Biomedical subjects

Y Takada

Publications and source records attributed to Y Takada.

At least 91 records · Page 5Linked to original sources

PubMed2000

Urokinase-type plasminogen activator receptor (CD87) is a ligand for integrins and mediates cell-cell interaction.

Binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR/CD87) regulates cellular adhesion, migration, and tumor cell invasion. However, it is unclear how glycosyl phosphatidylinositol-anchored uPAR, which lacks a transmembrane structure, mediates signal transduction. It has been proposed that uPAR forms cis-interactions with integrins as an associated protein and thereby transduces proliferative or migratory signals to cells upon binding of uPA. We provide evidence that soluble uPAR (suPAR) specifically binds to integrins alpha4beta1, alpha6beta1, alpha9beta1, and alphavbeta3 on Chinese hamster ovary cells in a cation-dependent manner. Anti-integrin and anti-uPAR antibodies effectively block binding of suPAR to these integrins. Binding of suPAR to alpha4beta1 and alphavbeta3 is blocked by known soluble ligands and by the integrin mutations that inhibit ligand binding. These results suggest that uPAR is an integrin ligand rather than, or in addition to, an integrin-associated protein. In addition, we demonstrate that glycosyl phosphatidylinositol-anchored uPAR on the cell surface specifically binds to integrins on the apposing cells, suggesting that uPAR-integrin interaction may mediate cell-cell interaction (trans-interaction). These previously unrecognized uPAR-integrin interactions may allow uPAR to transduce signals through the engaged integrin without a hypothetical transmembrane adapter and may provide a potential therapeutic target for control of inflammation and cancer.

Animals↗

PubMed2000

Pharmacologic graft protection without donor pretreatment in liver transplantation from non-heart-beating donors.

BACKGROUND: Non-heart-beating donors (NHBDs) are considered potential sources of transplant organs in an effort to alleviate the problem of donor shortage in clinical liver transplantation. We investigated the possibility of pharmacologic protection of hepatic allograft function from NHBDs without donor pretreatment. METHODS: Orthotopic liver transplantation was performed using pigs. In donors, cardiac arrest was induced by stopping the respirator. Forty-five minutes after cessation of the respirator, the liver was flushed with cold lactated Ringer's solution including heparin and with the University of Wisconsin (UW) solution, and then preserved for 8 hr at 4 degrees C in the UW solution. The pigs were divided into two groups: a control group and a treated group. In the treated group, an endothelin antagonist TAK-044 was added to the UW solutions (10 mg/L), and TAK-044 (10 mg/kg body weight) and a platelet activating factor antagonist E5880 (0.3 mg/kg body weight) were also administered to the recipients. RESULTS: TAK-044 and E5880 treatment significantly increased the 7-day survival rate of the recipients (100% vs. 17%, P<0.05). In the treated group, portal venous pressure immediately after reperfusion of the graft was significantly lower than in the control group, and postoperative increase in serum concentrations of glutamic oxaloacetic transaminase and total bilirubin was attenuated. Moreover, the energy charge and adenosine triphosphate concentration of the liver were rapidly restored after reperfusion. CONCLUSIONS: Pharmacologic modulation with TAK-044 and E5880 avoiding donor pretreatment can improve the viability of hepatic allografts procured from NHBDs.

Animals↗

PubMed2000

Selective stimulation by ceramide of the expression of the alpha isoform of retinoic acid and retinoid X receptors in osteoblastic cells. A role of sphingosine 1-phosphate-mediated AP-1 in the ligand-dependent transcriptional activity of these receptors.

Recent studies have demonstrated that sphingosine 1-phosphate (SPP) plays a functional role as a signaling molecule in gene expression in several kinds of cells. The present study demonstrates selective expression by ceramide of retinoic acid receptor-alpha (RAR-alpha) and retinoic X receptor-alpha (RXR-alpha) in osteoblastic MC3T3-E1 cells and a functional role of SPP-mediated AP-1 in the signaling mechanism of ligand-dependent transcriptional activity of heterodimers of these receptors in the cells. C(2)- and C(6)-ceramides selectively stimulated the expression of RAR-alpha and RXR-alpha genes, but not that of beta and gamma isoform genes of RAR and RXR, in the cells. The C(2)-ceramide-induced stimulation was clearly inhibited by dl-threo-dihydrosphingosine, an inhibitor of sphingosine kinase. SPP also selectively stimulated the expression of both receptors and increased the specific binding of the nuclear proteins to direct repeat 5 (DR-5), a consensus sequence of RAR-RXR. In addition, SPP markedly stimulated transient chloramphenicol acetyltransferase (CAT) activity of retinoic acid-dependent transcriptional activity in the cells transfected with a DR-5-CAT reporter gene. The SPP stimulation was activation protein-1 (AP-1)-dependent, because the SPP stimulatory action toward these nuclear gene expressions and the transient CAT activity were inhibited by antisense c-fos and c-jun oligonucleotides. We observed that SPP actually stimulated AP-1 transcriptional activity in the cells. This study suggests an important role of SPP-mediated AP-1 in the selective expression of RAR-alpha and RXR-alpha in osteoblastic cells via the sphingosine pathway.

