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K Sakimura

Publications and source records attributed to K Sakimura.

At least 55 records · Page 3Linked to original sources
PubMed1995

Upstream and intron regulatory regions for expression of the rat neuron-specific enolase gene.

Neuron-specific enolase (NSE) occurs in mature neurons and paraneurons. We have isolated the genomic clone coding for rat NSE and clarified its gene structure. In order to analyze the regulatory sequence in the 5'-upstream region and introns, we carried out transient expression experiments of NSE genomic DNA fragments fused to chloramphenicol acetyltransferase (CAT) gene which were transfected into several cultured cells. The used cells were primary cultured rat neurons, PC12, neuroblastoma 35, neuroblastoma 103, C6, primary cultured rat glial cells and HeLa cells. The promoter sequence (190 bp) upstream to the transcription initiation site was important in the expression of CAT gene in these cells. From the experiments with external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in the primary cultured neuron and PC12 cells was found to be localized at upstream 500 bp sequence of the intron 1 and 1.5 kbp upstream sequence of the transcription initiation site. In the upstream important sequences, there were the nearest sequences for AP-1 binding motif, AP-2 binding element, SP-1 binding sequence, cAMP response element, half site of glucocorticoid receptor (GRE) binding sequence, half site of thyroid hormor receptor (TR) or retinoic acid receptor (RAR) binding sequence and MTF-1 binding sequence. Furthermore, Octamer-6 binding motifs also were found. In the intron 1, 5' end upstream 50 bp and downstream 100 bp were the most important sequences. We found the nearest sequences for cAMP response element, E2F binding sequence, early growth response (EGR)-1 binding motif, half site of TCF-1 binding sequence and a neuron-specific element-like sequence in the intron 1.

Animals↗
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↗
PubMed1994

Expression and characterization of the alpha 2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptor channel in a baculovirus system.

Using a baculovirus expression vector system, the alpha 2 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptor (GluR) channel was expressed in Spodoptera frugiperda insect cells. Immunoblotting using the antibody made to the synthetic peptide corresponding to the C-terminus of GluR alpha 2 and [35S]methionine/[35S]cysteine metabolic radiolabeling revealed the major 102-kDa and the minor 98-kDa protein bands. Metabolic radiolabeling with tunicamycin suggested that the two bands correspond to glycosylated and unglycosylated forms, respectively. The recombinant GluR alpha 2 proteins expressed in insect cells were also identified by immunofluorescence staining. The results of [3H]AMPA binding assay using whole cells suggested that, in infected Sf21 cells, binding sites of the GluR alpha 2 proteins were possibly located on the extracellular side. Scatchard analysis of AMPA binding showed the following parameters: Kd = 16 nM, Bmax = 1.9 x 10(5) binding sites per cell or 1 pmol/mg protein in the total particulate fraction. The ligand binding characteristics of the receptors expressed in insect cells were examined. From the effect of various agonists on [3H]AMPA binding of the receptors expressed in insect cells, the rank order potency of agonists was quisqualate > AMPA > L-glutamate > kainate. Thus, the baculovirus-insect cell expression system provides high-efficiency expression of the receptor sufficient to permit structural and functional analyses.

Animals↗
PubMed1994

A novel mutation in Cu/Zn superoxide dismutase gene in Japanese familial amyotrophic lateral sclerosis.

Recently, several missense mutations in the Cu/Zn superoxide dismutase gene (SOD1) have been reported as a putative cause of chromosome-21q-linked familial amyotrophic lateral sclerosis (FALS). We have discovered a novel missense mutation (substitution of Thr for Ala4) in exon 1 (GCC to ACC) in two FALS patients from one Japanese FALS family. No mutations were found in 17 cases of sporadic ALS. The enzyme activity of recombinant fusion protein containing the Cu/Zn superoxide dismutase (SOD) with the Ala4-to-Thr mutation was significantly reduced in E. coli. On the other hand, in the expression system in insect cells using Baculovirus, the mutant SOD expressed an enzyme activity as high as wild-type SOD. These results suggest that the stability of SOD with the Ala4-to-Thr mutation is disrupted especially in the fusion protein. Autopsy was carried out on one of the two patients, and the pathological findings were typical of FALS with posterior column involvement. These results raise the possibility that mutation of the SOD1 is responsible for FALS with broader pathological involvement.

Amino Acid Sequence↗
PubMed1994

Familial amyotrophic lateral sclerosis with a mutation in the Cu/Zn superoxide dismutase gene.

Several missense mutations within exons 1, 2, 4 and 5 of the gene for Cu/Zn-binding superoxide dismutase (SOD1) have been discovered to be involved in the development of chromosome 21q-linked familial amyotrophic lateral sclerosis (FALS). We describe here an autopsied patient with FALS, in whom we have recently identified a novel missense mutation in exon 1 of the SOD1 gene. The neuropathological findings were compatible with those described previously in patients with FALS with posterior column involvement. This suggests that mutations of the SOD1 gene may be responsible for this form of FALS.

