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K Ikenaka

Publications and source records attributed to K Ikenaka.

At least 109 records · Page 6Linked to original sources
PubMed1990

Long-term enhancement of synaptic transmission by synthetic mast cell degranulating peptide and its localization of binding sites in hippocampus.

A bee venom, mast-cell-degranulating (MCD) peptide, was synthesized by stepwise formation of the two disulfide bridges. This synthetic MCD peptide induced long-term potentiation (LTP) in the CA1 region of a hippocampus slice at concentrations ranging from 10(-7) to 10(-5) M. The potentiating effect of MCD was not lost by biotinylation of its N-terminus, and thus it became possible to investigate localization of its binding site at a cellular level in the hippocampus slice at a LTP-inducible concentration (1 microM). We found that even after brief application of N-biotinyl-MCD (1 microM) to perfusate, its presence was observed in pyramidal cell bodies in the CA1 and CA3 regions, but not in granule cells in the dentate gyrus of the hippocampus slice. This observation does not coincide with the localization of its high-affinity binding sites revealed by 125I-MCD binding, suggestive that another type(s) of MCD binding site, such as low-affinity binding sites, are present in the hippocampus.

Animals↗
PubMed1990

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain.

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.

Amino Acids↗
PubMed1990

Reliable transient promoter assay using fluorescein-di-beta-D-galactopyranoside substrate.

The promoter region of the mouse myelin proteolipid protein (PLP) gene was cloned into a promoter testing vector, pIP111. The pIP111 vector is a promoterless derivative of pCH110 (SV40 early region promoter-lacZ) and contains the Escherichia coli lpp transcription terminator sequence at the 5' end of the cloning site. The newly constructed PLP-lacZ fusion plasmid (pWP) was transfected into PLP-nonproducing NIH-3T3 fibroblasts or PLP-producing C6 cells. When the measured beta-galactosidase activity in the pWP-transfected cells was normalized to the pCH110-transfected cells (an appropriate control if the SV40 early region promoter functions constitutively in various cell lines), the results suggested that the promoter region of the PLP gene contains the information necessary for initiation of transcription in a C6 cell-specific manner. However, the beta-galactosidase produced in viable cells was also detected by fluorescein-di-beta-D-galactopyranoside (FDG) treatment followed by image analysis using inverted fluorescent microscopy, which allowed the transfection efficiency to be calculated, and the beta-galactosidase activity obtained by the regular ONPG method was normalized with the value obtained. This procedure indicated that the promoter region of the PLP gene did not show C6-specific expression, because the SV40 early-region promoter was 10 times more active in NIH-3T3 cells than in C6 cells. Thus, the standard experiment gave misleading results. As our detection method is simple and can be used to analyze the promoter activity in a single cell, many applications should be possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Chimeric and molecular genetic analysis of myelin-deficient (shiverer and mld) mutant mice.

The shiverer and myelin-deficient (mld) mutants are two allelic mutations. Both are characterized by hypomyelination in the CNS and deficient expression of the MBP gene. Chimeric analysis of the pathogenesis of shiverer showed that shiverer mutation acts intrinsic to the oligodendrocyte, which is the only cell type expressing the MBP gene in the CNS. Molecular genetic studies by several groups demonstrated that shiverer is a deletion mutation in the MBP gene. Consequently, no MBP is produced in the shiverer mutant. In mld, however, partial expression of the MBP gene was observed. Interestingly, MBP was expressed in a mosaic fashion in the CNS of mld mice. Molecular genetic studies revealed that the mld mutant has duplicated MBP genes in tandem on a distal part (E2-4) of chromosome 18q. The reduced expression was based on the level of mRNA. A large portion is inverted in the upstream copy, and an intact copy is located downstream. We showed in mld mutants that antisense RNA corresponding to the inverted segment is transcribed, and it forms RNA duplex, with the RNA transcribed from the normal gene located downstream.

Animals↗
PubMed1988

Analysis of tissue glycoprotein sugar chains by two-dimensional high-performance liquid chromatographic mapping.

Excellent separation of 45 pyridylamino derivatives of oligosaccharides were achieved by the two-dimensional combination of reversed-phase and size-fractionation high-performance liquid chromatography. The sugar chains of brain glycoproteins were derivatized into a mixture of pyridylamino-oligosaccharides from lyophilized brain tissue without any purification steps, and they were well separated by the system used. The pattern obtained was reproducible, and inter-individual variation was negligible. This finding demonstrated the possibility that this method could be applied to the detection of differences in the structure of glycoprotein sugar chains in crude preparations.

Animals↗
PubMed1988

Analysis of transcription control elements of the mouse myelin basic protein gene in HeLa cell extracts: demonstration of a strong NFI-binding motif in the upstream region.

