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Biomedical subjects

F Ozawa

Publications and source records attributed to F Ozawa.

15 recordsLinked to original sources
PubMed1996

mRNA differential display reveals Krox-20 as a neural plasticity-regulated gene in the rat hippocampus.

The prolonged maintenance of hippocampal long-term potentiation depends on de novo protein and RNA synthesis, indicating an involvement of altered gene expression in long-lasting plastic changes in synaptic efficacy. We have used an mRNA differential display technique to identify a set of genes that are induced by neural activity in the rat hippocampus. Sixteen independent cDNAs were isolated whose mRNA level was markedly modulated by convulsive seizure. One of these encodes Krox-20, a zinc finger DNA binding protein. High frequency tetanic stimulation of perforant pathway, which elicited a persistent long-term potentiation (>10 h), rapidly induced expression of krox-20 mRNA in the hippocampus of urethane-anesthetized rat. The increase in krox-20 mRNA was transient and NMDA receptor-dependent. These results suggest a role for krox-20 in the maintenance of long-term potentiation.

Amino Acid Sequence↗
PubMed1996

Increase in activin beta A mRNA in rat hippocampus during long-term potentiation.

We have used mRNA differential display to isolate genes that are induced by neural activity in rat hippocampus. One of these encodes activin beta A subunit. Convulsive seizure caused by kainate significantly induced the expression of activin beta A mRNA. Furthermore high frequency stimulation (HFS) of perforant pathway, which produced a persistent long-term potentiation (LTP) (>10 h), caused a marked increase at 3 h in the level of activin beta A mRNA at the dentate gyrus of urethane-anesthetized rat. The increase was NMDA receptor-dependent. By contrast the level of inhibin alpha mRNA was not changed following the induction of LTP. THe results suggest a role for activin in maintenance of neural plasticity in the adult brain.

Activins↗
PubMed1996

Localization of Menkes gene expression in the mouse brain; its association with neurological manifestations in Menkes model mice.

Menkes gene (Mc1 or MNK, encoding putative copper-transporting ATPase) expression was investigated and compared in normal and macular mutant mouse brain. Northern blot analysis showed a distinct 8.3-kb transcript and no obvious difference in size or extent in normal mice and macular mutants on postnatal days 0, 4, 7, 10 or 13. In situ hybridization revealed that certain specific populations of cells in the brain express Menkes mRNA, and that their localization in normal and mutant mice did not differ and was conserved on days 4, 10 and 13. The most intense hybridization signals were observed in the hippocampal CA1 region and dentate gyrus, the olfactory bulb nuclei, the cerebellar granular cell layer, the choroid plexus and the ependyma, with less intense signals in the hippocampal CA3 region and cerebellar Purkinje cells. In addition, necrotic neuronal cell death was predominantly observed in the CA3 region and the Purkinje cells of macular mice after postnatal day 10. The finding that the regions that had lower expression level of Menkes mRNA corresponded to those showing neuronal necrosis suggests that the Menkes gene may be responsible for the neuronal degeneration in some specific portions of the brain and clinical manifestations in this mutant.

Adenosine Triphosphatases↗
PubMed1994

Murine cerebellar neurons express a novel gene encoding a protein related to cell cycle control and cell fate determination proteins.

We cloned cDNAs of a novel protein (designated V-1) that has been identified from among the developmentally regulated proteins in the rat cerebellum. Protein sequencing analysis (Taoka, M., Yamakuni, T., Song, S.-Y., Yamakawa, Y., Seta, K., Okuyama, T., and Isobe, T. (1992) Eur. J. Biochem. 207, 615-620) and cDNA sequence analysis revealed that the V-1 protein consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, which was originally found in the products of the cell cycle control genes of yeasts and the cell fate determination genes in Drosophila and Caenorhabditis elegans. In situ hybridization histochemistry revealed that the expression of the V-1 gene is transiently increased in postmigratory granule cells during postnatal rat cerebellar development and thereafter is markedly suppressed, whereas Purkinje cells constitutively express V-1 mRNA. In contrast, cerebellar granule cells of the staggerer mutant mouse continue to express the V-1 gene even when the granule cells of the normal mouse have ceased to express the V-1 gene, suggesting that the expression of the V-1 gene in granule cells is regulated through the interaction with Purkinje cells. On the basis of these results, we postulate that the V-1 protein has a potential role in the differentiation of granule cells.

Amino Acid Sequence↗
PubMed1993

Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae.

