Biomedical subjects

S Yoshida

Publications and source records attributed to S Yoshida.

At least 127 records · Page 7Linked to original sources

PubMed2001

O-helix mutant T664P of Thermus aquaticus DNA polymerase I: altered catalytic properties for incorporation of incorrect nucleotides but not correct nucleotides.

Previous studies indicate that the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) plays an important role in the replication fidelity of the enzyme. This study examines the role of Thr-664, which lies in the middle of the O-helix of Taq pol I. A mutant of Taq Pol I with a proline substitution of Thr-664 (T664P) exhibits much lower replication fidelity than the wild type enzyme in a forward mutation assay. T664P produces base substitution, single-base deletion, and single-base insertion errors at 20-, 5, and 50-fold higher rates than wild type, respectively. In specific activity and steady-state kinetic experiments, T664P was catalytically robust for insertion of correct nucleotides. In contrast, it incorporated incorrect nucleotides 6.1- to 10-fold more efficiently than wild type at a template dC. Mismatched primer termini were extended by T664P 4.2- to 9.5-fold more efficiently than wild type. These data imply that the O-helix with a proline at position 664 functions like wild type Taq pol I for correct nucleotide incorporations, but bends and enlarges the catalytic pocket of the enzyme and increases the rate of nucleotide misincorporation.

Base Sequence↗

PubMed2001

Search for rare and forbidden Charm Meson decays D0 --> Vl+l- and hhll.

We report results of a search for flavor-changing neutral current (FCNC), lepton flavor, and lepton-number violating decays of the D0 (and its antiparticle) into three and four bodies. Using data from Fermilab charm hadroproduction experiment E791, we examine modes with two leptons (muons or electrons) and a rho(0), K( *0), or straight phi vector meson or a nonresonant pi(pi), Kpi, or KK pair of pseudoscalar mesons. No evidence for any of these decays is found. Therefore, we present branching-fraction upper limits at 90% confidence level for the 27 decay modes examined (18 new).

Journal Article↗

PubMed2001

Selective interaction of triazole derivatives with DWF4, a cytochrome P450 monooxygenase of the brassinosteroid biosynthetic pathway, correlates with brassinosteroid deficiency in planta.

Brassinazole, a synthetic chemical developed in our laboratory, is a triazole-type brassinosteroid biosynthesis inhibitor that induces dwarfism in various plant species. The target sites of brassinazole were investigated by chemical analyses of endogenous brassinosteroids (BRs) in brassinazole-treated Catharanthus roseus cells. The levels of castasterone and brassinolide in brassinazole-treated plant cells were less than 6% of the levels in untreated cells. In contrast, campestanol and 6-oxocampestanol levels were increased, and levels of BR intermediates with hydroxy groups on the side chains were reduced, suggesting that brassinazole treatment reduced BR levels by inhibiting the hydroxylation of the C-22 position. DWF4, which is an Arabidopsis thaliana cytochrome P450 isolated as a putative steroid 22-hydroxylase, was expressed in Escherichia coli, and the binding affinity of brassinazole and its derivatives to the recombinant DWF4 were analyzed. Among several triazole derivatives, brassinazole had both the highest binding affinity to DWF4 and the highest growth inhibitory activity. The binding affinity and the activity for inhibiting hypocotyl growth were well correlated among the derivatives. In brassinazole-treated A. thaliana, the CPD gene involved in BR biosynthesis was induced within 3 h, most likely because of feedback activation caused by the reduced levels of active BRs. These results indicate that brassinazole inhibits the hydroxylation of the C-22 position of the side chain in BRs by direct binding to DWF4 and that DWF4 catalyzes this hydroxylation reaction.

Arabidopsis Proteins↗

PubMed2001

Stimulation of DNA polymerase alpha activity by cdk2-phosphorylated Rb protein.

We propose a new role of retinoblastoma protein as a cell growth activator in its phosphorylated form. The hyper-phosphorylated retinoblastoma protein generated by the action of cdk2/cyclin E strongly stimulated the activity of DNA polymerase alpha, but did not stimulate DNA polymerases delta, epsilon, or primase. But, cdk4/cyclin D-phosphorylated retinoblastoma protein showed little stimulation. Hyper-phosphorylated retinoblastoma protein interacted with the catalytic subunit of DNA polymerase alpha, and stabilised DNA polymerase alpha from heat inactivation at 45 degrees C. These results suggest that in G1 phase, hypo-phosphorylated retinoblastoma protein suppresses the progression of cell cycle as a transcription inhibitor, but that after phosphorylation by cdk2/cyclin E at the G1/S boundary, hyper-phosphorylated retinoblastoma protein acts as a cell-cycle promoter by optimising the DNA polymerase alpha reaction.

