Biomedical subjects

T Matsui

Publications and source records attributed to T Matsui.

At least 19 recordsLinked to original sources

PubMed1997

[A case of advanced gastric cancer that responded to long-term administration of low-dose 5'-DFUR].

A 83-year-old female suffering from advanced gastric cancer with paraaortic lymph node metastases was administered low-dose 5'-DFUR (600 mg/day) orally. The primary tumor reduced in size fifty days after the start of the treatment, and the swelled lymph node disappeared on abdominal CT scan. Specimens obtained by endoscopical biopsy were highly susceptible to pathological degeneration of the cancer. Low-dose 5'-DFUR was effective both for the primary lesion of gastric cancer and for the lymph node metastases in this case.

Aged↗

PubMed1996

Inhibition of metastasis by a dialysable factor in fetal bovine serum in B16 melanoma cells.

Fetal bovine serum (FBS) supplemented to the culture medium inhibited the metastasis of B16BL6 melanoma cells in a dose-dependent manner. This metastasis-inhibiting activity was accompanied by growth-promoting activity, up to the concentration of 10% FBS. However, among the clones isolated from B16BL6 cells, there were clones in which FBS significantly inhibited the metastasis without promoting their growth. Dialysis (cutoff M.W. 8000 Da) removed the metastasis-inhibiting activity but not the growth-promoting activity from FBS. These results indicate that FBS has metastasis-inhibiting activity which is independent of growth-promoting activity, and that the metastasis-inhibiting activity is carried by molecules removed by dialysis.

Animals↗

PubMed1996

The alpha3beta3gamma subcomplex of the F1-ATPase from the thermophilic bacillus PS3 with the betaT165S substitution does not entrap inhibitory MgADP in a catalytic site during turnover.

The hydrolytic properties of the mutant alpha3(betaT165S)3gamma and wild-type alpha3beta3gamma subcomplexes of TF1 have been compared. Whereas the wild-type complex hydrolyzes 50 microM ATP in three kinetic phases, the mutant complex hydrolyzes 50 microM ATP with a linear rate. After incubation with a slight excess of ADP in the presence of Mg2+, the wild-type complex hydrolyzes 2 mM ATP with a long lag. In contrast, prior incubation of the mutant complex under these conditions does not affect the kinetics of ATP hydrolysis. The ATPase activity of the wild-type complex is stimulated 4-fold by 0. 1% lauryl dimethylamine oxide, whereas this concentration of lauryl dimethylamine oxide inhibits the mutant complex by 25%. Compared with the wild-type complex, the activity of the mutant complex is much less sensitive to turnover-dependent inhibition by azide. This comparison suggests that the mutant complex does not entrap substantial inhibitory MgADP in a catalytic site during turnover, which is supported by the following observations. ATP hydrolysis catalyzed by the wild-type complex is progressively inhibited by increasing concentrations of Mg2+ in the assay medium, whereas the mutant complex is insensitive to increasing concentrations of Mg2+. A Lineweaver-Burk plot constructed from rates of hydrolysis of 20-2000 microM ATP by the wild-type complex is biphasic, exhibiting apparent Km values of 30 microM and 470 microM with corresponding kcat values of 26 and 77 s-1. In contrast, a Lineweaver-Burk plot for the mutant complex is linear in this range of ATP concentration, displaying a Km of 133 microM and a kcat of 360 s-1.

Adenosine Diphosphate↗

PubMed1996

Binding sites for Mg(II) in H(+)-ATPase from Bacillus PS3 and in the alpha 3 beta 3 gamma subcomplex studied by one-dimensional ESEEM and two-dimensional HYSCORE spectroscopy of oxovanadium(IV) complexes: a possible role for beta-His-324.

