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Biomedical subjects

Y Takai

Publications and source records attributed to Y Takai.

At least 163 records · Page 9Linked to original sources
PubMed1998

Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae.

We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method. The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.

Actins↗
PubMed1998

Involvement of Rabphilin3 in endocytosis through interaction with Rabaptin5.

Rabphilin3 and rabaptin5 are downstream target molecules of the Rab3 and -5 subfamily small G proteins that are implicated in exocytosis and endocytosis, respectively. We examined here the physical and functional relationship between the Rab3-rabphilin3 and Rab5-rabaptin5 systems. Rabphilin3 interacted with rabaptin5 at the N-terminal region (amino acids 1-280), which GTP-Rab3A interacted with. The interaction of rabphilin3 with rabaptin5 was inhibited by guanosine 5'-(3-O-thio)triphosphate-Rab3A. Overexpression of the N-terminal fragment of rabphilin3 (amino acids 1-280) inhibited the receptor-mediated endocytosis of transferrin, and this inhibition was overcome by co-transfection with a dominant active mutant of Rab3A or rabaptin5 in PC12 and HeLa cells. These results suggest that rabphilin3, free of GTP-Rab3A, regulates endocytosis through interaction with rabaptin5 after rabphilin3 complexed with GTP-Rab3A regulates exocytosis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP.

Cdc42 is a small GTPase of the Rho family which regulates the formation of actin filaments to generate filopodia. Although there are several proteins such as PAK, ACK and WASP (Wiskott-Aldrich syndrome protein) that bind Cdc42 directly, none of these can account for the filopodium formation induced by Cdc42. Here we demonstrate that before it can induce filopodium formation, Cdc42 must bind a WASP-related protein, N-WASP, that is richest in neural tissues but is expressed ubiquitously. N-WASP induces extremely long actin microspikes only when co-expressed with active Cdc42, whereas WASP, which is expressed in haematopoietic cells, does not, despite the structural similarities between WASP and N-WASP. In a cell-free system, addition of active Cdc42 significantly stimulates the actin-depolymerizing activity of N-WASP, creating free barbed ends from which actin polymerization can then take place. This activation seems to be caused by exposure of N-WASP's actin-depolymerizing region induced by Cdc42 binding.

3T3 Cells↗
PubMed1998

Sensitization of cells to ionizing radiation by chlorin e6Na.

We have investigated whether a hydrophilic photosensitizer, chlorin e6Na (Ce6Na), can sensitize cells to ionizing radiation. When V79-1 cells were pretreated with Ce6Na for 2 hr before receiving a dose of 2-14 Gy of irradiation, the cells became sensitive to X-irradiation. The sensitizing effect of Ce6Na depended on the dose of Ce6Na. The sensitizing effect also depended on the length of the treatment period before X-irradiation up to 4 hr, but not on the length of a treatment period after X-irradiation. Intracellular concentrations of Ce6Na were increased linearly after incubation with Ce6Na for periods of up to 4 h. The dose-modifying factor calculated from the survival curve was 1.25.

Animals↗
PubMed1998

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats.

S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S10100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0-5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands.

Animals↗
PubMed1998

Localization of Ca(2+)-binding S100 proteins in epithelial tumours of the skin.

The Ca(2+)-binding proteins S100A1, S100A2, S100A4, S100A6 and S100B were evaluated immunohistochemically in normal skin and skin appendage tumours. Epidermal basal cells, epithelial cells of sebaceous glands, hair follicle sheet epithelia and eccrine duct reacted strongly with an antiserum against human S100A2 but were nonreactive or weakly reactive to S100A1, S100A4, S100A6 and S100B. Varying types of skin appendage tumours and most peripheral cells in tumour nests of basal cell carcinoma and squamous cell carcinoma showed positive S100A2 immunoreactivity in neoplastic cells corresponding to basal cells but were nonreactive or faintly reactive for other S100 proteins. Langerhan's cells and melanocytes were labelled by S100B. Basophilic cells of calcifying epithelioma were occasionally stained with S100A2 antiserum. Eccrine poroma did not react with any S100 antiserum. Mixed tumours of the skin containing neoplastic myoepithelial cells stained strongly for S100A2 and S100B but only faintly for S100A1, S100A4, S100A6. This is the first report on selective evaluation of different S100 proteins in normal skin. These antibodies are valuable tools for better characterization of skin appendage tumours.

