Biomedical subjects
Publications and source records attributed to Satoshi Fujii.
An artificial phospholipid, possessing saturated alkyl chains as a membrane anchor and protein recognition site as well as an Fe(III)-EDTA moiety as a protein cleavable polar head group, was designed and synthesized based on the amidite method for the purpose of examination of cleavage of integral membrane proteins.
Alzheimer's disease is characterized by numerous amyloid-beta peptide (Abeta) plaques surrounded by microglia. Here we report that Abeta induces the proliferation of the mouse microglial cell line Ra2 by increasing the expression of macrophage colony-stimulating factor (M-CSF). We examined signal cascades for Abeta-induced M-CSF mRNA expression. The induction of M-CSF was blocked by a phosphatidylinositol 3 kinase (PI3-kinase) inhibitor (LY294002), a Src family tyrosine kinase inhibitor (PP1) and an Akt inhibitor. Electrophoretic mobility shift assays showed that Abeta enhanced NF-kappaB binding activity to the NF-kappaB site of the mouse M-CSF promoter, which was blocked by LY294002. These results indicate that Abeta induces M-CSF mRNA expression via the PI3-kinase/Akt/NF-kappaB pathway.
OBJECTIVE: We sought to determine the etiologic mechanism of proinflammatory cytokine, interleukin-6 (IL-6), and statin as regulators of synthesis of plasminogen activator inhibitor-1 (PAI-1), the physiological fibrinolysis inhibitor and an acute-phase reactant. METHODS AND RESULTS: Transient transfection and luciferase assay in HepG2 human hepatoma-derived cells demonstrated that IL-6 increased PAI-1 promoter activity and mevastatin decreased IL-6-inducible response. Systematic deletion assay of the promoter demonstrated that the region (-239 to -210 bp) containing a putative CCAAT/enhancer-binding protein (C/EBP) binding site was necessary. Point mutation in this site abolished the IL-6-inducible response. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that C/EBPalpha, C/EBPbeta, and C/EBPdelta were involved in protein-DNA complex formation in intact cells. Deoxyribonuclease (DNase) I footprinting analysis revealed that 5' flanking region (-232 to -210 bp) is acute-phase response protein-binding site. C/EBPdelta binding activity was increased by IL-6 and attenuated by mevastatin. Mevastatin attenuated IL-6-mediated increase of C/EBPdelta protein in the nuclear extracts. IL-6 also increased PAI-1 and C/EBPdelta mRNA in mouse primary hepatocytes. CONCLUSIONS: IL-6 increases hepatic PAI-1 expression mediated by the -232- to -210-bp region of the promoter containing a C/EBPdelta binding site. Vascular protection by statins may be partly mediated through regulation of CEBPdelta and consequent modulation of PAI-1 expression.
Coronary vasomotor response to acetylcholine infusion was studied in a 69-year-old currently smoking man with enhanced insulin response to oral glucose load. Coronary angiogram showed no significant stenoses. Immediately after 20 microg acetylcholine infusion in the right coronary artery, angiographic no-flow, ischemic electrocardiographic changes, and anginal pain developed in the absence of epicardial coronary obstruction. While intracoronary infusion of isosorbide dinitrate was only partially effective, intracoronary infusion of nicorandil, an agent known to improve coronary microcirculation, completely resolved these changes. This is the first case reported so far suggesting that enhanced insulin response may be associated with acetylcholine-induced microvascular vasoconstriction. Microvascular vasoconstriction must be considered when a patient with insulin resistance presents with chest pain.
