Biomedical subjects

A Kikuchi

Publications and source records attributed to A Kikuchi.

At least 73 records · Page 4Linked to original sources

PubMed2000

Inhibition of Wnt signaling pathway by a novel axin-binding protein.

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin.

Adenomatous polyposis coli gene product (APC) functions as a tumor suppressor and its mutations in familial adenomatous polyposis and colorectal cancers lead to the accumulation of cytoplasmic beta-catenin. The molecular mechanism by which APC regulates the stability of beta-catenin was investigated. The central region of APC, APC-(1211-2075), has the beta-catenin- and Axin-binding sites and down-regulates beta-catenin. Glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylated beta-catenin slightly in the presence of either APC-(1211-2075) or Axin(delta)(beta)(-catenin), in which the beta-catenin-binding site is deleted, and greatly in the presence of both proteins. The enhancement of the GSK-3 beta-dependent phosphorylation of beta-catenin was eliminated by the APC-binding site of Axin. Axin down-regulated beta-catenin in SW480 cells, but not Axin(delta)(beta)(-catenin). In L cells where APC is intact, Axin(delta)(beta)(-catenin) inhibited Wnt-dependent accumulation of beta-catenin but not Axin-(298-832)(delta)(beta)(-catenin) in which the APC- and beta-catenin-binding sites are deleted. These results indicate that the complex formation of APC and Axin enhances the phosphorylation of beta-catenin by GSK-3 beta, leading to the down-regulation of beta-catenin.

Adenomatous Polyposis Coli Protein↗

PubMed2000

A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation.

beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.

Amino Acid Sequence↗

PubMed2000

Ribozyme oligonucleotides against transforming growth factor-beta inhibited neointimal formation after vascular injury in rat model: potential application of ribozyme strategy to treat cardiovascular disease.

BACKGROUND: Because the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in transforming growth factor (TGF)-beta, a selective decrease in TGF-beta may have therapeutic value. Thus, we used the ribozyme strategy to actively cleave the targeted gene to selectively inhibit TGF-beta expression. METHODS AND RESULTS: We constructed ribozyme oligonucleotides (ONs) targeted to the sequence of the TGF-beta gene that shows 100% homology among the human, rat, and mouse species. The specificity of ribozyme against TGF-beta gene was confirmed by selective inhibition of TGF-beta mRNA in cultured vascular smooth muscle cells as well as balloon-injured blood vessels in vivo. Importantly, the marked decrease in TGF-beta resulted in significant inhibition of neointimal formation after vascular injury in a rat carotid artery model (P:<0.01), whereas DNA-based control ONs and mismatched ribozyme ONs did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by (1) a reduction in collagen synthesis and mRNA expression of collagen I and III and (2) a significant decrease in DNA synthesis as assessed by proliferating cell nuclear antigen staining. Moreover, we modified ribozyme ONs containing phosphorothioate DNA and RNA targeted to the TGF-beta gene. Of importance, modified ribozyme ONs showed a further reduction in TGF-beta expression. CONCLUSIONS: Overall, this study provides the first evidence that selective blockade of TGF-beta resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that modification of ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.

Animals↗

PubMed2000

Assignment of functional amino acids around the active site of human DNA topoisomerase IIalpha.

An expression library for active site mutants of human topoisomerase IIalpha (TOP2alpha) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2alpha mutants, no substitution was allowed at Tyr(805), the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu(794), Asp(797), Ala(801), and Arg(804). Thus, these 5 residues are critical for human TOP2alpha activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser(803) and Lys(798), which confer drug resistance, lie adjacent to this plane.

Amino Acid Sequence↗

PubMed2000

Alpha-glycosylceramides enhance the antitumor cytotoxicity of hepatic lymphocytes obtained from cancer patients by activating CD3-CD56+ NK cells in vitro.

Alpha-glycosylceramides, such as alpha-galactosylceramide and alpha-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, V alpha 14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by alpha-glycosylceramides. We recently reported that V alpha 24 TCR+ NKT cells, the human homologues of murine V alpha 14 TCR+NK1.1+ cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether alpha-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of alpha-glycosylceramides in cancer immunotherapy in humans. Here, we show that alpha-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3-CD56+ NK cells. Even though V alpha 24 TCR+NKT cells proliferated remarkably in response to alpha-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of alpha-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3-CD56+ NK cells in the liver.

