Biomedical subjects

A Kikuchi

Publications and source records attributed to A Kikuchi.

At least 181 records · Page 10Linked to original sources

PubMed1998

The P2 protein of rice dwarf phytoreovirus is required for adsorption of the virus to cells of the insect vector.

Intact particles of rice dwarf phytoreovirus adsorbed to and entered monolayer-cultured cells of the insect vector Nephotettix cincticeps and multiplied within the cells. Particles that lacked the P2 protein neither attached to nor infected such cells. Furthermore, P2-free particles obtained from a transmission-competent isolate of the virus were unable to infect insect vectors that had been allowed to feed on these virus particles through a membrane. However, when such virus particles were injected into insects via a glass capillary tube they successfully infected the insects, which became able to transmit the virus. These results support the hypothesis that, while P2-free particles can neither interact with nor infect cells in the intestinal tract of the insect vector, they do retain the ability to infect such cells when physically introduced into the hemolymph by injection.

Adsorption↗

PubMed1998

Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3beta and beta-catenin and inhibits axis formation of Xenopus embryos.

Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3beta (GSK-3beta). This protein had 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway. We designated this protein Axil for Axin like. Like Axin, Axil ventralized Xenopus embryos and inhibited Xwnt8-induced Xenopus axis duplication. Axil was phosphorylated by GSK-3beta. Axil bound not only to GSK-3beta but also to beta-catenin, and the GSK-3beta-binding site of Axil was distinct from the beta-catenin-binding site. Furthermore, Axil enhanced GSK-3beta-dependent phosphorylation of beta-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Two-dimensional manipulation of confluently cultured vascular endothelial cells using temperature-responsive poly(N-isopropylacrylamide)-grafted surfaces.

Temperature-responsive hydration/dehydration changes in surface-grafted poly(N-isopropylacrylamide) (PIPAAm) were utilized for hydrophilic/hydrophobic surface property alterations in cell culture. In this report, we utilized PIPAAm-grafted surfaces to recover confluently-cultured vascular endothelial cells as coherent monolayers from this cell culture substrate and to transfer to new cell culture substrates. For this purpose, we used two different methods to recover and transfer cell monolayer cultures: (1) chitin membranes used as an apical side cell support during cultured cell transfer, allowing cell basal side reattachment to new culture substrates after transfer; and (2) a cell culture insert (porous PET) used as both a support as well as new substrate, allowing basal surfaces of cultured cells to be exposed to the medium after transfer. In both cases, all cells grown on PIPAAm-grafted surfaces detach completely with maintenance of basement membrane-like structure. Recovered cells attach to the second culture surfaces, covering more than 60% of the new substrate, and retain approximately 90% viability and their original function as judged from tissue-type plasminogen activator secretion. This technique could be utilized to prepare novel bioartificial organs as well as cell co-culture systems by multi-layering different cell types to mimic tissue structures for tissue engineering.

Acrylic Resins↗

PubMed1998

Retinal pigmented epithelium cultures on thermally responsive polymer porous substrates.

A cross-linkable co-polymer of UV-sensitive 4-(N-cinnamoylcarbamide)methylstyrene (CCMS) and N-isopropylacrylamide (NIPAAm), was applied to porous tissue culture inserts. Surface chemical analyses of the inserts show an introduction of a thermally responsive polymer comparable to that on similarly incorporated non-porous polystyrene surfaces. Contact angle measurements as well as atomic force microscopy show a surface change in response to changing temperature in an aqueous environment, from hydrophilic, extended polymer chains below 32 degrees C to a dense hydrophobic film above 32 degrees C. Cell growth on porous inserts allowed measurement of cell expression, such as transepithelial resistance and fluid transport, which are not observable on cells from non-porous surfaces. Cultures of retinal pigmented epithelium (RPE) were able to restore an environment similar to in vivo by forming a tight junction barrier membrane upon confluence at 37 degrees C, as observed by changes in morphology, transepithelial resistance, and directionally-specific fluid transport. In addition, cells cultured on these surfaces detached as an oriented polarized sheet when the inserts were brought to 20 degrees C. This cell sheet was transplanted to other tissue culture surface without polymer detachment or dissolution, or cell damage caused by traditional detachment methods using proteolytic enzymes.

Acrylic Resins↗

PubMed1998

Spontaneous regression of localized neuroblastoma detected by mass screening.

