Biomedical subjects

T Yamazaki

Publications and source records attributed to T Yamazaki.

At least 271 records · Page 15Linked to original sources

PubMed1998

NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ.

Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology. The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region. The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.

Amino Acid Sequence↗

PubMed1998

ZAP-70 association with T cell receptor zeta (TCRzeta): fluorescence imaging of dynamic changes upon cellular stimulation.

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRzeta chain expressed as a chimeric protein (TTzeta and Tzetazeta), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tzetazeta fused to green fluorescent protein (ZAP-70 GFP and Tzetazeta-GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRzeta chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTzeta was indicated using mutant ZAP-70 GFPs and a truncated zeta chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tzetazeta- GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRzeta-ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.

Actins↗

PubMed1998

Resistance of Fc receptor- deficient mice to fatal glomerulonephritis.

Immune complex-mediated inflammation is a common mechanism of various autoimmune diseases. Glomerulonephritis (GN) is one of these diseases, and the main mechanism of the induction of GN has been unclear. We examined the contribution of Fc receptors in the induction of nephrotoxic GN by establishing and analyzing mice deficient in the Fc receptor gamma chain (FcRgamma). Whereas all wild-type mice died from severe glomerulonephritis with hypernitremia by administration of anti-glomerular basement membrane (GBM) antibodies, all FcRgamma-deficient mice survived. Histologically, wild-type mice showed glomerular hypercellularity and thrombotic changes, whereas the renal tissue in FcRgamma-deficient mice was almost intact. Deposition of anti-GBM antibody as well as complement components in the GBM were equally observed in both wild-type and knockout mice. These results demonstrate that the triggering of this type of glomerulonephritis is completely dependent on FcR+ cells.

Animals↗

PubMed1998

Liquid chromatographic determination of myocardial interstitial epinephrine.

This study describes a high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) for monitoring of epinephrine (Epi) in the myocardial interstitial space. The in vitro detection limit for Epi was 200 fg in a 50-microl injection. Using a cardiac dialysis technique, 60-microl dialysates were sampled from the myocardial interstitial space (6-min fractions). After an alumina procedure, the dialysate Epi concentration was measured using the HPLC-ED system. Although the basal Epi concentration was undetectable, local administration of desipramine increased Epi concentration of the dialysate to 38.1+/-18.5 pg/ml. This system affords a new possibility for estimating myocardial interstitial Epi level.

Animals↗

PubMed1998

IR Spectroscopic Study of Adsorption of Binary Gases over Ion-Exchanged ZSM-5 Zeolites.

Adsorption of binary gases (CH4/CO, CO/NO, and CO/CO2) on alkali metal (Li, Na, K, Rb, and Cs) ion-exchanged ZSM-5 zeolites were studied using IR spectroscopy. IR absorption bands of preadsorbed CH4, particularly that of nu1 (induced band), decreased with the increase in the partial pressure of CO, indicating the preferential adsorption of CO on the cation site of zeolite. In addition, IR band profiles of adsorbed CO changed in a different manner when CH4, NO, or CO2 was added to the system, suggesting that the selectivity of adsorption was governed not only by the polarity of adsorbates but also by the nature of sites present in the ZSM-5 pore bearing different electric field strengths. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Solution structure of thermostable cytochrome c-552 from Hydrogenobacter thermophilus determined by 1H-NMR spectroscopy.

