[Three-dimensional CT diagnosis of cerebrovascular pathology].
Explore the source record for details and available documents.
Aged↗
Biomedical subjects
H Hayashi
Publications and source records attributed to H Hayashi.
Fluoromicroscopic detection of myc-tagged GLUT4 on the cell surface. Co-localization of the translocated GLUT4 with rearranged actin by insulin treatment in CHO cells and L6 myotubes.
We earlier developed a novel method to detect translocation of glucose transporter type 4 (GLUT 4) directly, quantitatively and simply using c-MYC epitope-tagged GLUT4 (GLUT4myc) Kanai F, Nishioka Y, Hayashi H, Kamohara S, Todaka M, Ebina Y: J Biol Chem 268: 14523-14526, 1993). We further developed the method to visualize GLUT4myc on the cell surface++ by fluorescence microscope using a highly sensitive immunochemical detection system in tissue culture cells stably expressing GLUT4myc. The translocation of GLUT4myc was observed on stimulation with insulin in 3T3-L1 adipocytes, CHO cells and L6 myotubes stably expressing GLUT4myc. Platelet-derived growth factor (PDGF), norepinephrine and bradykinin also triggered GLUT4 translocation in CHO-GLUT4myc cells stably expressing each receptor. To observe the distribution of GLUT4 and actin after insulin treatment, double staining for GLUT4myc and actin was performed. Translocated GLUT4myc on the cell surface was co-localized with rearranged actin in CHO cells and L6 myotubes. This result suggests that a correlation exists between GLUT4 translocation and actin rearrangement.
3T3 Cells↗
Electrocardiographic changes induced by diesel exhaust particles (DEP) in guinea pigs.
We have previously reported that diesel exhaust particles (DEP) caused a negative inotropic effect that was followed by cardiac arrest in the isolated atrial preparation of guinea pigs. The purpose of this study was to examine the systemic effects of DEP on electrocardiographic (ECG) changes using guinea-pigs. We found that intravenously administered dimethyl sulfoxide (DMSO) extract of DEP solution induced arrhythmias and deaths via complete atrioventricular (AV) block in guinea pigs. The LD of DEP solution was 132.0 +/- 7.2 mg/kg. The coefficient of variance (CV) of LD measured by the modified Hatcher-Magnus method was relatively small (5.5%). Fractions of DEP extracted by hexane, ethanol or methanol, 4-hydroxyphthalic acid 2-methyl ester, a compound isolated from methanol extract of DEP did not induce significant ECG changes in guinea pigs. As compared with fresh DEP solution, the DMSO/DEP solution used in the present study induced similar cardiac toxicity after being stored in a freezer at 4 degrees C for 3 days. These results suggest that stable and water-soluble fractions of DEP may be responsible for cardiotoxicity.
Animals↗
The density of a homogeneous population of cells controls resetting of the program for swarmer formation in the unicellular marine microorganism Noctiluca scintillans.
Noctiluca scintillans is a luminescent marine dinoflagellate. The life cycle of Noctiluca consists of a vegetative stage and a swarmer stage. The swarmer stage of Noctiluca is initiated by formation of a swarmer-mother cell instead of binary fission of vegetative cells. We studied the formation of swarmers under various conditions and became convinced that the cells have a strict program for the formation of swarmers which starts to operate in every cell after a defined number of cell cleavages. The probability that the program will be executed appeared to be affected by the presence of other cells. In other words, a high density of cells suppressed the expression of the program. Suppression was achieved by resetting the mechanism and was related to the number of cell divisions. Our findings provide one of the simplest examples of a mechanism by which a large population produces individuality in a group of genetically homogeneous organisms.
Animals↗
The imine-pyridine torsion of the pyridoxal 5'-phosphate Schiff base of aspartate aminotransferase lowers its pKa in the unliganded enzyme and is crucial for the successive increase in the pKa during catalysis.
