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Biomedical subjects

A Kikuchi

Publications and source records attributed to A Kikuchi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Hypoperfusion in the supplementary motor area, dorsolateral prefrontal cortex and insular cortex in Parkinson's disease.

The changes of regional cerebral blood flow (rCBF) in Parkinson's disease (PD) were investigated. Because of individual differences in brain volume and the extent of brain atrophy, previous functional imaging studies involved potential methodological difficulties. In this study, using the statistical parametric mapping technique, 99mTechnetium-labeled hexamethylpropyleneamineoxime brain single-photon emission computed tomography images from 18 patients with PD were transformed into standard brain-based stereotaxic coordinate spaces and then compared with such images for 11 control subjects matched for age and extent of brain atrophy. A rCBF decrement in the supplementary motor area (SMA) and such decrement in the dorsolateral prefrontal cortex (DLPFC) were observed in the summarized PD images as compared with controls (p<0.005). In a subgroup in the Hoehn-Yahr III/IV stage (11 cases), the rCBF decrement was demonstrated not only in the SMA, but also in the DLPFC and insular cortex (p<0.001). There was a correlation between the degree of the rCBF decrement in the DLPFC or the insular cortex and the score of the unified Parkinson's disease rating scale (p<0.05), while the rCBF decrement in the SMA showed no relationship with the severity of disease. The function of the SMA is closely associated with the nigro-striatal pathway and its impairment can explain the basic akinetic symptoms in PD, which are responsive to L-DOPA treatment. On the other hand, the DLPFC and insular cortex may play key roles in specific symptoms of impairment at advanced stages, such as impaired working memory, postural instability and autonomic dysfunction. We hypothesize that the impairment of the DLPFC and insular function is correlated with the progression of the disease and is related to DOPA-refractory symptoms, which are major problems in the care of patients with advanced PD.

Adult↗
PubMed2001

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2001

In vitro anti-tumour activity of alpha-galactosylceramide-stimulated human invariant Valpha24+NKT cells against melanoma.

alpha-galactosylceramide (KRN 7000, alpha-GalCer) has shown potent in vivo anti-tumour activity in mice, including against melanoma and the highly specific effect of inducing proliferation and activation of human Valpha24+NKT-cells. We hypothesized that human Valpha24+NKT-cells activated by alpha-GalCer might exhibit anti-tumour activity against human melanoma. To investigate this, Valpha24+NKT-cells were generated from the peripheral blood of patients with melanoma after stimulation with alpha-GalCer pulsed monocyte-derived dendritic cells (Mo-DCs). Valpha24+NKT-cells did not exhibit cytolytic activity against the primary autologous or allogeneic melanoma cell lines tested. However, proliferation of the melanoma cell lines was markedly suppressed by co-culture with activated Valpha24+NKT-cells (mean +/- SD inhibition of proliferation 63.9 +/- 1.3%). Culture supernatants of activated Valpha24+NKT-cell cultures stimulated with alpha-GalCer pulsed Mo-DCs exhibited similar antiproliferative activities against melanoma cells, indicating that the majority of the inhibitory effects were due to soluble mediators rather than direct cell-to-cell interactions. This effect was predominantly due to release of IFN-gamma, and to a lesser extent IL-12. Other cytokines, including IL-4 and IL-10, were released but these cytokines had less antiproliferative effects. These in vitro results show that Valpha24+NKT-cells stimulated by alpha-GalCer-pulsed Mo-DCs have anti-tumour activities against human melanoma through antiproliferative effects exerted by soluble mediators rather than cytolytic effects as observed against some other tumours. Induction of local cytokine release by activated Valpha24+NKT-cells may contribute to clinical anti-tumour effects of alpha-GalCer.

Adjuvants, Immunologic↗
PubMed2001

Phylogenetic relationship and mode of evolution of yeast DNA topoisomerase II gene in the pathogenic Candida species.

