Search PubMed⌕ Search

Biomedical subjects

Y Takai

Publications and source records attributed to Y Takai.

At least 235 records · Page 13Linked to original sources
PubMed1996

Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells.

We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulin-secreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in beta-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunofluorescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decreased by the overexpression of wild-type Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human beta-cells, a different Rab3 isoform but with homologous function may replace Rab3A.

Animals↗
PubMed1996

[Analysis of locally controlled esophageal carcinomas treated with radiotherapy].

Of 227 esophageal carcinomas treated with a radiation dose of 60 Gy or more, 100 patients had no tumor or ulceration (with or without stenosis) of the esophagus after irradiation. We analyzed local control factors of these 100 patients to determine the need for further treatment. The cumulative local control rate at five years was 40% in all cases, 37% in 21 cases without any stenosis of the esophagus and 40% in 79 cases with stenosis. The presence of stenosis of the esophagus after irradiation was not a critical factor in predicting final local control. Local recurrence of tumors with findings of Borrmann III or Borrmann IV by the pretreatment esophageal barium study, tumors controlled after a total dose of more than 80 Gy, tumors without low dose rate telecobalt therapy (LDRT; 1 Gy/hour, 5 to 7Gy/day, a total dose of 12 to 15 Gy) as boost therapy, and apparently controlled tumors with a stenotic ratio of 60% or more or with 5 cm or more length of stenosis of the esophagus after irradiation was significantly higher than that of the others (p < 0.05). Multivariate analysis revealed that findings of pretreatment barium study, total dose, with or without LDRT, and length of stenosis of the esophagus after irradiation were significantly important factors in local control. Members of the high risk group of apparently controlled tumors should undertake surgical treatment or further intensive chemotherapy.

Aged↗
PubMed1996

[Epidemiological analysis of Epstein-Barr virus infection].

We analysed the characteristics of 372 Epstein-Barr virus (EBV) infected patients visiting our hospital from 1981 to 1995 classifying the phase I (from 1981 to 1988), the phase II (from 1989 to 1991) and the phase III (from 1992 to 1995). The incidence of infectious mononucleosis (IM) as EBV primary infection increased especially in the patients over 30 years old in the phase II and III. Immunological data on EBV specific antibodies showed twice positivity for anti EBV VCA-Igm antibody and the patients with anti-EBNA antibody decreased in the phase II and III. It is indicative of early diagnosis on primary EBV infection. EBV also causes atypical diseases, non-IM in this report, including hematological or neurological disorders. In hematological ones, one of 4 cases with virus associated hemophagocytic syndrome (VAHS) took fatal courses in the phase I, others had recovered by early diagnosis and adequate therapies (pulse therapy of steroids, exchange transfusion etc.). We indicated here the increased number and advanced age of patients with IM and improvement of the early diagnosis in recent 6 years compared with the previous 8 years.

Adolescent↗
PubMed1996

[Home care that meets the wishes of patients with terminal stage cancer and their families].

As our ward is a general surgical ward, we formerly had difficulties providing appropriate care for patients with terminal cancer. To solve the problem, we have formed a nursing study group in order to provide home care that would meet the wishes of such patients and their families. Our specific approach was as follows: 1) preparation of a form on which patient information is collected; 2) a support system for home care (such as pain control and home parenteral nutrition; and 3) a solid collaboration system between ward nurses and visiting nurses. Such a program enables us to understand the wishes of patients and families and to provide various support where needed. The project helps to lessen the anxiety and the burden that patients and families feel in home care, and improve the quality of life (QOL).

Aged↗
PubMed1995

Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules.

Rab3A small GTP-binding protein and its putative target protein, named Rabphilin-3A, are implicated in neurotransmitter release. We have investigated here the function of the lipid modifications of Rab3A (Mr approximately 25,000) in its interaction with Rabphilin-3A (Mr approximately 80,000). Lipid-modified GTP-Rab3A dimerized and lipid-unmodified one monomerized. Lipid-modified GTP-Rab3A (Mr approximately 50,000) formed a heterotetramer (Mr approximately 210,000) with two Rabphilin-3A molecules, whereas lipid-unmodified GTP-Rab3A formed a heterodimer (Mr approximately 105,000) with one Rabphilin-3A molecule. These results indicate that two lipid-modified GTP-Rab3A molecules form a heterotetramer with two Rabphilin-3A molecules.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Molecular cloning of an isoform of Doc2 having two C2-like domains.

