Biomedical subjects

Y Okano

Publications and source records attributed to Y Okano.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of a Gly19-->Val mutant of ram p25, a low Mr GTP-binding protein: loss of GTP/GDP-binding activity in the mutated ram p25.

A substitution of Gly for Val at position 19, which corresponds to oncogenic Gly13-->Val mutation of ras p21, was introduced in a low Mr GTP-binding protein, ram p25. The protein was expressed in cytosolic fraction of Escherichia coli and purified by using specific antibody raised against ram p25. The mutated protein had no guanine nucleotide-binding activity although [Val13]ras p21 was reported to have. The analysis of guanine nucleotide composition of the purified [Val19]ram p25 revealed that the protein was free of nucleotide whereas the normal ram p25 bound about 1 mol of GDP per mol of protein. These results strongly suggested that some part(s) of variable regions as well as the consensus regions are important for the biochemical properties of ram p25.

Amino Acid Sequence↗

PubMed1992

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of a low M(r) GTP-binding protein, ram p25, expressed by baculovirus expression system.

The ram gene was isolated from rat megakaryocyte cDNA library with an oligonucleotide probe which is specific for a low M(r) GTP-binding proteins c25KG purified from human platelets. Its gene product (ram p25) is a monomeric 25-kDa guanine nucleotide-binding protein. The protein was expressed by using baculovirus transfer vector, pAcYM1, which allowed the production at a high level of soluble recombinant ram p25 in Spodoptera frugiperda (Sf9) cells under the control of polyhedrin promoter. The expressed protein in cytosol of Sf9 cells was purified to near homogeneity by a combination of DEAE-Toyopearl 650(S) and hydroxyapatite HCA-100S column chromatography. The purified ram p25 bound approx. 0.8 +/- 0.02 mol of guanosine 5'-O-1-thiotriphosphate (GTP gamma S)/mol of protein with a Kd value of 340 +/- 4.91 nM in a reaction mixture containing 10 microM of free magnesium ions. In the presence of 5 mM Mg2+, [3H]GDP was dissociated from ram p25 at the rate of 0.015 +/- 0.0010 min-1 and the dissociation was greatly enhanced by addition of 250 mM (NH4)2SO4. The rate of [gamma-32P]GTP-hydrolysis for ram p25 was 0.010 +/- 0.0012 min-1. Thus, it was indicated that the GTP-hydrolysis reaction is a rate-limiting step in the guanine nucleotide turnover of ram p25. ram p25 shares 23 and 80% amino-acid homology with the Ha-ras p21 and c25KG protein, respectively, and is similar to them in GTP gamma S binding activity in a time- and dose-dependent manner. But it differs from ras p21 in the rate-limiting step of the guanine nucleotide turnover.

Baculoviridae↗

PubMed1992

Mass contents of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol in human platelets stimulated with a thromboxane analogue and thrombin.

Mass contents of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) were measured in U46619-stimulated human platelets. 1 microM of U46619 induced maximum responses in aggregation, 5-hydroxytryptamine (5HT) secretion and increase in intracellular free Ca2+ concentration ([Ca2+]i). Aggregation was almost comparable to that induced by maximal dose (1 U/ml) of thrombin, while 5HT release was almost half. The initial [Ca2+]i peak in response to U46619 was about half of thrombin stimulation. Production of IP3 and DG was, however, less than one tenth of that seen in thrombin stimulation. The profile (time course and concentration-dependency) of IP3 formation did not correlate with that of [Ca2+]i, suggesting that U46619 stimulates IP3-dependent and -independent Ca2+ mobilization. DG production was small but sustained for more than 5 min. These findings support the recent hypothesis that aggregation is regulated by a delayed accumulation of DG. The low level of 5HT secretion could be explained by the low production of second messengers, IP3 and DG.

Blood Platelets↗

PubMed1992

Abolishment of bradykinin-induced calcium oscillations in ras-transformed fibroblasts by the expression of 80 kDa diacylglycerol kinase.

Our previous study showed bradykinin-induced periodic Ca2+ changes (Ca2+ oscillations) in v-Ki-ras-transformed NIH/3T3 (DT) cells in which protein kinase C (PKC) activity is partially down-regulated by a sustained high level of 1,2-diacylglycerol (DAG) [FEBS Lett. (1991) 281, 263-266]. In the present study, DAG kinase with 80 kDa mass (80K DGK) has been successfully transfected in DT cells, which exhibited enhanced cellular DAG kinase activities, decreased cellular DAG contents, and increased PKC activities compared to the control vector-transfected cells. Furthermore, these DGK-transfectants showed strong inhibition in bradykinin-induced Ca2+ oscillations. The results suggest that the sustained DAG increase down-regulates the PKC activity, thereby leading to the induction of Ca2+ oscillations in DT cells.