BACKGROUND: Myocardial viability is not synonymous with contractile reserve and identifiable in a significant percentage of dysfunctional myocardial segments without contractile reserve. The usefulness of ultrasonic tissue characterization by the phase-corrected magnitude of cyclic variation of integrated backscatter (MVIB) in chronic coronary artery disease is not fully validated. Thus, whether MVIB predominantly reflects the contractile reserve or myocardial viability of chronically dysfunctional myocardium was determined. METHODS: The MVIB of severely dysfunctional interventricular septum or posterior wall was measured in 34 consecutive patients with previous myocardial infarction. Dobutamine stress echocardiography and fluorine-18 fluorodeoxyglucose positron emission tomography were used as the standards of contractile reserve and myocardial viability, respectively. RESULTS: Among 44 dysfunctional segments, only 15 were judged as having contractile reserve and 29 were judged as not by dobutamine stress echocardiography, whereas 26 segments showed myocardial viability using fluorine-18 fluorodeoxyglucose positron emission tomography and 18 did not. MVIB was greater in segments with than in those without contractile reserve (4.7 +/- 2.2 vs -1.4 +/- 4.9 dB, P < .0001), but there was considerable overlap between the groups. On the other hand, MVIB of segments with and without myocardial viability (4.1 +/- 2.6 vs -4.3 +/- 3.3 dB, P < .0001) was distinctly different and predicted myocardial viability with a sensitivity of 92% and a specificity of 94%. CONCLUSIONS: For patients with chronic coronary artery disease, MVIB better reflects myocardial viability than it does contractile reserve. Ultrasonic tissue characterization, in concordance with fluorine-18 fluorodeoxyglucose positron emission tomography, is a sensitive method for detecting myocardial viability.
Conformational change of nucleic acids induced by a small molecule is essential for many biological reactions and useful for biotechnology. To construct a spermine-responsive nucleic acid, we designed DNA sequences which can potentially form a unimolecular hairpin loop structure and a bimolecular duplex by itself. On the basis of UV melting and native PAGE experiments, we found four DNA oligomers of d16cg, d14cg, d12cg, and d14ta adopting different secondary structures depending on the spermine concentration. Their dissociation constants (Kd) with spermine were estimated in the order of 10(-6).
DNA/RNA molecules with the specific three-dimensional structure, express the specific structural and biological functions. The knowledge integration based on three dimensional structures can be used to explain biochemical observations, to predict biological functions and to design drugs specific to a given complex system. The database cataloging the interaction motifs of nucleic acid moieties has been developed. Skew matrix, a kind of kink parameter, affords the structural description between the adjacent moieties. The proper values in skew matrix have the good properties, i.e., impregnable and flexible presentation. The geometrical parameters about hydrogen bond and base stacking would also provide the tolerant aspect for stereochemical bioinformatics. The tentative database including these parameters with the species and physical properties of the surrounding nucleic acid components and amino acids has been constructed.
BACKGROUND: Type 2 diabetes and insulin resistance have been associated with several cardiac features including diastolic heart failure, impaired ventricular function, perimicrovascular fibrosis and increased expression of plasminogen activator inhibitor type-1 (PAI-1) in coronary arterial walls. Insulin receptor substrate-1 (IRS-1) is one mediator of insulin signaling. This study was performed to determine whether induction of cardiac manifestations typical of insulin resistance and increased PAI-1 expression occur developmentally or post-developmentally as a result of loss of IRS-1-mediated signaling. METHODS: IRS-1 knockout mice were studied at 20 weeks of age. RESULTS: Compared with control mice, the left ventricular (LV) and cardiac mass to body weight ratios were increased. Diastolic and systolic LV diameters and decreased fractional shortening were indicative of LV dilatation and dysfunction. Coronary perimicrovascular fibrosis, an increased coronary arterial wall to lumen ratio, increased immunoreactivity of PAI-1 in the tunica media and increased PAI-1 mRNA expression were present consistent with constrained mural proteo(fibrino)lysis. These cardiac manifestations were similar to those we and others have observed previously in insulin-resistant mice without impaired IRS-1-mediated signaling. CONCLUSIONS: Loss of IRS-1 signaling is sufficient to induce cardiac manifestations typical of insulin resistance including increased coronary arterial mural PAI-1 expression. Furthermore, increased PAI-1 expression is not dependent on IRS-1 signaling.