We previously isolated a novel protein having two C2-like domains known to interact with Ca2+ and phospholipid, and named Doc2 (Double C2). Doc2 is predominantly expressed in brain and is implicated in Ca(2+)-dependent neurotransmitter release. We have isolated here an isoform of Doc2 and named the original one Doc2 alpha and the new one Doc2 beta. Doc2 beta alsp has two C2-like domains and is 61% identical to Doc2 alpha at the amino acid level. In contrast to Doc2 alpha, the Doc2 beta mRNA is expressed ubiquitously. These results indicate that there are at least two isoforms of Doc2, and suggest that Doc2 beta is involved in Ca(2+)-dependent intracellular vesicle trafficking in various types of cells.

Adaptor Proteins, Signal Transducing↗
PubMed1995

A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae.

The RHO1 gene in Saccharomyces cerevisiae encodes a homolog of the mammalian RhoA small GTP-binding protein, which is implicated in various actin cytoskeleton-dependent cell functions. In yeast, Rho1p is involved in bud formation. A yeast strain in which RHO1 is replaced with RhoA shows a recessive temperature-sensitive growth phenotype. A dominant suppressor mutant was isolated from this strain. Molecular cloning of the suppressor gene revealed that the mutation occurred at the pseuodosubstrate site of PKC1, a yeast homolog of mammalian protein kinase C. Two-hybrid analysis demonstrated that GTP-Rho1p, but not GDP-Rho1p, interacted with the region of Pkc1p containing the pseudosubstrate site and the C1 domain. MKK1 and MPK1 encode MAP kinase kinase and MAP kinase homologs, respectively, and function downstream of PKC1. A dominant active MKK1-6 mutation or overexpression of MPK1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of the RhoA mutant. The dominant activating mutation of PKC1 suppressed the temperature sensitivity of two effector mutants of RHO1, rho1(F44Y) and rho1(E451), but not that of rho1(V43T). These results indicate that there are at least two signaling pathways regulated by Rho1p and that one of the downstream targets is Pkc1p, leading to the activation of the MAP kinase cascade.

Amino Acid Sequence↗
PubMed1995

Cloning of a brain-type isoform of human Rab GDI and its expression in human neuroblastoma cell lines and tumor specimens.

Rab proteins, a family of Ras-related small GTP-binding proteins, play a key role in regulating intracellular vesicle trafficking. Rab GDP dissociation inhibitor (GDI3) forms a soluble complex with Rab proteins and thereby prevents the exchange of GDP for GTP. Recently, two isoforms of Rab GDI cDNA were isolated from rats and mice. In this study, we have isolated a brain-type isoform of human Rab GDI cDNA and examined its expression in neuroblastoma. We tentatively designate it as human Rab GDI alpha (hu GDI alpha) and another human Rab GDI, as human Rab GDI beta (hu GDI beta). Hu GDI alpha cDNA encodes a protein of 447 amino acids with a deduced molecular weight of 50,200. Northern blot analysis revealed that hu GDI alpha gene is expressed abundantly in the brain but much less in other tissues, while hu GDI beta gene is ubiquitously expressed. All human neuroblastoma cell lines and tumor specimens examined express hu GDI alpha gene to various extents, while a human T cell leukemia cell line, MOLT3, does not. The levels of both hu GDI alpha and beta mRNA were constant in a human neuroblastoma cell line, NB1, during its neuronal differentiation, while Rab3A and neurofilament-L gene expression and the number of neurosecretory granules were elevated at this condition. These results suggest that hu GDI alpha gene expression is not related to the differentiation state of neuronal cells.

Amino Acid Sequence↗
PubMed1995

The farnesyl group of H-Ras facilitates the activation of a soluble upstream activator of mitogen-activated protein kinase.

To study the function of the farnesyl modification of Ras, the farnesyl group and a variety of its structural analogs, which lack one or more double bonds and/or the methyl groups, were enzymatically incorporated into recombinant H-Ras in vitro. These proteins were used in a cell- and membrane-free, Ras-dependent mitogen-activated protein kinase (MAP kinase) activation system derived from Xenopus laevis eggs to examine the contribution of the farnesyl group toward the activation of the kinase. Whereas non-farnesylated H-Ras is unable to activate MAP kinase, farnesylation of H-Ras alone, in the absence of further processing, is sufficient to cause the activation of MAP kinase in this system. All of the analogs of the farnesyl group, when incorporated into H-Ras, support the activation of the kinase to variable extents. These results suggest a direct but fairly nonspecific interaction of the farnesyl moiety of H-Ras with a soluble upstream activator of MAP kinase.

Animals↗
PubMed1995

Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21, and its inhibition by Clostridium botulinum C3 exoenzyme.