Animals↗

PubMed2000

Clinical outcomes of orbital irradiation combined with or without systemic high-dose or pulsed corticosteroids for Graves' ophthalmopathy.

PURPOSE: To evaluate the outcomes of orbital irradiation with or without high-dose or pulsed corticosteroids in patients with Graves' ophthalmopathy (GO). METHODS AND MATERIALS: One hundred and twenty-one patients with moderate to severe GO who received orbital irradiation from 1987 to 1997 were retrospectively analyzed. A total dose of 20 Gy in 10 fractions was delivered to the bilateral retrobulbar volume. Eighty-six patients were treated in combination with high-dose or pulsed corticosteroids and irradiation. Univariate and multivariate analyses were performed to assess the prognostic variables. RESULTS: The median follow-up period was 26 months. The overall clinical response was evaluated as excellent in 17 patients (14%), good in 65 (54%), fair in 31 (25%), no response in 7 (6%), and worse in 1 (1%). The best responses were noted for soft-tissue signs, extraocular muscle involvement, and sight loss, while a limited response was noted for proptosis. Multivariate analysis revealed that the use of high-dose corticosteroid or pulsed corticosteroids, female gender, and a shorter duration of ophthalmopathy before radiotherapy were significantly correlated with favorable outcomes. No long-term complications related to radiotherapy were observed. CONCLUSION: Orbital irradiation combined with high-dose or pulsed corticosteroids is an effective treatment for moderate to severe GO, especially in cases with major manifestations of soft-tissue signs, extraocular muscle impairment, or sight loss.

Adolescent↗

PubMed2000

The personal identification of many samples recovered from under the sea.

An automatic and rapid DNA typing system was employed for personal identification, using fragmentary tissue samples from victims in an airplane accident. Two victims were crushed into small pieces, and 33 samples suspected to belong to them were recovered from under the sea. From each sample, 10 mg was used for testing. The parents' bloods of two presumptive victims were also examined. DNA extraction from samples was performed by the NaI method, and the obtained DNA samples were analyzed with the ABI PRISM system. Among 33 samples, 31 samples were identified to be human tissues, possibly from two victims. The other two samples seemed to be parts of marine animals. ABO blood group, STR polymorphism, and mitochondrial DNA polymorphism typing were possible in every examined human sample. Two victims' fragmentary tissues were identified by determining ABO genotype, STR type and mitochondrial DNA type. The system we employed enabled an accurate typing of many fragmentary samples in a short time, thus contributing to the fast and secure identification of many victims in such cases as big air accidents.

ABO Blood-Group System↗

PubMed2000

The role of alpha and beta chains in ligand recognition by beta 7 integrins.

Integrins alpha(E)beta(7) and alpha(4)beta(7) are involved in localization of leukocytes at mucosal sites. Although both alpha(E)beta(7) and alpha(4)beta(7) utilize the beta(7) chain, they have distinct binding specificities for E-cadherin and mucosal addressin cell adhesion molecule-1 (MAdCAM-1), respectively. We found that mutation of the metal ion-dependent adhesion site (MIDAS) in the alpha(E) A-domain (D190A) abolished E-cadherin binding, as did mutation F298A on the A-domain surface near the MIDAS cleft. A docking model of the A-domain with E-cadherin domain 1 indicates that coordination of the alpha(E) MIDAS metal ion by E-cadherin Glu(31) and a novel projection of Phe(298) into a hydrophobic pocket on E-cadherin provide the basis for the interaction. The location of the binding site on the alpha(E) A-domain resembles that on other integrins, but its structure appears distinctive and particularly adapted to recognize the tip of E-cadherin, a unique integrin ligand. Additionally, mutation of the beta(7) MIDAS motif (D140A) abolished alpha(E)beta(7) binding to E-cadherin and alpha(4)beta(7)-mediated adhesion to MAdCAM-1, and alpha(4) chain mutations that abrogated binding of alpha(4)beta(1) to vascular cell adhesion molecule-1 and fibronectin similarly reduced alpha(4)beta(7) interaction with MAdCAM-1. Thus, although specificity can be determined by the integrin alpha or beta chain, common structural features of both subunits are required for recognition of dissimilar ligands.