The binding sites for Mg2+ in wild type F1 ATPase (TF1) and in the alpha 3 beta 3 gamma subcomplex from the thermophilic bacterium Bacillus PS3 have been studied by EPR and by ESEEM and HYSCORE spectroscopy of complexes with the oxovanadium cation VO2+. Complexes of metal-depleted TF1 and substoichiometric amounts of VO2+ display low-temperature EPR signals with spectral parameters g parallel = 1.947 and g perpendicular = 1.980, and hyperfine couplings with 51V, A parallel = 169 x 10(-4) cm-1 and A perpendicular = 61 x 10(-4) cm-1, that are indicative of a binding site for VO2+ with nitrogen ligands from the protein. This binding site is probably identical with the metal binding site with strong affinity M1 that has been characterized using Mn2+ in a previous study [Buy, C., Girault, G., & Zimmermann, J. L. (1996) Biochemistry 35, 9880-9891]. The three-pulse ESEEM spectrum of the VO2+ complex with TF1 shows a frequency pattern with spectral properties that are evidence for two nitrogen ligands to the VO2+ with hyperfine couplings A1 = 4.75 MHz and A2 = 6.5 MHz and nuclear quadrupole parameters e2Qq1 = 2.8-3.2 MHz and e2Qq2 = 2.0-2.3 MHz. The ligands are identified as a lysine terminal amine and a histidine imidazole, which are proposed as Lys-164 and His-324 from a beta subunit. The HYSCORE data obtained for the VO.TF1 complex show correlations within each pair of the ESEEM nu dq peaks from the 14N nuclei, confirming the interpretation of the one-dimensional spectra. Evidence for the formation of a ternary complex by addition of VO2+ and ATP to metal-depleted TF1 is shown in the EPR and ESEEM spectra and in the contour plots of the HYSCORE data. Two pairs of correlation patterns are resolved in addition to the peaks from the two 14N ligands, which are interpreted as hyperfine couplings with 31P beta and 31P gamma of the ATP that binds the VO2+ cation. The assignment of the two hyperfine couplings to the specific phosphates, A(31P beta) = 15.5 MHz and A(31P gamma) = 8.7 MHz, in the VO.TF1.ATP complex is proposed by comparison with those measured for VO2+ in solution with ATP at pH 6.3 and 2.3. These results are discussed in light of the previous data with the analogous Mn.TF1 complex, and a model is proposed in which the native Mg2+ in the M1 site is coordinated by the side chain of beta-Lys-164 and is in close proximity to a histidine residue (probably beta-His-324) that may have a critical role. Additional coordination by two phosphates from ATP (probably the beta- and gamma-phosphates) is observed in the ternary complex VO.TF1.ATP. ESEEM and HYSCORE data are also obtained for the analogous complexes VO. alpha 3 beta 3 gamma and VO. alpha 3 beta 3 gamma .ATP that show very similar properties in terms of coordination of the divalent metal cation, except for the lysine ligand that is found to be lost in the ternary complex with ATP. It is suggested that this observation may reflect changes in the metal and nucleotide active sites that are associated with the absence of the delta and epsilon subunits in the subcomplex.

Adenosine Triphosphate↗

PubMed1996

Unusual pKa of the carboxylate at the putative catalytic position of the thermophilic F1-ATPase beta subunit determined by 13C-NMR.

Glutamic acid-190 in the beta subunit of F1-ATPase from thermophilic Bacillus PS-3 (TF1) was reported to be essential for the ATPase activity. The mutant TF1beta subunit in which Glu-190 had been substituted by cysteine was carboxymethylated with 13C-labeled monoiodoacetic acid. The pKa value of the carboxymethylene group at the 190 position was determined as 5.6 +/- 0.4 by 13C-NMR. On the basis of this value, the pKa of the carboxylate of Glu-190 of the TF1beta subunit was estimated to be 6.8 +/- 0.5. The unusually high pKa could play a role in the catalytic mechanism of F1-ATPase.

Adenosine Diphosphate↗

PubMed1996

G protein-coupled cholecystokinin-B/gastrin receptors are responsible for physiological cell growth of the stomach mucosa in vivo.

Many peptide hormone and neurotransmitter receptors belonging to the seven membrane-spanning G protein-coupled receptor family have been shown to transmit ligand-dependent mitogenic signals in vitro. However, the physiological roles of the mitogenic activity through G protein-coupled receptors in vivo remain to be elucidated. Here we have generated G protein-coupled cholecystokinin (CCK)-B/gastrin receptor deficient-mice by gene targeting. The homozygous mice showed a remarkable atrophy of the gastric mucosa macroscopically, even in the presence of severe hypergastrinemia. The atrophy was due to a decrease in parietal cells and chromogranin A-positive enterochromaffin-like cells expressing the H+,K(+)-ATPase and histidine decarboxylase genes, respectively. Oral administration of a proton pump inhibitor, omeprazole, which induced hypertrophy of the gastric mucosa with hypergastrinemia in wild-type littermates, did not eliminate the gastric atrophy of the homozygotes. These results clearly demonstrated that the G protein-coupled CCK-B/gastrin receptor is essential for the physiological as well as pathological proliferation of gastric mucosal cells in vivo.

Animals↗

PubMed1996

Preparation and reactions of myoglobin mutants bearing both proximal cysteine ligand and hydrophobic distal cavity: protein models for the active site of P-450.

