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Biomedical subjects

Y Okano

Publications and source records attributed to Y Okano.

At least 199 records · Page 11Linked to original sources

Calcium oscillation associated with reduced protein kinase C activities in ras-transformed NIH3T3 cells.

We show here novel intracellular Ca2+ oscillation in v-K-ras-transformed NIH3T3 cells induced by mitogenic peptide hormones, bradykinin and bombesin, as well as fetal calf serum. Induction of the Ca2+ oscillation is strongly correlated with the malignant properties and inversely with PKC activities in vitro and in vivo. These results suggest that the mitogen-induced Ca2+ oscillation is negatively regulated by PKC, which modulates Ca2+ influx in v-K-ras-transformed NIH3T3 cells.

Animals↗

Founder effect of a prevalent phenylketonuria mutation in the Oriental population.

A missense mutation has been identified in the human phenylalanine hydroxylase [PAH; phenylalanine 4-monooxygenase; L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] gene in a Chinese patient with classic phenylketonuria (PKU). A G-to-C transition at the second base of codon 413 in exon 12 of the gene results in the substitution of Pro413 for Arg413 in the mutant protein. This mutation (R413P) results in negligible enzymatic activity when expressed in heterologous mammalian cells and is compatible with a classic PKU phenotype in the patient. Population genetic studies reveal that this mutation is tightly linked to restriction fragment length polymorphism haplotype 4, which is the predominant haplotype of the PAH locus in the Oriental population. It accounts for 13.8% of northern Chinese and 27% of Japanese PKU alleles, but it is rare in southern Chinese (2.2%) and is absent in the Caucasian population. The data demonstrate unambiguously that the mutation occurred after racial divergence of Orientals and Caucasians and suggest that the allele has spread throughout the Orient by a founder effect. Previous protein polymorphism studies in eastern Asia have led to the hypothesis that "northern Mongoloids" represented a founding population in Asia. Our results are compatible with this hypothesis in that the PKU mutation might have occurred in northern Mongoloids and subsequently spread to the Chinese and Japanese populations.

Asian People↗

Diacylglycerol formation and DNA synthesis in endothelin-stimulated rat C6 glioma cells: the possible role of phosphatidylcholine breakdown.

Stimulation of C6 cells with endothelin-1 (ET) caused a biphasic sn-1,2-diacylglycerol (1,2-DG) generation, which occurred not only via phosphatidylinositol 4,5-P2 (PIP2) hydrolysis by specific phospholipase C action, but also through the breakdown of phosphatidylcholine (PC) induced by either PC phospholipase C or D. ET also stimulated DNA synthesis in serum-starved C6 cells. However, in the presence of 1-(5-isoquinolynylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C (PKC) inhibitor, the [3H]thymidine incorporation was markedly inhibited, which was concurrent with the 1,2-DG formation: the second peak was reduced. These findings suggest that brain glial cells might be an important target for ET. In addition to other possible roles, ET may also mediate mitogenesis in the brain, presumably through a PKC-dependent activation of phospholipase C and/or D hydrolyzing PC.

Animals↗

Newly identified U4/U6 snRNP-binding proteins by serum autoantibodies from a patient with systemic sclerosis.

We found serum autoantibodies directed against the proteins binding exclusively to U4/U6 of Sm small nuclear ribonucleoprotein particle (snRNP) in serum from a patient (MaS) with systemic sclerosis. Their specificity, called anti-MaS, is distinct from that of known antibodies against U snRNP. The U4 and U6 small nuclear RNA from a 32P-labeled HeLa cell extract and five proteins with Mr 150,000, 120,000, 80,000, 36,000, and 34,000, in addition to Sm core proteins (B, B', D, E, F, and G) from an [35S] methionine-labeled extract, were immunoprecipitated by anti-MaS in isotonic solution. However, the Sm core proteins and U4 and U6 small nuclear RNA were separated from the protein-A-Sepharose facilitated MaS immunoprecipitate by incubation in a solution containing 500 mM NaCl. In immunoblots, anti-MaS antibodies reacted with one protein of Mr 150,000 from a HeLa cell nuclear extract that was fractionated by SDS-PAGE and transferred to a nitrocellulose sheet. The monospecific immunoaffinity purified antibody eluted from the immunoblot band immunoprecipitated U4 and U6 small nuclear RNA and reblotted the protein with Mr 150,000. These data indicate that anti-MaS antibodies recognize at least one antigenic protein that binds exclusively to the U4/U6 snRNP.

Adult↗

A prevalent missense mutation in Northern Europe associated with hyperphenylalaninaemia.

A missense mutation has been identified in the phenylalanine hydroxylase (PAH) gene of a Danish patient with hyperphenylalaninaemia (HPA). An A-to-G transition at the second base of codon 414 results in the substitution of Cys for Tyr in the mutant PAH protein. In in vitro expression studies, the Tyr414-to-Cys414 mutant construct produced a protein which exhibited a significant amount of normal PAH enzyme activity, which is consistent with both in vitro and in vivo measurements of PAH activity in HPA patients. Population genetic studies reveal that this mutation is present on 50% of mutant haplotype 4 chromosomes in the Danish population. Together with the previously reported codon 158 mutation, these two mutant alleles comprise over 90% of all mutant haplotype 4 chromosomes in the Northern European population. Thus, two allele-specific oligonucleotide probes can detect most mutant haplotype 4 chromosomes in Northern Europe.

