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Biomedical subjects

H Hayashi

Publications and source records attributed to H Hayashi.

At least 631 records · Page 35Linked to original sources

Chaotic and phase-locked responses of the somatosensory cortex to a periodic medial lemniscus stimulation in the anesthetized rat.

Field potential responses of the somatosensory cortex to a periodic medial lemniscus (ML) fiber stimulation were investigated in anesthetized rats. Since the field potential responses of the cortex depend on the depth of anesthesia, two criteria were introduced to control the depth of anesthesia. One criterion is that spindle oscillations are caused by a single shock to ML fibers, and the other is that the dominant frequency of spontaneous field potential rhythm is in the frequency range of the delta wave. Phase-lockings and chaotic responses occurred depending on stimulus parameters under the above conditions. Trajectories of the chaotic responses in the two-dimensional phase space (V, dV/dt) reconstruct strange attractors, and stroboscopic cross-sections of each attractor show stretching and folding process. Each one-dimensional strobomap of the chaotic responses is a noninvertible function with an unstable fixed point. This is undoubted evidence for chaotic responses of the somatosensory cortex in vivo. Power spectra well characterize the periodic and the chaotic responses. Positive Lyapunov exponents and low, noninteger correlation dimensions of the chaotic responses are consistent with the above evidence. Consequently, four kinds of chaotic responses were classified. A periodic-chaotic transition sequence was observed at a relatively low stimulus current when the frequency of the stimulation was varied. A cascade of period-doubling bifurcations was also observed on a route from the region of the 1:1 phase-locking to one of the regions of the chaotic responses.

Anesthesia↗

Analysis of the substrate-recognition mode of aromatic amino acid aminotransferase by combined use of quasisubstrates and site-directed mutagenesis: systematic hydroxy-group addition/deletion studies to probe the enzyme-substrate interactions.

Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromatic amino acids. Because both reactions are supposed to occur in a single reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intriguing question. This was probed by spectroscopic analysis of the complexes of beta-hydroxylated substrates and the wild-type and [Tyr70-->Phe] mutant enzymes. Both L-erythro-3-hydroxyaspartate and L-erythro-3-phenylserine reacted with the wild-type ArAT to give an absorption maximum at around 500 nm, reflecting the formation of the quinonoid intermediate. When the hydroxy group of Tyr70 of ArAT was deleted by replacement of the residue with phenylalanine, the 500-nm absorption greatly decreased in either of the ArAT-beta-hydroxy amino acid complexes, showing the presence of specific interactions, which stabilize the 500-nm absorbing quinonoid intermediates, between the phenolic hydroxy group of Tyr70 and the beta-hydroxy groups of the two quasisubstrates. From these results, it was concluded that the conformations of the two quasisubstrates are essentially identical in their enzyme-bound forms. This implies that the phenyl group of the substrate phenylalanine occupies the same region as that occupied by the beta-carboxyl group of the substrate aspartate, and the region should be near Arg292, the residue that binds the beta-carboxylate group of substrates. The [Arg292-->Ala] or [Arg292-->Leu] mutation increased the Km values for aromatic amino acids 5-10 fold, and the [Arg292-->Lys] mutation increased these values 10-100-fold, without affecting the kcat values. This shows that the side chain of Arg292 is partially involved in the binding of the aromatic ring of substrates to ArAT.

Amino Acid Sequence↗

Roles of insulin, guanosine 5'-[gamma-thio]triphosphate and phorbol 12-myristate 13-acetate in signalling pathways of GLUT4 translocation.

Insulin, guanosine 5'-[gamma-thio]triphosphate (GTP[S] and phorbol 12-myristate 13-acetate (PMA) trigger the translocation of Gl UT4 (type 4 glucose transporter; insulin-sensitive glucose transporter) from an intracellular pool to the cell surface. We have developed a highly sensitive and quantitative method to detect GLUT4 immunologically on the surface of intact 3T3-L1 adipocytes and Chinese hamster ovary (CHO) cells, using c-myc epitope-tagged GLUT4 (GLUT4myc). We examined the roles of insulin, GTP[S] and PMA in the signalling pathways of GLUT4 translocation in the CHO cell system. Among small molecular GTP-binding proteins, ras, rab3D, rad and rho seem to be candidates as signal transmitters of insulin-stimulated GLUT4 translocation. Overexpression of wild-type H-ras and the dominant negative mutant H-rass17N in our cell system respectively enhanced and blocked insulin-stimulated activation of mitogen-activated protein kinase, but did not affect insulin-stimulated GLUT4 translocation. Overexpression of rab3D or rad in the cells did not affect GLUT4 translocation triggered by insulin, GTP[S] or PMA. Treatment with Botulinum C3 exoenzyme, a specific inhibitor of rho, had no effect on GLUT4 translocation induced by insulin, GTP[S] or PMA. Therefore these small molecular GTP-binding proteins are not likely to be involved in GLUT4 translocation. In addition, insulin, GTP[S] and PMA apparently stimulate GLUT4 translocation through independent pathways.

