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Biomedical subjects

A Kikuchi

Publications and source records attributed to A Kikuchi.

At least 379 records · Page 21Linked to original sources
PubMed1989

Small molecular weight GTP-binding proteins in human platelet membranes. Purification and characterization of a novel GTP-binding protein with a molecular weight of 22,000.

When guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding activity was assayed in the particulate and cytosol fractions of human platelets, most activity was found in the particulate fraction. GTP-binding proteins (G proteins) were extracted from the particulate fraction by sodium cholate and purified by several column chromatographies. At least three G proteins with Mr values of about 21,000, 22,000, and 24,000 (21K G, 22K G, and 24K G, respectively) were separated in addition to the stimulatory (Gs) and inhibitory (Gi) regulatory GTP-binding proteins of adenylate cyclase. Among them, the amount of 22K G was more than 10-fold of those of other G proteins. 22K G was purified to near homogeneity and characterized. 22K G specifically bound GTP gamma S, GTP, and GDP, with a Kd value for GTP gamma S of about 50 nM. [35S]GTP gamma S binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. 22K G hydrolyzed GTP to liberate Pi, with a turnover number of 0.01 min-1. 22K G was not copurified with the beta gamma subunits of Gs and Gi and was not recognized by the antibodies against the ADP-ribosylation factor for Gs and the ras protein. The peptide map of 22K G was different from those of the smg-25A and rho proteins, which we have purified from bovine brain membranes. 21K G was identified to be the c-ras protein, but 24K G was unidentified. These results indicate that there are multiple G proteins in platelet membranes and that a novel G protein (22K G) is a major G protein in platelets.

Blood Platelets↗
PubMed1989

Changes of subcellular localization of Thy-1 antigen during thymic myoid cell differentiation.

Using a quantitative enzyme immunoassay, Thy-1 antigen expressed by a rat myoid cell line R615B2 was detected mainly on the cell surface at a single cell stage, whereas at the stage of forming myotubes, Thy-1 was found predominantly in the cytoplasm. The muscle specific creatine kinase activity also increased in association with the shift of Thy-1 from the cell surface to the cytoplasm, suggesting biological significance of Thy-1 redistribution in muscle differentiation from single cells to multinucleated cells.

Animals↗
PubMed1989

Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain.

We have purified to near homogeneity and characterized three small molecular weight (Mr) GTP-binding proteins, the c-Ki-ras protein (c-Ki-ras p21), the rho protein (rho p20) and a novel smg-25A protein (smg p25A), from bovine brain crude membranes. In the present studies, the intrasynaptosomal distribution of these 3 small Mr G protein has been investigated using bovine brain. ras p21 and rho p20 are found in the synaptosomal membrane fraction but not in the synaptosomal soluble fraction. In contrast, smg p25A is found in both the synaptosomal membrane and soluble fractions. These results indicate that the intrasynaptosomal distribution of these small Mr G proteins is different and suggest that they are involved in different neuronal functions.

Animals↗
PubMed1989

Chronic sinusitis and woodworking as risk factors for cancer of the maxillary sinus in northeast Japan.

In the period 1983 to 1985, 66 patients presented to six Japanese university hospitals with squamous cell carcinoma of the maxillary sinus. Using self-administered questionnaires, a case-control study was conducted to examine history of nasal diseases, occupational exposures, and other possible risk factors for this disease. For each patient, two controls were selected from the general population, matched to the patient by sex, age (+/- 5 years), and district of residence. A history of chronic sinusitis was associated with a 2.3-fold increase in risk (p = 0.05). A high relative risk was also observed in males with an occupational history of woodworking or joinery, particularly when these jobs involved sanding or lathing practices (RR = 7.5, p = 0.02). No association between cigarette smoking and maxillary sinus cancer was observed in this study and no evidence was found that indoor air pollution in the home is involved in cancer development.

Adult↗
PubMed1989

[ras p21-like small MW GTP-binding proteins in transmembrane signaling].

