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Biomedical subjects

K Fujimoto

Publications and source records attributed to K Fujimoto.

At least 73 records · Page 4Linked to original sources
PubMed2001

A novel synthetic Arg-Gly-Asp-containing peptide cyclo(-RGDf==V-) is the potent inhibitor of angiogenesis.

Arg-Gly-Asp (RGD)-containing peptide is a ligand for integrin alpha(V)beta3 and acts as an angiogenic inhibitor. A novel cyclic RGD peptide, cyclo(-RGDf==V-) (f==V), was synthesized and its biological activities were characterized and compared with its analogs, cyclo(-RGDfV-) (fV) and cyclo(-RGDf-MeV-) (fMeV). It bound to integrin alpha(V)beta3 with almost the same affinity as the fV and fMeV analogs. All three compounds inhibited the adhesion and growth of HUVEC cells in a dose-dependent manner in vitro. Out of three, fMeV had the strongest effect, f==V was almost as strong as fMeV, and fV had the least effect. However, in vivo, f==V significantly decreased the intratumoral microvessel density (MVD) in the DLD-1 (human colon cancer cell) inoculated mice, while fMeV had little effect. These results suggest the potential usefulness of the cyclo(-RGDf==V-) as an antiangiogenic agent for clinical use in the future.

Animals↗
PubMed2001

Soluble interleukin-6 receptor alpha inhibits the cytokine-Induced fractalkine/CX3CL1 expression in human vascular endothelial cells in culture.

Soluble form of IL-6 receptor alpha (sIL-6R) is known to serve as an agonist, without exogenous IL-6, on endothelial cells which do not express IL-6R but have only IL-6 receptor beta chain, gp130. We investigated the effect of sIL-6R on fractalkine expression in human umbilical vein endothelial cells (HUVECs) in culture. sIL-6R markedly inhibited HUVEC fractalkine/CX3CL1 expression induced by interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma. IL-1alpha-induced fractalkine expression was inhibited by sIL-6R in time- and concentration-dependent manners. The experiment using actinomycin D indicated that sIL-6R lowered the stability of fractalkine mRNA. The inhibitory effect of sIL-6R was reversed by anti-gp130 neutralizing antibody. sIL-6R inhibited adhesion of mononuclear cells (MNCs) to HUVEC monolayers stimulated with IFN-gamma, but it did not inhibit the adhesion to monolayers stimulated with IL-1alpha. MNC chemotactic activity of conditioned medium of HUVEC stimulated with IL-1alpha or IFN-gamma was inhibited by co-treatment with sIL-6R. sIL-6R may play a regulatory role in immune responses by modulating the interaction between leukocytes and the vascular endothelium.

Cell Adhesion↗
PubMed2001

Decreased acetylcholine content and choline acetyltransferase mRNA expression in circulating mononuclear leukocytes and lymphoid organs of the spontaneously hypertensive rat.

It has been confirmed that the neurotransmitter acetylcholine (ACh) is present in blood; it is synthesized in T-lymphocytes by choline acetyltransferase (ChAT) and released upon T-lymphocyte activation. Both muscarinic and nicotinic ACh receptors have been identified on lymphocytes isolated from thymus, lymph node, spleen and blood, and their stimulation by muscarinic and nicotinic agonists elicits a variety of functional and biochemical effects, providing a strong argument that ACh synthesized and released from T-lymphocytes acts as an autocrine and/or paracrine factor regulating immune function. In the present study, we compared ACh levels in the blood, circulating mononuclear leukocytes (MNLs), thymus and spleen of spontaneously hypertensive rats (SHRs), which exhibit immune deficiencies related to the emergence of natural thymocytotoxic autoantibody, age-related decline of T-cell function and morphological changes in immune organs, with ACh levels in age-matched, normotensive Wistar Kyoto rats. In each case, ACh levels in 5-, 10- and 20-week-old SHRs were significantly lower than in WKYs. ChAT mRNA expression in MNLs was also significantly depressed in the SHRs. These results suggest that diminished synthesis and release of ACh from MNLs into blood and lymphoid organs likely reflects an immune deficiency related to T-cell dysfunction.

Acetylcholine↗
PubMed2001

Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation.

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.