Amino Acid Sequence↗

PubMed2000

Molecular basis of ligand recognition by integrin alpha5beta 1. II. Specificity of arg-gly-Asp binding is determined by Trp157 OF THE alpha subunit.

Different beta(1) integrins bind Arg-Gly-Asp (RGD) peptides with differing specificities, suggesting a role for residues in the alpha subunit in determining ligand specificity. Integrin alpha(5)beta(1) has been shown to bind with high affinity to peptides containing an Arg-Gly-Asp-Gly-Trp (RGDGW) sequence but with relatively low affinity to other RGD peptides. The residues within the ligand-binding pocket that determine this specificity are currently unknown. A cyclic peptide containing the RGDGW sequence was found to strongly perturb the binding of the anti-alpha(5) monoclonal antibody (mAb) 16 to alpha(5)beta(1). In contrast, RGD peptides lacking the tryptophan residue acted as weak inhibitors of mAb 16 binding. The epitope of mAb 16 has previously been localized to a region of the alpha(5) subunit that contains Ser(156)-Trp(157). Mutation of Trp(157) (but not of Ser(156) or surrounding residues) to alanine blocked recognition of mAb 16 and perturbed the high affinity binding of RGDGW-containing peptides to alpha(5)beta(1). The same mutation also abrogated recognition of the alpha(5)beta(1)-specific ligand peptide Arg-Arg-Glu-Thr-Ala-Trp-Ala (RRETAWA). Based on these findings, we propose that Trp(157) of alpha(5) participates in a hydrophobic interaction with the tryptophan residue in RGDGW, and that this interaction determines the specificity of alpha(5)beta(1) for RGDGW-containing peptides. Since the RGD sequence is recognized predominantly by amino acid residues on the beta(1) subunit, our results suggest that Trp(157) of alpha(5) must lie very close to these residues. Our findings therefore provide new insights into the structure of the ligand-binding pocket of alpha(5)beta(1).

Animals↗

PubMed2000

Previous exposure to footshock stress attenuates nicotine-induced serotonin release in rat striatum during the subsequent stress.

We have analyzed the effects of chronic or repeated footshock stress on the release of serotonin (5-hydroxytryptamine: 5-HT) in the striatum of rats that received nicotine by using a microdialysis technique. Neither local infusion of nicotine alone nor stress application alone changed 5-HT release. Local infusion of 1 mM nicotine to the striatum, however, significantly increased 5-HT release in the striatum to 145.9 +/- 30.8 pg/dialysate during simultaneous stress application. These increases of extracellular 5-HT release induced by the combination of nicotine and stress application were also observed in rats that had received daily chronic footshock. However, the previously administered footshock induced the reduced release of 5-HT from the striatum to 33.5 +/- 8. 6 (repeated footshock) and 10.0 +/- 3.6 pg/dialysate (daily footshock) when footshock was given together with nicotine infusion. These results suggest that previous exposure to stress attenuated the nicotine-induced 5-HT release in the striatum during the subsequent stress.

Animals↗

PubMed2000

Serotonergic neurons projecting to hippocampus activate locomotion.

Although the role of brain serotonergic neurons in locomotion has been extensively studied, their influence may vary depending upon the terminal areas. Thus, using microdialysis and microinjection techniques, we examined the relationship between serotonin (5-HT) levels in striatum, hippocampus or prefrontal cortex (PFC) and motor activity in rats. The systemic injection (10 mg/kg i.p.) of monoamine oxidase inhibitor, tranylcypromine (TC), significantly elevated 5-HT levels in the striatum, hippocampus and PFC accompanied by a parallel increase in motor activity of the rats. This effect was mimicked by microinfusions of TC (1.0 mM) or 5-HT (1. 0 mM) into the hippocampus and to some extent into PFC (the response delayed in time), but not into striatum. The increase in motor activity produced by local infusions of TC either into the hippocampus or PFC could be prevented by pretreatment with 10 microM tetrodotoxin infused into the hippocampus. However, tetrodotoxin infused to PFC failed to prevent hyperlocomotion produced by intrahippocampal infusion of TC, although the response was delayed in time. Thus, we conclude that serotonergic neurons projecting to the hippocampus are involved in locomotor activity and PFC serotonergic fibers may facilitate hippocampal control of locomotion.