We have separated multiple GTP-binding proteins (G proteins) having Mr values of about 20,000 (small MW G proteins) from bovine brain membranes, purified to near homogeneity and characterized two novel G proteins designated as smg p25A and smg p21, c-Ki-ras p21 and rho p20. smg p25A is specifically found in neural tissue and adrenal medulla. This G protein is also found in rat pheochromocytoma PC-12 cells, and its mRNA level increases after differentiation of the cells into neuron-like cells in response to nerve growth factor or dibutyryl cyclic AMP. These results suggest that smg p25A plays an important role in the regulation of neural functions. In contrast, smg p21 is found in most tissues. This G protein has the same putative effector domain as ras p21s, suggesting that smg p21 exerts actions similar and/or antagonistic to those of ras p21s. In fact, smg p21 has been found to be identical with the ras-suppressor gene, designated as Krev-1, recently isolated by Noda's group. On the other hand, rho p20 is ADP-ribosylated by botulinum toxin. This toxin, known to be a neurotoxin, has recently been shown to induce the morphological changes similar to those induced by ras p21 in fibroblasts. Thus, ras p21-like small Mr G proteins are part of a huge network of intracellular regulatory systems and play important roles in the regulation of various cell functions including cell transformation, proliferation and differentiation.

Amino Acid Sequence↗
PubMed1989

[Internal mammary lymph node metastases and parasternal recurrences in breast cancer].

The observed incidence (about 5%) of parasternal recurrence of breast cancer treated with radical mastectomy, is much lower than the rate (20-30%) of histological involvement of internal mammary lymph node examined by extended radical mastectomy. There are several explanations for this observation, but the correct reason remains unexplained. So the incidence of parasternal recurrences in cases of breast cancer autopsied should be examined more meticulously. Little mention has been made of clinically detectable parasternal recurrences in Japan, since they appears infrequently and have not been well documented. Our experience and present guidelines as to detection and management of parasternal recurrences are reviewed.

Breast Neoplasms↗
PubMed1988

Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products.

We have purified to near homogeneity a Mr 22,000 GTP-binding protein from human platelet membranes and identified it as the smg-21 gene product (smg p21), having the same putative effector domain as the ras gene products, which we have purified to near homogeneity from bovine brain membranes and characterized. This purified human platelet smg p21 was phosphorylated by cyclic AMP-dependent protein kinase. About one mol of phosphate was maximally incorporated into one mol of the protein. Only serine residue was phosphorylated. Both the guanosine 5'-(3-O-thio)-triphosphate (GTP gamma S)-bound and GDP-bound forms were phosphorylated with the same reaction velocity. The phosphorylation of smg p21 affected neither its GTP gamma S-binding nor GTPase activity. Human platelet smg p21 was not phosphorylated by protein kinase C. A Mr 24,000 GTP-binding protein partially purified from human platelet membranes was not phosphorylated by cyclic AMP-dependent protein kinase or protein kinase C.

Animals↗
PubMed1988

Identification of a platelet Mr 22,000 GTP-binding protein as the novel smg-21 gene product having the same putative effector domain as the ras gene products.

We have recently purified a Mr 22,000 GTP-binding protein (G protein) to near homogeneity from human platelet membranes and characterized it (Ohmori, T., Kikuchi, A., Yamamoto, K., Kim, S. and Takai, Y. (1989) J. Biol. Chem. in press). This platelet G protein was present most abundantly among several G proteins in platelets and showed a Mr of about 22,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This platelet G protein showed kinetic and physical properties very similar to those of the novel smg-21 gene product, having the same putative effector domain as the ras gene products, which we have recently purified to near homogeneity from bovine brain membranes and characterized (Kawata, M., Matsui, Y., Kondo, J., Hishida, T., Teranishi, Y. and Takai, Y. (1988) J. Biol. Chem. in press). Moreover, the peptide map of the platelet G protein was identical with that of the smg-21 gene product and the partial amino acid sequence of the platelet G protein was identical with that of the smg-21 gene product. These results indicate that this human platelet G protein is the smg-21 gene product.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes.

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.

Amino Acid Sequence↗
PubMed1988

ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1.

We have separated at least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 from bovine brain crude membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ideda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). Three of these G proteins were copurified with the proteins ADP-ribosylated by botulinum toxin type C1. One G protein ADP-ribosylated by this toxin was identified to be the bovine brain rho protein (rho p20) which was purified to near homogeneity (Yamamoto, K., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 9926-9932). rho p20 was ADP-ribosylated by botulinum toxin type C1 in time- and dose-dependent manners. About 0.4 mol of ADP-ribose was maximally incorporated into 1 mol of rho p20. The ADP-ribosylation of rho p20 was dependent on the presence of Mg2+. GTP enhanced the ADP-ribosylation in the presence of a low concentration (50 nM) of Mg2+ but not in the presence of a high concentration (0.5 mM) of Mg2+. The high concentration of Mg2+ fully stimulated the ADP-ribosylation even in the absence of GTP. The ADP-ribosylation of rho p20 did not affect its GTP gamma S-binding and GTPase activities. These results indicate that there are at least three G proteins ADP-ribosylated by botulinum toxin type C1 in bovine brain crude membranes and that one of them is rho p20. Two other G proteins have not yet been identified, but neither the c-ras protein, ADP-ribosylation factor for Gs, nor a G protein with a Mr of 24,000 was ADP-ribosylated by this toxin.