3T3 Cells↗

Effects of aging on the microclimate pH of the rat jejunum.

The acidic microclimate layer in the vicinity of the cell surface of mammalian jejunum is important for absorption of some nutrients, such as small peptides and folate. The present study was undertaken to investigate the effect of aging on the cell surface pH (microclimate pH) of the jejunum of rats. The microclimate pH was measured in vitro in superfused preparations using single-barreled pH-sensitive microelectrodes filled with a liquid ion exchanger. The thickness of the microclimate layer was estimated by reading the distance of microelectrode advancements. The existence of a microclimate pH in the jejunum was confirmed in the senescent rats, but the value of the microclimate pH was significantly higher in the senescent (24 mo) rats (6.52 +/- 0.02) than in the young-adult (6 mo) rats (6.09 +/- 0.01) (P < 0.01). Na+ removal from the perfusate or the addition of amiloride elevated the pH in the senescent rats as well as in the young-adult rats. The microclimate layer was slightly thinner in the senescent rats than in the young-adult rats. The acidity of the microclimate layer of intestinal surface is lower in senescent animals than in the young-adult ones. One of reasons for this is the thinner mucus layer in senescent animals.

Aging↗

Temperature-controlled release property of phospholipid vesicles bearing a thermo-sensitive polymer.

As a novel temperature-sensitive liposome, dioleoylphosphatidylethanolamine vesicles bearing poly(N-isopropylacrylamide), which shows a lower critical solution temperature (LCST) near 32 degrees C, were designed. Poly(N-isopropylacrylamide) having long alkyl chains which are anchors to the lipid membranes was prepared by radical copolymerization of N-isopropylacrylamide and octadecyl acrylate using azobisisobutyronitrile as the initiator. The copolymer obtained revealed the LCST at about 30 degrees C in an aqueous solution. Dioleoylphosphatidylethanolamine vesicles coated with the copolymer was prepared and release property of the copolymer-coated vesicles was investigated. While release of calcein encapsulated in the copolymer-coated vesicles was limited below 30 degrees C, the release was drastically enhanced between 30 and 35 degrees C. Complete release from the vesicles was achieved within several seconds at 40 degrees C. This temperature-controlled release property of the vesicles can be attributable to stabilization and destabilization of the vesicle membranes induced by the copolymer fixed on the vesicles below and above the LCST, respectively. Moreover, the fluorometric measurement using dioleoyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethan ola mine suggested that the extensive release of calcein observed above the LCST is resulted from the bilayer to HII phase transition of the vesicle membranes. Since LCST of the copolymer is controllable, these vesicles might have potential usefulness as a drug delivery system with high temperature-sensitivity.

Acrylic Resins↗

Purification, cDNA cloning, and regulation of lysophospholipase from rat liver.

A lysophospholipase was purified 506-fold from rat liver supernatant. The preparation gave a single 24-kDa protein band on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, and 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8. The purified enzyme was used for the preparation of antibody and peptide sequencing. A cDNA clone was isolated by screening a rat liver lambda gt11 cDNA library with the antibody, followed by the selection of further extended clones from a lambda gt10 library. The isolated cDNA was 2,362 base pairs in length and contained an open reading frame encoding 230 amino acids with a Mr of 24,708. The peptide sequences determined were found in the reading frame. When the cDNA was expressed in Escherichia coli cells as the beta-galactosidase fusion, lysophosphatidylcholine-hydrolyzing activity was markedly increased. The deduced amino acid sequence showed significant similarity to Pseudomonas fluorescence esterase A and Spirulina platensis esterase. The three sequences contained the GXSXG consensus at similar positions. The transcript was found in various tissues with the following order of abundance: spleen, heart, kidney, brain, lung, stomach, and testis = liver. In contrast, the enzyme protein was abundant in the following order: testis, liver, kidney, heart, stomach, lung, brain, and spleen. Thus the mRNA abundance disagreed with the level of the enzyme protein in liver, testis, and spleen. When HL-60 cells were induced to differentiate into granulocytes with dimethyl sulfoxide, the 24-kDa lysophospholipase protein increased significantly, but the mRNA abundance remained essentially unchanged. Thus a posttranscriptional control mechanism is present for the regulation of 24-kDa lysophospholipase.