Alleles↗

Species-specific antibodies of Tetrahymena acid alpha-glucosidase.

1. Tetrahymena acid alpha-glucosidases A and B were purified from T. pyriformis W and T. thermophila 399, respectively. The acid alpha-glucosidases A and B were different in immunological properties and thermostability. 2. The acid alpha-glucosidases A and B reflected specific distribution between T. pyriformis and T. thermophila. 3. Type A and B of taurolipid showed a species-specific distribution pattern between T. pyriformis and T. thermophila.

Animals↗

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.

Base Sequence↗

Missense mutations prevalent in Orientals with phenylketonuria: molecular characterization and clinical implications.

Two missense mutations in the phenylalanine hydroxylase (PAH) genes of Orientals with phenylketonuria (PKU) have been identified. A G-to-A transition in exon 7 of the gene results in the substitution of Gln243 for Arg243 (R243Q) and accounts for 18% of all PKU chromosomes among Chinese. An A-to-G transition in exon 6 of the gene results in the substitution of Cys204 for Tyr204 (Y204C) and identifies about 13 and 5% of all PKU chromosomes in the Chinese and Japanese populations, respectively. The R243Q construct produced less than 10% of normal PAH activity in in vitro expression analysis in a eukaryotic cell system, and patients homozygous for this substitution exhibit a severe clinical phenotype. These results are consistent with previous findings in this expression system. The Y204C construct, however, produced near normal levels of PAH enzyme activity and immunoreactivity in this in vitro expression system. Because this substitution is present only on PKU chromosomes, it is a valuable marker for identifying the corresponding mutant allele for carrier screening of PKU. With the characterization of these two substitutions, about 60% of PKU alleles in China can now be identified. The continuing search for additional PKU mutations will permit effective carrier screening and prenatal gene diagnosis of PKU in East Asia.

Alleles↗

Intracellular Ca2+ changes in cultured vascular smooth muscle cells by treatment with various spasmogens.

In order to evaluate various spasmogens, which are candidates for cerebral vasospasm after subarachnoid haemorrhage, the intracellular calcium mobilizations were examined in cultured vascular smooth muscle cells preloaded with a fluorescent Ca2+ probe fura-2. Endothelin, oxyhaemoglobin, 5-hydroxytryptamine, norepinephrine, prostaglandin F2 alpha, leukotrienes C4 and D4 produced dose-dependent increases in intracellular Ca2+ concentration ([Ca2+]i). However, bilirubin did not induce any significant [Ca2+]i elevation. The maximal levels of [Ca2+]i peak attained by endothelin or oxyhaemoglobin were higher than those of other compounds. Endothelin was the most potent in that it induced a high sustained [Ca2+]i elevation at much lower concentrations compared with others. The combination of oxyhaemoglobin and endothelin induced a transient increase in [Ca2+]i followed by a sustained lower plateau, then the [Ca2+]i level was again increased slowly followed by a sustained higher plateau which lasted for more than 10 min after the exposure. These results suggest that endothelin and/or oxyhaemoglobin may play a crucial role in contraction of vascular smooth muscle after subarachnoid haemorrhage.

Animals↗

Cytosolic calcium changes in cultured rat aortic smooth-muscle cells induced by oxyhemoglobin.

To clarify the mechanism of contractive effects in arteries caused by oxyhemoglobin, changes in the concentration of cytosolic calcium [( Ca++]i) before and after exposure to oxyhemoglobin were measured in vitro in cultured vascular smooth-muscle cells obtained from rat aorta. This was accomplished by preloading these cells with a fluorescent intracellular Ca++ probe fura-2/AM. Oxyhemoglobin induced a significant elevation of [Ca++]i in vascular smooth-muscle cells which was sustained for 10 minutes. This response was completely abolished by chelating extracellular calcium with ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetra-acetic acid (EGTA). Oxyhemoglobin induced no accumulation of mass content of inositol 1,4,5-trisphosphate (IP3(1,4,5]. The oxyhemoglobin-induced elevation of [Ca++]i was not blocked by verapamil, a calcium antagonist. Serotonin induced a rapid, transient increase of [Ca++]i followed by a sustained elevation above baseline for 5 minutes. Additions of EGTA or verapamil had a small effect on the peak height of serotonin-induced [Ca++]i elevation, but the [Ca++]i level declined more quickly to the basal level in treated compared with control cells. These results indicate that oxyhemoglobin-induced [Ca++]i elevation is caused by the influx of extracellular calcium, which is independent of the verapamil-blocked voltage-gated calcium channel. The long-lasting high elevation of [Ca++]i caused by oxyhemoglobin suggests that oxyhemoglobin may contribute to the production of abnormal contractions and/or irreversible damage in vascular smooth-muscle cells.