Adjuvants, Immunologic↗

PubMed2000

Haemopoietic biglycan produced by brain cells stimulates growth of microglial cells.

We have recently found that soluble biglycan purified from rat thymic myoid cells had haemopoietic activity capable of inducing preferential growth and differentiation of monocytic lineage cells from various haemopoietic sources, including brain microglial cells. In the present study, to understand developmental mechanisms of microglial/monocytic cells in the brain, we have attempted to identify haemopoietic activity of the brain biglycan. The mRNA and the immunological epitope of biglycan were detected in the rat brain homogenates and several rat glial cell lines. Immunohistochemical study showed that several different types of brain cells produced biglycan. During development biglycan synthesis in the brain appeared to be increased. The brain haemopoietic biglycan was easily separated by DEAE-Sepharose chromatography from the macrophage colony stimulating factor (M-CSF) which was concomitantly produced from the brain cells. The brain haemopoietic biglycan, purified through immunoaffinity column, indeed stimulated growth of primarily cultured microglial cells. Taken together, these results suggest that the haemopoietic biglycan plays an important role in generating brain-specific circumstances for development of microglial/monocytic cells.

Animals↗

PubMed2000

Regulation of complex formation of POB1/epsin/adaptor protein complex 2 by mitotic phosphorylation.

RalBP1 and POB1, the downstream molecules of small GTP-binding protein Ral, are involved in receptor-mediated endocytosis together with Epsin and Eps15. The regulation of assembly of the complex of these proteins was examined. RalBP1, POB1, Epsin, and Eps15 formed a complex with alpha-adaptin of AP-2 in Chinese hamster ovary cells, but the formation was reduced in mitotic phase. RalBP1, POB1, Epsin, and Eps15 were all phosphorylated in mitotic phase. The phosphorylated forms of POB1 and Epsin were recognized by the antibody MPM2, which is known to detect mitotic phosphoproteins. POB1 and Epsin were phosphorylated by p34(cdc2) kinase in vitro. Their phosphorylation sites (Ser(411) of POB1 and Ser(357) of Epsin) were determined. Phosphorylated Epsin and Epsin(S357D) formed a complex with alpha-adaptin less efficiently than wild type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, Epsin(S357D) but not Epsin(S357A) lost the effect of Epsin on the insulin-dependent endocytosis. These results suggest that phosphorylation of Epsin in mitotic phase inhibits receptor-mediated endocytosis by disassembly of its complex with POB1 and alpha-adaptin.

Adaptor Proteins, Vesicular Transport↗

PubMed2000

Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion.

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.

Adenosine Triphosphate↗

PubMed2000

NE-dlg, a mammalian homolog of Drosophila dlg tumor suppressor, induces growth suppression and impairment of cell adhesion: possible involvement of down-regulation of beta-catenin by NE-dlg expression.

Membrane-associated guanylate kinases (MAGUKs) are known to function as scaffolds for forming multiprotein complexes at the synaptic junctions of neuronal cells and at sites of epithelial cell-cell contact. In Drosophila, mutations of the lethal (1)-discs large (dlg) gene, which encodes a MAGUK protein, leads to post-synaptic structure defects in neuronal cells and neoplastic overgrowth of epithelial cells. We previously showed that NE-dlg (neuronal and endocrine dlg), a human homolog of the dlg, plays a crucial role in formation of synaptic structure in human neuronal cells. Here we demonstrate that NE-dlg, similar to Drosophila dlg, is involved in regulation of cell cycle progression and adhesive ability of non-neuronal cells. Overexpression of NE-dlg in proliferating cells including various cancer cell lines induced growth suppression and impairment of cell adhesive ability. Furthermore, NE-dlg overexpression caused the down-regulation of beta-catenin in cancer cells regardless of mutations in the APC (adenomatous polyposis coli) gene. The PDZ domains of NE-dlg were found to be essential for the growth suppression, loss of adhesive property and down-regulation of beta-catenin. We propose that NE-dlg regulates the cell growth and adhesive ability by controlling the level of beta-catenin through an APC-independent pathway. Inactivation of NE-dlg may therefore contribute to development and/or progression of human neoplasms.

