Biomedical subjects

Y Takai

Publications and source records attributed to Y Takai.

At least 361 records · Page 20Linked to original sources

PubMed1992

Molecular cloning of the human cDNA for a stimulatory GDP/GTP exchange protein for c-Ki-ras p21 and smg p21.

We have previously purified smg GDP dissociation stimulator (GDS) from bovine brain and isolated its cDNA from a bovine brain cDNA library. smg GDS stimulates the GDP/GTP exchange reaction of a group of small GTP-binding proteins (G proteins), including at least c-Ki-ras p21, smg p21A, smg p21B, rhoA p21 and rhoB p21, by stimulating the dissociation of GDP from and the subsequent binding of GTP to each small G protein. In this study, we have isolated and sequenced the cDNA of smg GDS from a human brain cDNA library using the cloned bovine smg GDS cDNA. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,122. Human smg GDS shares 93% nucleotide and 96% amino acid sequence homologies with bovine smg GDS. The isolated cDNA is expressed in Escherichia coli, and the encoded protein shows the physical and functional properties similar to those of bovine smg GDS.

Amino Acid Sequence↗

PubMed1992

Constitutive expression of leukemia inhibitory factor (LIF) mRNA and production of LIF by a cloned murine thymic stromal cell line.

We have found that normal mouse thymus expressed mRNA for leukemia inhibitory factor (LIF), suggesting the presence of cell component(s) in the thymus that produces this cytokine. High levels of mRNA for LIF were observed to be constitutively expressed in the murine thymic stromal cell clone, MRL104.8a. This stromal cell clone produced potent magnitudes of LIF activity in its culture supernatant. These observations imply that LIF may also exert its growth/differentiation-regulating effect in the thymus. Thus, this thymic stromal cell clone provides a tool for investigating potential roles of LIF in the intrathymic T cell development.

Animals↗

PubMed1992

[Abnormalities of coagulation, fibrinolysis and kallikrein-kinin system in 2 cases with remarkable eosinophilia].

We evaluated an initial activation of hemostasis (coagulation, fibrinolysis and kallikrein-kinin system) in two remarkable eosinophilia cases. The first case was a 29-year-old male who complained of a high fever, general exanthema and numbness of the limbs. He had peripheral blood eosinophilia (61.075/microliters) unknown etiology, and hypereosinophilic syndrome (HES) was diagnosed. The second case was a 60-year-old female who complained of numbness in her lower limbs. She also presented eosinophilia (10,379/microliters), and had clinical features of allergic granulomatous angitis (AGA). The administration of prednisolone led to clinical remission in both cases, including rapid improvement of eosinophilia, normalization of hypercoagulability and correction of platelet dysfunction. In hemostatic parameters, the titer of the soluble fibrin monomer complex (SFMC) became negative, and each value of FDP D-dimer, fpB beta 15-42, thrombin-antithrombin III complex (TAT) decreased, whereas the value of prekallikrein increased. A decrease in platelet aggregation with ADP, collagen and epinephrine was normalized by prednisolone administration. Hemostatic data in both cases indicate a possible relationship between the initial activation of hemostasis and the eosinophils, as well as the possibility of general and/or organic inflammations and intravascular thrombi formations in eosinophilia such as HES and AGA.

Adult↗

PubMed1992

[Small GTP-binding protein and platelet function].

In platelets, nearly ten small GTP-binding proteins (G proteins) are identified. Among these small G proteins, smg p21 is the most abundant. The C terminus of smg p21 is processed by three post-translational modifications: geranylgeranylation, removal of three amino acids and carboxyl methylation. There are two regulatory proteins for smg p21, smg p21 GTPase activating protein (GAP) and smg GDP dissociation stimulator (GDS). Moreover, smg p21 is phosphorylated by A-Kinase and this phosphorylation stimulates the action of smg GDS. C-Kinase and Ca2+ induce platelet activation, while A-Kinase antagonizes this activation. Since smg p21 is the substrate of A-Kinase, it is possible that smg p21 mediates the A-Kinase action and inhibits the platelet function, such as aggregation and secretion. The mode of action of and the possible function of smg p21 in platelets are discussed.

Alkyl and Aryl Transferases↗

PubMed1991

[Low-dose rate telecobalt therapy as a boost against esophageal carcinomas].

The results of treatment of 54 esophageal carcinomas managed with low-dose-rate telecobalt therapy (LDRT) as a boost were compared with those of 89 esophageal carcinomas treated with conventionally fractionated irradiation alone (CFI). The LDRT (1 Gy/hr, 5-7 Gy/day, to a total dose of 14-20 Gy) was boosted about 10 days after the CFI dose of 60 Gy. Although the LDRT group included more advanced cases than the CFI group, local effects and survival rate of the LDRT group, especially those with tumorous X-P and serrated types, were better than those of the CFI group. Late complications were more severe in the LDRT group. However, they were acceptable when the total dose in the LDRT group was brought under 80 Gy.