Amino Acid Sequence↗

Interleukin 1 (IL-1) production is not essential for acquired resistance of human A375 melanoma cells to anti-proliferative effect of IL-1.

The proliferation of human melanoma cell line A375-6 is inhibited by interleukin l (IL-l). However, the cells acquired resistance to IL-l after a long period of culture. We have reported that 2 resistant subclones, A375-R8 and -R19, produced IL-l alpha constitutively and that IL-l induced IL-6 production in an autocrine manner. Therefore, we supposed that IL-l alpha production renders the cells resistant to IL-l. To investigate the relationship between IL-l alpha production and IL-l resistance, we transfected the IL-l alpha expression plasmid to the IL-l-sensitive clone, A375-6, and the anti-sense mRNA expression plasmid to IL-l-resistant cells, A375-R8 and -R19. A375-6MS, a transfectant of mature IL-l alpha expression plasmid, expressed IL-l alpha mRNA and produced IL-l activity at a level comparable to the resistant cells. The transfectant also produced IL-6 and exhibited augmented expression of Mn-SOD mRNA. However, IL-l sensitivity of this transfectant was not affected. With respect to sensitivity to anti-proliferative effects of other cytokines, such as IL-6 and TNF alpha, there was no difference between the transfectant and parent cells. Although A375-R8PH10 and -R19PH10, transfectants of IL-l alpha anti-sense mRNA expression plasmid, exhibited a decrease in the level of IL-l production, their IL-l sensitivity did not differ from parent cells. These results, therefore, suggest that IL-l alpha production is not essential or sufficient for the acquisition of resistance to the anti-proliferative effect of IL-l.

Antineoplastic Agents↗

Superinduction of mouse epidermal ornithine decarboxylase activity by repeated 12-o-tetradecanoylphorbol-13-acetate treatments.

A correlation of the levels of epidermal protein kinase C (PKC) isozymes, steady state levels of ornithine decarboxylase (ODC) mRNA, and ODC antizyme with the induction of ornithine decarboxylase (ODC) activity by a second repeat 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment to mouse skin was determined. A single application of TPA to female CD-1 mouse skin leads to a dramatic induction of ODC activity (approximately 3 nmol CO2/60 min/mg protein) which peaks at about 5 h after treatment. However, a superinduction of ODC activity (approximately 13 CO2/60 min/mg protein) is observed upon the second TPA application at 48 or 72 h after the first TPA treatment. Prior application of a tumor initiating dose of 7,12-dimethylbenz[a]anthracine to mouse skin did not influence the degree of induction of ODC by a repeat TPA treatment. Western Blot analyses using antibodies specific to PKC alpha, beta, gamma, delta and epsilon indicate detectable levels of PKC alpha, beta, delta and epsilon in mouse epidermal extracts. A time course of the effects of a single topical application of 20 nmol of TPA to the mouse skin indicate that none of PKC isozymes (alpha, beta, gamma, delta and epsilon) were completely downregulated at times (72 h) when ODC was overinduced by TPA. TPA-induced steady state levels of ODC mRNA did not correlate with the degree of superinduction of ODC activity by TPA. The second TPA treatment, 72 h after the first TPA treatment, which leads to superinduction of ODC activity did not decrease the levels of the ODC-antizyme. The results indicate that superinduction of mouse epidermal ODC activity is regulated in part post-transcriptionally and may not be the result of either a loss of PKC isoform(s) or a decrease in the levels of ODC antizyme.

9,10-Dimethyl-1,2-benzanthracene↗

Exchange of aspartate and alanine. Mechanism for development of a proton-motive force in bacteria.