PURPOSE: To clarify whether and when neuroblastomas identified through screening do regress, and to ascertain how to treat them appropriately, we observed screened patients who had localized tumors, without any therapeutic intervention. PATIENTS AND METHODS: The criteria for the observation program were as follows: disease stage I or II; tumor less than 5 cm in diameter; no invasion to the intraspinal canal or growth to the great vessels; urinary vanillylmandelic acid (VMA) and homovanillic acid (HVA) less than 50 microg/mg creatinine; and informed consent. Of 25 patients identified through screening for 6-month-old infants in Saitama Prefecture, Japan between April 1994 and March 1996, 11 patients who met the criteria and one other patient with stage III tumor were enrolled onto the program. They were examined by abdominal ultrasonography (US) and their urinary VMA and HVA levels were assessed approximately once per month. The observation periods ranged from 4 to 27 months. RESULTS: The 11 tumors decreased in size, although one of these 11 tumors initially enlarged until the patient was 12 months of age and decreased in size thereafter. One other tumor slightly increased in size. Urinary VMA levels decreased in all patients. None of the tumors had completely disappeared by the last observation day. CONCLUSION: Our results suggest that regression of screened neuroblastoma is not a rare phenomenon. At present, it seems reasonable to adopt a wait-and-see strategy, with careful observation, for selected stage I or II tumors identified in infants screened at 6 months of age.

Homovanillic Acid↗

PubMed1998

Metabolic and endocrine responses to cold exposure in chronically incubated extrauterine goat fetuses.

To investigate developmental aspects of metabolic and endocrine responses to cold exposure in fetuses, we conducted experiments on six goat fetuses, three aged 95-116 d of gestation (dGA; group I), and three aged 122-134 dGA (group II), using an extrauterine fetal incubation system that provided arterio-venous extracorporeal membrane oxygenation (A-V ECMO). The fetuses were cannulated via the umbilical vessels, and their blood gas exchange was totally supported by A-V ECMO, while they were maintained in an isothermal incubator containing artificial amniotic fluid. After confirming that fetuses were in metabolically stable condition in the extrauterine incubation system, fetal core temperature was lowered by 2 degrees C over 2 h by decreasing the temperature of incubating fluid from 39.5 degrees C. During and after cold exposure, fetal heart rate and arterial blood pressure remained unchanged. We observed significant increases in oxygen consumption and plasma concentrations of norepinephrine, epinephrine, adrenocorticotropic hormone, and cortisol in group II but not in group I fetuses. In addition, based on regression analysis, maximal changes of these parameters during cold exposure were linearly correlated with gestational age significantly, and the regression lines were found to intersect the x (gestational age) axis at around 98-106 dGA. These results suggest that metabolic and endocrine responses to cold exposure develop with gestational age in the goat fetus, the responses being manifested around 100 dGA.

Animals↗

PubMed1998

Immunohistochemical and two-parameter flow cytometric studies of DNA topoisomerase II alpha in human epithelial ovarian carcinoma and germ cell tumor.

DNA topoisomerase II alpha (topo II alpha) is associated with active cell proliferation and is a target for chemotherapeutic agents administered to patients with ovarian cancer. To evaluate the biologic significance of topo II alpha expression in human ovarian carcinomas, we examined the expression of this protein immunohistochemically in tissue sections from 99 patients with ovarian cancer (85 common epithelial carcinomas, 14 germ cell tumors). We also measured topo II alpha and nuclear DNA content by two-parameter flow cytometry in 29 cases to evaluate possible qualitative changes of topo II alpha in the cell cycle of ovarian cancer cells. We observed a significant correlation of the labeling indices (LIs) of topo II alpha and Ki-67. The topo II alpha-to-Ki-67 ratio in germ cell tumors significantly exceeded that in common epithelial ovarian carcinomas (P = .038). Among the latter, the topo II alpha-to-Ki-67 ratio was significantly higher in serous cystadenocarcinomas than in mucinous cystadenocarcinomas. Two-parameter flow cytometric analysis revealed that topo II alpha expression was mainly observed in cells at the S to G2/M phases of the cell cycle, but, in some cases, topo II alpha positivity was detected in cells at G1. A significantly higher topo II alpha-to-Ki-67 ratio was detected in tumors with topo II alpha-positive cells at the G1 than in tumors in which topo II alpha-positive cells were not at G1. Results indicated that quantitative as well as quantitative changes in topo II alpha occur in human ovarian carcinomas.

Adult↗

PubMed1998

[TAL1 gene analysis in T-cell malignancies].

