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M Mishina

Publications and source records attributed to M Mishina.

At least 73 records · Page 4Linked to original sources
PubMed1998

Sensitivity of the N-methyl-D-aspartate receptor channel to butyrophenones is dependent on the epsilon2 subunit.

The effects of three kinds of butyrophenones, haloperidol, droperidol and spiperone, on the N-methyl-D-aspartate (NMDA) receptor channel were examined on the epsilon1/zeta1, epsilon2/zeta1, epsilon3/zeta1 and epsilon4/zeta1 heteromeric NMDA receptor channels, expressed in Xenopus oocytes. Micromolar concentrations of haloperidol selectively inhibited the epsilon2/zeta1 channel, whereas the epsilon1/zeta1, epsilon3/zeta1 and epsilon4/zeta1 channels were enhanced or minimally affected by higher concentrations of haloperidol. Similarly, droperidol and spiperone inhibited the epsilon2/zeta1 channel more strongly than the other epsilon/zeta channels, although sensitivities of the epsilon2/zeta1 channel to droperidol and spiperone were lower than those to haloperidol. These results suggest that the sensitivities of the NMDA receptor channels to butyrophenones are dependent on the epsilon2 subunit. Furthermore, the replacement with glutamine of the conserved asparagine residue in segment M2, which constitutes the Mg2+ block sites, of the epsilon2 and zeta1 subunits (the mutations epsilon2-N589Q and zeta1-N598Q, respectively) reduced the sensitivities to haloperidol. The mutation zeta1-N598Q reduced the sensitivities to haloperidol more effectively than the mutation epsilon2-N589Q. These results, together with previous findings, suggest that the haloperidol block sites of the NMDA receptor channel partially overlap the Mg2+ block sites.

Amino Acid Substitution↗
PubMed1998

Distribution and development of NMDA receptor activities at hippocampal synapses examined using mice lacking the epsilon1 subunit gene.

The effects of targeted disruption of the gene encoding N-methyl-D-aspartate (NMDA) receptor epsilon1 subunit were examined in hippocampal CA1 pyramidal cell synapses and compared with the effects in the CA3 region. The mutation resulted in the significant reduction of NMDA receptor activities at the synapses in the CA1 stratum oriens, as had been observed in the CA1 stratum radiatum which we reported before. This result was in sharp contrast to our previous observation that in the CA3 region, the epsilon1 mutation suppressed NMDA receptors at the synapses in the stratum radiatum but not in the stratum oriens. It is suggested that the subunit composition of NMDA receptors may not be determined simply by the location within a pyramidal cell, but by other factors such as properties of synaptic inputs. We also examined the postnatal development of long-term potentiation (LTP) in the CA3 region. The development of LTP at the CA3 stratum radiatum synapses closely followed the development of the epsilon1 subunit, and the epsilon1 mutation strongly suppressed this LTP, suggesting that the targeted disruption of the epsilon1 subunit may not be compensated by other epsilon subunits. The LTP at the CA3 stratum oriens synapses was not significantly affected by the mutation at any age.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1998

Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex.

In mammals, neurons are highly differentiated and play distinctive functions even in the same brain region. We found a novel cadherin-related neuronal receptor (Cnr) gene family by studying Fyn-binding activity in mouse brain. CNR1 protein is located in the synaptic junction and forms a complex with Fyn. Sequence analysis of eight Cnr products of approximately 20 genes indicates that these comprise a novel cadherin family of the cadherin superfamily. The expression patterns of each member of this novel family were grossly similar to each other but restricted to subpopulations of neurons of the same type. The diversity of the Cnr family genes suggests that there are molecular mechanisms that govern highly differentiated neural networks in the mammalian CNS.

Animals↗
PubMed1998

Role of the carboxy-terminal region of the GluR epsilon2 subunit in synaptic localization of the NMDA receptor channel.

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.

Afferent Pathways↗
PubMed1998

Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield.

