Cell cycle signaling and cardiovascular disease.
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Animals↗
Biomedical subjects
T Yoshimoto
Publications and source records attributed to T Yoshimoto.
Low density lipoprotein receptor-related protein is required for macrophage-mediated oxidation of low density lipoprotein by 12/15-lipoxygenase.
The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.
Animals↗
Preliminary crystallographic study of Thermus aquaticus glycerol kinase.
Glycerol kinase (GlpK) is an important enzyme which catalyzes the rate-limiting step in a central biochemical pathway involving glycerol metabolism. GlpK from the thermophile Thermus aquaticus has been overexpressed in glpK-deficient Escherichia coli and crystallized by the hanging-drop method. The crystal belongs to the cubic space group I23, with unit-cell parameters a = b = c = 163.94 (3) A. Native data were collected to 2.87 A resolution on a Cu Kalpha rotating-anode X-ray source.
Crystallization↗
Clear suppression of Th1 responses but marginal amelioration of autoimmune manifestations by IL-12p40 transgene in MRL-FAS(lprcg)/FAS(lprcg) mice.
To investigate the effects of overproduction of IL-12p40, a potent antagonist against IL-12, on lupus-like autoimmune disease in vivo, we generated p40 transgenic MRL-Fas(lprcg)/Fas(lprcg) mice. Serum p40 and IL-12 levels were 600- to 8000-fold and 3- to 20-fold higher in transgenic (p40-lpr(cg)) than nontransgenic (lpr(cg)) mice, respectively. Serum IFN-gamma levels increased after 3 months of age in lpr(cg) and this age-related increase was completely abrogated in p40-lpr(cg). Serum IL-4 levels were the same in both mice. Production of IgM and IgG anti-double-stranded DNA (dsDNA) antibodies was significantly lower in p40-lpr(cg). Anti-dsDNA antibodies decreased in Th1-dependent IgG2a but increased in the Th2-dependent IgG1 subclass significantly in p40-lpr(cg). Proteinuria, glomerulonephritis, and survival were only marginally ameliorated in p40-lpr(cg). The results suggest that excess p40 production in vivo may suppress Th1 responses in autoantibody and IFN-gamma production but lead to minimal improvement of clinical manifestations of autoimmune disease in this mouse model.
Aging↗
Enhancement of the thermal stability of pyroglutamyl peptidase I by introduction of an intersubunit disulfide bond.
From the comparison of the three-dimensional structure of mesophilic pyroglutamyl peptidase from Bacillus amyloliquefaciens and the thermophilic enzyme from Thermococcus litoralis, the intersubunit disulfide bond was estimated to be one of the factors for thermal stability. Since Ser185 was corresponded to Cys190 of the thermophilic enzyme by sequence alignment, the Ser185 residue was replaced with cysteine by site-directed mutagenesis. The S185C mutant enzyme appeared to form a disulfide bond, which was confirmed by SDS-PAGE with and without 2-mercaptoethanol. The mutant enzyme showed a catalytic efficiency equivalent to that of the wild-type enzyme for hydrolysis of a synthetic peptide substrate. However, the thermal stability of the S185C mutant was found to be 30 degrees C higher than that of wild-type. Thus the introduction of a disulfide bond enhanced thermal stability without changing the catalytic efficiency of the enzyme.
Amino Acid Sequence↗
A critical role of Fc receptor-mediated antibody-dependent phagocytosis in the host resistance to blood-stage Plasmodium berghei XAT infection.
Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR gamma-chain knockout (FcRgamma(-/-)) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcgammaRI, FcgammaRII, and FcgammaRIII, could not induce Ab-dependent phagocytic activity. These FcRgamma(-/-) mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-gamma by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRgamma(-/-) mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-gamma(-/-) mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-gamma production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.
Animals↗
Cyclosporin A, but not FK506, prevents the downregulation of phosphorylated Akt after transient focal ischemia in the rat.
The two immunosuppressants, cyclosporin A (CsA) and FK506, when given 1 and 3 h after the start of reperfusion following 2 h of middle cerebral artery (MCA) occlusion, reduce infarct volume to 30% of control. This suggests a common effect, e.g. one due to suppression of the activation of calcineurin. However, when given by the intracarotid (i.c.) route after only 5 min of recirculation CsA, but not FK506, reduced infarct volume even further, to 10% of control. This was attributed to the fact that CsA, but not FK506, block the in vitro assembly of a mitochondrial permeability transition (MPT) pore. The present experiments were undertaken to further characterize the anti-ischemic effect of CsA, when given i.c. 5 min after recirculation and to explore why CsA, when given at that time, is more efficacious than FK506. It was established that the i.c. administration of CsA in a dose of 10 mg/kg increased the tissue concentration of CsA 2- to 3-fold, when compared to the i.v. administration. CsA proved to be effective in reducing infarct volume even when the tissue damage was assessed by histopathology after 7 days of recovery. MCA occlusion of 2 h duration caused a sustained decrease in the phosphorylation Akt at threonine 308. Since this down regulation of Akt was prevented by CsA, the results suggested a link between dephosphorylaltion of Bad, and cell death. Interestingly FK506 did not prevent down regulation of Akt, it thus seems unlikely that the anti-ischemic effect of CsA is related to its association with cytosolic cyclophilin.