Site-specific recombination of the TAL1 gene was analyzed by Southern blotting and polymerase chain reaction (PCR) in 44 cases of childhood T-cell acute lymphoblastic leukemia (T-ALL), 20 cases of childhood T-cell non-Hodgkin's lymphoma (T-NHL) and 35 cases of adult T-cell malignancies. This recombination was found in 10 (22.7%) of 44 childhood T-ALL patients, but in none of the T-NHL or adult T-cell malignancies. Recombination of the TAL1 gene was therefore suggested to be specific for childhood T-ALL. The immunophenotypic features of the 10 T-ALL patients with this recombination were CD1-, CD2+, CD4-, CD7+, CD10-, and they had a significantly better outcome than other T-ALL cases without the recombination. The PCR technique revealed minimal residual disease (MRD) in 2 patients. One showed persistent MRD, while in the other MRD was recognized only at initial diagnosis. Further investigation is needed whether T-ALL with this recombination constitutes a distinct clinical subgroup among childhood T-ALL patients.

Adolescent↗

PubMed1998

DNA topoisomerase: the key enzyme that regulates DNA super structure.

DNA is a macromolecule carrying all genetic information and it must be packed in the cell nucleus. DNA is a double helix where one strand coils around the other. The helix is unwound and rewound every now and then to take out its information, which requires local alteration of the helical structure, resulting in the super-coiling of DNA. For replication, all the coils must be unwound at least once, and two daughter molecules are often catenated to each other. To solve these problems caused by the helical structure of DNA, topoisomerase activity introducing the transient breaking and rejoining of DNA strand is essential to perform each DNA transaction. In this article, we first review the mechanistic aspect of topoisomerase activity, and then discuss its basic clinical importance.

Adenosine Triphosphate↗

PubMed1997

Colocalization of Ras and Ral on the membrane is required for Ras-dependent Ral activation through Ral GDP dissociation stimulator.

Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslationally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells. The RalGDS action on Ral was enhanced by an active form of Ras but not a Ras mutant which was not post-translationally modified in the cells. The RalGDS activity was inhibited by acidic membrane phospholipids such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral was incorporated alone in the liposomes, RalGDS did not stimulate the dissociation of GDP from Ral. When Ral was incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-bound form of Ras did not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS required the Ras-binding domain of RalGDS. Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap did not enhance the RalGDS action in COS cells. Taken together with our previous observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells.

Animals↗

PubMed1997

Regulation of cross-linking of actin filament by IQGAP1, a target for Cdc42.

We have previously shown that IQGAP1, a recently identified target for Cdc42 and Rac1 small GTPases, showed a distribution similar to that of cortical actin cytoskeleton at the membrane ruffling area induced by insulin and Rac1(val12) (Kuroda, S., Fukata, M., Kobayashi, K., Nakafuku, M., Nomura, N., Iwamatsu, A., and Kaibuchi, K. (1996) J. Biol. Chem. 271, 23363-23367). Here we identified an IQGAP1-interacting molecule with molecular mass of 43 kDa (p43) from bovine brain cytosol, using glutathione S-transferase (GST)-IQGAP1 affinity column chromatography. The amino acid sequencing of the protein revealed that p43 was identical to beta- and gamma-actin. IQGAP1 was cosedimentated with filamentous actin (F-actin). The amino-terminal domain (amino acids 1-216) of IQGAP1 was responsible for the interaction with F-actin. Falling ball viscometry assay revealed that IQGAP1 cross-linked the F-actin. This IQGAP1 activity was further enhanced by guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).GST-Cdc42 but not by GDP.GST-Cdc42. The gel filtration analysis of IQGAP1 revealed that IQGAP1 appeared as oligomers and that GTPgammaS.GST-Cdc42 but not GDP.GST-Cdc42 enhanced the oligomerization of IQGAP1. These results strongly suggest that IQGAP1, acting downstream of Cdc42, can cross-link the actin filament through its oligomerization.

Actins↗

PubMed1997

Induction of ubiquitin conjugating enzyme activity for degradation of topoisomerase II alpha during adenovirus E1A-induced apoptosis.

Topoisomerase (topo) II alpha is degraded via polyubiquitination during adenovirus E1A-induced apoptosis in MA1 cells, a derivative of the human epidermoid carcinoma cell line KB. Topo II alpha ubiquitination activity in MA1 cells increased nearly 10-fold after induction of E1A in response to dexamethasone. To identify a topo II alpha ubiquitination factor(s), the S100 fractions prepared from apoptosis-induced (42 h) and uninduced (0 h) MA1 cells were first fractionated by ubiquitin-Sepharose columns. The ubiquitination activity induced by E1A was predominantly eluted with 20 mM AMP. Further fractionation of the AMP eluates on Resource-Q columns and the thiolester formation of the proteins resolved by electrophoresis with biotinylated ubiquitin revealed that a species of E2 isozyme recovered in the QFT2 fraction increased markedly in MA1 cells after E1A expression. These results indicate that a ubiquitination factor(s) specific to topo II alpha is induced during E1A-induced apoptosis in MA1 cells.