Adaptor Proteins, Signal Transducing↗

PubMed2000

NBP1 (Nap1 binding protein 1), an essential gene for G2/M transition of Saccharomyces cerevisiae, encodes a protein of distinct sub-nuclear localization.

Nap1p is identified in mammalian cell extract by its intrinsic activity to facilitate nucleosome assembly in vitro in the physiological ionic condition. The homologous proteins are present in most eukaryotes, and their functional analyses in vitro have suggested that they are necessary to keep proper nucleosome structures in transcription and replication. This protein is also identified for its interaction with Clb2p in vitro. To address the function of Nap1p in vivo, we have surveyed for proteins to interact with Nap1p by two-hybrid system and obtained two genes, NBP1 and NBP2 (Nap1 Binding Protein 1 and 2). NBP1 is an essential gene and encodes a novel protein consisting of 319 amino acids, with a coiled-coil structure in the center of the predicted amino acid sequence. Several A-kinase dependent phosphorylation sites and Cdc28p kinase-dependent sites are also observed. By isolating the temperature-sensitive mutant, we demonstrate that the nuclear division at a non-permissive temperature is delayed and that the population of cells with a large bud carrying a single nucleus with a short spindle are increased. This mutant also confers resistance against benomyl, a microtubule-destabilizing agent. Judging from the green fluorescent protein (GFP) signal fused with Nbp1p, this protein localizes in the nucleus as one or two tiny dots.

Amino Acid Sequence↗

PubMed2000

Regulation of beta-catenin signaling in the Wnt pathway.

beta-Catenin not only regulates cell to cell adhesion as a protein interacting with cadherin, but also functions as a component of the Wnt signaling pathway. The Wnt signaling pathway is conserved in various organisms from worms to mammals, and plays important roles in development, cellular proliferation, and differentiation. Wnt stabilizes cytoplasmic beta-catenin and then beta-catenin is translocated into the nucleus where it stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. The amounts and functions of beta-catenin are regulated in both the cytoplasm and nucleus. Its molecular mechanisms are becoming increasingly well understood.

Animals↗

PubMed2000

GSK-3beta-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by beta-catenin and protein phosphatase 2A complexed with Axin.

Axin forms a complex with adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3beta (GSK-3beta), and beta-catenin through different binding sites and downregulates beta-catenin. GSK-3beta-dependent phosphorylation of APC-(1211-2075) which has the Axin-binding site was facilitated by Axin, but that of APC-(959-1338) which lacks the Axin-binding site was not. Axin-(298-506) or Axin-(298-832), which has the GSK-3beta- and beta-catenin- but not APC-binding sites, did not enhance GSK-3beta-dependent phosphorylation of either APC-(1211-2075) or APC-(959-1338). Furthermore, beta-catenin stimulated the phosphorylation of APC-(959-1338) and APC-(1211-2075) by GSK-3beta in the presence of Axin. Consistent with these in vitro observations, expression of beta-catenin or Axin in COS cells promoted an SDS gel band shift of APC. These results indicate that APC complexed with Axin is effectively phosphorylated by GSK-3beta and that beta-catenin may modulate this phosphorylation. In addition, the heterodimeric form of protein phosphatase 2A (PP2A) directly bound to Axin, and PP2A complexed with Axin dephosphorylated APC phosphorylated by GSK-3beta. Taken together, these results suggest that GSK-3beta-dependent phosphorylation of APC can be modulated by beta-catenin and PP2A complexed with Axin.

Adenomatous Polyposis Coli Protein↗

PubMed2000

Rapid cell sheet detachment from poly(N-isopropylacrylamide)-grafted porous cell culture membranes.