Animals↗

[Assessment of therapeutic effects of cisplatin-phosphatidyl-choline-lipiodol (CPL) suspension for hepatocellular carcinoma].

Sixty-three patients with unresectable hepatocellular carcinoma (HCC) were treated with cisplatin-phosphatidyl-choline-Lipiodol (CPL) suspension. Partial response (PR) and minor response (MR) were obtained in 3 of 14 cases (21.4%) by one shot therapy, and in 13 of 43 cases (30.2%) by TAE therapy. AFP decreased in 11 of 15 patients (73.3%) by one shot therapy, and in 32 of 33 patients (97%) by TAE therapy. PIVKA II also decreased. The one-year survival rate was 74% in TAE therapy, and 52% in one shot therapy. The two-year survival rate was 53% in TAE therapy, and 28% in one shot therapy. Nausea, vomiting and fever were noted in most cases as adverse effects, but they were slight. The concentration of free-CDDP in the peripheral venous blood was lower and continued longer than that of CDDP on the market. These results suggest that CPL was useful as an anticancer agent for arterial chemotherapy or TAE therapy for unresectable HCC.

Carcinoma, Hepatocellular↗

Identification of a novel phenylketonuria (PKU) mutation in the Chinese: further evidence for multiple origins of PKU in Asia.

A novel mutation has been identified in the human phenylalanine hydroxylase (PAH) gene of a Chinese patient with classical phenylketonuria (PKU). It is a single base transition of G to A at the last base in intron 4 of the gene, which abolishes the 3'-acceptor site of the intron. Population screening indicates that this mutation constitutes about 8% of all PKU chromosomes in Chinese but is absent in Japanese and Caucasian PKU patients. It is prevalent in southern China but rare in northern China, providing additional evidence that there were multiple founding populations of PKU in east Asia.

Alleles↗

The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library.

A novel low Mr GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000-17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras-related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H-ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells.

Amino Acid Sequence↗

Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.

Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or EGTA (1 mM), the peak [Ca2+]i declined more rapidly to the resting level in VIC-stimulated NG108-15 cells, indicating that the receptor-mediated intracellular Ca2+ mobilization is followed by Ca2+ influx through the nifedipine-sensitive Ca2+ channel. Pretreatment with pertussis toxin only partially decreased Ins(1,4,5)P3 generation as well as the [Ca2+]i transient induced by VIC, whereas these events induced by endothelin-1 were not affected by the toxin, suggesting involvement of distinct GTP-binding proteins. The VIC-induced transient Ins(1,4,5)P3 formation coincident with the first early peak of DAG formation suggested that PtdIns(4,5)P2 is a principal source of the first DAG increase. Labelling studies with [3H]myristate, [14C]palmitate and [3H]choline indicated that in neuroblastoma cells phosphatidylcholine (PtdCho) was hydrolysed by a phospholipase C to cause the second sustained DAG increase. Down-regulation of protein kinase C (PKC) by prolonged pretreatment with phorbol ester markedly prevented the VIC-induced delayed DAG accumulation. Furthermore, chelation of intracellular CA2+ completely abolished the second sustained phase of DAG production. These findings suggest that PtdCho hydrolysis is responsible for the sustained production of DAG and is dependent on both Ca2+ and PKC.

Animals↗

Enhancement of GTP gamma S-binding activity by cAMP-dependent phosphorylation of a filamin-like 250 kDa membrane protein in human platelets.

The treatment of human platelets with the dibutyryl cyclic AMP (dbcAMP) revealed the presence of a 250 kDa protein which enhanced its GTP-binding activity. This protein was purified from platelet membranes by successive chromatographies on DEAE-cellulose, Ultrogel AcA34, Mono Q, HCA-hydroxyapatite, and TSK-3000SW columns. The positive cross-reaction of the 250 kDa protein with the anti-filamin antibody indicated that this protein is filamin or very close to it. The GTP gamma S-binding activity of this protein, when phosphorylated with cyclic AMP-dependent protein kinase (A-kinase), showed an over tenfold increase, with the specific activity being 3.6 nmol/mg protein. Dephosphorylation of the phosphorylated protein with alkaline phosphatase reduced the GTP gamma S-binding activity to the control untreated level.

Blood Platelets↗

Effects of medial preoptic area lesions on sleep and wakefulness in unrestrained rats.

Bilateral radiofrequency lesions of the medial preoptic area (MPOA) in unrestrained male rats resulted in a significant decrease in slow wave sleep (SWS) in the light period throughout two postoperative weeks, although the night-active pattern of circadian rhythms was little affected. Both diurnal and nocturnal paradoxical sleep (PS) gradually increased after the lesions. Within one week, however, the daily amount of total sleep (SWS + PS) was recovered to the normal level, since the loss of diurnal SWS was compensated by an increase in nocturnal sleep at the expense of wakefulness. The MPOA lesions brought about a transient elevation of brain temperature, which lasted only for the diurnal period of the day of lesioning. It is speculated that the MPOA plays a definite role in the passage of sleep-regulatory information, especially concerning the circadian distribution of sleep.

Animals↗