Acetylcysteine↗
PubMed2001

Molecular cloning of rabbit hyaluronic acid synthases and their expression patterns in synovial membrane and articular cartilage.

cDNAs for hyaluronic acid synthases (HAS2 and HAS3) were cloned from a cDNA library of cultured rabbit synovial membrane cells. The cDNA encoding the open reading frame of rabbit HAS2 and HAS3 was 1659 nucleotides in length with a predicted molecular mass of about 63 kDa. The amino acid sequence showed that the rabbit HAS2 was 98.7 and 98.4%, and HAS3 was 98.2 and 97.5% identical with human and mouse forms of the proteins, respectively. The predicted sequences for hyaluronic acid (HA) binding motifs and the catalytic domains related to beta 1-4 and beta 1-3 linkages, essential for HA synthesis, were almost conserved in both rabbit HAS2 and HAS3, similarly to human and mouse HASs. RT-PCR analysis and in situ hybridization revealed that the mRNA of HAS2 was highly expressed in the synovial membrane and articular cartilage, whereas the expression of HAS3 mRNA was slightest in these tissues. Thus, it is demonstrated that rabbit HASs are highly conserved in sequence content as compared to the human and mouse homologues described previously, and that HAS2 is predominantly expressed in the synovial membrane and articular cartilage, but HAS3 is not.

Amino Acid Sequence↗
PubMed2001

Transforming growth factor-beta1 promotes invasiveness after cellular transformation with activated Ras in intestinal epithelial cells.

Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial-fibroblastoid conversion associated with loss of cell-cell adhesion receptors such as E-cadherin and beta-catenin. We report here that TGF-beta1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell-cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-beta1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-Ras(Val12) (RIE-iRas cells) were used in this study. Induced expression of activated Ha-Ras(Val12) caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of beta-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-beta1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of beta-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-beta receptor type II (TbetaRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-beta1, was strongly induced by TGF-beta1 despite the marked downregulation of TbetaRII. The induction of Ha-Ras(Val12) markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-beta1 treatment. Expression of a dominant-negative form of TbetaRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-beta1. Collectively, these results suggest that TGF-beta1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of beta-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-beta1 occurs via TbetaRII.

Adherens Junctions↗
PubMed2001

Integrated natural radiation exposure studies in stable Yugoslav rural communities.

The results of field investigations of natural radiation exposures of the general population in two stable rural communities in Yugoslavia are presented. The principal emphasis was on exposures to contemporary indoor radon, but measurements of external penetrating radiation absorbed dose rates in air were carried out in the majority of cases. In addition, in a limited number of dwellings, measurements of thoron gas concentrations were made. By means of making a series of sequential 3-month radon measurements, both seasonal variations and annual average radon levels in the dwellings were determined. Using passive alpha track detectors, individual radon and thoron indoor concentrations as high as 9591 Bq m(-3) and 709 Bq m(-3), respectively, were detected while absorbed dose rates in air in the dwellings as high as 430 nGy h(-1) were recorded. On the basis of these different types of measurements, assessments could be made of the integrated natural radiation exposures being received by the populations. In addition to contemporary radon measurements, retrospective radon exposure assessments in most of the dwellings were made on the basis of measurements of 210Po concentrations in both surface (glass) traps and in volume (porous materials) traps. A description is given of the sampling strategies and protocols used in this field work. It is shown that at least one stable rural community receiving high natural radiation exposures, has been clearly identified and plans for future health investigations of the population there are outlined.

Air Pollutants, Radioactive↗
PubMed2001

Synergistic stimulation, by tumor necrosis factor-alpha and interferon-gamma, of fractalkine expression in human astrocytes.

Fractalkine is a CX3C chemonkine that appears to be a neuron-to-microglia signal molecule in the central nervous system. We studied the expression of fractalkine in normal human astrocytes in culture, by using semi-quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that tumor-necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) synergistically enhance the expression of fractalkine. The expression of both fractalkine mRNA and protein was increased in time- and concentration-dependent manners in the cells co-stimulated with TNF-alpha and IFN-gamma. Cycloheximide, an inhibitor of protein synthesis, and dexamethasone had no effect on the synergy of the stimulation of fractalkine expression. We conclude that normal human astrocytes produce fractalkine by co-stimulation with pro-inflammatory cytokines and it may serve as a potential signal for immune and inflammatory responses in the central nervous system.

Astrocytes↗
PubMed2001

Orphan G protein-coupled receptor, GPR41, induces apoptosis via a p53/Bax pathway during ischemic hypoxia and reoxygenation.