Animals↗

PubMed2001

Enhanced ribonucleotide incorporation by an O-helix mutant of Thermus aquaticus DNA polymerase I.

The O-helix of DNA polymerases has been implicated in substrate discrimination and replication fidelity. In this study, wild-type Thermus aquaticus DNA polymerase I (Taq pol I) and an O-helix mutant A661E was examined for their ability to discriminate between ribonucleotides and deoxyribonucleotides. Steady-state nucleotide extension kinetics were carried out using a template cytidine and each nucleotide dNTP and rGTP. Wild-type Taq pol I and A661E demonstrated similar Vmax and Km values for the correct nucleotide dGTP. However, A661E discriminated between incorrect and correct nucleotide less well than wild-type; discrimination was reduced by factors of 9.5-, 5.6- and 15-fold for dATP, dTTP and rGTP, respectively. These data suggest that A661E is efficient polymerases in the presence of the correct deoxynucleotide, dGTP, but it is impaired in ability to discriminate between correct and incorrect deoxyribonucleotides or between ribo- and deoxyribonucleotides. A structural model of Taq pol I is described in which the mutation A661E alters the interactions between the O-helix and the terminal two phosphate groups in the primer strand.

DNA Polymerase I↗

PubMed2001

Aminopeptidase N regulated by zinc in human prostate participates in tumor cell invasion.

Aminopeptidase N (AP-N) degrades collagen type IV and is proposed to play a role in tumor invasion. However, the precise functions of AP-N in tumor cells and the relationship of AP-N to prostate cancer remains unclear. In our study, we examined a possible role for zinc in the regulation of AP-N enzymatic activity in relation to tumor cell invasion in human prostate. AP-N purified from human prostate was irreversibly inhibited by low concentrations of zinc (Ki = 11.2 microM) and bestatin. AP-N, which has zinc in the active center, was also inhibited by the chelating agents, EDTA, o-phenanthroline and EGTA. EDTA was shown to remove zinc from the enzyme. When the effects of zinc and bestatin on invasion of PC-3 cells were investigated in vitro using a Transwell cell-culture chamber, zinc and bestatin effectively suppressed cell invasion into Matrigel at the concentration range of 50-100 microM. These results strongly suggest that the suppression of PC-3 cell invasion by zinc is based on the inhibition of AP-N activity by zinc. We also evaluated the expression of AP-N to investigate the relationship with the progression of prostate disease in human cancerous prostate. AP-N was found to be located at the cytoplasmic membranes of prostate gland epithelial cells and to be expressed more in prostate cancer, while the expression of prostate-specific antigen (PSA), which is a useful marker for prostate cancer, was shown in normal and cancer tissues, suggesting that AP-N is potentially a good histological marker of prostate cancer. Thus, highly expressed AP-N in human cancerous prostate probably plays an important role in the invasion and metastasis of prostate cancer cells.

Amino Acid Sequence↗

PubMed2001

Dual targeting of spinach protoporphyrinogen oxidase II to mitochondria and chloroplasts by alternative use of two in-frame initiation codons.

Protoporphyrinogen oxidase (Protox) is the final enzyme in the common pathway of chlorophyll and heme biosynthesis. Two Protox isoenzymes have been described in tobacco, a plastidic and a mitochondrial form. We isolated and sequenced spinach Protox cDNA, which encodes a homolog of tobacco mitochondrial Protox (Protox II). Alignment of the deduced amino acid sequence between Protox II and other tobacco mitochondrial Protox homologs revealed a 26-amino acid N-terminal extension unique to the spinach enzyme. Immunoblot analysis of spinach leaf extract detected two proteins with apparent molecular masses of 57 and 55 kDa in chloroplasts and mitochondria, respectively. In vitro translation experiments indicated that two translation products (59 and 55 kDa) are produced from Protox II mRNA, using two in-frame initiation codons. Transport experiments using green fluorescent protein-fused Protox II suggested that the larger and smaller translation products (Protox IIL and IIS) target exclusively to chloroplasts and mitochondria, respectively.