Animals↗
PubMed1998

Synaptic PDZ domain-containing proteins.

Synaptic junctions accumulate various molecules involved in neurotransmissions and synaptic plasticity. The proper organizations of these molecules at synaptic junctions sustain the interneuronal signal transmissions and the subsequent signal cascades underling learning and memory. Recent studies have revealed that a protein-interacting module named a PDZ domain plays important roles in the interactions among the components of synaptic junctions. In this article, we summarize synaptic PDZ domain-containing proteins and discussed their functions.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Animals↗
PubMed1998

Control of reorganization of the actin cytoskeleton by Rho family small GTP-binding proteins in yeast.

Accumulating evidence indicates that Rho family small GTP-binding proteins regulate reorganization of the actin cytoskeleton. There are members of the Rho family in the budding yeast Saccharomyces cerevisiae, in which powerful molecular genetical approaches are applicable. Recent identification of regulators and targets of the Rho family members has enhanced our understanding of the regulation and modes of action of Rho family members in reorganization of the actin cytoskeleton.

Actin Cytoskeleton↗
PubMed1998

Isolation and characterization of cortactin isoforms and a novel cortactin-binding protein, CBP90.

BACKGROUND: Cortactin is a major phosphotyrosyl protein in pp60v-src-transformed chicken embryo cells. Cortactin binds to actin filament (F-actin) through a unique region which consists of six tandem 37 amino acid repeats, named cortactin repeats. Furthermore cortactin has one src homology 3 (SH3) domain. RESULTS: In this study we have isolated two new isoforms of cortactin from the rat brain using a polymerase chain reaction (PCR) method, and have named the original cortactin as cortactin-A and the newly isolated forms as cortactin-B and -C. Cortactin-A, -B and -C had six, five, and four cortactin repeats, respectively. All the isoforms were able to bind to F-actin, but only cortactin-A demonstrated an F-actin-crosslinking activity. In addition, cortactin-A was able to bind along the side of F-actin. Next, using a blot overlay assay with glutathione S-transferase (GST)-cortactin-A, we identified a cortactin-A-binding protein with an Mr of approximately 90 kDa in rat brain and named it CBP90 (cortactin-binding protein with an Mr of approximately 90 KDa). CBP90 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. The deduced amino acid sequence of CBP90 had no significant similarity to any other protein, but it had a proline-rich domain at the C-terminal region. CBP90 was able to bind to all the cortactin isoforms. A deletion mutant analysis of cortactin-A and CBP90 revealed that the SH3 domain of cortactin-A was able to bind to the proline-rich region of CBP90. A Western blot analysis with an anti-CBP90 antibody indicated that, among the rat tissues examined, CBP90 was exclusively expressed in brain. Furthermore, its subcellular distribution and developmental expression patterns were similar to those of cortactin. CONCLUSION: These results suggest that cortactin interacts with CBP90 and plays a role in regulation of the actin cytoskeleton in brain.

Actins↗
PubMed1998

Fatal hemorrhage in irradiated esophageal cancer patients.

Between 1980 and 1994, 423 patients with esophageal cancer were given curative radiation therapy. Of these patients, 31 died of massive hemorrhage and were used as the subjects of analysis in this study. The incidence of massive hemorrhage in all patients was 7% (31/423). In the 31 patients who died of massive hemorrhage, 27 had local tumors and two had no tumors at hemorrhage (two unknown cases). The mean time interval from the start of radiation to hemorrhage was 9.2 months. In 9 autopsy cases the origin of hemorrhage was a tear of the aorta in 5 cases, necrotic local tumor in 3 cases and esophageal ulcer in 1 case. The positive risk factors for this complication seemed to be excess total dose, infection, metallic stent, and tracheoesophageal fistula. Chest pain or sentinel hemorrhage proceeding to massive hemorrhage was observed in about half of the patients.