Fabrication of functional tissue constructs using sandwiched layers of cultured cells could prove to be an attractive approach to tissue engineering. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheet and prevents functional damage. To accelerate the required culture substrate hydrophilic and hydrophobic structural changes in response to culture temperature alteration, poly(N-isopropylacrylamide) (PIPAAm) was grafted onto porous culture membranes by electron beam irradiation. Analyses by attenuated total reflection-Fourier transform IR and electron spectroscopy for chemical analysis revealed that PIPAAm was successfully grafted to surfaces of porous membranes. Atomic force microscopy (AFM) results showed that PIPAAm-grafted membranes had smoother surfaces than ungrafted controls while retaining their porous structure. The mean roughness of PIPAAm-grafted and -ungrafted porous membrane surfaces determined by digital AFM autocalculation was 4.40 +/- 0.4 and 5.9 +/- 0.4 nm, respectively. Tissue culture polystyrene (TCPS) dishes grafted with PIPAAm were compared with PIPAAm-grafted porous membranes in cell sheet detachment experiments. Approximately 75 min was required to completely detach cell sheets from PIPAAm-grafted TCPS surfaces compared to only 30 min to detach cell sheets from PIPAAm-grafted porous membranes. With porous membranes, the water accesses the PIPAAm-grafted surface from underneath and peripheral to the attached cell sheet, resulting in rapid hydration of grafted PIPAAm molecules and detachment of the cell sheet. With TCPS PIPAAm-grafted surfaces the water is supplied from only the periphery of a cell sheet, slowing detachment.

Acrylic Resins↗

PubMed2000

Temperature-responsive culture dishes allow nonenzymatic harvest of differentiated Madin-Darby canine kidney (MDCK) cell sheets.

We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37 degrees C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37 degrees C, trypsin digestion disrupted cell-cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20 degrees C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-beta-catenin antibody revealed that functional cell-cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20 degrees C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction.

Acrylic Resins↗

PubMed2000

DNA single-strand breaks are increased in muscle diseases with rimmed vacuoles.

Some pathological similarities between Alzheimer's disease and muscle diseases with rimmed vacuoles (RV) have been pointed out. For example, several pathological hallmark proteins have been reported to be immunopositive in the lesions of both diseases. Since apoptotic processes or primary DNA damage are suggested to play a role in the pathomechanism of Alzheimer's disease, we examined DNA double-strand breaks (DSB) and single-strand breaks (SSB) in the muscle biopsy specimens of several diseases, including muscle diseases with RV. Although no DSB-positive myonuclei were detected in any muscles examined, the number of SSB-positive myonuclei markedly increased in the muscles from cases with polymyositis and muscle diseases with RV. In polymyositis, SSB-positive myonuclei were observed in regenerating fibers and muscle fibers in the vicinity of inflammatory infiltrates, suggesting that the increase of SSB is due to muscle fiber regeneration following necrosis and inflammation. In muscle diseases with RV, however, SSB-positive myonuclei were observed in small angulated fibers and in morphologically normal fibers, regardless of necrosis, regeneration or inflammation. These findings suggest that muscle diseases with RV may share a common pathological process involving DNA damage.

Adolescent↗

PubMed2000

Release of adsorbed fibronectin from temperature-responsive culture surfaces requires cellular activity.

We have previously developed a temperature-responsive cell culture surface by grafting poly(N-isopropylacrylamide) that changes its surface hydrophobicity in response to temperature. While this surface shows similar hydrophobicity to that of commercial polystyrene cell culture surfaces and facilitates cell adhesion and proliferation at 37 degrees C, grafted polymer becomes hydrophilic below 32 degrees C and releases spread cultured cells without trypsin. Temperature-regulated cell detachment requires cell metabolic activity requiring ATP consumption, signal transduction, and cytoskeleton reorganziation. Precoating these surfaces with fibronectin (FN) improves spreading of less adhesive cultured hepatocytes and reducing culture temperature releases cultured cells from FN-adsorbed grafted surfaces. Immunostaining with anti-FN antibody revealed that only FN located beneath cultured cells is removed from culture surfaces after reducing temperature. FN adsorbed to surface areas lacking direct cell attachment remained surface-bound after reducing temperature. A novel concept of active cell detachment is also discussed.

Acrylic Resins↗

PubMed2000

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces.

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.

Acrylamides↗