Nerve growth factor (NGF) is a trophic agent that is essential for the development and survival of sympathetic and sensory nerves. A chemically-synthesized DNA fragment encoding human NGF (hNGF) and a cDNA encoding mouse NGF (mNGF) were engineered for expression in the yeast, Saccharomyces cerevisiae. Expression and secretion of hNGF and mNGF was attempted under the direction of the yeast PGK promoter and with various leader sequences. Among the leader sequences tested, that of the yeast alpha-factor successfully directed secretion of both hNGF and mNGF that were correctly processed. The content of the recombinant NGF (reNGF) in the culture supernatant was estimated to be 1 microgram/ml. The yeast-produced reNGF was able to bind to NGF receptors in rat pheochromocytoma (PC12) cells as efficiently as the standard mNGF, and partially purified reNGF could induce neurite outgrowth of PC12 cells. Thus, we have demonstrated that biologically active human and mouse reNGF can be produced in yeast cells.

Amino Acid Sequence↗
PubMed1992

Regulation of nerve growth factor and nerve growth factor receptor production by NMDA in C6 glioma cells.

The synthesis of nerve growth factor (NGF) and nerve growth factor receptor (NGFR) were studied in a C6 glioma cell line by Northern blot hybridization. In response to a glutamate agonist N-methyl-D-aspartic acid (NMDA), NGF mRNA increased by up to 2-fold after 4-12 h of culture. The non-NMDA receptor agonists, quisqualate and kainate, did not induce any increase of NGF mRNA, and kainate actually produced a decrease. The increase in NGF mRNA in response to NMDA was dose-dependent at 1, 5 and 10 microM. NGF receptor (NGFR) mRNA showed changes in expression which were similar to those for NGF mRNA, but were less marked. The specific glutamate antagonist 2-aminophosphonovaleric acid (APV) blocked the increase of NGF mRNA produced by NMDA. In the absence of Ca2+, an increase of NGF mRNA was still observed but in the presence of 1 mM ethylglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA), NGF mRNA production abolished. The mechanism producing an increase in NGF mRNA by NMDA may be mediated by cyclic AMP since intracellular cyclic AMP and NGF mRNA levels both increased following treatment with NMDA or dibutyryl cyclic AMP.

2-Amino-5-phosphonovalerate↗
PubMed1991

Enhanced secretion of human nerve growth factor from Saccharomyces cerevisiae using an advanced delta-integration system.

We have designed an advanced delta-integration system (integration of genes into the delta-sequence of yeast retrotransposon Ty) and used it for secretion of human nerve growth factor (hNGF) from Saccharomyces cerevisiae. The expression and secretion of hNGF was directed by the PGK promoter and MF alpha 1 prepro-signal. Using two selectable markers (URA3 and leu2-d), haploid yeast strains were constructed with approximately 20 copies of a delta-integrated hNGF expression cassette on four chromosomes. The strain secreted hNGF at levels 3-4 fold higher than a 2 micron-based plasmid. Northern and Western analyses revealed that the oversecretion was caused by an increased amount of mRNA. We also detected an unusual processing of the MF alpha 1 prepro-hNGF fusion protein that required the pep4 mutation. Application of this system for industrial purposes is discussed.

Biotechnology↗
PubMed1989

Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae.

The DNA coding for human nerve growth factor (hNGF) was chemically synthesized and introduced into Saccharomyces cerevisiae. Expression and secretion of hNGF was obtained by use of the yeast phosphoglycerate kinase-encoding gene promoter and the pre-pro sequence of the yeast alpha-mating factor. Immunoblotting with antiserum raised against a protein A-hNGF fusion protein, allowed the detection of an immunoreactive material secreted into the culture medium. A preparation from the culture medium, partially purified by ion-exchange column chromatography, stimulated neurite outgrowth from rat pheochromocytoma PC12h cells.

Amino Acid Sequence↗
PubMed1987

Expression of beta-nerve growth factor mRNA in rat glioma cells and astrocytes from rat brain.

A 50-base synthetic oligodeoxynucleotide complementary to a portion of mouse nerve growth factor (NGF) mRNA was used as a probe for analysis of the expression of NGF gene. Northern blot analysis showed the presence of a major 1.3 kb transcript, which was identical in size to mouse NGF mRNA, in both C6Bu1 cells and rat astrocytes cultured from newborn rat brain. Further, the rearrangement of DNA sequence in and around the NGF gene locus of C6Bu1 cells was not detected by Southern blot analysis. These results indicate the expression of NGF mRNA in both C6Bu1 cells and astrocytes from rat brain, suggesting that astrocytes may produce NGF protein in the rat brain, especially in developing rat brain.

Animals↗
PubMed1987

Molecular cloning of an amylase gene of Bacillus circulans.

An amylase gene of Bacillus circulans was cloned in B. subtilis and its nucleotide sequence was determined. The putative proamylase consists of 528 amino acids, which correspond to a molecular weight of 58,776. Homologous regions with other amylases of Bacillus species were found. A sigma 55-type promoter is located at about 250 bp upstream from the starting codon. This promoter was also functional in Escherichia coli, and able to express beta-galactosidase activity.

Amino Acid Sequence↗
PubMed1981

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.

Ascomycota↗