Hippocampal synapses express two distinct forms of the long-term potentiation (LTP), i.e. NMDA receptor-dependent and -independent LTPs. To understand its molecular-anatomical basis, we produced affinity-purified antibodies against the GluRepsilon1 (NR2A), GluRepsilon2 (NR2B), and GluRzeta1 (NR1) subunits of the N-methyl-D-aspartate (NMDA) receptor channel, and determined their distributions in the mouse hippocampus. Using NMDA receptor subunit-deficient mice as the specificity controls, section pretreatment with proteases (pepsin and proteinase K) was found to be very effective to detect authentic NMDA receptor subunits. As the result of modified immunohistochemistry, all three subunits were detected at the highest level in the strata oriens and radiatum of the CA1 subfield, and high levels were also seen in most other neuropil layers of the CA1 and CA3 subfields and of the dentate gyrus. However, the stratum lucidum, a mossy fibre-recipient layer of the CA3 subfield, contained low levels of the GluRepsilon1 and GluRzeta1 subunits and almost excluded the GluRepsilon2 subunit. Double immunofluorescence with the AMPA receptor GluRalpha1 (GluR1 or GluR-A) subunit further demonstrated that the GluRepsilon1 subunit was colocalized in a subset, not all, of GluRalpha1-immunopositive structures in the stratum lucidum. Therefore, the selective scarcity of these NMDA receptor subunits in the stratum lucidum suggests that a different synaptic targeting mechanism exerts within a single CA3 pyramidal neurone in vivo, which would explain contrasting significance of the NMDA receptor channel in LTP induction mechanisms between the mossy fibre-CA3 synapse and other hippocampal synapses.

Animals↗
PubMed1998

Bilateral representation of language function. Agenesis of corpus callosum by Wada and PET activation.

A 33-year-old right-handed man with intermittent headache was found to have agenesis of the corpus callosum. He underwent magnetic resonance imaging, positron emission tomography (PET), and detailed neuropsychologic tests, including a Wada test. There was bilateral representation of language, and a PET activation study with word repetition revealed bilateral areas of activation that were not completely symmetric. These findings and the literature concerning agenesis of the corpus callosum are discussed as is the possible compensatory mechanism for absence of the corpus callosum, which is important for cross-communication.

Adult↗
PubMed1998

Non-invasive blood pressure measurements in cats: clinical significance of hypertension associated with chronic renal failure.

The systolic, mean and diastolic pressures as well as the heart rate were measured using the oscillometric method, on a total of 104 cats (60 cats in the normal group, and 44 in the renal disease group) which were brought into Azabu University Animal Hospital. The blood pressure in the normal group was systolic: 115.4 +/- 10.1 mmHg, mean: 96.2 +/- 12.2 mmHg, and diastolic: 73.7 +/- 10.7 mmHg. Although no difference in heart rate, the renal disease group showed significantly (p < 0.05) higher values for systolic, mean, and diastolic pressure when compared with the normal group. Moreover, when plasma renin activity, angiotensin I and II, and aldosterone concentrations were measured in other cats (11 normal and seven with chronic renal failure), all cats with chronic renal failure showed significantly (p < 0.05) higher values than the normal group. It is, therefore, indicated that hypertension due to stimulating renin-angiotensin-aldosterone system may have manifested in cats with renal dysfunction.

Animals↗
PubMed1997

Impaired parallel fiber-->Purkinje cell synapse stabilization during cerebellar development of mutant mice lacking the glutamate receptor delta2 subunit.

The glutamate receptor delta2 subunit (GluRdelta2) is specifically expressed in cerebellar Purkinje cells (PCs) from early developmental stages and is selectively localized at dendritic spines forming synapses with parallel fibers (PFs). Targeted disruption of the GluRdelta2 gene leads to a significant reduction of PF-->PC synapses. To address its role in the synaptogenesis, the morphology and electrophysiology of PF-->PC synapses were comparatively examined in developing GluRdelta2 mutant and wild-type cerebella. PCs in GluRdelta2 mutant mice were normally produced, migrated, and formed spines, as did those in wild-type mice. At the end of the first postnatal week, 74-78% of PC spines in both mice formed immature synapses, which were characterized by small synaptic contact, few synaptic vesicles, and incomplete surrounding by astroglial processes, eliciting little electrophysiological response. During the second and third postnatal weeks when spines and terminals are actively generated, the percentage of PC spines forming synapses attained 98-99% in wild type but remained as low as 55-60% in mutants, and the rest were unattached to any nerve terminals. As a result, the number of PF synapses per single-mutant PCs was reduced to nearly a half-level of wild-type PCs. Parallelly, PF stimulation less effectively elicited EPSCs in mutant PCs than in wild-type PCs during and after the second postnatal week. These results suggest that the GluRdelta2 is involved in the stabilization and strengthening of synaptic connectivity between PFs and PCs, leading to the association of all PC spines with PF terminals to form functionally mature synapses.