Adult↗
PubMed1994

Hepatic myelopathy: an unusual neurological complication of advanced hepatic disease.

Encephalopathy is widely known as one of the neurological complications of chronic hepatic disease. Recently, the occurrence of progressive myelopathy in patients with advanced hepatic disease has been well documented and differentiated from encephalopathy. We describe a 76-year-old man with decompensatory liver cirrhosis due to hepatitis C virus infection who suffered from progressive paraplegia. Postmortem examination revealed demyelination of the lateral column of the spinal cord, especially of the thoracic segment. No evidence of spontaneous portosystemic shunts was found. These findings suggest that the patient had been affected with hepatic myelopathy, which is a rare complication of liver cirrhosis.

Aged↗
PubMed1993

Distinct distributions of five N-methyl-D-aspartate receptor channel subunit mRNAs in the forebrain.

The distributions of five NMDA receptor channel subunit mRNAs in the mouse forebrain at postnatal day 21 were semiquantitatively examined by in situ hybridization with subunit-specific oligonucleotide probes. In contrast to ubiquitous distribution of the zeta 1 subunit mRNA throughout the forebrain, distributions of four epsilon subunit mRNAs were highly variable from nucleus to nucleus. The telencephalon (except for the septum) expressed the epsilon 1 and epsilon 2 subunit mRNAs. Various combinations of the epsilon 1, epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs were present in different nuclei of the septum, the olfactory bulb, and the thalamus. In the hypothalamus, the suprachiasmatic nucleus expressed distinct signals for the epsilon 3 subunit mRNA alone, whereas other nuclei showed faint signals for the epsilon 1, epsilon 2, and epsilon 4 subunit mRNAs. Moreover, different signal levels of the epsilon subunit mRNAs were found in various regions. The hippocampal CA1 region expressed higher signals for the epsilon 1 and epsilon 2 subunit mRNAs than the CA3 region and the dentate gyrus. In the cerebral cortex, signal levels of the epsilon 1 subunit mRNA were higher in the laminae II/III, IV, and VI than the lamina V, whereas those of the epsilon 2 subunit mRNA were highest in laminae II/III and lowest in laminae IV and V. Different signal levels between the epsilon 1 and epsilon 2 subunit mRNAs were also discerned in the amygdala, the caudate-putamen, and the thalamus. The distinct anatomical distributions and differential signal levels of the epsilon subunit mRNAs strongly suggest different subunit organizations of the NMDA receptor channel in different forebrain neurons, which may result in functional diversity of the channel in vivo.

Animals↗
PubMed1993

Chromosomal localization of the epsilon 1, epsilon 3 and zeta 1 subunit genes of the human NMDA receptor channel.

Partial complementary DNAs for the epsilon 1, epsilon 3 and zeta 1 subunits of the human N-methyl-D-aspartate (NMDA) receptor channel were cloned using the corresponding mouse subunit cDNA clones as probes. Genomic DNA clones for the human epsilon 1, epsilon 3 and zeta 1 subunit genes were isolated using the corresponding partial cDNA clones as probes. By the fluorescence in situ hybridization, we mapped the genes for the epsilon 1, epsilon 3 and zeta 1 subunits of the human NMDA receptor channel to chromosomes 16p13, 17q25 and 9q34, respectively.

Adult↗
PubMed1993

Developmental study of the gene expression for alpha and gamma subunits of enolase in the rat brain by in situ hybridization histochemistry.

The gene expression for alpha and gamma subunits of enolase, a dimeric enzyme in the glycolytic pathway, was examined in the developing brain of rats by in situ hybridization. The expression for the gamma subunit of enolase was first detected in post-mitotic neurons settled in the mantle zone at E13, and it increased progressively until the adult stage. Expression signals for the alpha subunit were discerned in two discrete regions showing different developmental changes: the signals in the proliferative ventricular zone were intense at E13 and decreased and eventually disappeared around birth, whereas the signals in the mantle zone persisted until the adult stage. In the adult brain, mRNAs for the alpha and gamma subunits were expressed widely in neurons, resulting in almost similar temporal patterns in the brain except for the cerebellum. Expression levels of the alpha subunit in adult glial cells were below the detection threshold of the in situ hybridization analysis. These findings suggest that both alpha and gamma enolase subunits participate in energy production in neurons of the mature brain and that marked changes in the subunit composition of enolase occur according to both neuron type and maturation.

Animals↗
PubMed1992

Cloning and expression of the epsilon 4 subunit of the NMDA receptor channel.

The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon 4, has been revealed by cloning and sequencing the cDNA. The epsilon 4 subunit shares high amino acid sequence identity with the epsilon 1, epsilon 2 and epsilon 3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon 4 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon 4/zeta 1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon 4 subunit is thus distinct in functional properties from the epsilon 1, epsilon 2 and epsilon 3 subunits, and contributes further diversity of the NMDA receptor channel.

