temperatures, with the maximum reduction in the thickness being twice as large as that in other magnesium alloys containing Ca.