Animals↗
Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat.
Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia.
Animals↗
Localization of mRNAs for six ARFs (ADP-ribosylation factors) in the brain of developing and adult rats and changes in the expression in the hypoglossal nucleus after its axotomy.
ADP-ribosylation factors (ARFs) play crucial roles in the intracellular vesicular transport and in regulation of phospholipid-modifying enzyme activities and cytoskeletons. Using in situ hybridization histochemistry, the gene expression for six isoforms of ARF was examined during normal development of rats and in the hypoglossal nucleus following its axotomy. In the embryonic brain, the expression for ARF-1, -4, -5, -6 mRNAs was distinct in the ventricular germinal zone while that for ARF-3, -4, -5 in the mantle zone. In early postnatal brain, the expression for six ARFs was seen widely in various loci of the gray matter with different intensity, and the expression of ARF-4, -5, -6 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for all ARF isoforms decreased more or less in most gray matters and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression levels of ARF-2 and -4 mRNAs in affected hypoglossal nucleus increased after 24 h up to 7 days following axotomy of the hypoglossal nerve, while no such changes were seen in the expression levels for the other ARFs. The present findings suggest that ARFs are differentially involved in some processes essential to nerve regeneration as well as neuronal differentiation and maturation.
ADP-Ribosylation Factor 1↗
The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis.
Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O.H-N:Inh and Gln71:N-H.OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k(cat) value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k(cat)/K(m)) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.
Amino Acid Sequence↗
Cyclooxygenase-2 stimulates production of amyloid beta-peptide in neuroblastoma x glioma hybrid NG108-15 cells.
Cyclooxygenase (COX) synthesizes bioactive prostaglandins from arachidonic acid, and there are COX-1 and COX-2 isoforms with distinct pathophysiological functions. Recent studies demonstrated that COX-2 expression was up-regulated in the brain of patients with Alzheimer's disease. We established mouse neuroblastoma x rat glioma hybrid NG108-15 cells stably expressing human COX-2. The COX-2-expressing cells showed 3- to 4-fold increases in both COX activity and prostaglandin E(2) production. The mRNA level of amyloid precursor protein (APP) was elevated by approximately 2-fold in the COX-2-expressing cells compared with mock-transfected cells. Amyloid beta-peptide and a secreted form of APP, both derived from APP by proteolysis was also increased. Interestingly, neurite outgrowth was stimulated in the COX-2-expressing cells with concomitant reduction of the cell proliferation rate. A selective COX-2 inhibitor (JTE-522) and a nonselective COX inhibitor (indomethacin) suppressed production of amyloid beta-peptide and a secreted form of APP by inhibition of APP mRNA level, suggesting that COX-2 plays important roles in the neurodegenerative processes of Alzheimer's disease.
Amyloid beta-Peptides↗
Judging semantic and episodic incongruity: a magnetoencephalographic study.
Evoked magnetic fields over the entire head were measured during a semantic judgment task, in which conditions of the pre-test learning session were manipulated. Subjects learnt related and unrelated category-noun pairs, and were then asked to judge the relationship between category and noun. Unrelated pairs evoked activation around 400 ms in the left temporal area. In addition, newly presented related pairs, of which the categories were used to learn unrelated pairs, also evoked similar activities. Our findings suggest that these activities are not only related to the semantic incongruity between a pair of words as in previous studies, but also to the episodic incongruity between the learning and measurement sessions.
Adult↗
Neuromagnetic localization of N15, the initial cortical response to lip stimulus.
The initial cortical response of the trigeminal somatosensory evoked magnetic fields was measured for electrical stimulation of the lower lip in eleven subjects. The stimulus frequency was 0.7 Hz and stimulus intensity was nine times sensory threshold. The initial contralateral response was detected in 20 hemispheres at a latency of 14.6+/-1.3 ms and was named N15m. The equivalent current dipole of N15m was localized at the posterior bank of the central sulcus with anterior-superior orientation, and inferior to the dipole of N20m for median nerve stimulation.
Adult↗
Reduction of copper, zinc-superoxide dismutase in knockout mice does not affect edema or infarction volumes and the early release of mitochondrial cytochrome c after permanent focal cerebral ischemia.
Copper,zinc-superoxide dismutase (SOD1) was shown to be highly protective against ischemia/reperfusion injury in the brain. We have recently reported that SOD1 prevents the release of mitochondrial cytochrome c and subsequent apoptosis after ischemia/reperfusion in mice. To investigate its dose dependent effect on permanent focal cerebral ischemia, we examined neurological deficit scores, infarction volume, and the amount of hemisphere enlargement after 24 h of focal cerebral ischemia in both knockout mutants of SOD1 (Sod1 -/+ and Sod1 -/-) and wild-type littermates. We also examined the release of cytochrome c and subsequent DNA fragmentation after ischemia. There were no differences in the neurological deficit scores, infarction volumes and edema formation. There was also no difference of the amount cytosolic cytochrome c at 2 h and of the amount of DNA fragmentation at 24 h after focal cerebral ischemia. The results indicate that the SOD1 enzyme does not appear to affect cerebral infarction, cerebral edema nor the mitochondrial signaling pathway for apoptosis following permanent focal cerebral ischemia where there is no reperfusion injury.
