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Biomedical subjects

M Mishina

Publications and source records attributed to M Mishina.

At least 109 records · Page 6Linked to original sources
PubMed1996

[Benzodiazepine receptor and cerebral blood flow in early Alzheimer's disease--SPECT study using 123I-iomazenil and 123I-IMP].

This study was designed to investigate benzodiazepine receptors (BZR) and cerebral blood flow (CBF) in patients with early Alzheimer's disease. Imaging of BZR and measurement of CBF were performed by SPECT using 123I-Iomazenil (IMZ) and 123I-IMP respectively, in seven patients with early Alzheimer's disease and five patients with unilateral left cerebral infarction as controls. The values for the normal cerebral hemisphere (ratio to the contralateral cerebellum) in patients with cerebral infarction were adopted as control values. In patients with Alzheimer's disease, the CBF (ratio to cerebellum) decreased significantly in the frontal cortex and the parietal cortex compared with the control values. There was no significant difference in late IMZ SPECT counts (ratio to cerebellum) and washout (the ratio of late-to-early IMZ SPECT counts) between patients with Alzheimer's disease and the controls. However, the late IMZ SPECT counts and washout decreased in one patient with moderate dementia. There was a significant correlation between the severity of dementia and the late IMZ SPECT counts in the temporal cortex and the parietal cortex. These results suggest that benzodiazepine binding sites are relatively well preserved in patients with early Alzheimer's disease, and reduction of the CBF is caused by neuronal dysfunction rather than by neuronal loss. IMZ SPECT study is useful and necessary for clarifying the pathophysiological state in Alzheimer's disease.

Aged↗
PubMed1996

[A quantitative study of regional cerebral blood flow in patients with dementia of Alzheimer type using single photon emission computed tomography--clinical application of the IMP autoradiographic method (IMP ARG method)].

We quantitatively measured regional cerebral blood flow (rCBF) in 37 patients with dementia of Alzheimer type (DAT) to investigate the clinical utilities of the N-isopropyl-p-[123I]iodoamphetamine autoradiographic method (IMP ARG method) that is a quantitative method more simplified and less invasive for IMP-SPECT developed by Iida et al. A given standard input function and a given value of distribution volume (Vd) used for the rCBF measurement of this method were calculated from the dynamic study by six normal volunteers. Mean values [SD] of rCBF (ml/ 100 g/min) in the Cerebral Cortex were 49.0 [6.0] in the controls (n = 20), 42.6 [5.9] in mild DAT group (n = 14), 36.7 [5.5] in moderate DAT group (n = 12), and 26.4 [7.5] in severe DAT group (n = 11), respectively. These values were significantly different between each neighboring group. Moreover, the correlations between the score by the Hasegawa dementia scale (HDS-R) and each rCBF were significant in the temporal, parietal, and frontal cortex. These findings suggest that the rCBF measurement in IMP-SPECT using this method is useful for the diagnosis of the clinical severity in patients with DAT.

Aged↗
PubMed1995

Retarded vestibular compensation in mutant mice deficient in delta 2 glutamate receptor subunit.

The delta 2 subunit of ionotropic glutamate receptors is expressed only in the cerebellar Purkinje cell. In mutant mice deficient in the delta 2 protein, cerebellar long-term depression and motor coordination are impaired. We examined behavioural plasticity in these mutant mice after unilateral vestibular destruction. After intratympanic injection of sodium arsanilate, the mice showed roll head tilt and their righting response under a rotation load was impaired. These symptoms improved with time. However, compensation of the righting response was retarded in the mutant mice. These results suggest that motor learning of the delta 2 mutant mice is disturbed, and that the static and dynamic components of vestibular compensation may be controlled by different neuronal mechanisms.

Animals↗
PubMed1995

The sensitivity of AMPA-selective glutamate receptor channels to pentobarbital is determined by a single amino acid residue of the alpha 2 subunit.