Amino Acid Sequence↗

PubMed2001

BRI1 is a critical component of a plasma-membrane receptor for plant steroids.

Most multicellular organisms use steroids as signalling molecules for physiological and developmental regulation. Two different modes of steroid action have been described in animal systems: the well-studied gene regulation response mediated by nuclear receptors, and the rapid non-genomic responses mediated by proposed membrane-bound receptors. Plant genomes do not seem to encode members of the nuclear receptor superfamily. However, a transmembrane receptor kinase, brassinosteroid-insensitive1 (BRI1), has been implicated in brassinosteroid responses. Here we show that BRI1 functions as a receptor of brassinolide, the most active brassinosteroid. The number of brassinolide-binding sites and the degree of response to brassinolide depend on the level of BRI1 protein. The brassinolide-binding activity co-immunoprecipitates with BRI1, and requires a functional BRI1 extracellular domain. Moreover, treatment of Arabidopsis seedlings with brassinolide induces autophosphorylation of BRI1, which, together with our binding studies, shows that BRI1 is a receptor kinase that transduces steroid signals across the plasma membrane.

Arabidopsis↗

PubMed2001

Carbon dioxide pneumoperitoneum reduces levels of TNF-a mRNA in the brain, liver, and peritoneum in mice.

BACKGROUND: Cytokines are important regulators of the biological response to surgical stress. The aim of this study was to determine whether the CO(2) pneumoperitoneum would change the expression of TNF-a mRNA in the visceral organs, including the brain, in mice. METHODS: Mice were randomly assigned to one of six groups: control, anesthesia alone, insufflation with carbon dioxide, insufflation with air, laparotomy by short incision, or laparotomy by long incision. The brain, liver, jejunum, and peritoneum were harvested either 3 or 24 h after surgery. Levels of TNF-a mRNA in each tissue was measured by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The air insufflation group showed higher TNF-a mRNA levels in the brain and liver than the short-incision group. Levels of TNF-a mRNA in the brain, liver, and peritoneum were lower in the CO(2) pneumoperitoneum group than in the air insufflation group. Plasma IL-6 and catecholamine in the urine were lower in the CO(2) pneumoperitoneum group than the air insufflation group. CONCLUSION: Reduced synthesis of TNF-a in the visceral organs, including the brain, is correlated with a less marked biologic response to laparoscopic surgery.

Animals↗

PubMed2001

Structural analysis of La/Al2O3 catalysts by La K-edge XAFS.

Structural analysis of La/Al2O3 catalyst materials was carried out by means of XRD and La K-edge XAFS. Influences of loading amounts and treatment temperature on the local structure around La were investigated. La atoms are supported on gamma-Al2O3 in a highly dispersed form when a loading amount is less than 0.2 mmol g(Al2O3)(-1), and structural transformation of the La species does not take place in the temperature ranges of 873-1273 K. La species of highly loaded catalysts start to change their structure from oxide-like form to the aluminate at 1073 K. Formation of LaAlO3 perovskite on the catalyst surface requires a concentration of La atoms above 0.5 mmol g(Al2O3)(-1) (7.5 wt% as La2O3).

Journal Article↗

PubMed2001

Local structure analyses of Ce0.5Zr0.5O2 mixed oxides by XAFS.

Three types of CeO2-ZrO2 with the same composition (Ce/Zr = 1) were prepared by different methods, exhibited the different oxygen storage/release capacity (OSC). To investigate the relationship between the OSC and the local structure, the Ce and Zr K-edges XAFS spectra for these samples were measured. The features of Fourier transforms of these samples were different from each other. This suggested that the OSC was remarkably exerted by the local structure around Ce and Zr. The quantitative curve-fitting analysis of EXAFS was applied, and it was concluded that homogeneous Ce(0.5)Zr(0.5)O2 solid solution at atomic level exhibited the highest OSC among these CeO2-ZrO2 with the same composition (Ce/Zr = 1).

Journal Article↗

PubMed2001

XANES spectroscopy of a single neuron from a patient with Parkinson's disease.

