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Biomedical subjects

T Miyashita

Publications and source records attributed to T Miyashita.

At least 19 recordsLinked to original sources
PubMed2001

Distinctive tissue distribution and phosphorylation of IRSp53 isoforms.

An insulin-receptor substrate of 53-kDa protein (IRSp53) is an adapter protein, which interacts with the Rho-family of GTPases and mediates neurite outgrowth. It also binds to DRPLA protein, a product of the gene responsible for a polyglutamine disease, dentatorubral-pallidoluysian atrophy (DRPLA). Isoforms of human IRSp53 have been reported, each with a unique amino acid sequence at the C-terminal end. Here we report the distinctive tissue distribution and phosphorylation of three isoforms (L, S, and T-forms). Western blotting analyses with isoform-specific antibodies demonstrated that the L and S-forms were expressed in the brain, whereas the T-form was not present in any tissues examined, but was found in a cancer cell line. The L and S-forms were phosphorylated upon stimulation with insulin, and the T-form with IGF-I. Since phospho-acceptor sites were localized to the common portion, the difference in phosphorylation seems to be due to the unique C-terminal sequence.

Amino Acid Sequence↗
PubMed2001

Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family.

Bim protein is one of the BH3-only proteins, members of the Bcl-2 family that have only one of the Bcl-2 homology regions, BH3. BH3-only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. Bim(EL), Bim(L) and Bim(S). Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimalpha1, alpha2, and beta1-beta4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C-terminal hydrophobic region. Among the novel isoforms, only Bimalpha1 and alpha2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein-fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.

Amino Acid Sequence↗
PubMed2001

Calcium-sensitive nonselective cation channel identified in the epithelial cells isolated from the endolymphatic sac of guinea pigs.

We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 +/- 1.3 pS (n = 8) in our standard solution. The relative ionic permeability of the channel was in the order K+ = Na+ > Ca2+ >> Cl-. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 microm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 microm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 microm to 10 microm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.

Adenosine Triphosphate↗
PubMed2001

Observation of rho/omega meson modification in nuclear matter.

We measured the invariant mass spectra of electron-positron pairs in the target rapidity region of 12-GeV p+A reactions. We have observed a significant difference in the mass spectra below the omega meson between p+C and p+Cu interactions. This difference indicates that the spectral shape of mesons is modified at normal nuclear-matter density.

Journal Article↗
PubMed2001

ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase.

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.

Acyl Coenzyme A↗
PubMed2001

Comprehensive studies on subcellular localizations and cell death-inducing activities of eight GFP-tagged apoptosis-related caspases.

By using green fluorescent protein fusion, we investigated the subcellular localization of all the caspases that have been cloned from humans and implicated in the execution of apoptosis. We divided these caspases into three groups according to subcellular localization. The first group includes caspase-1, -3, -6, -7, and -9, which are expressed mainly in the cytoplasm with various levels of nuclear localization depending on the cell type. The second group has a single member, caspase-2, which is primarily localized in the nucleus. The nuclear localization was demonstrated to be mediated by a nuclear localization signal near the NH(2)-terminus of the prodomain. The third group includes caspase-8 and -10, which have a cytoplasmic distribution. These two members have potent, rapid cell death-inducing activity and are prone to make aggregates when overexpressed. Their prodomains formed marked fibrous structures in the cytoplasm whose localization seemed distinct from organelles or cytoskeletons. None of the GFP-caspases examined in this study showed a predominant mitochondrial localization as has been reported for some caspases.

Apoptosis↗
PubMed2001

Molecular cloning and characterization of Bif-1. A novel Src homology 3 domain-containing protein that associates with Bax.