Aged↗
PubMed1998

Rabphilin-3A: a multifunctional regulator of synaptic vesicle traffic.

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH2-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and beta-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH2 terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at the bud site and regulation of the actin cytoskeleton in Saccharomyces cerevisiae.

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213-1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826-987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

Actins↗
PubMed1998

Rho and Rab small G proteins coordinately reorganize stress fibers and focal adhesions in MDCK cells.

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.

Actins↗
PubMed1998

Prenatal diagnosis of Fukuyama-type congenital muscular dystrophy by microsatellite analysis.

We applied microsatellite analysis to prenatal diagnosis of Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive severe muscular dystrophy associated with brain malformations. Recent identification of the FCMD gene locus at 9q31-q33 provided the basis for prenatal diagnosis and carrier detection. We recently developed new microsatellite markers which are closer to the FCMD gene and improved the phenotype probability. Nine fetuses in eight unrelated FCMD families, including a twin pregnancy, were analysed using the newly developed markers. Four fetuses showed over 99% probability of being healthy either as normal homozygote (n = 1) or heterozygote carrier (n = 3) and were born without signs of FCMD. The other five fetuses were diagnosed with a probability of FCMD of 99% or greater; all of the latter parents decided to terminate the pregnancies. Brain malformations characteristic of FCMD in one of the aborted fetuses confirmed the diagnosis of FCMD at 19 weeks of gestation.

Adult↗
PubMed1998

Disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes during embryogenesis.

Parthenogenetic embryos, which contained one genome from a neonate-derived non-growing oocyte and the other from a fully grown oocyte, developed to day 13.5 of gestation in mice, 3 days longer than previously recorded for parthenogenetic development. To investigate the hypothesis that disruption of primary imprinting during oocyte growth leads to the modified expression of imprinted genes and this parthenogenetic phenotype, we have examined Peg1/Mest, Igf2, Peg3, Snrpn, H19, Igf2r and excess p57KIP2. We show that paternally expressed genes, Peg1/Mest, Peg3 and Snrpn, are expressed in the parthenotes, presumably due to a lack of maternal epigenetic modifications during oocyte growth. In contrast, the expression of Igf2, which is repressed in a competitive manner by transcription of the H19 gene, was very low. Furthermore, we show that the maternally expressed Igf2r and p57KIP2 genes were repressed in the alleles of the non-growing oocyte indicating maternal modifications during oocyte growth are necessary for its expression. Thus, our results show that primary imprinting during oocyte growth exhibits a crucial effect on both the expression and repression of maternal alleles during embryogenesis.

Animals↗
PubMed1998

Improved survival of children with advanced tumors by myeloablative chemotherapy and autologous peripheral blood stem cell transplantation in complete remission.

Five children with neuroblastoma (NB) stage IV and five children with rhabdomyosarcoma (RMS) stage III were treated with myeloablative chemotherapy and autologous peripheral blood stem cell transplantation (MCT/PBSCT) in the state of complete remission (CR) achieved by conventional therapy. PBSCs were collected in CR status using a cell separator with blood access through a double-lumen central venous catheter. PBSCs with 1.9+/-0.8x10(5) of CFU-GM per patient weights (kg) were infused following MCT after a period of conventional therapy for 11.1+/-2.1 or 9.7+/-0.9 months in NB or RMS patients, respectively. Regimen-related toxicity of MCT was tolerable and peripheral white blood cell count recovered beyond 1.0x10(3)/microl 10-12 days after infusion of PBSCs in all patients. All of RMS patients and three of five NB patients survived for an average of 31.6 months (ranged 10.8-58.1). The survival rate of these patients was improved as compared with our historical controls, and presumably, with that of conventional chemotherapy previously reported. Despite a limited number of patients, it appears that MCT/PBSCT may be effective in improving survival by preventing relapse which may occur thereafter if treated with conventional therapy alone. Furthermore, MCT/PBSCT reduced the duration of treatment, as compared with that of conventional chemo-therapy. Therefore, this study may suggest the feasibility and promise of the therapy including MCT/PBSCT for children with advanced stages of NB and RMS.

Antineoplastic Agents↗