Animals↗

PubMed2000

Accumulation of GTP-bound RhoA during cytokinesis and a critical role of ECT2 in this accumulation.

We developed a new pull-down assay for GTP-Rho and examined its level during cell cycle. HeLa cells were arrested in the S phase by thymidine and were enriched in the prometaphase, metaphase, telophase, and G(1) phase by collecting at 0, 45, 90, and 180 min after the release from the nocodazole arrest, respectively. The level of GTP-Rho did not change significantly from the S phase to the prometaphase, but increased thereafter, peaking in the telophase, and returned to the original level in the G(1) phase. The GDP-GTP exchange activity for Rho measured in cell lysates in parallel increased also during the mitosis with a peak in the metaphase. Using this system, we examined a role of ECT2, an exchanger for Rho GTPases, suggested to be involved in cytokinesis (Tatsumoto, T., Xie, X., Blumenthal, R., Okamoto, I., and Miki., T. (1999) J. Cell. Biol. , 147, 921-928). Expression of the dominant negative form of ECT2 completely suppressed both the rise of GTP-Rho in the telophase and the increased GDP-GTP exchange activity in the mitotic cell extracts. These results suggest a critical role of ECT2 in Rho activation during cytokinesis.

Animals↗

PubMed2000

Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3.

Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.

Amino Acid Sequence↗

PubMed2000

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.

Alleles↗

PubMed2000

Multiple discontinuous ligand-mimetic antibody binding sites define a ligand binding pocket in integrin alpha(IIb)beta(3).

Integrin alpha(IIb)beta(3), a platelet fibrinogen receptor, is critically involved in thrombosis and hemostasis. However, how ligands interact with alpha(IIb)beta(3) has been controversial. Ligand-mimetic anti-alpha(IIb)beta(3) antibodies (PAC-1, LJ-CP3, and OP-G2) contain the RGD-like RYD sequence in their CDR3 in the heavy chain and have structural and functional similarities to native ligands. We have located binding sites for ligand-mimetic antibodies in alpha(IIb) and beta(3) using human-to-mouse chimeras, which we expect to maintain functional integrity of alpha(IIb)beta(3). Here we report that these antibodies recognize several discontinuous binding sites in both the alpha(IIb) and beta(3) subunits; these binding sites are located in residues 156-162 and 229-230 of alpha(IIb) and residues 179-183 of beta(3). In contrast, several nonligand-mimetic antibodies (e.g. 7E3) recognize single epitopes in either subunit. Thus, binding to several discontinuous sites in both subunits is unique to ligand-mimetic antibodies. Interestingly, these binding sites overlap with several (but not all) of the sequences that have been reported to be critical for fibrinogen binding (e.g. N-terminal repeats 2-3 but not repeats 4-7, of alpha(IIb)). These results suggest that ligand-mimetic antibodies and probably native ligands may make direct contact with these discontinuous binding sites in both subunits, which may constitute a ligand-binding pocket.

Amino Acid Sequence↗

PubMed2000

Bovine milk kininogen fragment 1.2 promotes the proliferation of osteoblastic MC3T3-E1 cells.

The active component on the proliferation of osteoblastic MC3T3-E1 cells was purified and identified from bovine milk. The growth-promoting activity was measured by [(3)H]thymidine incorporation on the cell. The purified protein showed a molecular size of 17 kDa on SDS-PAGE. Its amino-terminal amino acid sequence was very similar to the internal sequence of bovine high molecular weight (HMW) kininogen, which comprises fragment 1.2. The promotion of proliferation was specific for osteoblastic MC3T3-E1 cells, not for fibroblast BALB/3T3 cells. In blood coagulation, HMW kininogen is considered to be cleaved by a specific enzyme kallikrein. HMW kininogen then releases two peptides, a biologically active peptide bradykinin and fragment 1.2, but the fate of fragment 1.2 is unknown. This milk-derived protein that comprises to fragment 1.2 showed a growth-promoting activity of osteoblasts. We propose the possibility that milk plays an important role in bone formation by supplying the active agent for osteoblasts as well as supplying calcium.