An activator of rat brain phospholipase D (PLD) that is distinct from the already identified PLD activator, ADP-ribosylation factor (ARF), was partially purified from bovine brain cytosol by a series of chromatographic steps. The partially purified preparation contained a 22-kDa substrate for Clostridium botulinum C3 exoenzyme ADP-ribosyltransferase, which strongly reacted with anti-rhoA p21 antibody, but not with anti-rac1 p21 or anti-cdc42Hs p21 antibody. Treatment of the partially purified PLD-activating factor with both C3 exoenzyme and NAD significantly inhibited the PLD-stimulating activity. These results suggest that rhoA p21 is, at least in part, responsible for the PLD-stimulating activity in the preparation. Recombinant isoprenylated rhoA p21 expressed in and purified from Sf9 cells activated rat brain PLD in a concentration- and GTP gamma S (guanosine 5'-O-(3-thiotriphosphate))-dependent manner. In contrast, recombinant non-isoprenylated rhoA p21 (fused to glutathione S-transferase) expressed in Escherichia coli failed to activate the PLD. This difference cannot be explained by a lower affinity of non-isoprenylated rhoA p21 for GTP gamma S, as the rates of [35S]GTP gamma S binding were very similar for both recombinant preparations and the GTP gamma S-bound form of non-isoprenylated rhoA p21 did not induce PLD activation. Interestingly, recombinant isoprenylated rhoA p21 and ARF synergistically activated rat brain PLD; a similar pattern was seen with the partially purified PLD-activating factor. The synergistic activation was inhibited by C3 exoenzyme-catalyzed ADP-ribosylation of recombinant isoprenylated rhoA p21 in a NAD-dependent manner. Inhibition correlated with the extent of ADP-ribosylation. These findings suggest that rhoA p21 regulates rat brain PLD in concert with ARF, and that isoprenylation of rhoA p21 is essential for PLD regulation in vitro.

ADP Ribose Transferases↗
PubMed1995

Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells: comparison with the effect of Rab3AL peptide.

Most guanine nucleotide binding proteins (G-proteins) possess an S-prenylated C-terminal cysteine whose carboxyl group can be reversibly methylated. The prenylcysteine analog N-acetyl-S-geranylgeranyl-cysteine (AGGC) (50 microM), a competitive inhibitor of prenylcysteine methyl transferases, introduced into streptolysin-O permeabilized HIT-T15 cells doubled the rate of basal (0.1 microM Ca2+) and of stimulated (10 microM Ca2+ or 100 microM GTP gamma S) insulin secretion in a reversible and ATP-dependent manner. N-acetyl-S-farnesylcysteine (AFC) was less potent while N-acetyl-S-geranyl-cysteine was inactive. Prenylcysteine action on exocytosis did not involve inhibition of G-protein methylation, since (1) the methyl ester derivative of AFC, an inefficient inhibitor of methyltransferases in HIT-T15 cell fractions, was as potent as AGGC in stimulating exocytosis; (2) S-adenosyl-homocysteine, a general inhibitor of methylation reactions, did not alter basal or GTP gamma S-triggered secretion while inhibiting Ca(2+)-induced insulin release. The binding of G-proteins to Rab/GDP-dissociation inhibitor, Rab3A/GTPase activating protein or rabphilin-3A was not affected by the prenylcysteine analogs. AGGC or AFC had the same effect on insulin release as a synthetic peptide mimicking the amino acid residues 52-67 of the G-protein Rab3A (Rab3AL). Moreover, the action on secretion of the combination of Rab3AL and prenylcysteines was not additive. We propose that the prenylcysteines and the Rab3AL peptide influence exocytosis by affecting the association of Rab3A with different proteins of the exocytotic machinery of insulin-secreting cells.

Acetylcysteine↗
PubMed1995

Evidence that the Rab3a-binding protein, rabphilin3a, enhances regulated secretion. Studies in adrenal chromaffin cells.

Rabphilin3a had been identified in brain as a Rab3a-binding protein and may serve as an effector for Rab3a function. We have cloned a splice variant of brain-Rabphilin3a from a bovine adrenal chromaffin cell cDNA library and investigated the function of the protein in regulated exocytosis in bovine chromaffin cells. The predicted amino acid sequence of chromaffin cell (c-) Rabphilin3a was identical with that of brain (b-) Rabphilin3a except for a 6-amino-acid insert VFSLSA in the amino-terminal half of the protein. An antibody directed against a carboxyl-terminal peptide recognized an 85-kDa protein in COS7 cells transfected with the cDNA in a mammalian expression vector. A band of similar mobility was enriched in a fraction of highly purified chromaffin granule membranes, consistent with the Rabphilin3a being associated with chromaffin granule membranes. Overexpression of either chromaffin cell or brain Rabphilin3a by transfection with the corresponding cDNAs in mammalian expression vectors enhanced DMPP-induced secretion of co-expressed human growth hormone (GH) approximately 30%. Chromaffin cells transfected with a plasmid with the entire coding sequence of c-Rabphilin3a inserted in the antisense orientation inhibited secretion of co-expressed GH by approximately 30%. Rabphilin3a mutants lacking one or both of the carboxyl-terminal C2 domains strongly inhibited DMPP-stimulated exocytosis. The single C2 domain deletion also strongly inhibited Ca(2+)-dependent secretion from digitonin-permeabilized cells. These data indicate that Rabphilin3a is a positive regulator of exocytosis. Because the C2 deletion mutants contain the amino-terminal Rab3a-GTP binding domain, they may inhibit secretion by competing with endogenous Rabphilin3a for interaction with Rab3a-GTP without being able to mimic the functional effects of full-length Rabphilin3a.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows.