Promoter elements of the mouse myelin basic protein (MBP) gene were analyzed by in vitro transcription using HeLa cell extracts. We demonstrated the MBTE (MBP transcription element), GC-box core and TATA-box elements, at -130, -93 and -34, respectively. The TATA-box was indispensable for the promoter function. The GC-box was suggested to function co-operatively with far upstream sequences including the MBTE. The MBTE was crucial to direct maximal transcription, and also functioned with a heterologous promoter irrespective of its orientation. We identified a ubiquitous binding factor which interacted specifically with the MBTE and activated transcription. Intensive foot-printing studies demonstrated that the MBTE had a NFI-binding sequence. The MBTE was considered to be one of the strongest NFI-binding motif among known cellular genes. Interestingly, similar strong NFI-binding motifs were suggested to be present in the enhancer of JC virus whose gene is expressed like the MBP gene, in the nervous system.

Animals↗
PubMed1988

Myelin proteolipid protein gene structure and its regulation of expression in normal and jimpy mutant mice.

The mouse proteolipid protein (PLP) gene was cloned into the lambda bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region. Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5'-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5'-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system. PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5'-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.

Amino Acid Sequence↗
PubMed1988

Gene organization and transcription of duplicated MBP genes of myelin deficient (shi(mld)) mutant mouse.

A hereditary dysmyelinating mutation, named myelin deficient (shi(mld)), is characterized by reduced expression of myelin basic protein (MBP). In shi(mld), the MBP gene is duplicated and its reduced expression is mainly determined by the level of mRNA. We have characterized the structure and function of the promoter regions of the duplicated MBP genes in shi(mld). Among the lambda clones containing promoter regions of the duplicated MBP genes in shi(mld), one (gene 1) had the same restriction enzyme pattern as that in control mice, but another (gene 2) had a rearrangement on a distal part of the promoter. A 712-bp nucleotide sequence upstream of the first exons of both of the duplicated MBP genes of shi(mld) was completely consistent with that of the control. Promoter activities of 1.3-kb 5'-flanking regions from respective genes of shi(mld) measured by in vitro run-off assay using HeLa whole-cell extracts were indistinguishable from that of the control MPB gene. Chromosomal mapping by in situ hybridization suggested that the duplicated MBP genes were located closely to each other at the distal part of chromosome 18. A recombinational event including the inversion seemed to have occurred within gene 1 and its possible relationship to the reduced expression of MBP is discussed.

Animals↗
PubMed1988

Recombination within the upstream gene of duplicated myelin basic protein genes of myelin deficient shimld mouse results in the production of antisense RNA.

The myelin deficient shimld mouse is an autosomal recessive mutant, characterized by hypomyelination in the central nervous system. The expression of the myelin basic protein (MBP) gene is inhibited transcriptionally. The MBP gene is duplicated tandemly in mld, and exons 3 to 7 of the upstream copy is inverted. In the present studies, we determined the approximate position of the 5' boundary and the nucleotide sequence surrounding the 3' boundary of the inversion and found a number of sequences homologous to the switching regions of mouse immunoglobulin heavy chain gene and J regions of human T cell receptor genes. Antisense RNA complementary to exons 3 and 7, which correspond to the inverted segment, was detected by RNase protection studies. This abnormal transcript was also shown to elongate through the inverted segment to reach the transcription initiation site of the downstream gene.

Animals↗
PubMed1988

A dominant mutation in Escherichia coli OmpR lies within a domain which is highly conserved in a large family of bacterial regulatory proteins.

We have fortuitously created an in-frame insertion mutation in the cloned ompR gene of Escherichia coli in the course of an experiment involving linker insertion mutagenesis. According to the DNA sequence, the mutant protein has an insertion at the 53rd amino acid residue, which replaced the original valine, with the sequence Ala-Leu-Glu. The expression level of the mutant protein, OmpRX6, in a minicell system, is similar to that of the wild-type protein and the size of the mutant is slightly larger than the wild type by approximately 300 daltons. This mutant was completely unable to activate porin expression as the wildtype does, and in addition, this phenotype was shown to be dominant over the wild type. Comparison of the amino acid sequence of OmpRX6 with those of a family of homologous bacterial regulatory proteins revealed that the mutation lies in a domain which is highly conserved among these proteins.

Amino Acid Sequence↗
PubMed1988

Pyridylamino sugar chain as an acceptor for galactosyltransferase.

Pyridylamino asialo-agalacto-biantennary sugar chain (PA-acceptor), prepared from human alpha 1-acid glycoprotein, was incubated with bovine milk galactosyltransferase. Transfer of galactose residues to PA-acceptor was detected by HPLC analysis, and thus PA-acceptor was shown to be useful for galactosyltransferase assay. Moreover, three species of products, i.e. PA-acceptor monogalactosylated on the Man alpha 1-3 branch of the trimannosyl core, PA-acceptor monogalactosylated on the Man alpha 1-6 branch, and digalactosylated PA-acceptor, were separated and identified by reversed-phase HPLC, so we could simultaneously determine the branch specificity (the ratio of galactosylation on Man alpha 1-3 branch to that on Man alpha 1-6 branch) of the galactosyltransferase. We fractionated the bovine milk galactosyltransferase on a DEAE-5PW column and confirmed that there was a heterogeneity in this enzyme preparation. Each fraction was assayed for acceptor specificity (the ratio of the activity towards N-acetylglucosamine to that towards PA-acceptor) and branch specificity using the PA-acceptor. However, we could not detect differences in the specificities among the fractions. In addition, we found that alpha-lactalbumin stimulated the galactosyltransferase activity towards PA-acceptor.