In aspartate aminotransferase, pyridoxal 5'-phosphate (PLP) forms a Schiff base with the epsilon-amino group of Lys258 (internal aldimine). The internal aldimine has a pKa value of 6.8. Binding of a substrate amino acid to the enzyme yields the Michaelis complex, in which PLP still forms the internal aldimine with Lys258. This is followed by a transaldimination process to form a Schiff base of PLP with the alpha-amino group of substrates (external aldimine). Kinetic analysis of the spectral changes during the reaction of the enzyme with a substrate analogue 2-methylaspartate showed that the aldimine is 6.4-8.6% protonated in the Michaelis complex and 32-43% in the external aldimine. The bases that accept protons from the aldimines are considered to be the substrate alpha-amino group in the Michaelis complex and the epsilon-amino group of Lys258 in the external aldimine. Therefore, the intrinsic pKa value of the aldimine is expected to increase over a range of 3 during transformation from the unliganded enzyme (pKa = 6.8) to the Michaelis complex (pKa = 8.8) and the external aldimine (pKa > 10). When the Lys258 side chain of the internal aldimine was "cleaved" by the construction of an enzyme in which Lys258 was replaced by Ala and the aldimine was reconstituted with methylamine, the pKa of the internal aldimine was increased to 9.6. This indicates that the low pKa value of the internal aldimine of the unliganded enzyme is provided by the side chain of Lys258 which destabilizes the planar conformation of the aldimine suitable for protonation. This strained conformation is partially relaxed in the Michaelis complex, and the pKa is moderately increased. On formation of the external aldimine, Lys258 is released and the aldimine is fixed to a near planar conformation and has a high pKa value. Thus, the aldimine pKa is modulated by a mechanism that exploits the conformational differences between the intermediate structures. The strain of the protonated internal aldimine is interpreted to enhance the catalytic ability of the enzyme by increasing the energy level of the free enzyme plus substrate at neutral pH relative to the transition state.
Alanine↗
Microsphere embolism-induced changes in noradrenaline uptake of the cerebral cortex in rats.
The present study was undertaken to elucidate pathophysiological changes in noradrenaline (NA) transporter and Na+/K+-ATPase, key regulators of cation gradient across the plasma membrane, in nerve terminals of the cerebral cortex after microsphere-induced cerebral embolism in rats. The Vmax value of NA uptake, when analyzed by the Eadie-Hofstee plot, tended to decrease on the 1st day and decreased on the 3rd and 7th days after the embolism without any change in the Km value. The NA content in cerebrocortical synaptosomes did not alter on the 1st day, but decreased on the 3rd and 7th days after the embolism. Ouabain (1 mM) inhibited NA uptake on the 1st day, but did not alter the uptake on the 3rd and 7th days after the embolism. The activity of Na+/K+-ATPase of cerebrocortical synaptosomes increased on the 1st day and gradually decreased up to the 7th day after the embolim. These results suggest that NA uptake in nerve terminals of the cerebral cortex decreased after microsphere embolism, which may be due to a reduction in function of NA transporters. The changes in Na+/K+-ATPase following microsphere embolism may represent a compensatory action to maintain ion homeostasis in nerve terminals at an early stage of ischemic injury.
Animals↗
Reconstitution of insulin signaling pathways in rat 3Y1 cells lacking insulin receptor and insulin receptor substrate-1. Evidence that activation of Akt is insufficient for insulin-stimulated glycogen synthesis or glucose uptake in rat 3Y1 cells.
Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4, 5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.
Amino Acid Sequence↗
Effect of growth hormone on the translocation of GLUT4 and its relation to insulin-like and anti-insulin action.
To elucidate the effect of growth hormone (GH) on the insulin signal transduction pathway leading to the translocation of glucose transporter-4 (GLUT4), we constructed Chinese hamster ovary cells that overexpressed GH receptor and GLUT4. Treatment with GH triggered GLUT4 translocation, and this translocation was completely inhibited by wortmannin. GH-induced GLUT4 translocation reached a maximum level after 30 min, and then gradually decreased and returned to the basal level after 2 h. Tyrosine phosphorylation of JAK2 also became maximal after 30 min and then gradually decreased. In contrast, GLUT4 translocation remained unchanged for 2 h after insulin treatment, and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) also remained constant for up to 2 h. Chronic GH treatment had almost no effect on insulin-stimulated Akt kinase activation and GLUT4 translocation. These results suggest that GH and insulin translocate GLUT4 in a similar manner, at least in part, and the difference in translocation depends on the difference in the tyrosine phosphorylation of JAK2 and IRS-1. The anti-insulin action of GH after chronic GH treatment does not appear to be mainly due to the inhibition of GLUT4 translocation.