Rho small GTP-binding protein regulates various cell functions, such as formation of stress fibers and focal adhesions, cell motility, membrane ruffling, cytokinesis and smooth muscle contraction in mammalian cells and bud formation in the yeast Saccharomyces cerevisiae. As to the functioning sites of Rho in Saccharomyces cerevisiae, we have recently shown that RHO1 protein, a homologue of mammalian RhoA, is concentrated to the growth region of the cells where cortical actin patches are clustered. However, in mammalian cells, the functioning sites of Rho have not yet been studied. In the present study, MDCK cell lines stably expressing myc-tagged RhoA (myc-RhoA) were prepared and localization of myc-RhoA was first immunohistochemically examined using an anti-myc antibody. In the resting cells, almost all of myc-RhoA was observed in the cytosol. When the cells were stimulated with phorbol ester or hepatocyte growth factor, membrane rufflings were induced and myc-RhoA was translocated to the membrane ruffling area. Moreover, myc-RhoA was translocated from the cytosol to the cell-cell adhesion sites when the cells were transferred from a low to normal Ca2+ medium. RhoA was also concentrated to the cleavage furrows during cytokinesis in Swiss 3T3 cells. Translocation of myc-RhoA to the membrane ruffling area was inhibited by prior microinjection into the cells of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, or of C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, indicating that both activation and functioning of Rho are essential for the translocation of Rho. The ERM (Ezrin, Radixin, Moesin) family members were colocalized with RhoA at all of these sites. However, RhoA was not apparently observed at the focal adhesion plaque where vinculin was localized. These results suggest that at least one of the functioning sites of Rho is the ERM family-controlled actin filament/plasma membrane association sites.

3T3 Cells↗
PubMed1995

Purification and characterization of Rab GDI beta from rat brain.

A new Rab GDI was purified from the synaptic soluble fraction of rat brain by several column chromatographies as a protein that inhibited the dissociation of [3H]GDP from Rab3A but was not recognized by an anti-Rab GDI alpha antibody. The partial amino acid sequence analysis revealed that it was identical with rat Rab GDI beta. Purified Rab GDI beta showed the kinetic properties similar to those of Rab GDI alpha, including the inhibitory effect on the dissociation of GDP from Rab3A, the substrate specificity, the requirement of the post-translational lipid modifications of Rab3A, the stoichiometric interaction with the GDP-bound form of Rab3A, the inhibitory effect on the binding of Rab3A to erythrocyte ghosts, and the stimulatory effect on the dissociation of Rab3A from the membrane.

Amino Acid Sequence↗
PubMed1995

Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins.

We previously purified a protein factor, named REKS (Ras-dependent Extracellular Signal-regulated Kinase (ERK)/mitogen-activated protein kinase Kinase (MEK) Stimulator), from Xenopus eggs by use of a cell-free assay system in which recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ki-Ras activates recombinant MEK. By use of this assay system, we purified here bovine REKS to near homogeneity from the cytosol fraction of bovine brain by successive chromatographies of Mono S, Mono Q, GTP gamma S-glutathione S-transferase-Ha-Ras-coupled glutathione-agarose, and Mono Q columns. It was composed of three proteins with masses of about 95, 32, and 30 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 95-, 32-, and 30-kDa proteins were identified by immunoblot analysis to be B-Raf protein kinase, 14-3-3 protein, and 14-3-3 protein, respectively. Moreover, the REKS activity was specifically immunoprecipitated by an anti-B-Raf antibody. Bovine REKS was activated by lipid-modified GTP gamma S-Ki-Ras far more effectively than by a lipid-unmodified one. Lipid-modified GDP-Ki-Ras was inactive. Exogenous addition of 14-3-3 proteins stimulated further the REKS activity both in the presence and absence of GTP gamma S-Ki-Ras. These results indicate that at least one of the direct targets of Ras is B-Raf complexed with 14-3-3 proteins in bovine brain.

14-3-3 Proteins↗
PubMed1995

Low molecular weight GTP-binding proteins in HL-60 granulocytes. Assessment of the role of ARF and of a 50-kDa cytosolic protein in phospholipase D activation.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.

ADP-Ribosylation Factors↗