3T3 Cells↗

PubMed2000

Topological scenario for stripe formation in manganese oxides

The spin-charge-orbital complex structures of manganites are studied using topological concepts. The key quantity is the "winding number" w associated with the Berry-phase connection of an e(g) electron parallel transported through Jahn-Teller centers, along zigzag one-dimensional paths in an antiferromagnetic environment of t(2g) spins. From these concepts, it is shown that the "bi-stripe" and "Wigner-crystal" states observed experimentally have different w's. Predictions for the spin structure of the charge-ordered states for heavily doped manganites are discussed.

Journal Article↗

PubMed2000

Differential effects of nicotine against stress-induced changes in dopaminergic system in rat striatum and hippocampus.

A number of studies have shown an increase in nicotine self-administration among smokers when exposed to stress. Since it is well known that nicotine or stress alter the dopaminergic system, we examined the effect of chronic nicotine administration on the dopamine level and its metabolism in the striatum and the hippocampus during stressful conditions in rats. Nicotine (0.4 mg/kg, i.p. for 14 days) increased the dopamine level in the striatum (P<0. 05) and decreased it in the hippocampus (P<0.05) in comparison with the effect of saline. Three hours of water-immersion restraint stress sharply elevated the dopamine level (P<0.05) and reduced the 3-methoxytyramine level (P ranged from 0.05 to 0.001 depending on the area and time point) in both brain regions studied, while dihydroxyphenylacetic acid and homovanilic acid levels were not altered. Nicotine pretreatment attenuated some of these changes in a region- and time-dependent manner. However, stress induced a decrease in dopamine turnover in the hippocampus (P<0.05) but not in the striatum, and nicotine failed to prevent this effect. Stress-induced alterations gradually returned toward normal during the 48-h observation period, and in some cases this was facilitated by nicotine. Thus, we demonstrated differential, region- and time-dependent protective effects of chronic nicotine administration against stress-induced changes in dopamine levels and release in brain regions critically affected by stress.

Animals↗

PubMed2000

Nicotine attenuates stress-induced changes in plasma amino acid concentrations and locomotor activity in rats.

It is known that stressor stimuli (both systemic and processive) and nicotine activate central nervous system. Surprisingly, numerous studies have demonstrated an increase in nicotine self-administration among smokers when exposed to stress in order to reduce the stress-related tension. Therefore, in the present study, we decided to investigate the influence of nicotine on both behavioral (i.e., on locomotor activity) and metabolic (i.e., on the level of amino acids in the plasma) changes following water immersion restraint stress in rats. As expected, the stress produced evident decline in locomotor activity of the rats (p < 0.001) and in the levels of all plasma amino acids studied (p < 0.05). Nicotine alone also significantly reduced locomotor activity (p < 0.05) and the levels of some plasma amino acids. However, when administered to rats subjected to water immersion and restraint, nicotine attenuated both stress-induced decrease in locomotor activity (p < 0.05) and in some plasma amino acids. Thus, this study demonstrated that the mode of action of nicotine is strongly dependent on the level of initial brain activity, which provide new evidence for arousal-modulation model of nicotine action.

Amino Acids↗

PubMed2000

TNF-alpha induced p21(WAF1) but not Bax in colon cancer cells WiDr with mutated p53: important role of protein stabilization.

The p21(WAF1)induces cell cycle arrest at G(1)and its expression is regulated by the functional p53. TNF-alpha induced expression of p21(WAF1)at protein and mRNA levels in a dose-dependent fashion with an association with G(1)-arrest in human colon cancer cells WiDr that carry mutated p53 at codon 273 (His(273)). However, TNF-alpha did not affect the levels of the Bax protein, which also has p53-binding sites on its promoter and causes apoptosis. Further experiments suggested that cycloheximide (CHX), a protein synthesis inhibitor, increased the levels of p21(WAF1)mRNA and the induction of p21(WAF1)mRNA by TNF-alpha did not require new protein synthesis. Co-transfection of the p53 His(273)expression construct with a luciferase gene controlled by the p21(WAF1)promoter showed that the p53 His(273)was inactive, although TNF-alpha increased the transcriptional rate of p21(WAF1)in these cells. Further study found that TNF-alpha markedly stabilized the p21(WAF1)protein. These findings suggest that TNF-alpha induces expression of p21(WAF1)through a distinct pathway from Bax and that protein stabilization is an important mechanism in the expression of p21(WAF1)independent of p53.

Apoptosis↗