We examined the idea that aspartate metabolism by Lactobacillus subsp. M3 is organized as a proton-motive metabolic cycle by using reconstitution to monitor the activity of the carrier, termed AspT, expected to carry out the electrogenic exchange of precursor (aspartate) and product (alanine). Membranes of Lactobacillus subsp. M3 were extracted with 1.25% octyl glucoside in the presence of 0. 4% Escherichia coli phospholipid and 20% glycerol. The extracts were then used to prepare proteoliposomes loaded with either aspartate or alanine. Aspartate-loaded proteoliposomes accumulated external [3H]aspartate by exchange with internal substrate; this homologous self-exchange (Kt = 0.4 mm) was insensitive to potassium or proton ionophores and was unaffected by the presence or absence of Na+, K+, or Mg2+. Alanine-loaded proteoliposomes also took up [3H]aspartate in a heterologous antiport reaction that was stimulated or inhibited by an inside-positive or inside-negative membrane potential, respectively. Several lines of evidence suggest that these homologous and heterologous exchange reactions were catalyzed by the same functional unit. Thus, [3H]aspartate taken up by AspT during self-exchange was released by a delayed addition of alanine. In addition, the spontaneous loss of AspT activity that occurs when a detergent extract is held at 37 degrees C prior to reconstitution was prevented by the presence of either aspartate (KD(aspartate) = 0.3 mm) or alanine (KD(alanine) > or = 10 mm), indicating that both substrates interact directly with AspT. These findings are consistent with operation of a proton-motive metabolic cycle during aspartate metabolism by Lactobacillus subsp. M3.

Adenosine Triphosphate↗

Inhibition of catalase activity in vitro by diesel exhaust particles.

The effect of diesel exhaust particles (DEP) on the activity of catalase, an intracellular antioxidant, was investigated because H2O2 is a cytotoxic oxidant, and catalase released from alveolar cells is an important antioxidant in the epithelial lining fluid in the lung. DEP inhibited the activity of bovine liver catalase dose-dependently, to 25-30% of its original value. The inhibition of catalase by DEP was observed only in the presence of anions such as Cl-,Br-, or thiocyanate. Other anions, such as CH3COO- or SO4-, and cations such as K+, Na+, Mg2+, or Fe2+, did not affect the activity of catalase, even in the presence of DEP extract. Catalase from guinea pig alveolar cells and catalase from red blood cells were also inhibited by DEP extracts, as was catalase from bovine liver. These results suggest that DEP taken up in the lung and located on alveolar spaces might cause cell injury by inhibiting the activity of catalase in epithelial lining fluid, enhancing the toxicity of H2O2 generated from cells in addition to that of O2- generated by the chemical reaction of DEP with oxygen.

Animals↗

Molecular cloning of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A.

Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Amino Acid Sequence↗

Body surface distribution of significant changes in QRST time-integral values after radiofrequency catheter ablation in patients with Wolff-Parkinson-White syndrome.

We analyzed 87-lead body surface QRST time-integral values (QRST values) in 29 patients with Wolff-Parkinson-White syndrome (group A, 17 patients with manifest left-sided accessory pathway; group B, 6 patients with manifest right-sided accessory pathway; and group C, 6 patients with concealed left-sided accessory pathway), before, 1 day after, and 1 week after radiofrequency catheter ablation (RCA). The number of leads with abnormal QRST values was significantly lower 1 week after RCA compared with those before RCA and 1 day after RCA in groups A and B (p < 0.05); there was no significant difference in QRST values before and 1 day after RCA in groups A and B. The QRST values over areas with preexisting repolarization abnormalities were significantly altered 1 week after RCA compared with before and 1 day after RCA in groups A and B (p < 0.01). However, there was no significant difference in the QRST values over areas without preexisting abnormalities before RCA. In group C, there were no significant differences in the QRST values or the number of leads with abnormal QRST values before, 1 day and 1 week after RCA. In conclusion, RCA did not significantly influence repolarization properties over areas without preexisting abnormalities, but gradually reduced preexisting repolarization abnormalities, which were closely related to the location of the accessory pathway in patients with manifest Wolff-Parkinson-White syndrome. Our results suggest that body surface QRST values are useful for assessment of repolarization abnormalities during the periablation period.

Adolescent↗

Cis-diamminedichloroplatinum(II) augments expression of tumor-associated antigens on human gastric cancer cell line KATO-3 and increases susceptibility and binding of tumor cells to various cytotoxic effector cells.