Androstadienes↗
Regulation of intracellular pH during H+-coupled oligopeptide absorption in enterocytes from guinea-pig ileum.
1. The mechanisms for regulating the intracellular pH (pHi) level during oligopeptide absorption were investigated in the enterocytes from guinea-pig ileum by identifying the acid-base transporters responsible for extruding H+ that enters the cell through the H+-oligopeptide cotransporter. 2. The pHi level was measured by microfluorometry in an isolated villus tip loaded with the pH-sensitive fluoroprobe 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oligopeptide-induced increment in the short-circuit current (Isc) was determined in a mucosal sheet in Ussing chambers. A CO2/HCO3--buffered solution was used. 3. The superfusion of glycylglycine (Gly-Gly, l0 mM) caused a decrease in pHi level, which returned to the basal level after removing Gly-Gly. This pHi recovery was strongly dependent on extracellular Na+. Amiloride partially inhibited the pHi recovery rate with an IC50 value of 41 microM, the maximum inhibition being approximately 70%. In the presence of amiloride at its maximum concentration (0.3 mM), the addition of 0.6 mM DIDS caused a further decrease, but did not abolish the pHi recovery rate. In the absence of CO2 and HCO3-, the pHi recovery was almost completely abolished by 0.3 mM amiloride. 4. The intracellular H+ accumulation induced by 0.3 mM amiloride or by 0.6 mM DIDS, as estimated from the pHi decrease and buffer capacity, was significantly greater during Gly-Gly superfusion than under resting conditions. 5. The increase in Isc induced by luminal glycylproline was attenuated by either removing serosal Na+ or by adding 0.5 mM amiloride or 0.6 mM DIDS to the serosal side. 6. We conclude that both Na+-dependent, amiloride-sensitive acid extrusion, probably by the Na+-H+ exchanger, and Na+- and HCO3--dependent, DIDS-sensitive acid extrusion, possibly by the Na+-HCO3- cotransporter, are involved in extruding H+ that enters cells by the H+-oligopeptide cotransport. It is proposed that these acid extrusion (or base loading) mechanisms are present in the basolateral membrane and are important for maintaining oligopeptide absorption, as well as the acid extrusion mechanism in the apical membrane.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
cDNA cloning and characterization of mouse nifS-like protein, m-Nfs1: mitochondrial localization of eukaryotic NifS-like proteins.
We have isolated a mouse cDNA which shows significant sequence similarity to the yeast nifS-like gene (y-NFS1), and termed it m-Nfs1. The deduced protein sequence (459 amino acids long) has several characteristic features common to those of bacterial NifS proteins, but distinct from them by its amino-terminal extension which contains a typical mitochondrial targeting presequence. m-Nfs1 was found to be a soluble 47-kDa protein in the matrix fraction of mouse liver mitochondria. The m-Nfs1 gene was ubiquitously expressed in most tissues, suggesting its housekeeping function in vivo. We also found that the gamma-NFS1 protein was localized in the mitochondrial matrix in yeast cells. These results suggest that both eukaryotic NifS-like proteins may play some roles in mitochondrial functions.
Amino Acid Sequence↗
Crystal structures of Paracoccus denitrificans aromatic amino acid aminotransferase: a substrate recognition site constructed by rearrangement of hydrogen bond network.