Bax is a proapoptotic member of the Bcl-2 protein family that commits the cell to undergo programmed cell death in response to apoptotic stimuli. To gain further insights into Bax mechanisms, we have identified a novel Bax-binding protein, termed Bif-1, by using a yeast two-hybrid cloning technique. Bif-1 is an evolutionarily conserved cytoplasmic protein that contains a predicted Src homology 3 (SH3) domain located near its C terminus but shares no significant homology with members of the Bcl-2 family. A Northern blot analysis indicates that Bif-1 is expressed in most tissues with abundant expression in heart and skeletal muscle. Bif-1 is capable of interacting with Bax as demonstrated by yeast two-hybrid, coimmunoprecipitation, and immunofluorescence studies. Induction of apoptosis in murine pre-B hematopoietic cells FL5.12 by interleukin-3 withdrawal results in increased association of Bax with Bif-1, which is accompanied by a conformational change in the Bax protein. Overexpression of Bif-1 promotes Bax conformational change, caspase activation, and apoptotic cell death in FL5.12 cells following interleukin-3 deprivation. Bif-1 thus represents a new type of regulator of Bax-mediated signaling pathways for apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Overexpression of a slit homologue impairs convergent extension of the mesoderm and causes cyclopia in embryonic zebrafish.

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.

Animals↗
PubMed2001

A method for detecting myocardial abnormality by using a total current-vector calculated from ST-segment deviation of a magnetocardiogram signal.

A simple method to determine the state of ischaemia or fibrosis of myocardial cells has been developed. This method uses the ST wave of 64-channel magnetocardiogram (MCG) signals to calculate three parameters from the current-arrow map of the normal component signal of the MCG. One parameter is a total current vector that is obtained through summation of all current arrows. Another is a variance current vector calculated from the differential vector of two total current vectors at different times. The third is a flatness factor between the magnitude of the total current vector and the variance current vector. The three parameters are independent of the distance between the heart and the gradiometers. We measured the MCG signals of 29 healthy subjects, twenty patients with coronary artery disease (ten with previous myocardial infarction (MI) and ten with angina pectoris (AP)), and eight patients with cardiomyopathy (four with hypertrophic cardiomyopathy (HCM), three with dilated cardiomyopathy (DCM), and one with restrictive cardiomyopathy (RCM)). With our method, none of the healthy subjects tested positive for myocardial abnormalities, while 80% of the MI patients, 50% of the AP patients, and 100% of the cardiomyopathy patients tested positive. Although further testing is needed, we feel this simple technique enables easy diagnosis of myocardial damage.

Cardiomyopathies↗
PubMed2001

A method for detecting myocardial abnormality by using a current-ratio map calculated from an exercise-induced magnetocardiogram.

A method for making a current-ratio map to determine the ischaemic area of angina pectoris (AP) patients has been developed. This method uses a current-arrow map calculated using a ORS wave from 64-channel magnetocardiogram (MCG) signals. The current-ratio map can be calculated from the ratio of an exercise-induced current vector to an at-rest current vector. The MCG signals of eight patients with angina pectoris (AP) (six patients with effort AP and two patients with variant AP) and four healthy volunteers were measured before and after a two-step exercise test. The current-ratio maps of the six patients with effort AP showed three distinct patterns: a left-circumflex-artery (LCX) pattern; a right-coronary-artery (RCA) pattern; and a left-anterior-descending (LAD) pattern. The maximum current ratios of these three patterns differed from those of normal patterns. The patterns of two patients with variant AP were similar to normal patterns. Furthermore, a comparison of the current-ratio map before and after percutaneous-transluminal-coronary-angioplasty (PTCA) treatment indicated that the cardiac ischaemia was reduced in all patients. An appropriate criterion to diagnose abnormality in a patient with an ischaemic myocardial area seems to be a maximum current ratio exceeding 0.4 to 0.5. Based on these preliminary results, it is believed that the location of an ischaemic area (the coronary artery part) can be estimated by using the ischaemic current-ratio map pattern.

Angina Pectoris↗
PubMed2001

Short-time ultrasound of head and neck squamous cell carcinoma under radiotherapy.