Chemical state of transition metals such as iron in a single neuron of the substantia nigra (SN) from a patient with Parkinson's disease (PD) was studied in this paper, using autopsy midbrain specimens including SN. Parkinson's disease is one of the major neurodegenerative diseases. The excessive accumulation of iron and its chemical states in SN neurons are related to the oxidative damage leading to neuronal cell death. X-ray absorption fine structure spectroscopy (XAFS) with SR micro beam (10 microm) was used to investigate the chemical state of iron in the SN neurons. X-ray absorption near-edge structure (XANES) spectroscopy results showed that the chemical state of iron in the neuromelanin granules within SN neurons changed from ferrous (Fe2+) to ferric (Fe3+) ion in the process of neuronal degeneration.

Humans↗

PubMed2001

Re-evaluation of acute neurotoxic effects of Cd2+ on mesencephalic trigeminal neurons of the adult rat.

The mechanism of Cd2+ neurotoxicity, which is considered to be secondary to changes in blood vessels, was re-evaluated in dissociated mesencephalic trigeminal (Me5) neurons of the adult rat. Cd2+ induced morphological changes in Me5 neurons at 0.1 and 1 mM but not at 0.01 mM. The changes appeared predominantly in the cytoplasm: destruction of the cytoplasmic organelles, swelling and vacuolization of the cell body, and finally resulted in cell lysis. These observations indicate necrosis rather than apoptosis, and no sign of degraded nuclear DNA, characteristic to apoptosis, was detected by the TUNEL technique. Using a Ca2+-sensitive dye Indo-1, Cd2+ was found to elevate the intracellular Ca2+ concentration [Ca2+](i) (both in the cytoplasm and the nucleus). Both the elevation in [Ca2+](i) and the morphological alteration were inhibited either by removing Ca2+-from the bathing medium or by the application of BAPTA/AM (10 microM), a membrane-permeable intracellular Ca2+ chelator. Furthermore, neither morphological changes nor elevation in [Ca2+](i) by Cd2+ occurred in the presence of Zn2+. It is concluded that (1) Cd2+ can directly affect nerve cells, (2) toxicity of Cd2+ on Me5 neurons is mediated by continuous elevation in [Ca2+](i), (3) Cd2+ induces necrotic cell death, and (4) Cd2+ neurotoxicity can be antagonized by Zn2+.

Animals↗

PubMed2001

Study of the D(+)(s)-->pi(-)pi(+)pi(+) decay and measurement of f(0) masses and widths.

From a sample of 848+/-44 D(+)(s)-->pi(-)pi(+)pi(+) decays, we find gamma(D(+)(s)-->pi(-)pi(+)pi(+))/gamma(D(+)(s)-->straight phipi(+)) = 0.245+/-0.028(+0.019)(-0.012). Using a Dalitz plot analysis of this three body decay, we find significant contributions from the channels rho(0)(770)pi(+), rho(0)(1450)pi(+), f(0)(980)pi(+), f(2)(1270)pi(+), and f(0)(1370)pi(+). We also present the values obtained for masses and widths of the resonances f(0)(980) and f(0)(1370).

Journal Article↗

PubMed2001

Experimental evidence for a light and broad scalar resonance in D(+) --> pi(-)pi(+)pi(+) decay.

From a sample of 1172 +/- 61 D(+)-->pi(-)pi(+)pi(+) decays, we find gamma(D(+)-->pi(-)pi(+)pi(+))/gamma(D(+)-->K-pi(+)pi(+)) = 0.0311 +/- 0.0018(+0.0016)(-0.0026). Using a coherent amplitude analysis to fit the Dalitz plot of these decays, we find strong evidence that a scalar resonance of mass 478(+24)(-23) +/- 17 MeV/c(2) and width 324(+42)(-40) +/- 21 MeV/c(2) accounts for approximately half of all decays.

Journal Article↗

PubMed2001

Increased expression of cyclooxygenase-2 in human pancreatic neoplasms and potential for chemoprevention by cyclooxygenase inhibitors.