Aminotransferase reversibly catalyzes the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5'-phosphate (PLP) as a cofactor. Various kinds of aminotransferases developing into catalysts for particular substrates have been reported. Among the aminotransferases, aromatic amino acid aminotransferase (EC 2.6.1. 57) catalyzes the transamination reaction with both acidic substrates and aromatic substrates. To elucidate the multiple substrate recognition mechanism, we determined the crystal structures of aromatic amino acid aminotransferase from Paracoccus denitrificans (pdAroAT): unliganded pdAroAT, pdAroAT in a complex with maleate as an acidic substrate analog, and pdAroAT in a complex with 3-phenylpropionate as an aromatic substrate analog at 2.33 A, 2. 50 A and 2.30 A resolution, respectively. The pdAroAT molecule is a homo-dimer. Each subunit has 394 amino acids and one PLP and is divided into small and large domains. The overall structure of pdAroAT is essentially identical to that of aspartate aminotransferase (AspAT) which catalyzes the transamination reaction with only an acidic amino acid. On binding the acidic substrate analog, arginine 292 and 386 form end-on salt bridges with carboxylates of the analog. Furthermore, binding of the substrate induces the domain movement to close the active site. The recognition mechanism for the acidic substrate analog in pdAroAT is identical to that observed in AspAT. Binding of the aromatic substrate analog causes reorientation of the side-chain of the residues, lysine 16, asparagine 142, arginine 292* and serine 296*, and changes in the position of water molecules in the active site to form a new hydrogen bond network in contrast to the active site structure of pdAroAT in the complex with an acidic substrate analog. Consequently, the rearrangement of the hydrogen bond network can form recognition sites for both acidic and aromatic side-chains of the substrate without a conformational change in the backbone structure in pdAroAT.
Bacterial Proteins↗
Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B'' (delta) regulatory subunit in vitro and identification of the phosphorylation sites.
Human erythrocyte protein phosphatase 2A, which comprises a 34-kDa catalytic C subunit, a 63-kDa regulatory A subunit and a 74-kDa regulatory B'' (delta) subunit, was phosphorylated at serine residues of B'' in vitro by cAMP-dependent protein kinase (A-kinase). In the presence and absence of 0.5 microM okadaic acid (OA), A-kinase gave maximal incorporation of 1.7 and 1.0 mol of phosphate per mol of B'', respectively. The Km value of A-kinase for CAB'' was 0.17 +/- 0.01 microM in the presence of OA. The major in vitro phosphorylation sites of B'' were identified as Ser-60, -75 and -573 in the presence of OA, and Ser-75 and -573 in the absence of OA. Phosphorylation of B'' did not dissociate B'' from CA, and stimulated the molecular activity of CAB'' toward phosphorylated H1 and H2B histones, 3.8- and 1.4-fold, respectively, but not toward phosphorylase a.
Animals↗
Effects of gamma-glutamylcysteine ethyl ester on heart mitochondrial creatine kinase activity: involvement of sulfhydryl groups.
To study the mechanism of the protective effect of gamma-glutamylcysteine ethyl ester, mitochondrial creatine kinase activity of rat heart was measured. Gamma-glutamylcysteine ethyl ester had a protective effect against the depression of creatine kinase activity induced by xanthine + xanthine oxidase or hydrogen peroxide. Gamma-glutamylcysteine ethyl ester also prevented the depression of creatine kinase activity induced by N-ethylmaleimide. It is suggested that the protective effect of gamma-glutamylcysteine ethyl ester is related to oxygen free radicals or to reduction of the sulfhydryl groups of the enzyme which were previously oxidized by oxygen free radicals.
Animals↗
Inelastic X-ray scattering in molecular liquids and electron correlation effects.
Inelastic X-ray scattering spectra of liquid water and cyclohexane have been measured with 2 eV resolution for a momentum transfer range between 0.69 and 2.77 au at BL16A of the Photon Factory, KEK, Tsukuba. Observed spectra are transformed to the dynamic structure factor which is normalized by using the Bethe sum rule, and the static structure factor is obtained. From a comparison with extended CI calculations by the use of various basis sets, correlation effects are proved to be of vital importance in inelastic X-ray scattering.
Journal Article↗
Spontaneous epileptiform bursts and long-term potentiation in rat CA3 hippocampal slices induced by chaotic stimulation of mossy fibers.
The relation between long-term potentiation (LTP) and spontaneous rhythm in CA3 was investigated using rat hippocampal slices. Field potential response of CA3 to mossy fiber stimulation consisted of a mono-synaptic positive potential and subsequent poly-synaptic negative potentials. LTP of both field potentials was induced by chaotic mossy fiber stimulation. Although CA3 did not show any spontaneous rhythm before LTP induction in a normal perfusing medium, CA3 spontaneously caused epileptiform bursts after LTP induction by chaotic mossy fiber stimulation. The amplitude of those epileptiform bursts and the inter-burst interval were not uniform. After LTP induction, the cross-correlation function of spontaneous field potentials simultaneously recorded at two sites approximately 300 micron apart in CA3 showed a large central peak. This indicates that neuronal activity at two sites is synchronized. These results suggest that epileptiform bursts in CA3 are caused by synchronization of spontaneous CA3 pyramidal cell activity due to LTP induced by chaotic burst stimulation.
