Search PubMed⌕ Search

Biomedical subjects

M Yoshida

Publications and source records attributed to M Yoshida.

At least 919 records · Page 51Linked to original sources
PubMed1997

Effect of ethanol pretreatment on mercury distribution in organs of fetal guinea pigs following in utero exposure to mercury vapor.

Ethanol (Et-OH), which is an inhibitor of catalase, reduces oxidation of mercury vapor (Hg0) into ionic mercury (Hg2+). Consequently, exposure of pregnant animals to Hg0 with pretreatment of Et-OH causes penetration of larger amount of Hg0 to the fetus. The fate of Hg0 in the fetus of pregnant guinea pigs, thus penetrated, was investigated. Et-OH pretreatment of the dams resulted in the transfer of more mercury to the fetuses and led to a marked increase in mercury in the fetal liver. Furthermore, according to the mercury distribution in the fetal organs, the animals in the Et-OH-pretreated group fell into two subgroups: a group of fetuses (subgroup 1) had higher mercury concentrations in the brain, heart and kidney compared with the group which was exposed to Hg0 without Et-OH-pretreatment (Hg0 group) and another group (subgroup 2) with similar organ mercury concentrations to that of the Hg0 group. Determination of metallothionein (MT) concentrations showed that MT concentration in the fetal liver of subgroup 2 was significantly higher than that of subgroup 1 and the Hg0 group. Further, the Sephadex G-75 chromatography of the cytosol of the fetal liver in subgroup 2 revealed that most of mercury, about 66%, was bound to metallothionein-like protein. On the other hand, in the cytosol of fetal liver of subgroup 1, a small amount, about 10% appeared in fractions corresponding to MT. These findings suggest that fetal hepatic MT plays a significant defense role against mercury crossing the placenta and is involved in regulating for the mercury distribution in the fetus.

Administration, Inhalation↗
PubMed1997

Active oxidants mediate IFN-alpha-induced microvascular alterations in rat mesentery.

The present study was designed to investigate the influences of IFN-alpha on the microcirculatory hemodynamics. The mesenteric microcirculation of male Wistar rats was observed through an intravital fluorescence microscopic system. The leukocyte behavior, RBC velocity, and albumin leakage were monitored simultaneously before and after a continuous infusion of IFN-alpha. In other rats, the oxidant-sensitive fluorescence probe dihydrorhodamine-123 (DHR) fluorescence was observed in the same set up. Administration of IFN-alpha increased the number of adherent and emigrated leukocytes and decreased the RBC velocity in mesenteric venules. Oxidative stress indicated by DHR fluorescence was exacerbated in microvessels of IFN-alpha-treated rats. Following the leukocyte recruitment and oxidative stress, an exaggerated albumin leakage was observed. Thrombus formation in venules and hemorrhage along venules were frequently observed in rats treated with IFN-alpha. N,N'-dimethylthiourea, a scavenger of hydrogen peroxide and a hydroxyl radical, largely prevented these microvascular responses. Pretreatment of rats with mAb directed against either CD18 or ICAM-1 also attenuated the IFN-alpha-induced microvascular alterations. It is concluded, therefore, that a high concentration of IFN-alpha stimulates CD18/ICAM-1-dependent adhesive interactions with endothelial cells and oxidant production of leukocytes, which leads to microcirculatory derangements characterized by decreased barrier function and reduced anticoagulant activity of venular endothelial cells.

Animals↗
PubMed1997

Amplification of recombinant adenoviral transgene products occurs by inhibition of histone deacetylase.

n-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5-5 mM, and that the amplification required an exposure of 12-24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level.

Adenoviruses, Human↗
PubMed1997

Chaperonin-mediated folding of green fluorescent protein.

Chaperonin-mediated folding of green fluorescent protein (GFP) was examined by real-time monitoring of recovery of fluorescence and by gel filtration high-performance liquid chromatography. Acid-denatured GFP can fold spontaneously upon dilution into the neutral buffer. When Escherichia coli GroEL/ES was present, folding of GFP was arrested. Folding was resumed by subsequent addition of 100 microM or 1 mM ATP, and native GFP was regenerated to 100% yield. When folding was resumed by 10 microM ATP (1.4 mol/mol GroEL subunit), about 60% of GFP recovered native structure, and one-half of them (30%) was found to be still bound to GroEL/ES, indicating the occurrence of folding in the central cavity of the GroEL ring underneath GroES (cis-folding). Because the overall rates of GroEL/ES-, ATP-mediated GFP folding were all similar to that of spontaneous folding, it was concluded that cis-folding proceeded as fast as spontaneous folding. The GroEL/ES-bound native GFP was observed only when both GroES and ATP (but not ADP) were present in the folding mixture. Holo-chaperonin from Thermus thermophilus, which was purified as a cpn60/10 complex, exhibited the similar cis-folding. Consistently, ATP-dependent exchange of cpn10 in the holo-chaperonin with free cpn10 was observed.