Aged↗

PubMed1991

Expression and localization of smg p25A (= rab3A) in cultured rat hippocampal cells.

We have studied the expression of smg p25A and synaptophysin in cultured hippocampal neurons isolated from 5-day-old rat brain by an immunocytochemical technique. In a dispersed cell culture seeded on astrocyte monolayers, well-branching neurite proliferation was observed along with age in culture. The synaptophysin immunoreactivity was present in the neuronal cell bodies and neurites at 1 and 5 days in vitro (DIV) and was eventually localized to discrete areas along neurites at 15 DIV while the immunoreactivity in cell bodies became less prominent. On the other hand, the smg p25A immunoreactivity was observed in the neuronal cell bodies and neurites at 1 through 15 DIV. The immunoreactivity for smg p25A or synaptophysin was not observed in astrocytes and this finding was confirmed by an immunoblot analysis. These results indicate that smg p25A as well as synaptophysin is present exclusively in neurons and suggest that these two synapse-associated proteins have different sites of function and different kinetics of synthesis, transport, and/or turnover in cultured hippocampal neurons.

Animals↗

PubMed1991

Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the geranylgeranylated synthetic peptides designed from its C-terminal region.

smg p21B, a member of the ras p21-like small GTP-binding protein superfamily, undergoes post-translational modifications, which are geranylgeranylation of the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids (QLL) and the subsequent carboxyl methylation of the exposed prenylated cysteine residue. smg p21B has a polybasic region upstream of the prenylated cysteine residue. We have previously proposed that these C-terminal structures of smg p21B are essential for the action of its stimulatory GDP/GTP exchange protein, named GDP dissociation stimulator (GDS). We studied here which structure of the C-terminal region of smg p21B is important for its interaction with smg p21 GDS. For this purpose, we synthesized a peptide according to the C-terminal structure of smg p21B, which was PGKARKKSSC-geranylgeranyl-carboxyl methyl, and its variously modified peptides and examined their ability to interact with smg p21 GDS and to interfere with the smg p21 GDS action to stimulate the GDP/GTP exchange reaction of smg p21B. The results indicate that the phosphorylated form of PGKARKKSSC-geranylgeranyl stoichiometrically interacts with smg p21 GDS, that the presence of the geranylgeranyl moiety is essential for, but not sufficient for, the smg p21 GDS action, and that the presence of the methyl moiety, removal of the three C-terminal amino acids, and the presence of the polybasic amino acids also affect the smg p21 GDS action. It is likely that all the steps of the post-translational processing and presence of the polybasic region in the C-terminal region of smg p21B are related to its interaction with smg p21 GDS.

Amino Acid Sequence↗

PubMed1991

A novel prenyltransferase for a small GTP-binding protein having a C-terminal Cys-Ala-Cys structure.

smg p25A/rab3A p25 is a member of the small GTP-binding protein superfamily which is implicated in intracellular vesicle transport. smg p25A has a cDNA-predicted C-terminal structure of Cys-Ala-Cys. The protein purified from bovine brain membranes is geranylgeranylated at both the two cysteine residues and carboxyl-methylated at the C-terminal cysteine residue. Two types of prenyltransferase for small GTP-binding proteins have thus far been reported: ras p21 farnesyltransferase (ras p21 FT) and rhoA p21 geranylgeranyltransferase (rhoA p21 GGT). Neither of them geranylgeranylated smg p25A having a C-terminal Cys-Ala-Cys structure. In this paper, a smg p25A GGT was partially purified from bovine brain cytosol and separated from the ras p21 FT and rhoA p21 GGT by column chromatographies. smg p25A GGT transferred the geranylgeranyl moiety from geranylgeranyl pyrophosphate to both the two cysteine residues in the C-terminal Cys-Ala-Cys structure of smg p25A. smg p25A GGT did not use farnesyl pyrophosphate as a substrate and was also inactive on c-Ha-ras p21 and rhoA p21 with either farnesyl pyrophosphate or geranylgeranyl pyrophosphate as a substrate. These results indicate that there are at least three types of prenyltransferase for small GTP-binding proteins in mammalian tissues.

Amino Acid Sequence↗

PubMed1991

A stimulatory GDP/GTP exchange protein for smg p21 is active on the post-translationally processed form of c-Ki-ras p21 and rhoA p21.