Clinical concentrations of pentobarbital inhibit the alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor (GluR) channels. Recently, the AMPA-selective GluR channels that contained the alpha 2 subunit were shown to be more sensitive to pentobarbital block than those without the alpha 2 subunit. Here we demonstrated that replacement by glutamine of the arginine residue in putative transmembrane segment M2 of the alpha 2 subunit (mutation alpha 2-R586Q) drastically reduced the pentobarbital sensitivity of the alpha 2 heteromeric channel to the level comparable to those of the alpha 1 and alpha 2-R586Q homomeric channels. These results suggest that the arginine residue in segment M2 of the alpha 2 subunit is the critical determinant of the sensitivities of the AMPA-selective GluR channels to pentobarbital.

Amino Acid Sequence↗
PubMed1995

Differential expression of two zebrafish emx homeoprotein mRNAs in the developing brain.

The complete amino-acid sequences of two zebrafish emx homeoproteins containing a homeodomain homologous to that of the Drosophila empty spiracles head gap gene product have been determined by cloning and sequencing of cDNAs. The zebrafish emx1 and emx2 homeoproteins consist of 233 and 247 amino acids, respectively. The zebrafish emx1 and emx2 mRNAs are present at 12 h after fertilization, when the presumptive brain is in a simple tubular structure, and before first primary neurons appear. During brain development, the emx1 mRNA is localized in the dorsal telencephalon, whereas the emx2 mRNA is distributed in the dorsal telencephalon, parts of the diencephalon and the otocyst. The differential expression patterns of the two emx homeoprotein mRNAs may define the subdivisions of the zebrafish telencephalon.

Amino Acid Sequence↗
PubMed1995

Stable production of mutant mice from double gene converted ES cells with puromycin and neomycin.

The antibiotic puromycin is an effective inhibitor of protein synthesis and puromycin N-acetyl transferase gene could be used as a dominant selection marker. We report the effective production of mutant mice from double gene-converted ES cells by selection with G418 and puromycin. We confirmed that (i) puromycin efficiently inhibited the growth of ES cells at a low-dose (0.1 microgram/ml) and for a short time (2 days), independent of G418 selection; (ii) when these selected ES cells were injected into eight-cell stage embryos, the cells produced chimeras with high levels of chimerism; (iii) these chimeric males were fertile and exclusively yielded ES cell-derived offspring; and (iv) each offspring contained both neomycin transferase and puromycin N-acetyl transferase genes.

Acetyltransferases↗
PubMed1995

Identification of four different forms of syntaxin 3.

cDNAs for four different forms of syntaxin 3 were cloned from a mouse brain cDNA library, and the proteins encoded by these clones were named syntaxin 3A (previous syntaxin 3), 3B, 3C and 3D. Syntaxin 3B contained a different sequence in the carboxyl terminal region from that of syntaxin 3A. The amino terminal region of syntaxin 3C contained an 18 amino acid sequence instead of a 34 amino acid sequence present in syntaxins 3A and 3B. Syntaxin 3D consisted of only 86 amino acids and lacked any putative transmembrane segments. These forms of syntaxin 3 are probably generated by alternative splicing of the primary transcript of syntaxin 3 gene. Cytoplasmic portions of syntaxins 3A and 3B but not of syntaxin 3C or 3D bound to Munc-18/n-sec1.

Alternative Splicing↗
PubMed1995

Effects of propofol on various AMPA-, kainate- and NMDA-selective glutamate receptor channels expressed in Xenopus oocytes.

Effects of a general intravenous anesthetic 2,6-diisopropylphenol (propofol) on various glutamate receptor (GluR) channels were examined on the alpha 1 and alpha 1/alpha 2 GluR channels selective for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), the beta 2/gamma 2 GluR channels selective for kainate, and the epsilon 2/zeta 1 and epsilon 3/zeta 1 N-methyl-D-aspartate (NMDA) receptor channels expressed in Xenopus oocytes. Propofol suppressed the current responses of the alpha 1/alpha 2, beta 2/gamma 2, epsilon 2/zeta 1 and epsilon 3/zeta 1 channels in a dose-dependent manner, whereas it enhanced the current responses of the alpha 1 channel. The extents of inhibition were in the order epsilon 2/zeta 1 > epsilon 3/zeta 1 > beta 2/gamma 2 > alpha 1/alpha 2 channels. During perfusion of 500 microM propofol, the alpha 1/alpha 2, beta 2/gamma 2, epsilon 2/zeta 1 and epsilon 3/zeta 1 channels were progressively suppressed. Furthermore, 10 min perfusion of 20 microM propofol inhibited the epsilon 2/zeta 1 channel by 24%. These results suggest that clinical concentrations (approximately 35 microM) of propofol suppress the NMDA receptor channels slightly.