3T3 Cells↗

PubMed1992

Novel human autoantibodies reactive with 5'-terminal trimethylguanosine cap structures of U small nuclear RNA.

A class of RNA-containing particles, U small nuclear/nucleolar ribonucleoprotein particles (U snRNP), are well known to be targets for sera from patients with various autoimmune diseases. In the most cases the protein components carry the antigenic determinants. We have identified serum autoantibodies from three patients with systemic sclerosis that were directed against U1-U5 snRNA by immunoprecipitation of deproteinized 32PO4 labeled HeLa cell total RNA. By competitive radioimmunoprecipitation assays, an experimentally induced anti-2,2,7-trimethylguanosine (TMG) cap structure mAb inhibited the reaction of these antisera. In addition, IgG isolated from the antisera inhibited the anti-TMG mAb reaction to the U snRNA. Furthermore, a structural analog, 7-methylguanosine-triphosphate, competitively inhibited the reaction of the antisera to the U snRNA. Thus we concluded that the TMG cap structure of the U snRNA could be a target for serum autoantibodies.

Autoantibodies↗

PubMed1992

Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown.

Bradykinin (BK) induced a biphasic increase in 1,2-diacylglycerol (DAG) in both K-ras-transformed fibroblasts (DT) and the parent NIH-3T3 cells. The first phase was coincident with the increase in Ins(1,4,5)P3 resulting from PtdIns(4,5)P2 hydrolysis, and the second, sustained, phase was derived from phosphatidylcholine (PtdCho) hydrolysis. In NIH-3T3 cells, stimulation by BK induced greater production of choline than phosphocholine in [3H]choline-labelled cells and appreciable phosphatidylethanol (PtdEtOH) formation in [3H]myristic acid-labelled cells, suggesting that PtdCho was hydrolysed mainly by a phospholipase D (PLD) activity. Pretreatment with propranolol, an inhibitor of phosphatidate phosphohydrolase, markedly diminished the second DAG accumulation, supporting the above notion. In DT cells, BK induced predominantly phosphocholine generation and little PtdEtOH formation, indicating that the PtdCho hydrolysis was due to a phospholipase C (PLC) activity. The BK-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) observed in single DT cells [Fu, Sugimoto, Oki, Murakami, Okano & Nozawa (1991) FEBS Lett. 281, 263-266] were detected as a sustained [Ca2+]i elevation when assayed in a cell suspension. A receptor-operated Ca2+ channel blocker, SK&F 96365, suppressed both the BK-induced phosphocholine generation and the sustained [Ca2+]i elevation in a similar dose-dependent manner. These results thus suggested that oscillations in [Ca2+]i are involved in the activation of PtdCho-specific PLC in DT cells.

3T3 Cells↗

PubMed1992

Autoantibody to U3 nucleolar ribonucleoprotein (fibrillarin) in patients with systemic sclerosis.

To determine the clinical significance of serum antibodies to the U3 small nuclear ribonucleoprotein particle ([U3]snRNP), we studied sera from 416 patients with systemic sclerosis (SSc) and 264 controls, using immunofluorescence and immunoprecipitation assays. The presence of serum anti-(U3)snRNP was highly specific to SSc, was found more frequently in blacks, and was associated with skeletal muscle disease and primary pulmonary arterial hypertension. These antibodies may identify one or more unusual clinical subsets of SSc.

Adult↗

PubMed1992

Serum autoantibody to the nucleolar antigen PM-Scl. Clinical and immunogenetic associations.

OBJECTIVE: The inflammatory myopathies are characterized by distinctive autoantibodies that are associated with certain clinical features and immunogenetic patterns. Anti-PM-Scl is one such antibody and is found in pure myositis, myositis in overlap, and systemic sclerosis (SSc). Our purpose was to describe the clinical and immunogenetic associations of the anti-PM-Scl antibody. METHODS: Serum samples from 617 patients with various connective tissue diseases were screened for anti-PM-Scl antibody by indirect immunofluorescence and Ouchterlony double immunodiffusion. Patients with anti-PM-Scl were serologically typed for HLA-DR and DQ, and the genes encoding DQ alpha and DQ beta were characterized by hybridization of sequence-specific oligonucleotide to amplified genomic DNA. RESULTS: Twenty-three patients (4%) had serum anti-PM-Scl. Sixteen had either pure myositis or myositis in overlap, 6 had SSc alone, and 1 had SSc and rheumatoid arthritis. Twenty of the antibody-positive patients had serologic HLA typing performed; 15 (75%) were HLA-DR3 positive, and 17 (85%) expressed the DQw2 allele. None of the 5 DR3 negative patients shared a unique DR or DQ antigen with the DR3 positive patients, and further DNA analysis of 10 patients (4 of whom were DR3 negative) did not reveal any unique DQ alleles. CONCLUSION: Anti-PM-Scl identifies a subset of patients with myositis, SSc, or an overlap of the two disorders, and this antibody has a strong but not exclusive immunogenetic association with the HLA-DR3 antigen.