We have purified a stimulatory GDP/GTP exchange protein for smg p21A and -B, ras p21-like small GTP-binding proteins (G proteins), cloned its cDNA, and named it GDP dissociation stimulator (smg p21 GDS). We show here that smg p21 GDS is active not only on smg p21A and -B but also on c-Ki-ras p21 and rhoA p21, all of which are post-translationally processed. Furthermore, we show that smg p21 GDS is inactive on the post-translationally unprocessed form of these proteins and on the post-translationally unprocessed form of c-Ha-ras p21 and smg p25A. All of the small G proteins recognized by smg p21 GDS have a cDNA-predicted C-terminal "CAAX" motif (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid) and a polybasic region upstream of this motif. These results suggest that smg p21 GDS is at least active on a group of small G proteins having these unique C-terminal structures. Moreover, they suggest that the C-terminal post-translational processing of these small G proteins, by farnesylation or geranylgeranylation of the C-terminal cysteine residue, removal of amino acids in positions denoted "AAX", and carboxyl methylation of the exposed cysteine residue, is important for the smg p21 GDS action.

Animals↗

PubMed1991

Transactivation of the TPA-responsive element by the oncogenic C-erbB-2 protein is partly mediated by protein kinase C.

The mutant c-erbB-2 gene encoding a protein with Glu instead of Val-659 in the transmembrane domain is able to transform NIH3T3 cells, while the wild type c-erbB-2 unless overexpressed does not. The mutant c-erbB-2 protein shows enhanced tyrosine kinase activity in vitro. Transient expression of this active c-erbB-2 stimulated the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element, serum response element, and cyclic AMP response element. Particularly, stimulation of the TPA response element by active c-erbB-2 was prominent. In contrast, transient expression of wild type c-erbB-2 stimulated none of these elements. Transactivation of the TPA response element was also observed in a cell line that stably expresses active c-erbB-2. The active c-erbB-2-induced transactivation of the TPA response element was partially prevented either by down-regulation of protein kinase C or by the protein kinase C inhibitor H7. These results indicate that protein kinase C is partly involved in oncogenic signalling of the active c-erbB-2 protein that leads to Jun/Fos-mediated transcriptional activation in nuclei.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester.

smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.

Adrenal Gland Neoplasms↗

PubMed1991

The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21.

rhoA p21, a ras p21-like small GTP-binding protein, has the same C-terminal consensus motif of Cys-A-A-X (A is an aliphatic amino acid and X is any amino acid) as ras p21s, which is posttranslationally processed. We here determine the posttranslationally processed C-terminal structure of the rhoA p21 purified from bovine aortic smooth muscle. Incubation of rhoA p21-expressing insect cells with exogenous [3H]mevalonolactone caused the labeling of rhoA p21, suggesting that rhoA p21 is prenylated. Consistently, Raney nickel treatment of rhoA p21 released a geranylgeranyl moiety as estimated by gas chromatography/mass spectrometry. No lipid moiety was released by KOH or NH2OH treatment. Extensive digestion of rhoA p21 with Achromobacter protease I yielded a C-terminal peptide, Ser-Gly-Cys190, that lacked the three C-terminal amino acids predicted from the cDNA but was geranylgeranylated and carboxyl methylated at the cysteine residue. Bovine brain cytosol geranylgeranylated the bacterial rhoA p21 having the three C-terminal amino acids predicted from the cDNA but not the protein lacking the three C-terminal amino acids. Bovine brain membranes methylated the synthetic C-terminal peptide with 10 amino acids of rhoA p21 which was geranylgeranylated at its C-terminal cysteine residue but not the peptide which was not geranylgeranylated. These results suggest that rhoA p21 is first geranylgeranylated followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the exposed cysteine residue.

Alcaligenes↗

PubMed1991

The smg GDS-induced activation of smg p21 is initiated by cyclic AMP-dependent protein kinase-catalyzed phosphorylation of smg p21.

We have shown that smg p21B is phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) and that membrane acidic phospholipids such as phosphatidic acid and phosphatidylinositol markedly inhibit the smg GDS-induced activation of smg p21B. However, we show here that phosphatidic acid and phosphatidylinositol exhibit a less inhibitory effect on the smg GDS-induced activation of the phosphorylated form of smg p21B. Thus, in the presence of membrane acidic phospholipids, the smg GDS-induced activation of smg p21B is totally dependent on the protein kinase A-catalyzed phosphorylation of smg p21B. Since smg p21B is located on the membranes in resting cells, it is likely that the smg GDS-induced activation of smg p21B is initiated by the protein kinase A activation.

Animals↗

PubMed1991

[Radiotherapy of intracranial germinomas].