The solution structure of a thermostable cytochrome c-552 from a thermophilic hydrogen oxidizing bacterium Hydrogenobacter thermophilus was determined by proton nuclear magnetic resonance spectroscopy. Twenty structures were calculated by the X-PLOR program on the basis of 902 interproton distances, 21 hydrogen bonds, and 13 torsion angle constraints. The pairwise average root-mean-square deviation for the main chain heavy atoms was 0.91 +/- 0.11 A. The main chain folding of the cytochrome c-552 was almost the same as that of Pseudomonas aeruginosa cytochrome c-551 that has 59% sequence identity to the cytochrome c-552 but is less thermostable. We found several differences in local structures between the cytochromes c-552 and c-551. In the cytochrome c-552, aromatic-amino interactions were uniquely formed between Arg 35 and Tyr 32 and/or Tyr 41, the latter also having hydrophobic contacts with the side chains of Tyr 32, Ala 38, and Leu 42. Small hydrophobic cores were more tightly packed in the cytochrome c-552 because of the occupancies of Ala 5, Met 11, and Ile 76, each substituted by Phe 7, Val 13, and Val 78, respectively, in the cytochrome c-551. Some of these structural differences may contribute to the higher thermostability of the cytochrome c-552.

Amino Acid Sequence↗

PubMed1998

Kinetic studies on bovine cytochrome p45011 beta catalyzing successive reactions from deoxycorticosterone to aldosterone.

The reactions for the synthesis of aldosterone from deoxycorticosterone were investigated kinetically in the membrane-reconstituted system with bovine cytochrome P45011 beta at 37 degrees C. Reaction rapid-quenching experiments for the metabolism of deoxycorticosterone by P45011 beta showed that aldosterone was produced via corticosterone, not via 18-hydroxydeoxycorticosterone. The kinetic analysis revealed that aldosterone was formed successively from fractions of intermediate metabolites which did not dissociate from P45011 beta. The rate of each reaction step in the successive reactions was estimated from the combination of results of the rapid-quenching experiments and the metabolism of deoxycorticosterone in the presence of an excess amount of substrate, in which the dissociation of final product, aldosterone, from the enzyme was the slowest step in the synthesis from deoxycorticosterone. Under steady-state reaction conditions, the interaction of P45011 beta with P450scc stimulates the production of corticosterone from deoxycorticosterone by about 10-fold but inhibits further reactions from corticosterone. The rapid-quenching experiments showed, however, that the rate constant for the 11 beta-hydroxylation of deoxycorticosterone for corticosterone production in the presence of P450scc was almost the same as that without P450scc. The interaction of P45011 beta with P450scc in the reaction system for deoxycorticosterone metabolism was found to slow the rate of the subsequent 18-hydroxylation of the produced corticosterone and to accelerate the dissociation of the corticosterone from P45011 beta, which stimulated the corticosterone production and inhibited the further reaction for aldosterone synthesis in the steady-state reaction.

Aldosterone↗

PubMed1998

Norepinephrine efflux evoked by potassium chloride in cat sympathetic nerves: dual mechanism of action.

Using a dialysis technique, prominent efflux of norepinephrine (NE) from cardiac sympathetic nerve endings was observed under local administration of potassium chloride (KCl, 100 mM). KCl induced NE efflux was suppressed by omega-conotoxin GVIA or desipramine but residual efflux of NE was still detectable. In the presence of omega-conotoxin GVIA, KCl induced efflux of NE was augmented by pretreatment with reserpine, indicating that this efflux of NE was derived from axoplasma with neurotransporter. These data suggest that a KCl induced brisk increase in dialysate NE levels might occur as a consequence of exocytotic NE release and carrier mediated outward NE transport from nerve endings.

Adrenergic Uptake Inhibitors↗

PubMed1998

The carboxyl-terminal region of protein C is essential for its secretion.