Adolescent↗

PubMed1992

Frequency and distribution of phenylketonuric mutations in Orientals.

The frequency and distribution of eight mutations (R111X, IVS4nt-1, Y204C, R243Q, IVS7nt-2, W326X, Y356X, and R413P) in the phenylalanine hydroxylase gene of Orientals in Japan and Korea were examined by allele-specific oligonucleotide hybridization. The mutant alleles comprised 54 and 55% of the phenylketonuria (PKU) chromosomes examined in 36 patients in Japan and 10 patients in Korea, respectively. The spectrum of PKU mutations in Japan was similar to that in China, particularly in northern China, but different from that in Korea. The IVS4nt-1 mutation had a high frequency in Korea and southern China, due to the result of the founder effect and genetic drift. The R413P mutation, which may have originated in the regions surrounding the Baikal, expanded to northern China and Japan. We did not find Caucasian mutations in the Japanese or Korean PKU chromosomes. Thus, PKU mutations occurred after racial divergence between Caucasians and Mongoloids, and there were different founding populations for PKU in the two populations.

Alleles↗

PubMed1992

Presence and some characterization of GDP dissociation inhibitors for a low Mr GTP-binding protein, ram p25, in rat spleen cytosol.

Two proteinous factors, designated here as ram p25 GDP dissociation inhibitor (I) and (II) (ram-GDI (I) and (II)), were detected in the cytosolic fraction of rat spleen, which inhibited the initial dissociation of GDP from ram p25 produced by E. coli by causing characteristic lag. They had very weak effects on the rate of dissociation of GDP after the lag, and did not affect the mode of the dissociation of 5'-(3-O-thio)triphosphate (GTP gamma S) from ram p25. By gel filtration, the molecular masses of ram-GDI (I) and (II) were calculated to be 90 KDa and 40 KDa, respectively. These ram-GDIs did not affect the GDP-dissociation of Ha-ras protein produced in E. coli.

Animals↗

PubMed1992

Different protein kinase C isozymes could modulate bradykinin-induced extracellular calcium-dependent and -independent increases in osteoblast-like MC3T3-E1 cells.

Effects of protein kinase C (PKC) on bradykinin (BK)-induced intracellular calcium mobilization, consisting of rapid Ca2+ release from internal stores and a subsequent sustained Ca2+ inflow, were examined in Fura-2-loaded osteoblast-like MC3T3-E1 cells. The sustained Ca2+ inflow as inferred with Mn2+ quench method was blocked by Ni2+ and a receptor-operated Ca2+ channel blocker SK&F 96365, but not by nifedipine. The short-term pretreatment with phorbol 12-myristate 13-acetate (PMA), inhibited BK-stimulated Ca2+ inflow, and the prior treatment with PKC inhibitors, H-7 or staurosporine, enhanced the initial internal release and reversed the PMA effect. Moreover, 6 h pretreatment with PMA caused similar effect on the BK-induced inflow to that obtained with PKC inhibitors, whereas 24 h pretreatment was necessary to affect the internal release. On the other hand, the translocation and down-regulation of PKC isozymes were examined after PMA treatment of MC3T3-E1 cells by immunoblot analyses of PKCs with the isozyme-specific antibodies. 6 h treatment with PMA induced down-regulation of PKC beta, whereas longer treatment was needed for down-regulation of PKC alpha. Taken together, it was suggested that the BK-induced initial Ca2+ peak and the sustained Ca2+ inflow through the activation of a receptor-operated Ca2+ channel, are differentially regulated by PKC isozymes alpha and beta, respectively, in osteoblast-like MC3T3-E1 cells.

Animals↗

PubMed1992

Evidence of the presence of GTPase inhibiting proteins for a low M(r) GTP-binding protein, ram p25, in rat spleen and pheochromocytoma PC12 cells.

1. GTPase inhibiting activity for a low M(r) GTP-binding protein, ram p25, was detected in the cytosolic fractions of rat spleen and PC12 cells. 2. The inhibitors were heat-labile and trypsin-sensitive, indicating that they were of proteinous nature. 3. The molecular mass of the inhibitor in the spleen appeared to be about 65 kDa on the elution profile from the gel filtration column.

Adenosine Triphosphate↗

PubMed1992

Identification of three novel PKU mutations among Chinese: evidence for recombination or recurrent mutation at the PAH locus.

Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.

