PubMed Health⌕ Search

Biomedical subjects

R Kuwano

Publications and source records attributed to R Kuwano.

At least 73 records · Page 4Linked to original sources
PubMed1988

Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases.

The 14-3-3 protein is a family of acidic proteins present exclusively in the brain and is believed to have a function in monoamine biosynthesis because of its ability to activate tyrosine hydroxylase and tryptophan hydroxylase in the presence of Ca2+/calmodulin-dependent protein kinase type II. In this study, we resolved bovine brain 14-3-3 protein into seven polypeptide components by means of reversed-phase chromatography and determined the amino acid sequence of one of these components (eta chain) by cloning its cDNA from a bovine cerebellum cDNA library. The eta-chain mRNA is 1.8 kilobases long and encodes a polypeptide of 246 amino acids and Mr 28,221. Computer-assisted analysis of the sequence indicates that the eta chain exhibits no internal sequence repeats, nor does it have significant sequence similarity to other proteins with known amino acid sequence. However, the eta chain appears to consist of two structural regions that are distinguishable in their clearly different charge characteristics: the almost neutral amino-terminal region and the strongly acidic carboxyl-terminal region. The structural features of the eta chain and the domain organization of tyrosine and tryptophan hydroxylases suggest that the 14-3-3 protein binds to the regulatory domain of the phosphorylated hydroxylases through its acidic carboxyl-terminal region and activates the hydroxylases by inducing an active conformation.

14-3-3 Proteins↗
PubMed1988

Developmental and regional changes of mRNA for a cerebellar protein (spot 35) in the rat brain.

Spot 35 protein is a cerebellar Ca-binding protein. Because Southern blot analysis showed evidence for the nucleotide sequence of spot 35 protein cDNA in the rat genome, we applied cDNA to quantitate the mRNA of rat spot 35 protein. The size of this mRNA was about 1,900 nucleotides in length, and that of mRNA from bovine cerebellum was larger. We also examined the developmental changes and regional distribution of spot 35 protein mRNA in rat brains by dot-blot analysis using cDNA as a probe. During postnatal days 1-20, a rapid increase of mRNA levels was observed. Further, the level of mRNA for spot 35 protein was found in the cerebellum and was negligible in the cerebral cortex, striatum, and hippocampus.

Animals↗
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 cDNA to mRNA for a cerebellar spot 35 protein.

The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca-binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1,714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5'-noncoding region, and the 801 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full-length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca-binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca-binding protein (28K).

Amino Acid Sequence↗
PubMed1987

ID sequences in the genes of three brain-specific proteins.

We characterized the brain-specific gene coding for rat S-100 protein beta-subunit and found three "brain identifier (ID)" elements, which have been proposed to regulate the gene expression in rat brain. The nucleotide sequences of these elements corresponded well with that of the consensus ID element and were clearly different from those of "ID-like" elements in rat beta B1-crystallin gene, etc. ID elements were also observed in the flanking regions of rat neuron-specific enolase and cholecystokinin genes, which were expressed in the neuronal cells. Direct repeats were observed in the regions flanking ID elements.

Animals↗
PubMed1987

Tissue distribution of rat S-100 alpha and beta subunit mRNAs.

Using Southern blot analysis it is demonstrated that S-100 alpha and beta genes were found as single copies in the rat genome. S-100 alpha mRNA was not found at all in rat brain during the developmental period, but it was slightly detectable in muscle and kidney. S-100 beta mRNA was detected in rat brain, and its mRNA level increased during the developmental period. The different localizations of the subunits may be due to the specific expression of each gene in various tissues.

Aging↗
PubMed1986

Molecular cloning of cDNA of S100 alpha subunit mRNA.

The primary structure of the bovine S-100 alpha mRNA on the basis of molecular cloning and sequence analysis of the cDNA are described. The sequence is composed of 532 bp which include the 282 bp of the complete coding region, 89 bp at the 5'-noncoding region, 161 bp at the 3'-noncoding region, polyadenylation signal, ATTAAA and poly(A) tail. Northern blot analysis shows that the size of S-100 alpha mRNA is about 700-800 bases long and a single mRNA occurs in bovine brain. Bovine brain contains both S100 alpha and beta subunits and their mRNAs. In contrast, the rat brain contains only S100 beta subunit and its mRNA.

Animals↗
PubMed1986

Developmental and regional changes of cholecystokinin mRNA in rat brains.

Since the nucleotide sequence of cholecystokinin (CCK) cDNA was found in the rat gene, we applied cDNA to quantitate the CCK mRNA. The size of the mRNA for a CCK precursor was 850 nucleotides in length using brain cytoplasmic RNA. There were no bands except CCK mRNA by Northern blot analysis. We also examined the developmental changes and regional distribution of CCK mRNA in rat brains by dot-blot and gel-blot hybridization using CCK cDNA as a probe. CCK mRNA was barely detectable in the fetal brain, but started to increase postnatally and attained the plateau level after 20-30 days. Further, the level of CCK mRNA was highest in the frontal cortex, followed by those of the hippocampus and striatum. The cerebellum contained only negligible CCK mRNA. These results are in agreement with those of CCK concentration in the corresponding brain areas and suggest a transcriptional control of CCK concentration.