We have previously reported a mutated protein C, designated protein C Nagoya (PCN), characterized by the deletion of a single guanine residue (8857G). This frameshift mutation results in the replacement of the carboxyl-terminal 39 amino acids of wild-type protein C (G381-P419) by 81 abnormal amino acids. This elongated mutant was not effectively secreted, and was retained in the endoplasmic reticulum. To determine why PCN is not secreted, we constructed a series of mutants from which some or all of the 81 amino acids were deleted. None of these shortened proteins were secreted from producing cells, indicating that the carboxyl-terminal extension is not mainly responsible for the intracellular retention of PCN, and that the 39 carboxyl-terminal amino acids of wild-type protein C are required for secretion. To determine which residues are essential for the secretion of protein C, deletion mutants of the carboxyl-terminal region (D401-P419) were prepared. Metabolic labeling showed that mutants of protein C truncated before W417, Q414, E411, or K410 were efficiently secreted. On the other hand, the mutants truncated before D409 were retained and degraded intracellularly. Immunofluorescence and immunoelectron microscopy showed that truncation before D409 blocks the movement from rough endoplasmic reticulum to the Golgi apparatus. To understand the conformational change in the carboxyl-terminal region, two models of truncated activated protein C were constructed using energy optimization and molecular dynamics with water molecules.

Amino Acid Sequence↗

PubMed1998

The gene for human protein Z is localized to chromosome 13 at band q34 and is coded by eight regular exons and one alternative exon.

Human protein Z is a vitamin K-dependent plasma glycoprotein, deficiency of which leads to a mild bleeding tendency. Protein Z appears to assist hemostasis by binding thrombin and promoting its association with phospholipid vesicles. In this study, to characterize the gene for protein Z, its organization and structure were determined by a combination of PCR amplification of leukocyte DNA and isolation of phage clones from a human genomic library. The gene spanned about 14 kb and consisted of 9 exons including one alternative exon. It was of note that the gene organization was essentially identical to that of other vitamin K-dependent proteins, such as factors VII, IX, and X and protein C. The nucleotides in introns at exon/intron boundaries for eight regular exons were the consensus GT-AG sequences. In contrast, the sequence at an optional exon/intron junction was found to be GC rather than GT. The extra exon inserts a unique peptide consisting of 22 amino acids in the prepro-leader sequence. A similar situation was previously observed in factor VII, but not in other vitamin K-dependent plasma proteins. We also assigned the gene for protein Z to chromosome 13 by PCR amplification of genomic DNAs from human/hamster cell hybrids. Fluorescence in situ hybridization, employing a genomic clone coding for human protein Z, further localized the gene to band q34, where the genes of three other vitamin K-dependent proteins are clustered. These genes may have evolved via duplication of an ancestral gene at this locus.

Alternative Splicing↗

PubMed1998

The operation of a superconducting wiggler at TERAS.

A superconducting wiggler has been successfully installed at the ETL 800 MeV electron storage ring facility (TERAS). The operation of the wiggler at magnetic field strengths of 5 T with electron beam energy of 750 MeV has been accomplished. The wiggler has been designed and constructed to produce synchrotron radiation with critical photon energy around 3 keV for scientific, industrial and medical applications. We report here experiments that demonstrate the possibility of stable operation of a superconducting wiggler in a small storage ring.

Journal Article↗

PubMed1998

Solution structure of the IRF-2 DNA-binding domain: a novel subgroup of the winged helix-turn-helix family.

BACKGROUND: The transcription of interferon (IFN) and IFN-inducible genes is mainly regulated by the interferon regulatory factor (IRF) family of proteins, which recognize a unique AAGTGA hexamer repeat motif in the regulatory region of IFN genes. A DNA-binding domain of approximately 100 amino acids has been commonly found in the IRF family of proteins, but it has no sequence homology to known DNA-binding motifs. Elucidation of the structures of members of the IRF family is therefore useful to the understanding of the regulation and evolution of the immune system at the structural level. RESULTS: The solution structure of the DNA-binding domain of interferon regulatory factor-2 (IRF-2) has been determined by NMR spectroscopy. It is composed of a four-stranded antiparallel beta sheet and three alpha helices, and its global fold is similar to those of the winged helix-turn-helix (wHTH) family of proteins. A long loop (Pro37-Asp51) is found immediately before the HTH motif, which is not found in other wHTH proteins. The NMR signals of residues in this long loop, as well as the second helix of the HTH motif, are strongly affected upon the addition of the hexamer repeat DNA, suggesting that these structural elements participate in DNA recognition and binding. CONCLUSIONS: The structural similarity of the DNA-binding domain of IRF-2 with those of proteins in the wHTH family shows that the IRF proteins belong to the wHTH family, even though there is no apparent sequence homology among proteins of the two families. The sequential structure alignment program (SSAP) shows that IRF-2 has a slightly different structure from typical wHTH proteins, mainly in the orientation of helix 2. The IRF family of proteins should therefore be categorized into a subfamily of the wHTH family. The evidence here implies that the evolutional pathway of the IRF family is distinct from that of the other wHTH proteins, in other words, the immune system diverged from an evolutional stem at an early stage.