Animals↗
PubMed1997

Lipid mediators modulate NMDA receptor currents in a Xenopus oocyte expression system.

We determined the modulatory effects of various lipid mediators on mouse N-methyl-D-aspartate (NMDA) receptor currents in the Xenopus oocyte expression system. Arachidonic acid, but not oleic acid potentiated NMDA receptor activity. The epsilon1/zeta1 heterodimer of the NMDA receptor was more sensitive to arachidonic acid than was the epsilon2/zeta1 heterodimer. Platelet-activating factor (PAF) and lysophosphatidic acid (LPA) both activated the NMDA currents, and the effects were more evident in the epsilon2/zeta1 heterodimer than in epsilon1/zeta1. These activations were abolished by treatment with protein kinase inhibitors, suggesting a possible phosphorylation of the receptor. Thus, lipid mediators do have modulatory effects on NMDA receptor currents, the potentiating effects of which differ depending on subtype of the NMDA receptor.

Animals↗
PubMed1997

Molecular cloning of Fyn-associated molecules in the mouse central nervous system.

Fyn tyrosine kinase is expressed extensively in the central nervous system (CNS) of mammals, and its genetic disruption in mouse displays several behavioral abnormalities with morphological and electrophysiological defects in the brain. To understand the signaling pathways in which Fyn is involved in the CNS, we screened molecules that directly associate with Fyn in neonatal mouse brain by using a two-hybrid yeast system. We isolated five cDNA clones with strong and reproducible Fyn-binding activity. Sequence analyses revealed that three of them are previously reported molecules, SON, tctex-1, and hnRNP K, and that two clones encode novel sequences. The hnRNP K has been shown to associate with Fyn, so our yeast system is appropriate to isolate Fyn-binding molecules. Northern hybridization analyses indicated that all isolated clones are expressed in the mouse brain and that the mRNA levels of the two molecules (tctex-1 and clone 82) change during development in the brain. A full-length cDNA of clone 82 was obtained and its deduced amino acid sequence was homologous to the RNA-binding proteins. Isolation of many Fyn-binding molecules suggest that, in the mouse CNS, Fyn mediates multiple signaling pathways by binding to multiple molecules and that some of these pathways play critical roles in determining a certain type of behavior.

Age Factors↗
PubMed1997

Detection of telomerase activity in exfoliated cells in urine from patients with bladder cancer.