Previous studies have demonstrated the immunomodulatory effects of cisplatin under certain conditions. The present study was designed to clarify whether cisplatin modulates the expression of surface antigens, especially human leukocyte antigen (HLA), on human tumor cell lines and/or augments the susceptibility and binding of tumor cells to cytotoxic effector cells. A human gastric cancer cell line, KATO-3, was employed. The expression of HLA and other tumor-associated antigens was analyzed by flow cytometry using FITC-conjugated monoclonal antibodies. The cytotoxicity of effector cells was determined by 51Cr release assay. The expression of HLA class I antigen, beta2-microglobulin, leukocyte function-associated antigen-1, and AC-81 adenocarcinoma-associated antigen on KATO-3 increased after exposure to cisplatin at 10 micrograms/ml for 3-6 hr; augmentation of HLA class I subtypes -B2 and -B27 was particularly prominent. Furthermore, the susceptibility and binding of KATO-3 to both lymphokine-activated killer cells and KATO-3-specific cytotoxic T lymphocytes significantly increased after cisplatin treatment. Cisplatin may modulate the expression of tumor-associated antigens on some human tumor cells. Tumor regression by cisplatin administration may depend on its direct cytotoxicity as well as on its modulating effects on the expression of tumor-associated antigens, subsequently leading to the activation of the immune surveillance system against the tumor.

Adjuvants, Immunologic↗

A large compressibility change of protein induced by a single amino acid substitution.

The adiabatic compressibility (beta s) was determined, by means of the precise sound velocity and density measurements, for a series of single amino acid substituted mutant enzymes of Escherichia coli dihydrofolate reductase (DHFR) and aspartate aminotransferase (AspAT). Interestingly, the beta s values of both DHFR and AspAT were influenced markedly by the mutations at glycine-121 and valine-39, respectively, in which the magnitude of the change was proportional to the enzyme activity. This result demonstrates that the local change of the primary structure plays an important role in atomic packing and protein dynamics, which leads to the modified stability and enzymatic function. This is the first report on the compressibility of mutant proteins.

Aspartate Aminotransferases↗

Radioimmunoassay of a newt sex pheromone, sodefrin, and the influence of hormones on its level in the abdominal gland.

A specific and sensitive radioimmunoassay (RIA) was developed for the measurement of sodefrin, a female-attracting decapeptide pheromone of the red-bellied newt, Cynops pyrrhogaster. Sodefrin synthesized according to the amino acid sequence of native sodefrin, isolated from the abdominal glands of the cloaca of the male newt, was used as a reference standard. An antiserum to sodefrin was produced by immunizing a rabbit with synthetic sodefrin that was extended on its C-terminus with Cys coupled to hemocyanin. For the radioligand, sodefrin N-terminally extended with Tyr was used. An aqueous extract of the abdominal glands of C. pyrrhogaster produced a displacement curve parallel to the sodefrin standard, whereas that from the sword-tailed newt (Cynops ensicauda) showed no inhibition of binding in this RIA. The sensitivity of the RIA was 30.5 +/- 3.4 pg/100 microliters assay buffer. Intraassay and interassay coefficients of variation were 1.6 and 4.5%, respectively. The RIA was used to determine sodefrin levels in the abdominal gland of the male newt. Hypophysectomy and castration greatly reduced the sodefrin content. Administration of testosterone propionate (TP) to the hypophysectomized and castrated newt increased the pheromone content in the abdominal gland. A combination of prolactin (PRL) and TP elevated the sodefrin content markedly, while PRL alone scarcely affected it. Immunoreactive sodefrin was observed in the epithelial cells of the abdominal gland of both hormone-treated and saline-injected newts. Among them, groups treated with PRL plus TP and TP alone exhibited strong immunoreactivity in their abdominal gland compared with PRL-treated and saline-injected groups.

Abdomen↗

Isolation of a novel lectin from the globiferous pedicellariae of the sea urchin Toxopneustes pileolus.

Sea urchin lectin-I (SUL-I), a 32 kDa lectin was purified from the large globiferous pedicellariae of the sea urchin, Toxopneustes pileolus by using gel permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. SDS-PAGE showed that SUL-I is a monomeric protein with a molecular mass of 32 kDa. Amino acid analysis indicates SUL-I to contain 294 residues. SUL-I was shown to have chemotactic properties for guinea-pig neutrophils at concentrations of 0.625 microgram/ml. These data suggest that a 32 kDa lectin from T. pileolus may be related to defensive role.

Animals↗