Amino Acid Sequence↗
PubMed1992

Mutations in a putative agonist binding region of the AMPA-selective glutamate receptor channel.

The region preceding putative transmembrane segment M1 of the glutamate receptor (GluR) channel is well conserved among subunits and has been proposed to constitute a part of the agonist binding site. The functional significance of this region was examined by introducing point mutations into charged residues of the alpha 1 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective GluR channel. The dose-response relationships of the mutant receptors were studied after expression in Xenopus oocytes by injection of the mutant alpha 1 subunit-specific mRNA together with the wild-type alpha 2-subunit-specific mRNA. Variable changes in the EC50 values for different agonists were found for the replacement of glutamic acid 398 by lysine and for the replacement of lysine 445 by glutamic acid. These residues may be involved in selective interaction of the GluR channel with agonists.

Amino Acid Sequence↗
PubMed1992

Molecular diversity of the NMDA receptor channel.

Two novel subunits of the mouse NMDA receptor channel, the epsilon 2 and epsilon 3 subunits, have been identified by cloning and expression of complementary DNAs. The heteromeric epsilon 1/zeta 1, epsilon 2/zeta 1 and epsilon 3/zeta 1 NMDA receptor channels exhibit distinct functional properties in affinities for agonists and sensitivities to competitive antagonists and Mg2+ block. In contrast to the wide distribution of the epsilon 1 and zeta 1 subunit messenger RNAs in the brain, the epsilon 2 subunit mRNA is expressed only in the forebrain and the epsilon 3 subunit mRNA is found predominantly in the cerebellum. The epsilon 1/zeta 1 and epsilon 2/zeta 1 channels expressed in Xenopus oocytes, but not the epsilon 3/zeta 1 channel, are activated by treatment with 12-O-tetradecanoylphorbol 13-acetate. These findings suggest that the molecular diversity of the epsilon subunit family underlies the functional heterogeneity of the NMDA receptor channel.

Amino Acid Sequence↗
PubMed1992

Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs.

The glutamate receptor (GluR) channel plays a key part in brain function. Among GluR channel subtypes, the NMDA (N-methyl-D-aspartate) receptor channel which is highly permeable to Ca2+ is essential for the synaptic plasticity underlying memory, learning and development. Furthermore, abnormal activation of the NMDA receptor channel may trigger the neuronal cell death observed in various brain disorders. A complementary DNA encoding a subunit of the rodent NMDA receptor channel (NMDAR1 or zeta 1) has been cloned and its functional properties investigated. Here we report the identification and primary structure of a novel mouse NMDA receptor channel subunit, designated as epsilon 1, after cloning and sequencing the cDNA. The epsilon 1 subunit shows 11-18% amino-acid sequence identity with rodent GluR channel subunits that have been characterized so far and has structural features common to neurotransmitter-gated ion channels. Expression from cloned cDNAs of the epsilon 1 subunit together with the zeta 1 subunit in Xenopus oocytes yields functional GluR channels with high activity and characteristics of the NMDA receptor channel. Furthermore, the heteromeric NMDA receptor channel can be activated by glycine alone.

Amino Acid Sequence↗
PubMed1992

Human glial fibrillary acidic protein (GFAP): molecular cloning of the complete cDNA sequence and chromosomal localization (chromosome 17) of the GFAP gene.

We isolated three glial fibrillary acidic protein (GFAP) cDNA clones from a glioma cell line, U-251 MG. One clone isolated from a U-251 MG cDNA library was long, but lacked both ends. Using poly(A)+ RNA and primers synthesized according to the sequence of this clone, we used the polymerase chain reaction-assisted rapid amplification of cDNA ends (PCR-RACE) method, which is a strategy to isolate cDNA ends, and obtained cDNA clones for the 5' and 3' ends. From the sequences of these overlapping clones, the complete nucleotide sequence of human GFAP cDNA was established. The start (ATG) and the stop (TGA) signals were seen at nucleotide positions 15 and 1311, respectively, and divided the entire sequence of 3027 bp into 14 bp of 5' non-coding, 1296 bp of coding and 1717 bp of 3' non-coding regions. Using cDNA probes made from both the coding and the 3' non-coding regions, Northern blot hybridization was performed with two different stringencies on RNAs from human and rodent brains and human GFAP-positive and -negative cells. It was shown that the 3' non-coding region probe was more specific for human GFAP than the coding region probe which was specific only under higher stringency conditions. This was also suggested by homology analysis of the sequence with those of various intermediate filament proteins. Based on these findings, we performed spot blot hybridization of sorted human chromosomes and Southern blot hybridization of PCR-amplified DNAs of a panel of hamster-human somatic cell hybrids and localized the human GFAP gene to chromosome 17.

Amino Acid Sequence↗