Diamond films with various crystal qualities were grown by chemical vapor deposition on silicon wafers. Their crystallinity was characterized by Raman scattering and electron backscattering diffraction. By fabricating a device structure for surface acoustic wave (SAW) using these diamond films, the propagation loss was measured at 1.8 GHz and compared with the crystallinity. It was found that the propagation loss was lowered in relatively degraded films having small crystallites, a narrow distribution in the diamond crystallite size, and preferential grain orientation. This experiment clarifies diamond film characteristics required for high-frequency applications in SAW filters.
A 72-year-old woman was admitted to our institution because of sudden chest pain. Emergency coronary angiography revealed thrombotic occlusion of the distal right coronary artery. A large cylindrical thrombus was retrieved from her distal right coronary artery using a thrombus aspiration catheter. IVUS showed minimal atherosclerosis and moderate ectatic change at the proximal right coronary artery. A reconstructed IVUS image also showed that a mural thrombus with abrupt ending was still retained at the ectatic segment. Based on this evidence, coronary ectasia was thought to be the primary cause for the thrombus formation and acute myocardial infarction in this case.
Although it is expected that a social dilemma is solved by giving people feedback information about others' cooperative behavior, previous studies demonstrated that the information may promote cooperative behavior in some situations but may promote non-cooperative behavior in other situations. The study hypothesize that the feedback information of the others' cooperative behavior promote cooperative behavior for those who feel high obligation to cooperate in a social dilemma situation, such as a problem of bicycle parking behavior on road, but promote defective behavior for those who have low moral obligation. This is because the former people are hypothesized to attribute the cause of other's cooperative behavior to their social and intrinsic motivation, but the latter are hypothesized to attribute it to the egoistic motivation. The data from an experiment (n = 126) supported the hypotheses.
We have investigated the potential role of CD1d-restricted natural killer T (NKT) cells in the development of atherosclerosis in mice. When fed an atherogenic diet (AD), NKT cell-deficient CD1d(-/-) mice had significantly smaller atherosclerotic lesions than AD-fed C57BL/6 (wild-type [WT]) mice. A significant reduction in atherosclerotic lesions was also demonstrated in AD-fed, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice reconstituted with CD1d(-/-) bone marrow cells compared with the lesions observed in Ldlr(-/-)mice reconstituted with WT marrow cells. In addition, repeated injections of alpha-GalCer or the related glycolipid OCH to apolipoprotein E knockout (apoE(-/-)) mice during the early phase of atherosclerosis significantly enlarged the lesion areas compared with mice injected with vehicle control. However, administering alpha-GalCer to apoE(-/-) mice with established lesions did not significantly increase the lesion area but considerably decreased the collagen content. Atherosclerosis development in either AD-fed WT or apoE(-/-) mice was associated with the presence of Valpha14Jalpha18 transcripts in the atherosclerotic arterial walls, indicating that NKT cells were recruited to these lesions. Thioglycolate-elicited macrophages pulsed with oxidized low-density lipoproteins expressed enhanced CD1d levels and induced NKT cells to produce interferon-gamma, a potentially proatherogenic T-helper 1 (TH1) cytokine. Collectively, we conclude that NKT cells are proatherogenic in mice.