BACKGROUND: Telomeres are specific structures located at the ends of chromosomes that help maintain chromosome stability. In most tissues, telomeres become shorter as cells divide, a phenomenon thought to be associated with limitations on normal cell proliferation. Almost all types of cancer cells, including bladder cancer cells, express the enzyme telomerase, which can maintain or extend telomere length. PURPOSE: We examined telomerase activity in tumor specimens from a cohort of patients with bladder cancer and determined whether telomerase could be detected in exfoliated cancer cells present in urine from these patients. METHODS: Spontaneously voided urine specimens and bladder-washing fluids (obtained by propelling normal saline into the bladder through a catheter and then withdrawing the liquid contents) were taken from 45 patients before they underwent surgery. Telomerase activity was examined by means of the TRAP (telomeric repeat amplification protocol) assay on extracts of tumor samples from 42 patients and extracts of exfoliated cells in urine and bladder-washing fluid from 42 and 43 patients, respectively. Standard cytologic examination (Pap staining) of urine specimens was also used to detect exfoliated cancer cells. RESULTS: Telomerase activity was found in 41 (98%; 95% confidence interval [CI] = 87%-100%) of the 42 tumor samples examined. In contrast, it was not detected in normal bladder tissue from two autopsied individuals who were free of bladder cancer and five of six individuals who had bladder cancer. Telomerase was detected in exfoliated cells in 23 (55%; 95% CI = 39%-70%) of the 42 spontaneously voided urine specimens and in 36 (84%; 95% CI = 69%-93%) of the 43 bladder-washing fluids examined. Considering voided urine specimens and bladder-washing fluids together, telomerase was detected in exfoliated cells from 40 (89%; 95% CI = 76%-96%) of the 45 patients. Telomerase activity was not detected in bladder-washing fluids from 12 cancer-free individuals. Cancer cells were detected by means of standard cytologic examination in the urine of 19 (42%; 95% CI = 28%-58%) of the 45 patients. Urine cytologic examination detected cancer cells in one (8%; 95% CI = 0%-38%) of 12 patients with grade 1 tumors and in 13 (46%; 95% CI = 28%-66%) of 28 patients with grade 2 tumors. In contrast, telomerase activity was detected in exfoliated cells (in voided urine or bladder-washing fluids) from nine (75%; 95% CI = 43%-95%) of 12 patients with grade 1 tumors and from 27 (96%; 95% CI = 82%-100%) of 28 patients with grade 2 tumors. CONCLUSION AND IMPLICATION: Telomerase activity can be detected in exfoliated cells in urine from patients with bladder cancer, and measurement of this activity appears to be more sensitive in detecting the presence of cancer than standard urine cytologic examination. These findings suggest that measuring telomerase activity in exfoliated cells would be useful in the diagnosis and follow-up of patients with bladder cancer, a possibility that warrants further study.

Aged↗
PubMed1997

Synapse-selective impairment of NMDA receptor functions in mice lacking NMDA receptor epsilon 1 or epsilon 2 subunit.

1. We have explored the effects of targeted disruption of the N-methyl-D-aspartate (NMDA) receptor epsilon 1 or epsilon 2 subunit gene on NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) and long-term potentiations (LTPs) at the two types of synapse in mouse hippocampal CA3 pyramidal neurons: those formed by the commissural/associational (C/A) and fimbrial (Fim) inputs. 2. Electrophysiological experiments were performed in hippocampal slices prepared from both wild-type and epsilon 1- or epsilon 2-disrupted mice using extracellular and whole-cell patch recording techniques. To assess the epsilon 1, epsilon 2 and zeta 1 subunit expression at cellular levels, we performed non-isotopic in situ hybridization with digoxigenin-labelled cRNA probes. 3. We could record EPSCs in response to the stimulations to either of the C/A and Fim afferents from a single CA3 pyramidal neuron. The epsilon 1, epsilon 2 and zeta 1 subunits were expressed together in individual CA3 neurons. 4. The epsilon 1 subunit disruption selectively reduced NMDA EPSCs and LTP in the C/A-CA3 synapse without significantly affecting those in the Fim-CA3 synapse, whereas the epsilon 2 subunit mutation diminished NMDA EPSCs and LTP in the Fim-CA3 synapse with no appreciable functional modifications in the C/A-CA3 synapse. 5. These results suggest that NMDA receptors with different subunit compositions function within a single CA3 pyramidal cell in a synapse-selective manner.

Animals↗
PubMed1997

Analysis of the glycine binding domain of the NMDA receptor channel zeta 1 subunit.

In an attempt to examine glycine binding domain of the zeta 1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel, we constructed N-terminal or C-terminal deletion mutants of the zeta 1 subunit cDNA and expressed them in Spodoptera frugiperda cells using a baculovirus system. Analysis of binding of a glycine binding site antagonist, 5,7-[3(-3)H]dichlorokynurenate ([3H]DCKA) to the deleted zeta 1 mutants provided the first direct experimental evidence that the regions of N-terminal 282 and C-terminal 102 amino acid residues do not significantly affect glycine binding, and that both the region of approximately 260 amino acid residues preceding the putative transmembrane segment M1 and the region between the segments M3 and M4 are required to form the glycine binding domain.

Animals↗
PubMed1997

Altered intracellular localization of the glutamate receptor channel delta 2 subunit in weaver and reeler Purkinje cells.