Animals↗
Molecular characterization of a glyoxysomal long chain acyl-CoA oxidase that is synthesized as a precursor of higher molecular mass in pumpkin.
A cDNA clone for pumpkin acyl-CoA oxidase (EC 1.3.3.6; ACOX) was isolated from a lambdagt11 cDNA library constructed from poly(A)+ RNA extracted from etiolated cotyledons. The inserted cDNA clone contains 2313 nucleotides and encodes a polypeptide of 690 amino acids. Analysis of the amino-terminal sequence of the protein indicates that the pumpkin acyl-CoA oxidase protein is synthesized as a larger precursor containing a cleavable amino-terminal presequence of 45 amino acids. This presequence shows high similarity to the typical peroxisomal targeting signal (PTS2). Western blot analysis following cell fractionation in a sucrose gradient revealed that ACOX is localized in glyoxysomes. A partial purification of ACOX from etiolated pumpkin cotyledons indicated that the ACOX cDNA codes for a long chain acyl-CoA oxidase. The amount of ACOX increased and reached to the maximum activity by day 5 of germination but decreased about 4-fold on the following days during the subsequent microbody transition from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA was already high at day 1 of germination, increased by about 30% at day 3, and faded completely by day 7. These data indicated that the expression pattern of ACOX was very similar to that of the glyoxysomal enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the beta-oxidation spiral, during germination and suggested that the expression of each enzyme of beta-oxidation is coordinately regulated.
Acyl-CoA Oxidase↗
Two vascular apoptosis-inducing proteins from snake venom are members of the metalloprotease/disintegrin family.
Hemorrhagic snake venom induces apoptosis in vascular endothelial cells. In a previous report, we described the purification of a vascular apoptosis-inducing protein (VAP) from Crotalus atrox [Masuda, S., Araki, S., Kaji, K. & Hayashi, H. (1997) Biochem. Biophys. Res. Commun. 235, 59-63]. We report here the identification of a second vascular apoptosis-inducing protein, VAP2, in venom from C. atrox. When we fractionated crude venom from C. atrox by isoelectric focusing, we found two proteins with apoptosis-inducing activity, one was basic and the other acidic. The basic protein corresponded to VAP, and we named the acidic protein VAP2. VAP2 was a monomeric protein with molecular mass of 55 kDa and an isoelectric point of 4.5. VAP2 killed vascular endothelial cells in culture, and the death of cells exhibited the characteristic features of apoptotic activity of VAP2 seemed to be specific to endothelial cells, as reported for VAP. The half-lethal doses of VAP and VAP2 were 0.3 microg/ml and 0.1 microg/ml, respectively. Analysis of the partial amino acid sequences of VAP and VAP2 revealed similarities to members of the metalloprotease/disintegrin family. The sensitivity of VAP2 to changes in pH and temperature was distinct from that of VAP. Our results suggest that VAP and VAP2 are members of the metalloprotease/disintegrin family.
Amino Acid Sequence↗
Dimethylarsinic acid, a main metabolite of inorganic arsenics, has tumorigenicity and progression effects in the pulmonary tumors of A/J mice.
The pulmonary tumorigenicity of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics, was examined in A/J mice fed with drinking water containing DMAA for 25 and 50 weeks. Mice fed with 400 ppm DMAA for 50 weeks produced more pulmonary tumors than untreated mice (mean number per animal 1.36 versus 0.50; P < 0.05). Histological examination revealed that the number of mice which bore adenocarcinomas or papillary adenomas correlated with the concentration of DMAA given (untreated versus 400 ppm; P = 0.002), suggesting that DMAA could promote tumorigenic processes. These results are consistent with the epidemiological studies on the pulmonary carcinogenesis of arsenics and suggest that DMAA alone can act as a carcinogen in mice.
Animals↗