Increased carotid artery intima-media thickness (IMT), but not necessarily peripheral vessel IMT, accompanies atherosclerosis. We hypothesized that IMT in a peripheral, muscular artery known to be resistant to atherosclerotic changes would increase with hypertension, thereby limiting increases in wall stress and potentially preserving endothelial cell function reflected by flow-mediated dilation (FMD). Plasminogen activator inhibitor type-1 (PAI-1) can inhibit vascular smooth muscle cell migration contributing to increased IMT. Thus, increased PAI-1 may attenuate the mural adaptive response. A high-resolution scanner designed to delineate brachial artery FMD and IMT was used in studies of previously untreated patients with essential hypertension (n = 18) and age- and gender-matched normotensive subjects (n = 15). Brachial IMT was increased with hypertension (0.36 +/- 0.07 vs 0.27 +/- 0.03 mm in controls, p <0.01), and FMD was lower (3.6 +/- 1.5% vs 7.8 +/- 3.6, p <0.01). PAI-1 antigen in blood was increased (40.5 +/- 31.8 vs 26.3 +/- 11.6 ng/ml, p <0.05). IMT and FMD correlated positively (r = 0.63, p <0.05) in hypertensive patients. FMD correlated inversely with wall stress (r = -0.57, p <0.05). IMT correlated inversely with PAI-1 (r = -0.61, p <0.05). These observations support the hypothesis that increased PAI-1 attenuated increases in neointimal vascular smooth muscle cell cellularity. Thus, increased PAI-1 may attenuate a mural, adaptive response to hypertension associated with preservation of endothelial cell function.
In CA1 neurons of guinea pig hippocampal slices, long-term depression (LTD) was induced in the field EPSP response in the absence of test synaptic inputs (one stimulus every 20 s) by application of the metabotropic glutamate receptor (mGluR) agonist, aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD). This effect was blocked and long-term potentiation (LTP) was induced by co-application of N-methyl-D-aspartate (NMDA) during ACPD perfusion (ACPD/NMDA-induced LTD). These results indicate that the state of NMDA receptor activation during ACPD perfusion determines whether LTP or LTD is induced in hippocampal CA1 neurons. Co-application of an inositol 1, 4, 5-trisphosphate (IP3) receptor inhibitor, 2-aminotheoxydiphenyl borate, during ACPD application had no effect on the ACPD/NMDA-induced LTP, but increased the magnitude of the ACPD-induced LTD, suggesting that the ACPD/NMDA-induced LTP involves NMDA receptors, but not IP3 receptors, whereas the converse applies to the ACPD-induced LTD.
The function of Aurora-C kinase, a member of the Aurora kinase family identified in mammals, is currently unknown. We present evidence that Aurora-C, like Aurora-B kinase, is a chromosomal passenger protein localizing first to centromeres and then to the midzone of mitotic cells. Aurora-C transcript is expressed at a moderate level albeit about an order of magnitude lower than Aurora-B transcript in diploid human fibroblasts. The level of Aurora-C transcript is elevated in several human cancer cell types. Aurora-C and Aurora-B mRNA and protein expressions are maximally elevated during the G2/M phase but their expression profiles in synchronized cells reveal differential temporal regulation through the cell cycle with Aurora-C level peaking after that of Aurora-B during the later part of the M phase. Aurora-C, like Aurora-B, interacts with the inner centromere protein (INCENP) at the carboxyl terminal end spanning the conserved IN box domain. Competition binding assays and transfection experiments revealed that, compared with Aurora-C, Aurora-B has preferential binding affinity to INCENP and co-expression of the two in vivo interferes with INCENP binding, localization, and stability of these proteins. A kinase-dead mutant of Aurora-C had a dominant negative effect inducing multinucleation in a dose-dependent manner. siRNA mediated silencing of Aurora-C and Aurora-B also gave rise to multinucleated cells with the two kinases silenced at the same time displaying an additive effect. Finally, Aurora-C could rescue the Aurora-B silenced multinucleation phenotype, demonstrating that Aurora-C kinase function overlaps with and complements Aurora-B kinase function in mitosis.