Adenosine Diphosphate Ribose↗
PubMed1988

Small molecular weight GTP-binding proteins in human erythrocyte ghosts.

GTP-binding proteins (G proteins) were extracted from human erythrocyte ghosts by sodium cholate and purified by gel filtration on an Ultrogel AcA-44 column followed by hydroxyapatite column chromatography. At least two peaks of G proteins were separated by hydroxyapatite column chromatography. The second peak contained G proteins recognized by the antibodies against the respective alpha subunits of Gs and Gi, and the ras protein, while the G protein of the first peak was not recognized by any of these antibodies. The G protein of the first peak was purified further by Mono Q HR5/5 column chromatography. The purified G protein showed a molecular weight of about 22 kDa on SDS-polyacrylamide gel electrophoresis. This G protein (22K G) specifically bound guanosine 5'-(3-O-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 50 nM. GTP gamma S-binding to 22K G was inhibited by pretreatment with N-ethylmaleimide. The G proteins recognized by the antibodies against the alpha subunit of Go and the ADP-ribosylation factor for Gs, designated as ARF, were not detected in human erythrocyte ghosts. These results indicate that there are at least two species of small molecular weight G proteins in human erythrocyte ghosts: one is the ras protein and the other is a novel G protein of 22K G.

Cholic Acid↗
PubMed1988

Multiple small molecular weight GTP-binding proteins in bovine brain cytosol. Purification and characterization of a 24KDa protein.

We have separated multiple small Mr GTP-binding proteins (G-proteins) from bovine brain crude membranes, purified a novel 24KDa G protein (smg p25A) to near homogeneity and characterized it. In this paper, we have studied these small Mr G proteins in the cytosol fraction of bovine brain. [35S]GTP gamma S-binding activity is detected in the cytosol fraction but this activity is one-sixth to one-eighth of that of the crude membrane fraction. When G proteins in the cytosol fraction are purified by successive chromatographies on DEAE-cellulose, Ultrogel AcA-44, hydroxyapatite and Mono Q HR5/5 columns, multiple small Mr G proteins are separated. One of these G proteins shows a Mr of about 24KDa. Its physical, immunological and kinetic properties are indistinguishable from smg p25A. These results indicate that there are also multiple small Mr G proteins in the cytosol fraction of bovine brain, and suggest that one of the cytosol G proteins is the soluble form of smg p25A.

Animals↗
PubMed1988

Purification and characterization of W-protein. A DNA-binding protein showing high affinity for the W chromosome-specific repetitive DNA sequences of chicken.

A protein component, which binds with high affinity to the W chromosome-specific XhoI family repetitive DNA of chicken (Tone, M., Sakaki, Y., Hashiguchi, T., and Mizuno, S. (1984) Chromosoma (Berl.) 89, 228-237), was detected in the 0.35 M NaCl extract of the female chicken liver nuclei. This protein, designated as W-protein, was substantially purified by phosphocellulose, hydroxyapatite, and DEAE-Toyopearl column chromatography. Molecular weight of W-protein was estimated to be about 72,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but it seems to form multimeric structure having apparent molecular weight of about 2.3 X 10(6) under nondenaturing conditions. W-Protein binds strongly to both 0.7- and 1.1-kb repeating units of the XhoI family, both of which show curved DNA characteristics, and weakly to the AATAT-satellite sequence of Drosophila melanogaster. Stable binding of W-protein requires greater than or equal to 300 base pairs of the 0.7-kilobases sequence, or more than 14 tandem repeats of the 21-base pair internal repeating unit of the 0.7-kilobase sequence. DNA footprint analysis and effects of some DNA-binding compounds suggest that the DNA double helix wraps around W-protein or its multimeric form contacting through A-T-rich minor grooves. A possible role of W-protein in the formation of W heterochromatic body is discussed.

Animals↗
PubMed1988

Mode of inhibitory action of cholecystokinin in amylase release from isolated rat pancreatic acini--inhibition of secretory process post to protein kinase C-calcium ion systems.