We have reported that H93C human myoglobin (Mb), in which proximal histidine (His93, F8) was replaced by cysteine, gave nearly identical spectroscopic features of P-450 [Adachi, S., Nagano, S., Ishimori, K., Watanabe, Y., Morishima, I., Egawa, T., Kitagawa, T., & Makino R. (1993) Biochemistry 32, 241-252]. More importantly, the thiolate ligand enhanced its oxygenation activities when supported by H2O2 due to the exclusive encouragement of heterolytic O-O bond cleavage of peroxides. While we have attributed the enhanced heterolysis to the electron donation from the thiolate ligand, possible participation of the distal histidine (H64, E7) in H93C Mb cannot be eliminated. In addition, the racemic product formation catalyzed by H93C Mb implied that its distal cavity could prevent substrates from accessing to the heme and the reactions may proceed other than by the P-450 type mechanism (ferryl oxygen transfer). In order to clarify whether the distal histidine is involved in the O-O bond cleavage step and to improve accessibility of substrates, the distal histidine of H93C Mb is replaced by smaller and nonpolar residues, glycine (H64G/H93C Mb) and valine (H64V/H93C Mb), by site-directed mutagenesis. Various spectroscopic studies on these double-mutated Mbs revealed the ligation of cysteine to the ferric heme as a thiolate form. In the reaction with cumene hydroperoxide, the anionic nature of the proximal cysteine in H64G/H93C and H64V/H93C Mbs was found to encourage the heterolytic O-O bond cleavage as observed for H93C Mb. The results clearly demonstrate that the distal histidine of H93C Mb is hardly involved in the O-O bond cleavage step and are in good agreement with the role of thiolate ligation for the formation of the reactive intermediate, equivalent to compound I, in the catalytic cycle of P-450 reactions. In the oxygenation of methyl p-tolyl sulfide, the ratios of ferryl oxygen transfer increased in H64G/H93C Mb (58%) and H64V/H93C Mb (78%) as compared to H93C Mb (53%). The increased ratios of ferryl oxygen transfer imply the active site of H64G/H93C and H64V/H93C Mbs being more accessible for substrates; however, the sulfoxidation by the ferric mutant Mbs/H2O2 system was much slower than that by H93C Mb. The poor activities of these mutant Mbs are attributed to the significantly discouraged binding of H2O2.

Acetophenones↗

PubMed1996

Autocrine loop through cholecystokinin-B/gastrin receptors involved in growth of human leukemia cells.

The cholecystokinin (CCK)-B/gastrin receptor binds two brain-gut hormones, CCK and gastrin, with high affinities. These peptides have a trophic effect on gastrointestinal cells expressing the receptor in vivo as well as in vitro. Recently, this receptor mRNA was reported to be expressed in immunocytes localized in the lamina propria of normal rat stomach mucosa. Here, we studied the receptor expression in human hematopoietic cells in order to determine whether they play a role in cell growth. The CCK-B/gastrin receptor mRNA was detectable in the polymorphonuclear (PMN) cells but not in the mononuclear cells of normal peripheral white blood cells by reverse transcription-polymerase chain reaction. The receptor transcript was, however, expressed in human leukemia cell lines (14 of 18 cell lines tested) derived from not only myeloid, but also T- and B- lymphoid lineages. The CCK-B/gastrin receptors on several leukemia cell lines were shown to be biologically active by demonstrating ligand-dependent cell proliferation in serum-deprived medium. Interestingly, a human CCK-B/gastrin receptor specific antagonist, YM022, but not its stereotype isoform, selectively inhibited the DNA synthesis of THP-1, MOLT-16, MOLT-14, and CCRF-CEM in the absence of exogenous peptide ligands. Further investigation revealed that these leukemia cell lines and normal PMN cells also expressed gastrin mRNA. These results suggest that growth of human leukemia cells is promoted by an autocrine mechanism through the CCK-B/gastrin receptors.

Cell Division↗

PubMed1996

Purification and characterization of bitiscetin, a novel von Willebrand factor modulator protein from Bitis arietans snake venom.

We have screened 20 snake venoms and purified a novel snake venom protein, named bitiscetin, from Bitis arietans venom that specifically binds to human von Willebrand factor (vWF) and induces platelet agglutination. Bitiscetin showed a heterodimeric structure composed of disulfide-linked alpha (16kDa) and beta (13kDa) subunits on SDS-PAGE and showed a basic nature with pI value of 9.1, in contrast to botrocetin (pI 4.6), a vWF modulator isolated from another snake (Bothrops jararaca) venom. Bitiscetin-induced platelet agglutination was dependent on vWF and platelet membrane glycoprotein (GP) Ib, but not on Ca2+ and GPIIb/IIIa. vWF bound to bitiscetin but not to botrocetin electroblotted to a PVDF membrane after SDS-PAGE and this binding was diminished after reduction of disulfide bonds of bitiscetin. Bitiscetin did not cross-react to anti-botrocetin monoclonal antibodies. These results suggest that bitiscetin directly interacts with vWF and requires the protein conformation for its interaction as well as botrocetin, but its interaction manner with vWF appears to be different from that of botrocetin.