Aminopyridines↗
PubMed1987

The isolation and characterization of RNA coded by the micF gene in Escherichia coli.

A new species of micF RNA, which contains 93 nucleotides (a 4.5S size), was isolated from Escherichia coli. The sequence of the 4.5S micF RNA corresponds to positions G82 through U174 of the micF gene. The 5' terminal end of this smaller micF RNA is triphosphorylated signifying that it is a primary transcript. Its promoter region, which is situated within the greater micF structural gene, has been identified and characterized by lacZ fusion analysis. A 6S micF RNA species, which has a base composition predicted for a transcript from the full length gene has also been detected; however, the 4.5S micF RNA is the predominant species. The work clearly shows by biochemical identification the presence of chromosomally encoded micF RNA.

Bacterial Outer Membrane Proteins↗
PubMed1987

The fifth exon of the myelin proteolipid protein-coding gene is not utilized in the brain of jimpy mutant mice.

The jimpy mouse, an X-linked recessive dysmyelinating and demyelinating mutant, has been shown to contain abnormal myelin proteolipid protein (PLP) mRNA. To understand the molecular basis of the mutation, we analyzed the structure of PLP mRNA by an RNase-mapping procedure, using the probes specific to each exon of the mouse PLP gene. We found that the fifth exon of the PLP gene is not utilized in the jimpy.

Animals↗
PubMed1987

Inefficient transcription of the myelin basic protein gene possibly causes hypomyelination in myelin-deficient mutant mice.

A hereditary dysmyelination mutation, named myelin deficient (mld), is considered to be allelic to shiverer, a deletion mutation of the myelin basic protein (MBP) gene. The present study showed that MBP expression is greatly reduced in mld, but that it is still detectable. Northern blot analysis revealed that the pronounced decrease in the MBP level in mld resulted from a reduced mRNA level and was not caused by deletion of a large portion of the MBP gene as in shiverer. Southern blot studies with BamHI-digested chromosomal DNA suggested some part of the MBP gene, at least the 5'-portion, was duplicated in mld. These results indicated that the mld and shiverer mutations were different from each other, even though genetic allelism between the two was reconfirmed. We also examined the developmental pattern of the gene expression of MBP and that of another protein, myelin proteolipid protein (PLP), specifically expressed in the oligodendrocyte, in mld by RNA dot blot study. The mRNA level of MBP in mld was greatly reduced during the active myelination stages, gradually increasing and remaining constant in the later stages. The PLP-mRNA content in mld was almost normal (60-80% that of control) at any stage of development. All these findings imply that the primary defect in mld is due to reduced transcriptional activity of the MBP gene.

Alleles↗
PubMed1986

Interaction of a transcriptional activator, OmpR, with reciprocally osmoregulated genes, ompF and ompC, of Escherichia coli.

The ompB locus, comprised of the genes ompR and envZ, regulates the expression of the genes ompF and ompC that encode the major porin proteins in the outer membrane of Escherichia coli K-12. OmpR is believed to activate transcription of ompF and ompC in a reciprocal manner depending upon the osmolarity of the culture medium. We were able to purify OmpR to homogeneity and to characterize its interaction with the porin gene promoters. The purified OmpR was shown to bind specifically to promoter fragments of both ompF and ompC. Deoxyribonuclease I footprinting was carried out in order to determine binding sites of OmpR in the promoter regions of the ompF and ompC. OmpR was found to protect the regions -105 to -60 of ompF and -102 to -78 of ompC, respectively. Two consensus sequences were found in these protected sites and are likely to play an important role in OmpR recognition of these promoter regions. The purified OmpR was also shown to be functionally active in transcription in vitro; OmpR activates the expression of ompF and inhibits the transcription of ompC from the most distal of its three tandem promoters, P3.

Bacterial Outer Membrane Proteins↗
PubMed1986

Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters.

ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.

Base Sequence↗
PubMed1985

Uncoupling of osmoregulation of the Escherichia coli K-12 ompF gene from ompB-dependent transcription.

The expression of the genes for the Escherichia coli K-12 outer membrane proteins, ompF and ompC, is subject to osmoregulation and responds to changes in the osmolarity of the growth medium. The transcription of these genes is dependent on the products of the regulatory locus ompB (comprising the genes ompR and envZ). The native promoter of ompF was replaced with an inducible lpp promoter to eliminate this transcriptional dependence of ompF expression on ompB. As a result, it was possible for the OmpF protein to be produced in an ompB mutant strain that does not normally express ompF. Surprisingly, the expression of ompF under the lpp promoter was still osmoregulated not only in the ompB+ strain but also in two ompB strains tested. These results indicate the involvement of a factor(s) besides the ompR and envZ gene products in the osmoregulation of OmpF production. This factor may interact with a sequence downstream of the ompF promoter. In addition, we show that the expression of ompF under the lpp promoter has no direct effect on ompC expression.

Bacterial Outer Membrane Proteins↗