Aging↗
PubMed1984

Molecular cloning and the complete nucleotide sequence of cDNA to mRNA for S-100 protein of rat brain.

The complete nucleotide sequence of mRNA for beta-subunit of rat brain S-100 protein was determined from recombinant cDNA clones. The sequence was composed of 1488 bp which included the 276 bp of the complete coding region, the 120 bp of the 5'-noncoding region and the 1092 bp of the 3'-noncoding region containing two polyadenylation signals. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was homologous to the amino acid sequence of bovine S-100 beta subunit except 4 residues showing species differences. From the viewpoint of evolutionary implications, the homology between the nucleotide sequence of S-100 and those of rat intestinal Ca-binding protein (ICaBP) and calmodulin (CaM) was examined. A dot-blot hybridization of poly(A) RNA from the developing rat brains using a labeled cDNA showed a rapid increase in S-100 mRNA at 10-20 postnatal days. The presence of S-100 mRNA in C-6 glioma cells is also described.

Age Factors↗
PubMed1984

Binding of S-adenosylhomocysteine to hydroxyindole O-methyltransferase.

Biochim Biophys Acta 1984 May 31;787(1 ) 1-7 (S-adenosyl-L-methionine:N-acetylserotonin O-methyltransferase, EC 2.1.1.4) purified from bovine pineal gland forms a complex with S-adenosylhomocysteine, one of the products of the reaction catalyzed by the enzyme. The binding of S-adenosylhomocysteine to the enzyme has been characterized by the use of S-[8-14C]adenosylhomocysteine or S-[U -14C]adenosylhomocysteine. The complex did not dissociate during filtration through Sephadex G-25. Long-term dialysis against ligand-free phosphate buffer did bot dissociate the bound S-adenosylhomocysteine. S-Adenosylhomocysteine co-migrated with hydroxyindole O-methyltransferase on disc polyacrylamide gel electrophoresis. Although the complex was stable even under nonequilibrium conditions, the bound S- adenosylhomocysteine was separated into adenosine and homocysteine in the presence of S- adenosylhomocysteine hydrolase. The binding of S-adenosylhomocysteine was optimal at pH 7.0, but was not dependent on temperature. Scatchard plots showed that Kd was 6.5 X 10(-9) M and the maximal binding was 1 mol of S-adenosylhomocysteine per subunit of the enzyme. Hydroxyindole O-methyltransferase cannot form a stable complex with S-adenosylmethionine, and the addition of excess amounts of S-adenosylmethionine impairs the binding of S-adenosylhomocysteine to the enzyme. The product inhibition by S-adenosylhomocysteine may be based on the binding of S-adenosylhomocysteine to the enzyme with high affinity and on the stable enzyme-product complex formation during the transmethylation reaction.

Acetylserotonin O-Methyltransferase↗
PubMed1984

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor.

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.

Amino Acid Sequence↗
PubMed1983

Immunocytochemical demonstration of hydroxyindole O-methyltransferase (HIOMT), neuron-specific enolase (NSE) and S-100 protein in the bovine pineal gland.

The immunocytochemical localization for hydroxyindole O-methyltransferase (HIOMT), neuron-specific enolase (NSE) and S-100 protein in the bovine pineal gland is described in this paper. Most of the pinealocytes showed immunoreactivities to a HIOMT antiserum and an NSE antiserum, simultaneously. Neither HIOMT-nor NSE-immunoreactivity was observed in a few pinealocytes. On the other hand, dispersed interstitial cells (glial cells) were stained only by an S-100 protein antiserum. No evident HIOMT-immunoreactivity was observed in the retina and intestinal mucosa, where HIOMT has been suggested to occur.

Acetylserotonin O-Methyltransferase↗
PubMed1983

Developmental changes in the translatable mRNA for beta subunit of S-100 protein in rat brain.

The presence of mRNA coding for beta subunit of S-100 protein was demonstrated in polyadenylated RNA from the rat brain in vitro translation in a reticulocyte lysate cell-free system. The products were identified with S-100 protein beta subunit using the immunoprecipitation of the reaction products with the specific antisera, comigration of the isolated, labelled peptide with the purified S-100 protein in SDS-polyacrylamide gel electrophoresis and fluorography and the same retention time of the labelled S-100 protein beta subunit with authentic S-100 beta subunit by high performance liquid chromatography. The size determination of mRNA for S-100 protein on sucrose density gradient centrifugation gave 6-8 S. The assay gave a linear response with increasing amounts of polyadenylated RNA, allowing quantitation of mRNA level for S-100 protein in polyadenylated RNA. During the prenatal period and 10 postnatal days, only minute amounts of mRNA for beta subunit of S-100 protein could be found, however a dramatic increase of mRNA for beta subunit of S-100 was observed within the period of 10 to about 30 days and the mRNA level maintained a plateau from 40 days to adult age. These date indicate that the development changes in the amount of S-100 protein in the rat brain found by other authors is strongly correlated with the changes in the level of its translatable mRNA.

Aging↗