Amino Acid Sequence↗

PubMed1996

Enhancement and repression of cyanide-insensitive respiration in Aspergillus niger.

The biosynthesis of the alternative oxidase protein consisting of cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in Aspergillus niger was investigated. Cyanide-insensitive respiration was enhanced by addition of antimycin A to the incubation mixture, but repressed by the addition of cycloheximide, emetine, puromycin (cytosolic translation inhibitors), carbonylcyanide-m-chlorophenylhydrazone (uncoupler) and actinomycin D (transcription inhibitor). These results show that the alternative oxidase protein of A. niger was synthesized de novo in cytosol and transported in mitochondria.

Antimycin A↗

PubMed1996

Intrinsic signaling function of APP as a novel target of three V642 mutations linked to familial Alzheimer's disease.

APP695 is a transmembrane precursor of Abeta amyloid. In familial Alzheimer's disease (FAD), three mutations V642I/F/G were discovered in APP695, which has been suggested by multiple studies to be a cell surface signaling receptor. We previously reported that normal APP695 encodes a potential GO-linked receptor with ligand-regulated function and that expression of the three FAD mutants (FAD-APPs), not normal APP, induces cellular outputs by GO-dependent mechanisms. This suggests that FAD-APPs are constitutively active GO-linked receptors. Here, we provide direct evidence for this notion. Reconstitution of either recombinant FAD-APP with GO vesicles induced activation of GO, which was inhibitable by pertussis toxin, sensitive to Mg2+ and proportional in quantity to the reconstituted amounts of FAD-APP. Consistent with the dominant inheritance of this type of FAD, this function was dominant over normal APP, because little activation was observed in APP695-GO vesicles. Experiments with antibody competition and sequence deletion indicated that His657-Lys676 of FAD-APP, which has been specified as the ligand-dependent GO-coupling domain of normal APP, was responsible for this constitutive activation, confirming that the three FAD-APPs are mutationally activated APP695. This study identifies the intrinsic signaling function of APP to be a novel target of hereditary Alzheimer's disease mutations, providing an in vitro system for the screening of potential FAD inhibitors.

Alzheimer Disease↗

PubMed1996

G protein-mediated neuronal DNA fragmentation induced by familial Alzheimer's disease-associated mutants of APP.

Missense mutations in the 695-amino acid form of the amyloid precursor protein (APP695) cosegregate with disease phenotype in families with dominantly inherited Alzheimer's disease. These mutations convert valine at position 642 to isoleucine, phenylalanine, or glycine. Expression of these mutant proteins, but not of normal APP695, was shown to induce nucleosomal DNA fragmentation in neuronal cells. Induction of DNA fragmentation required the cytoplasmic domain of the mutants and appeared to be mediated by heterotrimeric guanosine triphosphate-binding proteins (G proteins).

Alzheimer Disease↗

PubMed1996

Negative transactivation of cAMP response element by familial Alzheimer's mutants of APP.

In familial Alzheimer's disease (FAD), missense point mutations V642I/F/G, which co-segregate with the disease phenotype, have been discovered in amyloid precursor APP695. Here, we report that three FAD mutants (FAD-APPs) negatively regulated the transcriptional activity of cAMP response element (CRE) by a G(o)-dependent mechanism, but expression of wildtype APP695 had no effect on CRE. Experiments with various Galpha(s) chimeras demonstrated that Phe-APP coupled selectively to the C-terminus of Galpha(0). Again, wild-type APP695 had no effect on its C-terminus. These data indicate that FAD-APPs are gain-of-function mutants of APP695 that negatively regulate the CRE activity through G(o). This negative transactivation of CRE is the first biochemically analyzed signal evoked by the three FAD-APPs, but not by wild-type APP695, in a whole-cell system. We discuss the significance of constitutive CRE suppression by FAD-APPs, which is potentially relevant to synaptic malplasticity or memory disorders.

Alzheimer Disease↗

PubMed1996

Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway.

3T3 Cells↗

PubMed1996

Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers.