Animals↗
PubMed1995

Light- and electron-microscopic localization of the glutamate receptor channel delta 2 subunit in the mouse Purkinje cell.

The localization of the glutamate receptor channel delta 2 subunit was investigated by immunohistochemistry. The delta 2-immunoreactivity was observed exclusively in the molecular layer of the cerebellar cortex. The electron microscopic analysis showed that the delta 2 subunit was localized in the dendritic spines of the Purkinje cells. The immunopositive spines often formed synaptic contacts with parallel fiber varicosities. As for the Purkinje cells ectopically localized in the cerebellar nuclei and brain stem, the dendritic shafts and cell bodies were strongly labeled. These ectopic Purkinje cells also formed asymmetrical synapses at the delta 2-immunopositive dendritic spines. Considering the specific localization of the delta 2 subunit in the postsynaptic site of the Purkinje cells, the subunit is suggested to be involved in the excitatory synaptic transmission in the cells, as a component of the glutamate receptor channel.

Animals↗
PubMed1995

Suppression of LTD in cultured Purkinje cells deficient in the glutamate receptor delta 2 subunit.

Involvement of the glutamate receptor channel delta 2 subunit in cerebellar long-term depression (LTD) was studied in cultures prepared from wild-type and mutant mice deficient in the delta 2 subunit. LTD of the glutamate response was induced by pairing glutamate applications and depolarization of a Purkinje cell in wild-type culture. However, in cultured Purkinje cells prepared from mutant mice, the same conditioning failed to induce LTD. Immunocytological staining showed that mutant Purkinje cells develop and express calbindin (a marker protein for Purkinje cells) as do wild-type cells, but they express no delta 2 subunit protein. The results indicate that the glutamate receptor channel delta 2 subunit is involved in the postsynaptic down-regulation of glutamate sensitivity, presumably during cerebellar LTD.

Animals↗
PubMed1995

Cerebellum of the adult reeler mutant mouse contains two Purkinje cell populations with respect to gene expression for the N-methyl-D-aspartate receptor channel.

Recent studies have identified five NMDA receptor subunits, which exhibit distinct cellular expressions in the normal rodent brain. The purpose of this investigation is to clarify the molecular-anatomical organization in the cerebellum of the reeler mutant mouse, in which various categories of the Purkinje cells are present as to the cell position and synaptic connectivity. In comparison with the distribution of the inositol 1,4,5-trisphosphate receptor mRNA, a molecular marker specific to the Purkinje cells, the epsilon 1 subunit mRNA of the NMDA receptor channel was found in the adjacent sections to be expressed in a subset of the Purkinje cells. In the rostrocaudal extent, the Purkinje cells expressing the epsilon 1 subunit mRNA were distributed preferentially in the rostral cerebellum, irrespective of the normal and heterotopic positions. In the mediolateral extent, they formed segregated cell clusters, interposed by epsilon 1 subunit mRNA-negative clusters. Hybridizing signals for the zeta 1 subunit mRNA were found in all the Purkinje cell population, whereas those for the epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs were not detected in the cells. These findings suggest that the reeler cerebellum is topographically compartmentalized by two subpopulations of the Purkinje cells, one expressing the epsilon 1 and zeta 1 subunit mRNAs, and the other expressing the zeta 1 subunit mRNA alone.

Animals↗
PubMed1995

Expression and characterization of the zeta 1 subunit of the N-methyl-D-aspartate (NMDA) receptor channel in a baculovirus system.