The glutamate receptor (GluR) channel delta 2 subunit is expressed abundantly and specifically in cerebellar Purkinje cells. Our previous study demonstrated that the GluR is expressed as early as embryonic day 15 prior to Purkinje cell synaptogenesis, and its protein product accumulates in dendritic spines during normal Purkinje cell maturation. In this study, we examined expression and distribution of the GluR delta 2 in the weaver and reeler mutant cerebelli, which show abnormal cytoarchitecture and neural circuitry. In situ hybridization analysis showed that GluR delta 2 mRNA was expressed in entire Purkinje cells in both mutant mice. Immunohistochemical analysis revealed that intracellular localization of GluR delta 2 was altered in some region of mutant cerebelli. In the cortical surface where Purkinje cells from synapses with parallel fibers, GluR delta 2-immunoreactivity was restricted to dendritic spines of Purkinje cells as observed in normal mice. In contrast, in the subcortical region where granule cells and parallel fibers are absent, the immunoreactivity was found widely in Purkinje dendrites. Thus, the GluR delta 2 protein did not accumulate to the dendritic spines of Purkinje cells lacking synaptic contact with parallel fibers. These results suggest that the expression of both GluR delta 2 mRNA and protein is independent of abnormalities in the mutant cerebelli, but relocalization of the GluR delta 2 protein might depend on the formation of synapses between Purkinje cells and parallel fibers.

Animals↗
PubMed1997

Inducible expression of N-methyl-D-aspartate (NMDA) receptor channels from cloned cDNAs in CHO cells.

To develop a drug screening system, we introduced expression vectors carrying the mouse N-methyl-D-aspartate (NMDA) receptor channel epsilon1 and zeta1 subunit cDNAs under the promoter of the Drosophila heat shock protein hsp70 into Chinese hamster ovary (CHO) cells. We selected clonal cell lines by means of RNA blot hybridization and fura-2 fluorometry. One of these cell lines, ZE1-1, optimally expressed the epsilon1 and zeta1 subunit mRNAs when induced by an incubation at 43 degrees C for 2 h. Heated ZE1-1 cells exhibited the NMDA-induced intracellular Ca2+ elevation, whereas unheated they showed no such response. NMDA and L-glutamate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, induced an increase in the intracellular Ca2+ concentration. The response to the agonists was marginal in the absence of glycine, and diminished by Mg2+ and NMDA receptor antagonists. Furthermore, exposure to agonists of ZE1-1 cells expressing the epsilon1/zeta1 NMDA receptor channel resulted in the release of lactate dehydrogenase (LDH) activity in the culture medium indicating agonist-induced cell death. NMDA receptor antagonists inhibited the LDH activity release. These results suggest that ZE1-1 cells will provide a useful screening system for novel drugs acting on the epsilon1/zeta1 NMDA receptor channel.

Animals↗
PubMed1997

Calcium- and calmodulin-dependent phosphorylation of AMPA type glutamate receptor subunits by endogenous protein kinases in the post-synaptic density.

We have detected immunoreactivities of AMPA receptor subunits GluR1-4 in post-synaptic density (PSD) fraction and tested whether they can be phosphorylated by endogenous kinases. Incubation of PSD with Ca2+ and calmodulin increased phosphorylation of GluR1 and GluR2/3. The phosphorylation of GluR1 was largely blocked by a Ca2+/calmodulin-dependent protein kinase type II inhibitor. Thus Ca2+/calmodulin-dependent phosphorylation of glutamate receptor may be a mechanism underlying enhanced post-synaptic receptor responsiveness in LTP.

Animals↗
PubMed1997

Arginine-481 mutation abolishes ligand-binding of the AMPA-selective glutamate receptor channel alpha1-subunit.

Arginine-481 is located in the putative agonist-binding region preceding the putative transmembrane segment M1 of the alpha1-subunit of the AMPA-selective glutamate receptor (GluR) channel. This amino acid is completely conserved among GluR proteins. A site-directed mutagenesis study using a baculovirus expression system showed that substitution of glutamate, glutamine and lysine for arginine-481 of the recombinant alpha1-subunit protein abolishes binding to [3H]AMPA completely. The present study provides the first direct experimental evidence that the conserved charged arginine-481 residue is essential, directly or indirectly, for the acquisition of ligand-binding activity by the receptor protein.

Animals↗