Specific receptors for brain-gut peptide hormones, cholecystokinin (CCK) and gastrin, are expressed in a variety of human tumor cells. CCK and gastrin promote the growth of NIH3T3 cells into which the CCK-B/gastrin receptor had been introduced via a eukaryotic expression vector. In this study, we have examined the effect of CCK-8 on the actin cytoskeleton by using two mouse fibroblast cell lines expressing human CCK-B/gastrin receptors. Treatment with very low concentration of CCK-8 (10(-10) M) induced the formation of actin stress fibers within one minute. Stress fiber formation increased for 30 min. In contrast, a potent mitogen for fibroblasts, platelet-derived growth factor (PDGF), initially induced membrane ruffling and, later, a weak formation of stress fibers. Microinjection of rho GDP dissociation inhibitor or Clostridium botulinum ADP-ribosyltransferase C3 which is known to impair the function of a small GTP-binding protein, rho p21, inhibited the stress fiber formation by CCK-8 as well as by PDGF. These results indicate that CCK-B/gastrin receptor could regulate stress fiber formation in a rho p21-dependent manner. The signals from CCK-B/gastrin receptor might affect cell growth as well as cell motility or adhesion by regulating the actin cytoskeleton.

3T3 Cells↗

PubMed1996

Differential activation of the murine laminin B1 gene promoter by RAR alpha, ROR alpha, and AP-1.

RAR alpha, ROR alpha and AP-1 activated the transcription of the murine laminin B1 gene promoter, which consists of three core elements. Cotransfection experiments showed that AP-1-activated transcription is further stimulated by RAR alpha, but not by ROR alpha. Additionally, it was demonstrated by promoter mutagenesis that all of the three core elements are required for transactivation by RAR alpha and ROR alpha, while any one of them is dispensable for AP-1-activated transcription. These results suggest that the modes of transactivation of the laminin B1 promoter are different among the three transactivators.

Animals↗

PubMed1996

An attempt to convert noncatalytic nucleotide binding site of F1-ATPase to the catalytic site: hydrolysis of tethered ATP by mutated alpha subunits in the enzyme.

The alpha and beta subunits of F1-ATPase are homologous in primary structure and have similar folding topologies. The position of the essential Glu residue in the catalytic sites which reside in the beta subunits is occupied by a Gln residue in the noncatalytic nucleotide binding sites which reside in the alpha subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replaced the Gln-Lys sequence in the noncatalytic binding site of the alpha subunit with Glu-Arg and, reciprocally, the Glu in the catalytic site of the beta subunit with Gln. The resultant mutant alpha3beta3gamma complex lost steady-state ATPase activity. However, HPLC analysis of tryptic digests of the mutant alpha3beta3gamma complex which had been photolabeled with 2-N3-[8-3H]ATP revealed that ATP tethered to the noncatalytic binding side was hydrolyzed, indicating that a primitive catalytic ability was generated at the alpha subunit by the introduced Glu.

Adenosine Triphosphate↗

PubMed1996

Structural asymmetry of F1-ATPase caused by the gamma subunit generates a high affinity nucleotide binding site.

The alpha 3 beta 3 gamma and alpha 3 beta 3 complexes of F1-ATPase from a thermophilic Bacillus PS3 were compared in terms of interaction with trinitrophenyl analogs of ATP and ADP (TNP-ATP and TNP-ADP) that differed from ATP and ADP and did not destabilize the alpha 3 beta 3 complex. The results of equilibrium dialysis show that the alpha 3 beta 3 gamma complex has a high affinity nucleotide binding site and several low affinity sites, whereas the alpha 3 beta 2 complex has only low affinity sites. This is also supported from analysis of spectral change induced by TNP-ADP, which in addition indicates that this high affinity site is located on the beta subunit. Single-site hydrolysis of substoichiometric amounts of TNP-ATP by the alpha 3 beta 3 gamma complex is accelerated by the chase addition of excess ATP, whereas that by the alpha 3 beta 3 complex is not. We further examined the complexes containing mutant beta subunits (Y341L, Y341A, and Y341C). Surprisingly, in spite of very weak affinity of the isolated mutant beta subunits to nucleotides (Odaka, M., Kaibara, C., Amano, T., Matsui, T., Muneyuki, E., Ogasawara, K, Yutani, K., and Yoshida, M. (1994) J. Biochem. (Tokyo) 115, 789-796), a high affinity TNP-ADP binding site is generated on the beta subunit in the mutant alpha 3 beta 3 gamma complexes where single-site TNP-ATP hydrolysis can occur. ATP concentrations required for the chase acceleration of the mutant complexes are higher than that of the wild-type complex. The mutant alpha 3 beta 3 complexes, on the contrary, catalyze single-site hydrolysis of TNP-ATP rather slowly, and there is no chase acceleration. Thus, the gamma subunit is responsible for the generation of a high affinity nucleotide binding site on the beta subunit in F1-ATPase where cooperative catalysis can proceed.

Adenosine Diphosphate↗