We have determined the nucleotide sequences of about 55% of the region of the DNA topoisomerase II gene (approximately 2.3 kb) isolated from the pathogenic Candida species, C. dubliniensis, C. parapsilosis, C. tropicalis, C. krusei, C. kefyr, C. guilliermondii and C. lusitaniae. Evolutionary relationships among nine Candida species including those of C. albicans and C. glabrata were studied based on the DNA topoisomerase II gene. The nucleotide sequences of 2192 bp, which covered two catalytic domains, ATPase and cutting/resealing, were subjected to phylogenetic analysis. Sequence comparison and evolutionary analysis have revealed that the Candida species tested here are not monophyletic, and the two strains within the species C. tropicalis and C. parapsilosis are too diverse to be in a single species. A wide variety of divergence was observed among the functional domains of DNA topoisomerase II, suggesting that Candida species were in different evolutionary paths at least as regarding the DNA topoisomerase II gene. Sequence information and the observation on the species-specific manner of molecular evolution of DNA topoisomerase II in Candida will be applied to develop a method of identification and characterization of the Candida species in both natural and clinical isolates.

Candida↗
PubMed2001

Atypical multidrug resistance may be associated with catalytically active mutants of human DNA topoisomerase II alpha.

In human cells, atypical drug resistance was previously identified with reduced catalytic activity or nuclear localization efficiency of DNA topoisomerase II alpha (TOP2 alpha). We have shown two etoposide resistant hTOP2 alpha mutants, K798L and K798P confer resistance to etoposide. In this work, we showed these mutants are also resistant against doxorubicin and mAMSA in vivo in the yeast strain ISE2, rad52, top2-4 at the non-permissive temperature. We purified these mutants to characterize the drug resistant mechanism. Purified recombinant proteins were 8- to 12-fold more resistant to etoposide and doxorubicin than wild type TOP2 alpha, and 2-fold more resistant to amsacrine, as measured by accumulation of cleavable DNA. These data show that K798L and K798P may be intrinsically resistant against these drugs in vitro and that this character may confer atypical multidrug resistant phenotype in vivo in yeast.

Amino Acid Substitution↗
PubMed2001

Synergistic activation of the Wnt signaling pathway by Dvl and casein kinase Iepsilon.

Although casein kinase Iepsilon (CKIepsilon) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKIepsilon activates the Wnt signaling pathway in co-operation with Dvl. CKIepsilon and Axin associated with different sites of Dvl, and CKIepsilon and Dvl interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dvl or CKIepsilon alone did not accumulate beta-catenin, but their co-expression accumulated greatly. Dvl and CKIepsilon activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dvl mutant that binds to Axin but not to CKIepsilon activated Tcf, it did not synergize with CKIepsilon. Another Dvl mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKIepsilon. Furthermore, Dvl and CKIepsilon co-operatively induced axis duplication of Xenopus embryos. These results indicate that Dvl and CKIepsilon synergistically activated the Wnt signaling pathway and that the binding of the complex of Dvl and CKIepsilon to Axin is necessary for their synergistic action.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Follicle-stimulating hormone promotes the growth of human epithelial ovarian cancer cells through the protein kinase C-mediated system.

We have previously described that follicle-stimulating hormone (FSH) stimulated the growth of human epithelial ovarian cancer tissues and cells. In order to determine the signaling pathway on FSH action in ovarian cancer, we used an epithelial ovarian cancer cell line (HRA line) which constitutively FSH receptors (FSHRs). FSH significantly increased cell proliferation (230.1 +/- 20.5%, P < 0.05) and (3)H-thymidine uptake (443.5 +/- 35.1%, P < 0.01). 1-(5-Isoquinolinesulfonyl)-2-methyipiperazine (H7, 1 5 nM), staurosponine (STR, 5 nM) and calphostin C (5 nM), specific protein kinase C (PKC) inhibitors, significantly suppressed the FSH-stimulated cell growth (120.2-140.2%, P < 0.05) and (3)H-thymidine uptake (140.5-173.9%, P < 0.05), whereas N-(2-guanidinoethyl)-5-isoquinoline-sulfon-amide (HA1004, l5 nM), which is a derivant of H7 and inhibits most of protein kinases except PKC, showed no effect on the FSH-stimulated cell growth and (3)H-thymidine uptake. A pretreatment with 12-0-tetradecanoylphorbol-13 acetate (TPA, 100 ng/ml) or STR (20 nM) significantly suppressed the subsequent FSH-stimulated cell growth (TPA; 152.3 +/-10.3%, STR; 160.4 +/- 15.9%, P < 0.05) and (3)H-thymidine uptake (TPA; 250.4 +/-18.3%, STR; 208.7 +/- 15.9%, P < 0.05). STR abolished the suppression of TPA preincubation on the subsequent FSH-stimulated cell growth and (3)H-thymidine uptake. HRA cells constitutively expressed PKCalpha but not PKCbeta nor PKCgamma. The levels of either expression of PKCalpha protein and mRNA were significantly amplified by FSH. These data suggest that stimulation of PKCalpha transcription is involved in the FSH-stimulated cell growth and DNA synthesis in epithelial ovarian cancer cells.