To evaluate the efficacy of ultrasonography for patients with head and neck squamous cell carcinoma before and during radiotherapy (RT), consecutive patients with macroscopic lesions were examined before and during RT. Each scan was performed percutaneously in a short time (less than 5 min). The demonstration rate of primary tumors and the largest node of previously known metastatic cervical nodes were measured. Of all 190 patients, 91 primary tumors were detected (47.9%). Primary tumors in oral cavity, oropharynx, hypopharynx and cervical esophagus were determined in over 70% and, for those of nasopharynx and larynx, the demonstration rates were 30% or less. Nodal metastases were visualized in 78 of 80 patients with regional metastases (97.5%). Using the above information, 73 patients (38.4%) received the benefit of radiation therapy. Short-time sonography is a valuable modality for patients with selected sites of head and neck cancers.

Adolescent↗
PubMed2001

Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish.

Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of Islet-2 or the LIM-interacting domain of Ldb proteins prevented binding of Islet-2 to Ldb proteins in vitro and caused similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons as the defects induced by injection of Islet-2-specific antisense morpholino oligonucleotide. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains derived from two different Islet-1 family members, demonstrated that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors.

Animals↗
PubMed2001

Extended polyglutamine selectively interacts with caspase-8 and -10 in nuclear aggregates.

A growing number of inherited neurodegenerative disorders, including Huntington's disease, have been shown to be caused by the expansion of CAG/polyglutamine repeats. The molecular mechanism underlying these disorders, however, has yet to be clarified. We and others previously demonstrated that caspase-8 was activated by proteolysis in association with the expression of extended polyglutamine. Here, we further analyzed the selectivity of caspases in the process mediated by extended polyglutamine. Among upstream caspases, caspase-10, a close homolog of caspase-8, was also proteolytically activated, but caspase-9 was not. Caspase-8 and -10 were recruited into nuclear aggregates of extended polyglutamine, where at least a fraction of these caspases was converted to the activated forms. Caspase-8 and -10 were co-immunoprecipitated with polyglutamine only when the polyglutamine was pathologically extended, whereas caspase-2, -3, -6, -7 and -9 were not co-immunoprecipitated with polyglutamine regardless of its size. A dominant-negative form of caspase-8 with a mutation at the catalytic cysteine residue inhibited polyglutamine-mediated nuclear apoptotic phenotype. These results suggest that caspase-8 and -10 are autoactivated as a result of close proximity of the proforms of these molecules that occurs due to aggregate formation, which reveals a novel toxic gain-of-function mechanism for the pathogenesis of CAG-repeat disorders.

Apoptosis↗
PubMed2001

Effects of autologous fresh platelet concentrate on haemostasis in cardiac reoperations.

The aim of this study was to determine whether autologous fresh platelet concentrate (PC) significantly improves haemostasis in cardiac reoperations compared with autologous fresh whole blood (WB). Forty-eight patients who had elective cardiac reoperations with a low-dose aprotinin priming regimen were divided into two groups. The amount of allogeneic blood transfusion was less in patients who were harvested 15 units of PC (Group PC; n = 24) before cardiopulmonary bypass (CPB) than patients who were harvested 400 ml of WB (Group WB; n = 24). The amount of mediastinal drainage for 12 h in intensive care units were significantly reduced in Group PC compared with Group WB (435 +/- 273 ml in Group PC versus 909 +/- 209 ml in Group WB; P < 0.001). Platelet count and collagen-induced whole blood platelet aggregation increased significantly higher in Group PC than Group WB after reinfusion. In conclusion, autologous fresh PC improved haemostasis compared with autologous fresh whole blood in cardiac reoperations with a low-dose aprotinin priming regimen.

Aged↗
PubMed2001

Impedance magnetocardiogram.

We have developed an impedance magnetocardiogram (IMCG) system to detect the change of magnetic field corresponding to changes in blood volume in the heart. A low magnetic field from the electrical activity of the human heart--the so-called magnetocardiogram (MCG)--can be simultaneously detected by using this system. Because the mechanical and electrical functions in the heart can be monitored by non-invasive and non-contact measurements, it is easy to observe the cardiovascular functions from an accurate sensor position. This system uses a technique to demodulate induced current in a subject. A flux-locked circuit of a superconducting quantum interference device has a wide frequency range (above 1 MHz) because a constant current (40 kHz) is fed through the subject. It is shown for the first time that the system could measure IMCG signals at the same time as MCG signals.