Adenosine Diphosphate↗
PubMed1997

Lamprey fructose-1,6-bisphosphate aldolase: characterization of the muscle-type and non-muscle-type isozymes.

To study evolutionary aspects of fructose-1,6-bisphosphate (Fru-1,6-P2) aldolase during deuterostomian evolution, we have purified and characterized aldolases from the muscle and liver of lamprey (Entosphenus japonicus). Aldolase from the skeletal muscle and liver was identified to be the muscle-type isozyme and the non-muscle-type isozyme that was encoded by cDNAs M8 and L3, respectively, as described previously (Zhang, R., Yatsuki, H., Kusakabe, T., Iwabe, Miyata, T., Imai, T., Yoshida, M., and Hori, K., J. Biochem. 117, 545-553, 1995). The muscle-type isozyme has properties similar to vertebrate aldolase A, while the non-muscle-type isozyme shows a similarity to bacterial class I aldolase and vertebrate aldolase C but not to aldolase B, the liver-type aldolase, in terms of kinetic parameters: the Kcat values toward Fru-1,6-P2 and Fru-1-P, the Fru-1,6-P2/Fru-1-P activity ratio, and the Km values toward these substrates. The two enzymes have tetrameric forms with a molecular mass of approximately 160,000 and have similar pH optimum. The muscle-type and non-muscle-type isozymes from the tissues show different electrophoretic mobility; the muscle-type isozyme moves much faster than the non-muscle-type isozyme toward anodic side. The recombinant muscle-type and non-muscle-type aldolases gave similar characteristics as those from the tissues. The results presented in this paper, together with the data presented in the previous paper, strongly suggest that in lamprey it is possible to have two types of aldolase isozymes rather than one or three isozymes.

Amino Acid Sequence↗
PubMed1997

Biochemical differences between staurosporine-induced apoptosis and premature mitosis.

Apoptosis is morphologically related to premature mitosis, an aberrant form of mitosis. Staurosporine, a potent protein kinase inhibitor, induces not only apoptotic cell death in a wide variety of mammalian cells but also premature initiation of mitosis in hamster cells that are arrested in S phase by DNA synthesis inhibitors. Here we report on the biochemical differences between the two phenomena commonly caused by staurosporine. Rat 3Y1 fibroblasts that had been arrested in S phase with hydroxyurea underwent apoptosis by treatment with staurosporine, whereas S-phase-arrested CHO cells initiated mitosis prematurely when similarly treated with a low concentration of staurosporine. Chromosome condensation occurred in both apoptosis (3Y1) and premature mitosis (CHO). However, neither formation of mitotic spindles nor mitosis-specific phosphorylation of MPM-2 antigens was observed in apoptosis of 3Y1 cells, unlike premature mitosis of CHO cells. The p34cdc2 kinase activated in normal and prematurely mitotic cells remained inactive in the apoptotic cells, probably because the active cyclin B/p34cdc2 complex was almost absent in the S-phase-arrested 3Y1 cells. The absence of intracellular activation of p34cdc2 in apoptosis was confirmed by immunohistochemical analyses using a specific antibody raised against Ser55-phosphorylated vimentin which is specifically phosphorylated by p34cdc2 during M phase. Furthermore, phosphorylation of histones H1 and H3, which is associated with mitotic chromosome condensation, did not occur in the apoptotic cells. These results indicate that the two phenomena, staurosporine-induced apoptosis and premature mitosis, are different in their requirement for p34cdc2 kinase activation and histone phosphorylation.

Animals↗
PubMed1997

Thirteen-week subchronic toxicity study of thiamphenicol in F344 rats.

A 13-week subchronic toxicity study of thiamphenicol (TAP) was performed in F344 rats. The minimum lethal dose was estimated to be greater than 10 g/kg body weight, when a single dose of 4-10 g/kg of TAP was given orally. In the subchronic toxicity study, groups of 12 F344 rats of each sex were given solutions containing 0 (control), 125, 250 and 500 ppm of TAP as their drinking water ad libitum for 13 weeks. Body weight gain was significantly suppressed in both sexes of the 250 and 500 ppm groups. Slight suppression of erythropoiesis was observed in the highest-dose group along with slightly reduced spermatogenesis in the testes of the males. In addition, spermatogranulomas were found in the epididymis of both middle- and highest-dose groups. The no observed adverse effect level (NOAEL) was concluded to be 125 ppm (daily doses of 9.0 mg/kg in males and 11.6 mg/kg in females). From the above described results, doses of 250 and 125 ppm were selected as appropriate for a 2-year carcinogenicity study.

Animals↗
PubMed1997

Renal effects of a nitric oxide donor, NOC 7, in anesthetized rabbits.