Using a baculovirus expression vector system, the zeta 1 subunit of the mouse N-methyl-D-aspartate (NMDA) receptor channel was expressed in Spodoptera frugiperda insect cells. The peptide corresponding to the C-terminus of the zeta 1 subunit was synthesized by using the multiple antigen peptide (MAP) system, and an antibody to the synthetic peptide was produced. Immunoblotting using the newly developed antibody revealed the major 122-kDa and the minor 104-kDa protein bands. The effect of tunicamycin on the immunoblots and [35S]methionine/[35S]cysteine metabolic radiolabeling suggested that the two bands corresponded to glycosylated and non-N-glycosylated forms, respectively. Membranes prepared from insect cells infected with the recombinant virus had the binding activity of antagonist ligand 5,7-[3-3H]dichlorokynurenate (DCKA) of a glycine recognition domain of the receptor. Both immunofluorescence labeling and the [3H]DCKA binding assays also showed a greater level of expression (Bmax = 51 pmol/mg protein) in the insect cells. The ligand binding characteristics of the receptors expressed in insect cells suggested that the single zeta 1 subunit protein has glycine antagonist binding properties comparable to those of the native NMDA receptor channels. The lack of DCKA-binding activity of the non-N-glycosylated NMDA receptor expressed in the presence of tunicamycin suggested that N-linked oligosaccharide is essentially required for expression of a functional receptor in insect cells. This is the first report describing the importance of N-glycosylation for the acquisition of ligand binding to NMDA receptor channel subunit protein.

Animals↗
PubMed1995

Reduced spontaneous activity of mice defective in the epsilon 4 subunit of the NMDA receptor channel.

In an attempt to examine the functional significance of the molecular diversity of the N-methyl-D-aspartate (NMDA) receptor channel, we generated mutant mice defective in the epsilon 4 subunit by gene targeting technique. The epsilon 4 subunit mutant mice grew and mated normally. No epsilon 4 subunit protein was detected in the homozygous mutant mice, and the amount of the epsilon 4 subunit protein of 155 kDa was reduced in the heterozygous mice. The expressions of the other NMDA receptor channel subunit mRNAs were not appreciably affected by the mutation. The mutant mice exhibited no obvious histological abnormalities in the various brain regions and in the formation of whisker-related neuronal patterns (barrels, barreloids and barrelettes). In an open field test, however, the epsilon 4 subunit mutant mice showed a reduced spontaneous activity. No significant difference was found between the heterozygous and mutant mice in motor activity and anxiety tests. These results suggest that the epsilon 4 subunit of the NMDA receptor channel plays a role in vivo in controlling the spontaneous behavioral activity.

Analysis of Variance↗
PubMed1995

N-linked glycosylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-selective glutamate receptor channel alpha 2 subunit is essential for the acquisition of ligand-binding activity.

The N-linked glycosylation of the alpha 2 subunit of the mouse alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N-glycosylation sites. The recombinant receptor proteins were identified by [35S]methionine/[35S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [3H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 102 kDa and a minor species of approximately 98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N-glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N-glycosylation site is required for the formation and maintenance of the GluR alpha 2 subunit protein in an active conformation for ligand binding. Possible roles of N-glycosylation of GluR alpha 2 subunit protein are discussed.

Animals↗
PubMed1995

Contribution of chromosome 9p21-22 deletion to the progression of human renal cell carcinoma.

To investigate the possible role of genomic aberrations of chromosome 9p21-22 in the tumorigenesis of human renal cell carcinoma (RCC), 10 RCC cell lines, 55 primary RCCs and 5 metastatic lesions were studied by Southern blotting and polymerase chain reaction-based analysis. Nine of 10 RCC cell lines showed a homozygous deletion of MTSI/CDKN2/(p16), while only 1 in 55 primary tumors had this deletion. Loss of heterozygosity on 9p21-22 was observed in 5 of 10 informative primary RCCs from patients with metastasis, but in only 4 of 31 informative tumors (13%) without metastasis (P = 0.025). Futhermore, 3 of 5 metastatic tumors (60%) showed hemi- or homozygous deletion of MTSI/CDKN2. These results indicate that the 9p21-22 deletion may be a relatively late event in RCC tumorigenesis and could be associated with RCC metastasis.

Alleles↗
PubMed1995

[Activation of imprinted genes in human carcinogenesis].

Genomic imprinting is defined as a differential expression of genes depending on their parental origin. IGF2 gene on chromosome 11p, which is maternally imprinted in normal human development, has been demonstrated to be activated by loss of imprinting (LOI) during tumorigenesis of Wilms tumor or other kidney neoplasms in childhood. Although the molecular mechanism of genomic imprinting is not clarified yet, it is certain that disruption of the imprinting mechanism has a causative role in some human cancers.

Animals↗