Orphan receptors that couple to G protein without known ligands are considered to relate directly to drug discovery. Here, we examine the expression of various orphan receptors in H9c2 cells during ischemic hypoxia and reoxygenation. Among orphan receptors examined, the level of G protein-coupled receptor 41 (GPR41) mRNA increases significantly, with a peak at 2 h after reoxygenation, and recovers to the control level by 3 h after reoxygenation. The level of glyceraldehyde-3-phosphate dehydrogenase mRNA used as an internal control remains almost constant. The levels of c-fos and c-jun mRNA increase significantly with ischemic hypoxia and reoxygenation. The transfection of GPR41 into H9c2 cells results in a significant decrease in cell number, with DNA fragmentation observed by in vitro and in situ assay. The amount of p53 protein increases significantly in the nuclei of cells expressing GPR41, accompanying an increase in the transcriptional activity of p53. Consistent with the activation of p53, the level of bax mRNA is significantly increased, which leads to an increase in Bax protein. Furthermore, the expression of a deletion mutant of a GPR41, which lacks the G protein binding site and shows an attenuation of intracellular phosphorylation signals to H9c2 cells, inhibits cell death and the increase in p53 protein within 24 h after reoxygenation. These observations demonstrate that GPR41 is a novel receptor that activates p53 leading to apoptosis during reoxygenation after ischemic hypoxia in H9c2 cells. We have designated GPR41 as the hypoxia-induced apoptosis receptor, HIA-R.

Animals↗
PubMed2001

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endothelial cells in culture.

Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.

Blotting, Western↗
PubMed2001

Molecular cloning of rat transmembrane domain protein of 40 kDa regulated in adipocytes and its expression in H9c2 cells exposed to ischemic hypoxia and reoxygenation.

We clone a 1230 bp complementary DNA encoding rat transmembrane domain protein of 40 kDa regulated in adipocytes (TPRA40), an orphan receptor, by reverse transcription-polymerase chain reaction using H9c2 cells derived from embryonic rat heart. The deduced amino acid sequence of rat TPRA40 consists of 369 amino acids and has a longer carboxyl terminus than that of the mouse protein. The level of TPRA40 mRNA decreases significantly throughout ischemic hypoxia and reoxygenation.

Adipocytes↗
PubMed2001

Measurement of the CP violation parameter sin2 phi(1) in B(0)(d) meson decays.

We present a measurement of the standard model CP violation parameter sin2 phi(1) (also known as sin2beta) based on a 10.5 fb(-1) data sample collected at the Upsilon(4S) resonance with the Belle detector at the KEKB asymmetric e(+)e(-) collider. One neutral B meson is reconstructed in the J/psiK(S), psi(2S)K(S), chi(c1)K(S), eta(c)K(S), J/psiK(L), or J/psipi(0) CP-eigenstate decay channel and the flavor of the accompanying B meson is identified from its charged particle decay products. From the asymmetry in the distribution of the time interval between the two B-meson decay points, we determine sin2 phi(1) = 0.58(+0.32)(-0.34)(stat)+0.09-0.10(syst).

Journal Article↗
PubMed2001

Identification and quantification of molecular species of diacyl glyceryl ether by reversed-phase high-performance liquid chromatography with refractive index detection and mass spectrometry.

We have developed a method to identify and quantify the molecular species of diacyl glyceryl ether (DAGE) using high-performance liquid chromatography (HPLC) equipped with a refractive index detector and an electrospray ionization and time of flight mass spectrometer (LC-RI-MS). An octadecyl silica column with a mixture of acetonitrile and dichloromethane (65:35, v/v) as an eluant was used for the HPLC. When the LC-RI-MS method was applied to a mixture of synthetic DAGEs; 1-O-hexadecyl-2,3-dioleoylglycerol (O-16:0-18:1-18:18:1), 1-O-octadecyl-2,3-dioleoylglycerol (O-18:0-18:1-18:1), 1-O-octadecenyl-2,3-dioleoylglycerol (O-18:1-18:1-18:1), 1-O-octadecyl-2,3-didocodahexaenoylglycerol (O-18:0-22:6-22:6), and 1-O-octadecenyl-2,3-didocosahexaenoylglycerol (O-18:1-22:6-22:6), good separation and quantification were obtained on the refractive index chromatogram. A pseudo-molecular ion [M+NH4]+ and a monoacyl glyceryl ether ion [M-RCO2] + were observed for all synthetic DAGEs on the mass spectrum. It was found that the fatty acids and glyceryl ether in DAGE could be easily identified by these mass spectra. When this LC-RI-MS method was applied to the DAGEs extracted from muscle of Stromateus stellatus, approximately 18 peaks were observed on LC-RI-MS chromatograms and the major molecular species of DAGEs were identified as O-16:0-18:1-18:1.