Cell Division↗
PubMed2001

A distinct subnuclear localization of mammalian DNA topoisomerase IIbeta in yeast.

Mammalian topoisomerase II isoforms alpha and beta are diverged in their C-terminal domain (CTD), but both isoforms complement the yeast top2 mutation. In this study, mammalian topoisomerase IIalpha-CTD and IIbeta-CTD were tagged with yellow fluorescent protein (YFP), expressed in yeast cells, and their localization was examined. YFP tagged-topoisomerase IIalpha-CTD was distributed evenly throughout the nucleus, while YFP tagged-topoisomerase IIbeta-CTD was sequestered into a subnuclear compartment. Deletion analysis revealed that two regions (amino acids 1207-1234 and 1513-1573) of the topoisomerase IIbeta-CTD are essential for specific localization of the beta isoform: if either of the two regions is removed, the mutant topoisomerase IIbeta-CTD distributes evenly throughout the nucleus. The data suggest that yeast cells distinguish the nuclear and subnuclear localization signals associated with these two mammalian topoisomerase II isoforms.

Amino Acid Sequence↗
PubMed2001

Decreased topoisomerase IIalpha expression confers increased resistance to ICRF-193 as well as VP-16 in mouse embryonic stem cells.

To elucidate the relationship between topoisomerase (topo) II expression and sensitivity to anti-topo II drugs in mammalian cells, we generated mouse embryonic stem cell mutants heterozygous for the topo IIalpha gene by gene targeting. The level of topo IIalpha in the heterozygous cells reduced to one-half of that found in wild-type cells, while topo IIbeta levels were similar in both cell types. Importantly, the heterozygous cells exhibited an increased resistance to ICRF-193 as well as VP-16, suggesting that ICRF-193, like VP-16, exerts its cytotoxicity through converting topo II to a poison.

Animals↗
PubMed2001

Aqueous chromatography utilizing pH-/temperature-responsive polymer stationary phases to separate ionic bioactive compounds.

Cross-linked poly(N-isopropylacrylamide-co-acrylic acid) (poly(IPAAm-co-AAc))-grafted silica bead surfaces were prepared and applied as new column matrix materials that exploit temperature-responsive anionic chromatography to separate basic bioactive compounds, specifically catecholamine derivatives, in aqueous mobile phases. Since poly(IPAAm-co-AAc) has a well-known temperature-responsive phase transition and apparent pKa shift, polymer-grafted silica bead surfaces are expected to exhibit simultaneous hydrophilic/hydrophobic and charge density alterations under thermal stimuli. Elution behavior of catecholamine derivatives from a copolymer-modified bead packed column was monitored using aqueous mobile-phase HPLC under varying temperature and pH. Catecholamine derivatives had higher retention times on poly(IPAAm-co-AAc) columns at higher pH in comparison with those on noncharged PIPAAm reference columns, suggesting an electrostatic interaction as a separation mode. Temperature also affected the retention behavior of catecholamine derivatives. Optimal separation of four catecholamine derivatives was achieved at elevated temperature, 50 degrees C, and at pH 7.0. This is due to the increased hydrophobicity of the stationary phase as evidenced by the elution of a nonionic hydrophobic steroid. From these results, mutual influences of both electrostatic and hydrophobic interactions between basic catecholamine derivatives and pH-/temperature-responsive surfaces are noted. Consequently, elution of weakly charged bioactive compounds is readily regulated through the modulation of stationary-phase thermoresponsive hydrophilic/hydrophobic and charge density changes.