Obesity and insulin resistance confer increased risk for accelerated coronary disease and cardiomyopathic phenomena. We have previously shown that inhibition of angiotensin-converting enzyme (ACE) prevents coronary perimicrovascular fibrosis in genetically obese mice that develop insulin resistance. This study was performed to elucidate mechanism(s) implicated and to determine the effects of attenuation of angiotensin II (Ang) II. Genetically obese ob/ob mice were given ACE inhibitor (temocapril) or Ang II type 1 (AT(1)) receptor blocker (olmesartan) from 10 to 20 weeks. Cardiac expressions of plasminogen activator inhibitor (PAI)-1, the major physiologic inhibitor of fibrinolysis, and transforming growth factor (TGF)-beta(1), a prototypic profibrotic molecule, were determined and extent of perivascular coronary fibrosis was measured. Twenty-week-old obese mice exhibited increased plasma levels of PAI-1 and TGF-beta(1) compared with the values in lean counterpart. Perivascular coronary fibrosis in arterioles and small arteries was evident in obese mice that also showed increased left ventricular collagen as measured by hydroxyproline assay. Immunohistochemistry confirmed the deposition of perivascular type 1 collagen. Markedly increased PAI-1 and TGF-beta were seen immunohistochemically in coronary vascular wall and confirmed by western blotting. When obese mice were treated with temocapril or olmesartan from 10 to 20 weeks, both were equally effective and prevented increases in perivascular fibrosis, plasma PAI-1 and TGF-beta(1), left ventricular collagen and mural immunoreactivity for PAI-1, TGF-beta and collagen type 1. The c-Jun NH(2)-terminal kinase (JNK) activity was elevated in the left ventricle of obese mice (western) and blocked by temocapril and olmesartan. Ang II-mediated upregulation of PAI-1 and TGF-beta(1) with collagen deposition may explain the mechanism of perivascular fibrosis in obese mice. ACE inhibition and blockade of AT(1) receptor may prevent coronary perivascular fibrosis and collagen deposition even before development of overt diabetes. JNK activation may be a mediator of obesity-related cardiac dysfunction and a potential therapeutic target.
Nitric oxide (NO) is well known to have a wide variety of biological and physiological functions in animals. On the basis of the fact that Fe(II)-dithiocarbamates react with NO, a Fe(II)-N-(dithiocarboxy)sarcosine complex (Fe(II)-DTCS) was proposed as a trapping agent for endogenous NO. However, quantitative pharmacokinetic investigation for NO-Fe(II)-dithiocarbamate complexes in experimental animals has been quite limited. This paper describes the results on the quantitative pharmacokinetic features of a NO-Fe(II)-N-DTCS in both the blood and bile of rats following intravenous (i.v.) administration of the complex. For this purpose, we applied two in vivo methods, i.e. (1) in vivo blood circulation monitoring-electron spin resonance (BCM-ESR) which previously developed, and (2) in vivo biliary excretion monitoring-electron spin resonance (BEM-ESR). We monitored real-time ESR signals due to nitrosyl-iron species in the circulating blood and bile flow. The ESR signal due to NO-Fe(II)-DTCS was stable in biological systems such as the fresh blood and bile. In in vivo BCM- and BEM-ESR, the pharmacokinetic parameters were calculated on the basis of the two-compartment and hepatobiliary transport models. The studies also revealed that the compound is widely distributed in the peripheral organs and partially excreted into the bile. We named a kinetic method to follow spin concentrations as spinnokinetics and this method will be useful for detecting and quantifying the endogenously generated NO in Fe(II)-DTCS administered animals.
Protein-nucleic acid interactions and manners in which they control cellular communication are interesting subjects. Structural interaction data based on three-dimensional structure provide the valuable information for understanding these interactions. The database cataloging the interaction motifs of nucleic acid moieties has been developed. DNA/RNA molecules with the specific three-dimensional structure, express the specific structural and biological functions. The polymorphic nature of recognition interactions and/or motifs in protein-nucleic acid complex are generated from a variety of week forces, such as hydrogen bonds and hydrophobic interactions. The geometrical parameters about hydrogen bond and base stacking would provide the tolerant aspect for these characterizations. The tentative database including these geometrical parameters with the species and physical properties of the surrounding amino acids has been constructed. The user can obtain the selected list with several descriptors for the structure definition and sequence properties.