Animals↗
PubMed2001

Sensitive boundary condition dependence of noise-sustained structure.

Sensitive boundary condition dependence (BCD) is reported in a convectively unstable system with noise, where the amplitude of generated oscillatory dynamics in the downstream depends sensitively on the boundary value. This BCD is explained in terms of the decrease (relaxation) of the comoving Lyapunov exponent (characterizing the convective instability) from upstream to downstream [K. Fujimoto and K. Kaneko, Physica D 129, 203 (1999)]. It is shown that a fractal BCD appears if the dynamics that represent the spatial change of the fixed point includes transient chaotic dynamics, and if appropriate intensity of noise is presented. By considering as an example a one-way-coupled map lattice, this theory for BCD is demonstrated.

Journal Article↗
PubMed2001

Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a human macrophage cell line.

Prostaglandins (PGs) play regulatory roles in a variety of physiological and pathological processes, including the immune response, cytoprotection and inflammation. Desferrioxamine (DFX), an iron chelator, is known to reduce free radical-mediated cell injury and to upregulate certain inflammatory mediators. We investigated the effects of DFX on the production of PGs and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGs, using a human macrophage cell line, U937. Our results showed that COX-2 expression and PGE(2) production are upregulated by DFX treatment and that this upregulation is dependent on both COX-2 promoter activity and alteration of mRNA stability. COX-2 promoter activity may be, at least in part, mediated by activation of the extracellular signal-regulated kinase pathway. These findings suggest that iron metabolism may regulate inflammatory processes by modulating PGs as well as other inflammatory mediators.

Binding Sites↗
PubMed2001

Role of the conserved Ser-Tyr-Lys triad of the SDR family in sepiapterin reductase.

Sepiapterin reductase (EC 1.1.1.153; SPR) is an enzyme involved in the biosynthesis of tetrahydrobiopterin; and SPR has been identified as a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family based on its catalytic properties for exogenous carbonyl compounds and molecular structure. To examine possible differences in the catalytic sites of SPR for exogenous carbonyl compounds and the native pteridine substrates, we investigated by site-directed mutagenesis the role of the highly conserved Ser-Tyr-Lys triad (Ser and YXXXK motif) in SPR, which was shown to be the catalytic site of SDR-family enzymes. From the analysis of catalytic constants for single- and double-point mutants against the triad, Ser and YXXXK motif, in the SPR molecule, participate in the reduction of the carbonyl group of both pteridine and exogenous carbonyl compounds. The Ser and the Tyr of the triad may co-act in proton transfer and stabilization for the carbonyl group of substrates, as was demonstrated for those in the SDR family. But either the Tyr or the Ser of SPR can function alone for proton transfer to a certain extent and show low activity for both substrates.

Alcohol Oxidoreductases↗
PubMed2001

Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins.

DEC1 is a basic helix-loop-helix (bHLH) protein related to Drosophila Hairy, Enhancer of split and HES, and involved in the control of proliferation and/or differentiation of chondrocytes, neurons, etc. We report here the identification and characterization of human, mouse and rat DEC2, a novel member of the DEC subfamily. DEC2 had high (97%) and moderate (52%) similarities in the bHLH region and the Orange domain with DEC1, respectively. However, DEC2, but not DEC1, had alanine and glycine-rich regions in the C-terminal half. Unlike Hairy, Enhancer of split and HES, DEC2 lacked the WRPW motif for interaction with the corepressor Groucho. The DEC2 gene was mapped to human chromosome 12p11.23-p12.1, mouse chromosome 6 G2-G3 and rat chromosome 4q43 distal-q4, where the conserved linkage homology has been identified among these species. Unlike DEC1, which was broadly expressed in many tissues, DEC2 showed a more restricted pattern of mRNA expression. The DEC subfamily proteins may play an important role in tissue development.

Amino Acid Sequence↗