Adenovirus E1A Proteins↗

PubMed1997

Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain.

Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta. We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains. Several C-terminal deletion mutants of mouse topoisomerase IIalpha were constructed and expressed in yeast top2 cells. We found that the CTD of topoisomerase IIalphais dispensable for enzymatic activity in vitro but is required for nuclear localization in vivo. Interestingly, the CTD of topoisomerase IIbetawas also able to function as a signal for nuclear targeting. We therefore examined whether the CTD alone is sufficient for nuclear localization in vivo . The C-terminal region was fused to GFP (green fluorescent protein) and expressed under the GAL1 promoter in yeast cells. As expected, GFP signal was exclusively detected in the nucleus, irrespective of the CTD derived from either topoisomerase IIalphaor IIbeta. Surprisingly, when the upstream sequence of each CTD was added nuclear localization of the GFP signal was found to be cell cycle dependent: topoisomerase IIalpha-GFP was seen in the mitotic nucleus but was absent from the interphase nucleus, while topoisomerase IIbeta-GFP was detected predominantly in the interphase nucleus and less in the mitotic nucleus. Our results suggest that the catalytically dispensable CTD of topoisomerase II is sufficient as a signal for nuclear localization and that yeast cells can distinguish between the two isoforms of mammalian topoisomerase II, localizing each protein properly.

Amino Acid Sequence↗

PubMed1997

The post-translational modifications of Ral and Rac1 are important for the action of Ral-binding protein 1, a putative effector protein of Ral.

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and possesses the GTPase-activating activity for Rac1 and CDC42. We examined the roles of the post-translational modifications of Ral and Rac1 for the action of RalBP1. In COS cells, Ral(G23V), a constitutively active form, was mainly detected in the membrane fraction while most of Ral(G23V/C203S), a Ral mutant which is not post-translationally modified, was found in the cytosol fraction. When RalBP1 was expressed alone in COS cells, it was found in the cytosol but not in the membrane fraction. When RalBP1 was coexpressed with Ral(G23V), a part of RalBP1 was found in the membrane fraction. However, when RalBP1 was coexpressed with Ral(G23V/C203S), all of RalBP1 was recovered in the cytosol fraction. Although Ral bound to RalBP1 at a molar ratio of 1:1, the interaction of Ral with RalBP1 did not affect the GTPase-activating activity of RalBP1 for Rac1. Furthermore, RalBP1 was more active on the post-translationally modified form of Rac1 and CDC42 than the unmodified form. These results suggest that the post-translational modification of Ral is important for the subcellular localization of RalBP1 and that the interaction of Ral with RalBP1 is not essential for the activity of RalBP1 but plays a role in recruiting RalBP1 to the membrane where its substrates, Rac1 and CDC42, reside.

Animals↗

PubMed1997

Characterization of Ral GDP dissociation stimulator-like (RGL) activities to regulate c-fos promoter and the GDP/GTP exchange of Ral.

Ral GDP dissociation stimulator-like (RGL) has been identified to be a possible effector protein of Ras. RGL shares 50% amino acid identity with Ral GDP dissociation stimulator and contains the CDC25-like domain in the central region and the Ras-interacting domain in the C-terminal region. Since the modes of activation and action of RGL have not yet been clarified, in this paper we have analyzed the functions of RGL. In COS cells, RGL interacted with RasG12V/E37G (a Ras mutant in which Gly-12 and Glu-37 were changed to Val and Gly, respectively) which failed to bind to Raf, but not with RasG12V/T35S which bound to Raf. Raf did not inhibit the binding of RGL to RasG12V/E37G under the condition that Raf inhibited that of RGL to RasG12V. Expression of either RGL or Raf into NIH3T3 cells slightly activated c-fos promoter, while coexpression of both proteins greatly stimulated the c-fos promoter activity. RGL stimulated the GDP/GTP exchange of Ral and this action was enhanced by the post-translational modification of Ral. However, RGL was not active on Ras, Rac, CDC42, Rap, or Rho. Furthermore, this action of RGL to stimulate the GDP/GTP exchange of Ral was dependent on Ras in COS cells. These results suggest that RGL constitutes another Ras-signaling pathway which is distinct from the Raf pathway and indicate that the RGL pathway regulates the c-fos promoter activity and the GDP/GTP exchange of Ral.