Animals↗
Fas/Apo-1 (CD95)-mediated apoptosis of neutrophils with Fas ligand (CD95L)-expressing tumors is crucial for induction of inflammation by neutrophilic polymorphonuclear leukocytes associated with antitumor immunity.
To address how FasL-expressing tumors induce neutrophil emigration and abrogate tumorigenicity, we investigated the behavior of FasLcDNA-transfected hepatoma MH134 (G2) cells injected into wild-type (+) mice, lpr(cg)/lpr(cg) (lpr(cg)) mice with death domain (DD)-mutated Fas, and gld/gld lpr/lpr (gld/lpr) mice with defects in FasL/Fas. G2 cells were eradicated after extensive infiltration of neutrophils around them in + mice but formed tumors without such infiltration in lpr(cg) and gld/lpr mice. Abundant cell debris suggestive of apoptosis of infiltrating neutrophils was found among G2 tumor cells in + mice but a few neutrophils infiltrating among G2 cells were intact in lpr(cg) and gld/lpr mice. Collectively, these results indicate the crucial role of Fas DD in Fas-mediated apoptosis of neutrophils and suggest that apoptosis of neutrophils with FasL-expressing tumors may trigger the extensive infiltration of neutrophils, resulting in violent inflammation and ultimately in the eradication of tumor cells.
Animals↗
Enhancement of fibrinolytics with a laser-induced liquid jet.
BACKGROUND AND OBJECTIVE: There are several problems inherent in the treatment of cerebral embolisms, such as the narrow therapeutic time window and the severe side effects of fibrinolytic drugs. There is thus need of a new method of removing a cerebral thrombus more rapidly using smaller amounts of fibrinolytics. STUDY DESIGN/MATERIALS AND METHODS: The liquid-jet generator was made by insertion of an optical fiber (diameter: 0.6 mm) into a balloon catheter (6 Fr). A pulsed holmium (Ho) YAG laser (pulse duration time = 350 micros) was used as a laser source. The maximum penetration depth of a liquid jet generated with this device into a gelatin artificial thrombus was measured at various stand-off distances (L; distance between the optical fiber end and the catheter exit). Based on the result, a stand-off distance of 13 mm was chosen to investigate the enhancement of urokinase (UK) efficacy by only a single operation of the liquid-jet device in artificial thrombi made of human blood. RESULTS: Maximum penetration depth increased in proportion to L and reached a maximum value (9 mm) when L was around 13 mm. Fibrinolysis rates (%) after incubation with a small amount of UK for 10 and 30 minutes were predominantly raised by a single use of the laser-induced liquid jet (5.4 +/- 2.4 vs. 22.6 +/- 6.1 and 7.3 +/- 3.8 vs. 38.3 +/- 5.6, respectively (mean +/- SD, P < 0.001)). CONCLUSIONS: A laser-induced liquid jet effectively promoted fibrinolysis in vitro with use of only a small amount of fibrinolytics.
Analysis of Variance↗
IL-12 plays a pathologic role at the inflammatory loci in the development of diabetes in NOD mice.
Accumulating evidence suggests that CD4(+)T helper type 1 (Th1) cells play a major role in the development of insulin-dependent diabetes mellitus (IDDM) in the non-obese diabetic (NOD) mouse model. Interleukin (IL)-12 is a potent immunoregulatory molecule that is a key determinant of T-cell differentiation into Th1 cells, and has been implicated in the development of IDDM. To investigate the role of IL-12 that is locally produced by islet-infiltrating cells in the development of IDDM, we generated transgenic NOD mice in which the IL-12 p40 homodimer, a natural antagonist of IL-12, was produced exclusively in islets without affecting the levels of IL-12 p40 in the systemic circulation. We found that the incidence of diabetes was significantly reduced in these transgenic mice. These results clearly demonstrate that IL-12 locally produced by islet-infiltrating cells plays a critical role in the development of IDDM.
Animals↗
Movement-related magnetic fields to tongue protrusion.
Movement-related magnetic fields (MRFs) associated with tongue protrusion were measured in five normal subjects using a helmet-shaped magnetoencephalography system. Bihemispherical two-dipolar patterns appeared from approximately -2000 or -1000 to 0 ms to the trigger signal indicating when protrusion of the tongue tip reached the frontal part of the palate. Equivalent current dipoles (ECDs) for the MRFs were localized on the central sulcus, 14.4 +/- 6.1 mm inferior (P < 0.0001) and 7.6 +/- 6.9 mm anterior (P < 0.01) to the ECD for the N20m in the somatosensory evoked fields for median nerve stimuli. The ECD orientations of MRFs were anterior and perpendicular to the central sulcus. These results correspond to the movement-related potentials for tongue protrusion previously recorded from subdural electrodes in patients with epilepsy. Magnetoencephalography can be applied to analyze cortical functions related to tongue movement with high resolution in time and space in normal subjects.
Adult↗