The incubation of isolated rat pancreatic acini with low doses (1 x 10(-11)-1 x 10(-10) M) of cholecystokinin-octapeptide (CCK8) induced amylase release. This CCK8-induced amylase release has been shown to be mediated through the protein kinase C activation and the Ca2+ mobilization which are linked to the phospholipase C-mediated hydrolysis of phosphoinositides. However, the incubation of the acini with high doses (1 x 10(-9)-1 x 10(-7) M) of CCK8 reduced amylase release to the level less than that induced by the maximally effective dose (1 x 10(-10) M) of this secretagogue. Under the same conditions, the high doses of this secretagogue did not inhibit the phospholipase C-mediated hydrolysis of phosphoinositides. The stimulatory action of the maximally effective dose of CCK8 in amylase release was mimicked by the simultaneous addition of protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+ ionophore A23187. A high dose (1 x 10(-7) M) of CCK8 reduced the amylase release induced by the combination of TPA and A23187. These results suggest that the high doses of CCK8 inhibit the secretory process post to the protein kinase C-Ca2+ systems and thereby reduce the amylase release induced by the maximally effective dose of CCK8 in rat pancreatic acini.

Amylases↗
PubMed1988

Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain.

We have purified a novel GTP-binding protein (G protein) with a Mr of about 24,000 to homogeneity from bovine brain membranes (Kikuchi, A., Yamashita, T., Kawata, M., Yamamoto, K., Ikeda, K., Tanimoto, T., and Takai, Y. (1988) J. Biol. Chem. 263, 2897-2904). In the present studies, we have isolated and sequenced the cDNA of this G protein from a bovine brain cDNA library using oligonucleotide probes designed from the partial amino acid sequences. The cDNA of the G protein has an open reading frame encoding a protein of 220 amino acids with a calculated Mr of 24,954. This G protein is designated as the smg-25A protein (smg p25A). The amino acid sequence deduced from the smg-25A cDNA contains the consensus sequences of GTP-binding and GTPase domains. smg p25A shares about 28 and 44% amino acid homology with the ras and ypt1 proteins, respectively. In addition to this cDNA, we have isolated two other homologous cDNAs encoding G proteins of 219 and 227 amino acids with calculated Mr values of 24,766 and 25,975, respectively. These G proteins are designated as the smg-25B and smg-25C proteins (smg p25B and smg p25C), respectively. The amino acid sequences deduced from the three smg-25 cDNAs are highly homologous with one another in the overall sequences except for C-terminal 32 amino acids. Moreover, three smg p25s have a consensus C-terminal sequence, Cys-X-Cys, which is different from the known C-terminal consensus sequences of the ras and ypt1 proteins, Cys-X-X-X and Cys-Cys, respectively. These results together with the biochemical properties of smg p25A described previously indicate that three smg p25s constitute a novel G protein family.

Amino Acid Sequence↗
PubMed1988

Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes.

At least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 were extracted from the crude membrane fraction of bovine brain by sodium cholate and purified by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite, and Mono Q HR5/5. One G protein with a Mr of about 24,000 (24K G) was purified to near homogeneity and characterized. 24K G bound maximally about 0.7 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 46 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. 24K G hydrolyzed GTP to liberate Pi with a rate of about 40 mmol of Pi/mol of protein/min. 24K G did not associate with the beta gamma subunit of Go. 24K G was distinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the ras protein (ras p21) and ADP-ribosylation factor, the G protein with a Mr of about 21,000 serving as a cofactor for the cholera toxin-dependent ADP-ribosylation of Gs. 24K G was not recognized by the antibody against ras p21, ADP-ribosylation factor, the alpha subunit each of Gs, Gi, and Go, or tubulin. These results indicate that 24K G is a novel G protein and suggest that there are multiple forms of G proteins with small Mr values as is the case with the G proteins which have the alpha subunits with Mr values of about 40,000.

Animals↗
PubMed1988

A yeast gene required for the G1-to-S transition encodes a protein containing an A-kinase target site and GTPase domain.

A new temperature-sensitive mutant of Saccharomyces cerevisiae, gst1 (G1-to-S transition) was isolated. At nonpermissive temperature the mutant cells with large buds accumulated and DNA synthesis was substantially arrested. From the reciprocal experiment of temperature-shift and mating-factor treatment, it was shown that the execution point was post 'START'. This suggested that the mutation affected the G1-to-S phase transition in the cell cycle. A DNA clone complementing the gst1-1 mutation was isolated from a yeast gene library, and gst1 was mapped in chr4R, by Southern blotting of cloned sequence to the individual yeast chromosome DNA by OFAGE system and by genetic analysis. The gene product was tentatively assigned from DNA sequencing analysis, as a protein of mol. wt 76,565 which contained consensus sequences for a target site of cAMP-dependent protein kinase(s) and for GTPase with extensive homology to polypeptide chain elongation factor EF1 alpha.

Amino Acid Sequence↗