Amino Acid Sequence↗

PubMed1998

Role of ion channels and exchangers in mechanical stretch-induced cardiomyocyte hypertrophy.

We have previously reported that stretching of cardiomyocytes activates the phosphorylation cascade of protein kinases, including Raf-1 kinase and mitogen-activated protein (MAP) kinases, followed by an increase in protein synthesis partly through enhanced secretion of angiotensin II and endothelin-1. Membrane proteins, such as ion channels and exchangers, have been postulated to first receive extracellular stimuli and evoke intracellular signals. The present study was performed to determine whether mechanosensitive ion channels and exchangers are involved in stretch-induced hypertrophic responses. Neonatal rat cardiomyocytes cultured on expandable silicone dishes were stretched after pretreatment with a specific inhibitor of stretch-sensitive cation channels (gadolinium and streptomycin), of ATP-sensitive K+ channels (glibenclamide), of hyperpolarization-activated inward channels (CsCl), or of the Na+-H+ exchanger (HOE 694). Pretreatment with gadolinium, streptomycin, glibenclamide, and CsCl did not show any inhibitory effects on MAP kinase activation by mechanical stretch. HOE 694, however, markedly attenuated stretch-induced activation of Raf-1 kinase and MAP kinases by approximately 50% and 60%, respectively, and attenuated stretch-induced increase in phenylalanine incorporation into proteins. In contrast, HOE 694 did not inhibit angiotensin II-and endothelin-1-induced Raf-1 kinase and MAP kinase activation. These results suggest that among many mechanosensitive ion channels and exchangers, the Na+-H+ exchanger plays a critical role in mechanical stress-induced cardiomyocyte hypertrophy.

Angiotensin II↗

PubMed1998

Kinetic analysis of successive reactions catalyzed by bovine cytochrome p450(17alpha,lyase).

Bovine P450(17alpha,lyase) containing an additional four histidine residues at the COOH terminus was expressed in Escherichia coli and purified by one-step column chromatography using Ni-chelate resin. The membrane enzyme was incorporated into liposome membranes having similar lipid composition to that of the endoplasmic reticulum. In the presence of excess substrate, the P450-proteoliposomes metabolize pregnenolone (Delta5-steroid) to 17alpha-hydroxypregnenolone and further to dehydroepiandrosterone. The enzyme catalyzed 17alpha-hydroxylation of progesterone (Delta4-steroid) but did not form androstenedione from progesterone, although the proteoliposomes could catalyze the conversion of 17alpha-hydroxyprogesterone to androstenedione. The kinetic analysis of rapid quenching experiments showed that about 20% of the pregnenolone consumed was converted successively to dehydroepiandrosterone via a fraction of 17alpha-hydroxypregnenolone that did not dissociate from the enzyme. The rapid quenching experiments for progesterone metabolism by the proteoliposomes revealed that the dissociation rate of 17alpha-hydroxyprogesterone was 10 times faster than that of 17alpha-hydroxypregnenolone. The release of the intermediate metabolite of Delta4-steroid is sufficiently faster than the lyase reaction to prevent further reaction by the P450. It is concluded that the dissociation rates of the first hydroxylation metabolites regulate the successive reactions of P450(17alpha,lyase).