Adult↗
PubMed2001

Prenatal diagnosis of QT prolongation by fetal magnetocardiogram--use of QRS and T-wave current-arrow maps.

To determine the T wave of a fetal magnetocardiogram (FMCG), we have evaluated the T/QRS ratio and obtained current-arrow maps that indicate weak currents. We measured FMCG signals for 52 normal fetuses and two abnormal fetuses with prolonged QT waves by using three superconducting quantum interference device (SQUID) systems: a nine-channel system, a 12-channel vector system and a 64-channel system. The T/QRS ratio was calculated for all the normal fetuses from the maximum magnitudes of the QRS complex and the T wave. Current-arrow maps of the QRS complex (R wave) and T wave were obtained by using the 64-channel system, and the phase differences of the total-current vectors were calculated by using the current-arrow maps. The results showed that the T/QRS ratio had a wide variability of 0.35 for the normal fetuses. However, the magnitude of the prolonged T wave was as weak as the detection limit of the SQUID magnetometer. Although the T/QRS ratios for the fetuses with QT prolongation were within the normal range (< 0.35), the weak magnitude of the prolonged T wave could be evaluated. On the other hand, by comparing the current-arrow maps of the R and T waves for the normal fetuses, we found that the maximum-current arrows were indicated as either in the same direction or in opposite directions. These patterns could be identified clearly by the phase differences. Very weak prolonged T waves for the two abnormal fetuses could be determined by using these current-arrow maps and phase differences. Consequently, although the T/QRS ratios of FMCG signals have a wide distribution, we have concluded that the current-arrow map and phase difference can be used to determine the T wave of an FMCG signal.

Adult↗
PubMed2001

Reverse redistribution of 99m Tc-sestamibi after direct percutaneous transluminal coronary angioplasty in acute myocardial infarction: relationship with wall motion and functional response to dobutamine stimulation.

Reverse redistribution (RR) of 99mTc-sestamibi is observed after direct percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI). The purpose of this study was to clarify the functional characteristics of myocardial segments with RR after direct PTCA in AMI. Thirty patients with AMI who had undergone direct PTCA were examined. Myocardial perfusion tomography with 99mTc-sestamibi and low dose dobutamine echocardiography were performed within 2 weeks of the onset. The 99mTc-sestamibi images were obtained 1 and 3 h after tracer administration. The left ventricle was divided into nine segments, and regional 99mTc-sestamibi uptake and clearance were quantitatively evaluated in each segment. RR was defined as a decrease in 99mTc-sestamibi uptake of >10% on 3 h delayed images compared with the 1 h early images. The left ventricle in the echocardiographic images was also divided into nine segments corresponding to the scintigraphic images, and regional wall motion was assessed in the resting condition as the baseline and during dobutamine administration (5-10 microg x kg(-1) x min(-1)). Out of a total of 270 myocardial segments, 111 segments were perfused by the culprit coronary artery and were defined as ischaemic segments. There were 25 segments with RR and 86 segments without RR in the ischaemic myocardium. Enhanced clearance of 99mTc-sestamibi was observed in ischaemic segments with RR (P<0.001). Echocardiography demonstrated that 24 out of 25 segments with RR and 61 out of 86 segments without RR had wall motion abnormalities. Dobutamine infusion improved wall motion in 20 (83%) of the 24 dysfunctional segments with RR and 33 (54%) of the 61 dysfunctional segments without RR (P<0.02). These findings suggest that RR indicates reversible functional abnormalities associated with preserved contractile reserve in response to dobutamine. The early and delayed imaging of 99mTc-sestamibi provides useful information regarding the residual viability of the dysfunctional myocardium in AMI patients.

Acute Disease↗