BACKGROUND: Cyclooxygenase-2 (COX-2) is thought to be linked to carcinogenesis; however, very little is known about its expression in pancreatic neoplasms. The authors studied the expression of COX-2 in human pancreatic neoplasms and investigated the effect of COX inhibitors on the growth of human pancreatic carcinoma cells. METHODS: Expression of COX-2 protein was immunohistochemically examined in 42 human pancreatic duct cell carcinomas (PDCs) and in 29 intraductal papillary mucinous tumors (IPMTs [adenomas, 19; carcinomas, 10]) of the pancreas that were resected surgically at the National Cancer Center Hospital in Tokyo. The growth of four human pancreatic carcinoma cell lines also was evaluated in the presence of COX inhibitors. RESULTS: Marked COX-2 expression was observed in 57% (24 of 42) of PDCs, in 58% (11 of 19) of adenomas, and in 70% (7 of 10) of adenocarcinomas of IPMTs. However, there was no correlation between COX-2 expression and clinicopathologic indices of the patients. All four pancreatic cancer cell lines expressed COX-2 protein weakly or strongly, and the inhibitory effect of aspirin on cell growth was correlated with the expression of COX-2. CONCLUSIONS: COX-2 was expressed in adenomas of IPMTs as well as in carcinomas and might have played a role in the development of pancreatic tumors. In this study, COX inhibitors, as nonsteroidal anti-inflammatory drugs, were shown to be possible preventive agents against pancreatic neoplasms.

Adenocarcinoma, Mucinous↗

PubMed2001

Change of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task.

We have analyzed oligosaccharide chains in brain microsomes of rats fed an n-3 polyunsaturated fatty acid-deficient (safflower oil group; S group) or -rich (perilla oil group; P group) diet before and after brightness-discrimination learning tasks. The amount of concanavalin A-binding sites (mainly mannoside) of the brain microsomes was found to be significantly less in the S group than the P group before the learning task. Detailed analysis of glycoprotein glycans demonstrated that high mannose type oligosaccharides were dominant in brain microsomes before the learning task in both dietary groups, whereas multiantennary complex-type oligosaccharides became dominant after the learning task and especially a tetra-antennary glycan, that had a core structure of the glycan of neural cell adhesion molecule, was more increased in the S-group than the P group. When polysialylated glycans were analyzed on serotonin-conjugated HPLC column, the glycans in the S-group microsomes before the learning task contained larger amount of higher affinity-polysialylated glycans to serotonin column than those in the P-group, and also contained larger amount of phosphoglycans that showed also high affinity to serotonin column than the P-group. Removal of mannoside from microsomes by alpha-mannosidase-treatment changed the membrane surface physical property, especially permittivity, as revealed by analysis of the interaction with 1-anilinonaphthalene-8-sulfonate. These results suggest that high mannose content and several multiantennary glycans including polysialylated and phospho-glycans were changed by dietary n-3 fatty acid deficiency and learning task in rat brain microsomal glycoproteins and that these changes may affect membrane functions through changes of membrane surface physical properties and reactivity against serotonin.

Animals↗

PubMed2001

Diet-induced hyperhomocysteinemia exacerbates neointima formation in rat carotid arteries after balloon injury.

BACKGROUND: Increasing evidence indicates that elevated plasma homocysteine levels are associated with an increased risk of atherosclerosis and endothelial dysfunction, although little specific information on the mechanisms responsible for the atherogenic effects of homocysteine or on the in vivo contribution made by hyperhomocysteinemia to atherosclerosis is currently available. Because homocysteine is known to exert a direct inhibitory effect on endothelial cell growth in vitro, we hypothesized that this effect contributes to the progression of atherosclerotic lesions initiated by endothelial damage caused by mechanical injury. METHODS AND RESULTS: We prepared diet-induced hyperhomocysteinemic rats in which neointima formation after balloon injury to the common carotid artery was assessed. Moderate hyperhomocysteinemia (plasma homocysteine levels 3- to 4-fold higher than control) significantly exacerbated neointima formation. Oral administration of folate, which had a homocysteine-lowering effect, diminished neointima formation induced by moderate hyperhomocysteinemia. Furthermore, the attenuation of reendothelialization was shown in diet-induced hyperhomocysteinemic rats with Evans blue staining. CONCLUSIONS: Diet-induced hyperhomocysteinemia, even mild to moderate, exacerbates neointima formation after denuding injury, making hyperhomocysteinemia a likely risk factor for postangioplasty restenosis. It may be mediated through an inhibitory effect of homocysteine on reendothelialization. Homocysteine lowering with folate supplementation can effectively ameliorate the detrimental effects of moderate hyperhomocysteinemia. Clinical trials would seem to be warranted.

Administration, Oral↗