Animals↗

PubMed1998

Cell type-specific angiotensin II-evoked signal transduction pathways: critical roles of Gbetagamma subunit, Src family, and Ras in cardiac fibroblasts.

Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal-regulated kinases (ERKs). Ang II-induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the Gbetagamma subunit-binding domain of the beta-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II-induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II-induced ERK activation in cardiac myocytes. These findings suggest that Ang II-evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the Gbetagamma subunit of Gi protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas Gq and protein kinase C are important in cardiac myocytes.

Angiotensin II↗

PubMed1998

Effects of omega-conotoxin GVIA on cardiac sympathetic nerve function.

Using a cardiac dialysis technique, the effects of omega-conotoxin GVIA (N-type Ca2+ channel blocker) on cardiac sympathetic nerve function was examined in anesthetized cats. Dialysis probes were implanted in the left ventricular wall and the concentration of dialysate norepinephrine (NE) served as an indicator of NE output at cardiac sympathetic nerve endings. Administration of omega-conotoxin GVIA (10 microg/kg i.v.) suppressed dialysate NE responses to the nerve stimulation. The ouabain (1 microM) induced NE increment was less markedly suppressed by omega-conotoxin GVIA. Furthermore, omega-conotoxin GVIA neither influenced neuronal NE uptake nor tyramine induced release of NE from stores. These findings suggest that the neuronal effect of omega-conotoxin GVIA is attributable to a reduction of exocytotic NE release without alterations of neuronal NE uptake or storage. Cardiac dialysis with omega-conotoxin GVIA offers a new approach for the discrimination between Ca2+ dependent exocytotic and non-exocytotic NE release.

Adrenergic Uptake Inhibitors↗

PubMed1998

New ferromagnetic bone cement for local hyperthermia.

We have developed a ferromagnetic bone cement as a thermoseed to generate heat by hysteresis loss under an alternate magnetic field. This material resembles bioactive bone cement in composition, with a portion of the bioactive glass ceramic component replaced by magnetite (Fe3O4) powder. The temperature of this thermoseed rises in proportion to the weight ratio of magnetite powder, the volume of the thermoseed, and the intensity of the magnetic field. The heat-generating ability of this thermoseed implanted into rabbit and human cadaver tibiae was investigated by applying a magnetic field with a maximum of 300 Oe and 100 kHz. In this system, it is very easy to increase the temperature of the thermoseed in bone beyond 50 degrees C by adjusting the above-mentioned control factors. When the temperature of the thermoseed in rabbit tibiae was maintained at 50 to 60 degrees C, the temperature at the interface between the bone and muscle (cortical surface) surrounding the material rose to 43 to 45 degrees C; but at a 10-mm distance from the thermoseed in the medullary canal, the temperature did not exceed 40 degrees C. These results demonstrate that ferromagnetic bone cement may be applicable for the hyperthermic treatment of bone tumors.

Animals↗

PubMed1998

Molecular phylogeny of Dipetrocarpaceae in Southeast Asia based on nucleotide sequences of matK, trnL intron, and trnL-trnF intergenic spacer region in chloroplast DNA.

To obtain a refined molecular phylogeny of dipterocarp species in Southeast Asia, nucleotide sequences of matK, the intron of trnL, and intergenic spacer region between trnL and trnF in chloroplast DNA were determined in 16 species throughout 10 genera. In the resultant trees Southeast Asian dipterocarp species were divided into two clusters. One cluster consisted of Anisoptera, Vatica, Cotylelobium, and Upuna, all with the base chromosome number of x = 11. The second cluster consisted of Hopea, Shorea, Neobalanocarpus, Dryobalanops, Parashorea, and Dipterocarpus, mostly with the base chromosome number of x = 7. Dipterocarpus was the only genus that had the base chromosome number x = 11 in the latter cluster. This result suggests that the chromosome number changed from x = 11 to x = 7 after Dipterocarpus branched in the latter cluster. Other evolutionary changes of morphological characters are also discussed.

Asia, Southeastern↗