Animals↗

PubMed1997

Unstable expression of the multi-drug-resistant phenotype in Chinese hamster ovary cells resistant to okadaic acid.

A Chinese hamster ovary cell line resistant to okadaic acid (OA), OAR2-3 has a mutation of the protein phosphatase (PP) 2A alpha gene and expresses a multi-drug resistance (MDR) phenotype. In the present work, we isolated two additional OA-resistant variants, also showing MDR with a cross-resistance profile similar to that of OAR2-3, and with increased and decreased expressions of the P-glycoprotein (Pgp) and DNA topoisomerase (topo) II protein, respectively. Unlike OAR2-3, however, they had no mutation in the same region of the PP2A alpha gene. Except for OA-resistance in OAR2-3, the MDR was found to decrease in the absence of OA, and this decrease was again associated with changes in topo II- and Pgp-expressions. Thus, we conclude that 1) OA regulates the expressions of Pgp and topo II positively and negatively, respectively, resulting in reversible expression of MDR irrespective of genetic changes and 2) in OAR2-3, the mutation in the PP2A alpha gene confers stable resistance to OA. The MDR was also linked with collateral sensitivity to some drugs, like cisplatin and nitrogen mustard.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Temperature-responsive liquid chromatography. 2. Effects of hydrophobic groups in N-isopropylacrylamide copolymer-modified silica.

We recently reported the new concept of temperature-responsive liquid chromatography using temperature-responsive poly(N-isopropylacrylamide)-modified surfaces as high-performance liquid chromatography media with aqueous mobile phases. Incorporation of hydrophobic sites is an important factor to improve the efficacy (selectivity and retention) of temperature-responsive chromatography. Toward this goal, we have synthesized semitelechelic copolymers of N-isopropylacrylamide (IPAAm) and butyl methacrylate (BMA) having reactive terminal functional groups using telomerization. The lower critical solution temperatures of the copolymers shift to lower temperatures with increasing hydrophobic BMA content in the poly(IPAAm-co-BMA) relative to that of the IPAAm homopolymer. This temperature-responsive semitelechelic copolymer was grafted to the surface of (aminopropyl)silica through the reaction of activated ester-amine coupling. The polymer-modified silica was used as a column packing material. Separation of a mixture of five steroids having various hydrophobicities was investigated. Retention of steroids on poly(IPAAm-co-BMA)-modified columns is increased with an increase in column temperature. The capacity factors for steroids on the copolymer-modified silica beads was much larger than that on homopolymer PIPAAm-modified columns. The capacity factor for testosterone at 50 degrees C was 33.8 for poly(IPAAm-co-BMA) containing 5 mol% BMA, while that for the PIPAAm homopolymer was 15.0 at the same temperature. The influence of column temperature on steroid retention behavior on copolymer-modified stationary phases was significant compared with the case of homopolymer-modified columns. Furthermore, retention times for steroids increased remarkably with increasing BMA composition. The temperature-responsive elution behavior for the steroids was strongly affected by the hydrophobicity of the grafted polymer chains on silica surfaces. Possible protein separation in temperature-responsive liquid chromatography was explored using insulin chains A and B, and beta-endorphin fragment 1-27. On IBc-3.2-modified silica column, these three peptides were successfully separated at 30 degrees C with 0.5 M NaCl aqueous solution (pH 2.1) as mobile phase. The retention times of these peptides were related to the number of hydrophobic amino acid residues in the peptides. In the proposed chromatography system, elution of target substances is controlled only by a small change in column temperature without any further modification of the aqueous mobile phase.

Acrylamides↗

PubMed1997

Absence of human T-lymphotropic virus type I in Japanese patients with cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a disease entity characterized by a primary sporadic T-cell proliferation in the skin. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma. Recently, several authors have detected the HTLV-1 genome in genomic DNA from patients with CTCL and proposed a causal relation of HTLV-1 to CTCL. However, it remains controversial because these studies contain some problems in materials used to detect HTLV-1. We investigated both fresh and cultured T lymphocytes (128 specimens) derived from 50 Japanese patients with CTCL, where HTLV-1 is endemic, by using polymerase chain reaction with four sets of primers including gag, pol, env, and pX regions of HTLV-1 to elucidate the relationship between HTLV-1 and CTCL in Japan. However, none of the 128 DNA specimens revealed positive for HTLV-1 in contrast to the previous studies. We conclude that CTCL, which does not include HTLV-1, is present although the pathogenesis of CTCL may be different by areas or races.

Adult↗