Acrylic Resins↗
PubMed2001

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A.

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

Animals↗
PubMed2001

TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukemia cells.

Human Valpha24NKT cells are activated by alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor-mediated manner. Here, we demonstrate that CD4(+)V alpha 24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) when the cells are activated by alpha-GalCer-pulsed dendritic cells but not prior to activation. We also show that myeloid leukemia cells from patients with AML M4, but not from patients with AML M0 or M1, undergo apoptosis following culture with TRAIL-expressing autologous or allogeneic healthy donor V alpha 24NKT cells. Apoptosis of AML M4 leukemia cells from patient peripheral blood was almost completely blocked by a neutralizing monoclonal antibody against TRAIL, indicating that TRAIL on V alpha 24NKT cells is essential for the induction of apoptosis in AML M4 leukemia cells. A nonobese diabetic-severe combined immunodeficient human leukemia (AML M4) model showed that human activated CD4(+)V alpha 24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated V alpha 24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated V alpha 24NKT cells, or other strategies to increase activated V alpha 24NKT cells in vivo, may be of benefit to patients with AML M4. (Blood. 2001;97:2067-2074)

Adult↗
PubMed2001

Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH2-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys76 --> Ala76, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P2 is essential for endocytosis mediated by clathrin-coated pits.

Adaptor Proteins, Vesicular Transport↗
PubMed2001

Two-dimensional manipulation of differentiated Madin-Darby canine kidney (MDCK) cell sheets: the noninvasive harvest from temperature-responsive culture dishes and transfer to other surfaces.

A renal epithelial cell line, Madin-Darby canine kidney (MDCK) cells, adheres, spreads, and proliferates to confluency on our developed temperature-responsive culture dishes grafted with a poly(N-isopropylacrylamide) (PIPAAm) at 37 degrees C. In addition to other cell types, including hepatocytes and endothelial cells, MDCK cell sheets noninvasively were harvested from PIPAAm-grafted dishes merely by reducing the temperature. However, during the early stage of culture (up to 3 weeks), confluent MDCK cell detachment is greatly repressed. In the present study, we succeeded in the rapid harvest of confluent MDCK cell sheets and intact transfer to other culture dishes by utilizing hydrophilically modified poly(vinylidene difluoride) (PVDF) membranes as supporting materials. Immunocytochemistry with anti-beta-catenin antibody revealed that the functional cell-cell junctions were well organized in the transferred MDCK cell sheets. The viability assay showed that the transferred cells were not damaged during the two-dimensional cell-sheet manipulation. By transmission electron microscopy it was confirmed that the harvested MDCK cells retained differentiated phenotypes and had many microvilli and tight junctions at the apical and lateral plasma membranes, respectively. This two-dimensional cell-sheet manipulation technique promises to be useful in tissue engineering as well as in the investigation of epithelial cell sheets.

Animals↗
PubMed2001

Novel patterned cell coculture utilizing thermally responsive grafted polymer surfaces.

Here we demonstrate a novel cell coculture method without any apparent limitation in cell-type combinations that exploits thermally responsive polymer-grafted patterns to alter cell-cell and cell-surface interactions. Thermally responsive acrylamide polymer is first covalently patterned onto culture surfaces by masked electron beam irradiation. One cell type is then cultured to confluency at 37 degrees C. Reducing cell culture temperature below 32 degrees C selectively swells temperature sensitive polymer-grafted domains, detaching adherent cells only from these grafted patterns. Another cell type is then seeded over the same surface at 37 degrees C. These subsequently seeded cells adhere only to the now-exposed polymer-grafted domains. Initially seeded cells remaining adherent on nonpatterned surfaces and cells added in the second seeding are then cocultured at 37 degrees C in well-ordered patterns.

Acrylic Resins↗
PubMed2001

Co-localization of chicken DNA topoisomerase IIalpha, but not beta, with sites of DNA replication and possible involvement of a C-terminal region of alpha through its binding to PCNA.

Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.

Amino Acid Sequence↗