Between 1978 and 1988, 28 patients with intracranial germinoma, verified or presumed, were treated with radiation. The diagnosis was made based on histology in 6 cases, on cerebrospinal fluid (CSF) cytology in 2 cases and on clinical (response to radiation) and radiological findings in the remaining 20 cases. The target volume was the primary site plus whole brain in 23 cases, whole brain in 4 cases and the primary site only in 1 case. Whole spinal irradiation was undertaken for 14 patients, including 9 patients of high risk group, i.e., with positive findings in CSF cytology, suspected subarachnoid space seeding, multifocal tumors, infiltration to the ventricular wall or previous surgery for the tumor. The average dose was 52.8 Gy to the tumor, 28.7 Gy to the whole brain and 21 Gy to the spinal axis. Five and ten-years survival rate were 100% and 96%, respectively. No intracranial recurrence or spinal metastasis has been found so far. Therefore no spinal irradiation seems to be unnecessary for non-high risk group of patients. Approximately 20 Gy should be sufficient, if spinal irradiation is to be indicated for high risk group. The dose for the primary tumor and whole brain could have been diminished to 40 Gy and 20 Gy, respectively.

Brain Neoplasms↗

PubMed1991

Similarity of expression of low molecular weight G proteins smg p21A and ras p21 in normal and malignant human tissues.

We prepared monoclonal antibodies specific for smg p21A, one of the low molecular weight GTP-binding proteins and possibly a suppressor molecule for ras p21. Two monoclonal antibodies (T22 and T212) reacted with smg p21A but not with Ki-ras p21, both of which were produced by Escherichia coli. These two clones detected an Mr 21,000 band in one-dimensional immunoblotting of extracts of a human pancreatic cancer cell line which was indistinguishable from a band detected by RASK-3, a monoclonal antibody specific for ras p21. However, T22 and T212 detected a single spot in two-dimensional immunoblotting that was clearly different from the three spots detected in the same cellular extracts by RASK-3. A series of normal and malignant human tissues were examined for the expression of smg p21A and ras p21 by immunohistochemical methods utilizing T22 and RASK-3. In essentially all tissues examined, both normal and malignant, smg p21A and ras p21 were expressed with great similarity. Expression of both molecules in all malignant tissues examined was coincident with that in normal tissues except that gastric cancer showed increased expression of the two molecules in comparison with normal gastric tissue.

GTP-Binding Proteins↗

PubMed1991

Enhancement of the actions of smg p21 GDP/GTP exchange protein by the protein kinase A-catalyzed phosphorylation of smg p21.

We have previously shown that cyclic AMP-dependent protein kinase (protein kinase A) phosphorylates smg p21A and -B, ras p21-like small GTP-binding proteins. In the present study, we investigated the function(s) of this phosphorylation by use of the smg p21B purified from human platelets. smg p21B bound to plasma membranes and the protein kinase A-catalyzed phosphorylation of smg p21B reduced this binding. Moreover, the phosphorylation of smg p21B enhanced the two actions of its specific GDP/GTP exchange protein, named GDP dissociation stimulator, when tested in a cell-free system: one is the action to stimulate the GDP/GTP exchange reaction of smg p21B, and the other is the action to inhibit the binding of smg p21B to membranes. Consistently, smg p21B was translocated from the membranes to the cytoplasm when it was phosphorylated by protein kinase A in intact platelets in response to prostaglandin E1 or dibutyryl cyclic AMP. The protein kinase A-catalyzed phosphorylation of smg p21B affected neither its basal GDP/GTP exchange reaction, basal GTPase activity, nor the GTPase activity stimulated by its specific GTPase activating protein. On the other hand, we have recently clarified that the structure of the C-terminal region of the post-translationally processed human platelet smg p21B is Lys-Lys-Ser-Ser-all-trans-geranylgeranyl Cys181 methyl ester, and that this modification of the C-terminal region is essential for smg p21B to bind to membranes. We furthermore determined here that protein kinase A phosphorylated Ser179 in this C-terminal region of smg p21B. These results indicate that protein kinase A-catalyzed phosphorylation of smg p21B makes smg p21B sensitive to the actions of smg p21 GDP dissociation stimulator.

Blood Platelets↗

PubMed1991

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6.

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.

Animals↗

PubMed1991

A geranylgeranyltransferase for rhoA p21 distinct from the farnesyltransferase for ras p21S.

We have clarified that rhoA p21 purified from bovine aortic smooth muscle is geranylgeranylated at the cysteine residue in the C-terminal CAAX motif (A is an aliphatic amino acid and X is any amino acid). In this paper, a geranylgeranyltransferase for rhoA p21 (rhoA p21 GGT) was partially purified from bovine brain cytosol. This enzyme transferred a geranylgeranyl moiety from geranylgeranyl pyrophosphate to rhoA p21 having the CAAX motif (rhoA p21-CAAX) but not to rhoA p21 lacking the AAX portion. rhoA p21 GGT was separated from the previously reported farnesyltransferase for ras p21s (ras p21 FT) by column chromatographies and did not geranylgeranylate or farnesylate c-Ha-ras p21-CAAX. ras p21 FT did not geranylgeranylate or farnesylate rhoA p21-CAAX. These results indicate that rhoA p21 GGT distinct from ras p21 FT is present in bovine brain cytosol.

Alkyl and Aryl Transferases↗