Asian People↗

PubMed1992

Effects of several growth factors on cultured neurofibroma cells.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by abnormalities affecting multiple tissues derived from the neural crest. The peripheral neurofibromas are numerous and sometimes reach several hundred in number. In this study, the possible involvement of several growth factors in neurofibroma growth was investigated in vitro. When explants of neurofibroma tissue were cultured, macrophage-like cells with pseudopodia migrated out first, and later took on a slender fusiform shape. These cells contained S-100 protein and were identified as Schwann cells. They did not proliferate under standard culture conditions. Nerve growth factor (NGF) was helpful in maintaining the differentiated phenotype of Schwann cells, but did not stimulate their proliferation. Immunohistochemical staining for type IV collagen revealed that some large flattened polygonal cells had a mesh of type IV collagen on the surface. These cells were perineurial cells. The proliferation of cells derived from neurofibroma was stimulated by basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-alpha). In comparison with skin fibroblasts, the cells derived from neurofibroma responded to these growth factors at considerably lower concentrations. Stimulation by EGF at physiological concentrations indicated the possible involvement of EGF in the development of neurofibromas.

Cell Division↗

PubMed1992

DNA synthesis and intracellular calcium elevation in porcine cerebral arterial smooth muscle cells by cerebrospinal fluid from patients with subarachnoid haemorrhage.

To understand the molecular mechanism of the pathogenesis of cerebral vasospasm following subarachnoid haemorrhage, we analysed the effect of cerebrospinal fluid from patients with subarachnoid haemorrhage on DNA synthesis and cytosolic-free calcium elevation in cultured porcine cerebral smooth muscle cells. Cerebrospinal fluid from patients on day 2 after subarachnoid haemorrhage induced transient elevation in cytosolic-free calcium levels. In contrast, the maximal elevation of cytosolic-free calcium levels induced by cerebrospinal fluid from control patients (without subarachnoid haemorrhage) was significantly lower than that induced by cerebrospinal fluid from patients with subarachnoid haemorrhage. In cultured porcine cerebral arterial smooth muscle cells, cerebrospinal fluid from patients with subarachnoid haemorrhage promoted levels of [3H]-thymidine incorporation (DNA synthesis) more than 2.5-fold higher than that promoted by cerebrospinal fluid from control patients without subarachnoid haemorrhage. However, in cultured aortic smooth muscle cells, there was no significant difference in [3H]-thymidine incorporation between cerebrospinal fluid from patients with subarachnoid haemorrhage and that by control cerebrospinal fluid. From these results in cerebral arterial smooth muscle cells, cerebrospinal fluid from patients following subarachnoid haemorrhage may play not only constrictive functions, evidenced by cytosolic-free calcium elevations, but also proliferative functions, demonstrated by promotion of [3H]-thymidine incorporation. The relevance of these factors to vasospasm will be discussed.

Animals↗

PubMed1992

Activation of phospholipase D by platelet-derived growth factor (PDGF) in rat C6 glioma cells: possible role in mitogenic signal transduction.

The effects of platelet-derived growth factor (PDGF) on phospholipase D (PLD) activity and deoxyribonucleic acid (DNA) synthesis in rat C6 glioma cells have been investigated. Pretreatment of serum-starved C6 cells with PDGF results in enhanced choline production and the phosphatidylethanol (PEt) formation in the presence of ethanol, indicating the activation of PLD acting on phosphatidylcholine (PC). The dose-response curve for choline generation and DNA synthesis were comparable. In addition, the effects of PDGF on both PEt formation and [3H]thymidine incorporation into acid-precipitable material was blocked by the potent protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) but not by N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA1004), a relatively weak inhibitor of PKC, suggesting that PDGF plays an important role as a positive regulator of glioma cell growth via a PLD-mediated mitogenic signal transduction cascades, which depends largely on the activation of PKC.

Animals↗

PubMed1992

Occurrence and biochemical characterization of GTP-binding proteins in Candida albicans.

The activities of GTP-binding and GTPase in Candida albicans were present in the cytosol, KCl- and cholate-extractable fractions. At least two kinds of GTP-binding proteins were found in the cytosolic fraction; the major one with a molecular mass of about 30 kDa and the other about 500 kDa. The former specifically bound guanine nucleotides and was most likely to bind GDP since guanosine 5'-O-(thio) triphosphate (GTP gamma S)-binding was accelerated by addition of (NH4)2SO4. The latter showed no specificity in nucleotide binding and could also bind adenine nucleotides. The proteins were not ADP-ribosylated by either pertussis toxin or cholera toxin. These results indicate that ras-like monomeric, low molecular mass GTP-binding proteins distinct from heterotrimeric G proteins such as Gi, Go and Gs are present in C. albicans.

Adenosine Diphosphate↗