Intrarenal arterial infusion of angiotensin II (4 ng/kg per min) reduced glomerular filtration rate and urinary Na+ excretion without affecting fractional Na+ excretion. Infusion of norepinephrine (30 ng/kg per min) reduced both urinary Na+ excretion and fractional Na+ excretion with a slight hypofiltration. The angiotensin II- and the norepinephrine-induced renal responses were suppressed during simultaneous infusion of a spontaneous nitric oxide donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl 1-triazene (NOC 7, 30 ng/kg per min) which itself had little influence on the renal parameters. The results suggest that in the rabbit kidney in vivo NOC 7 can interfere with the angiotensin II-induced hypofiltration and norepinephrine-evoked tubular reabsorption and thereby suppresses their antinatriuretic actions.

Anesthesia↗
PubMed1997

Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions.

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K a = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (p K a = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.

Base Composition↗
PubMed1997

A novel HSP70 gene of Schizosaccharomyces pombe that confers K-252a resistance.

A new gene encoding a heat shock protein 70 family protein of Schizosaccharomyces pombe (Sp), named sks2+, was cloned as a weak suppressor for the K-252a-sensitive mutation, ucm1. The nucleotide sequence of sks2+ revealed an open reading frame of a 613-amino-acid (aa) protein. The deduced aa sequence of sks2+ showed significant homology with Saccharomyces cerevisiae (Sc) Ssb1p and Ssb2p responsible for protein synthesis by non-organelle-localized ribosomes, as well as with other proteins of the HSP70 family. The cells lacking the functional sks2+ gene were viable and showed no increased sensitivity to K-252a but grew slowly with an elongated morphology. These results suggest that the sks2+ gene product plays a role in the cell cycle progression and is able to confer drug resistance in a multicopy state.

Amino Acid Sequence↗
PubMed1997

Cloning and sequencing of the gene encoding the plasma membrane H(+)-ATPase from an acidophilic red alga, Cyanidium caldarium.

A cDNA containing an open reading frame encoding the putative plasma membrane H(+)-ATPase in an acidophilic red alga, Cyanidium caldarium, was cloned and sequenced by means of PCR and Southern hybridization based on homologous sequences of P-type ATPases found in other organisms. The cloned cDNA is 3300 bp in length, containing a 2865 bp open reading frame encoding a polypeptide of 955 amino acids which has a predicted molecular mass of 105,371. The deduced amino acid sequence was found to be more homologous to those of P-type H(+)-ATPases from higher plants than that from the green alga Dunaliella bioculata.

Amino Acid Sequence↗
PubMed1997

Catalytic activity of the alpha3beta3gamma complex of F1-ATPase without noncatalytic nucleotide binding site.

A mutant alpha3beta3gamma complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 microM; Km(2), 135 microM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below approximately 300 microM.

Adenosine Diphosphate↗
PubMed1997

ADP-fluoroaluminate complexes are formed cooperatively at two catalytic sites of wild-type and mutant alpha3beta3gamma subcomplexes of the F1-ATPase from the thermophilic Bacillus PS3.

Addition of Al3+ and F- to the alpha3beta3gamma subcomplex of the TF1-ATPase containing MgADP in one catalytic site causes slow, complete inactivation as the ADP-fluoroaluminate complex is formed. This conflicts with the "bisite" stochastic model suggested earlier (Issartel, J. P., Dupuis, A., Lunardi, J. & Vignais, P. V. (1991) Biochemistry 30, 4726-4733] on the finding that complete inactivation of the bovine mitochondrial F1-ATPase by Al3+, F-, Mg2+, and excess ADP occurs as ADP-fluoroaluminate complexes form in two catalytic sites. When Al3+ and F- were added to alpha3beta3gamma containing MgADP in two catalytic sites, inactivation accelerated 8-fold, indicating catalytic to catalytic site cooperativity. When added to alpha3beta3gamma containing MgADP bound to one or two catalytic sites prior to addition of Al3+ and F-, phosphate inhibits formation of the ADP-fluoroaluminate complex. When introduced after adding 200 microM ADP plus Mg2+ to alpha3beta3gamma, but before adding Al3+ and F-, phosphate accelerated formation of the ADP-fluoroaluminate complex 3-fold. Sulfite accelerated formation of the ADP-fluoroaluminate complex 9-fold when 200 microM ADP plus Mg2+ was added to alpha3beta3gamma before adding Al3+ and F-. The accelerations induced by phosphate or sulfite in the presence of excess ADP and Mg2+ suggest noncatalytic to catalytic site cooperativity. When Al3+ and F- were added to the (alphaD261N)3beta3gamma subcomplex containing MgADP in a single catalytic site, the ADP-fluoroaluminate complex formed at least 10-fold more slowly than observed with wild-type under the same conditions. Therefore, the catalytic site containing MgADP recognizes the alphaD261N substitution when noncatalytic sites are empty. Cross-linking alpha to gamma or beta to gamma by oxidizing the (alphaA396C)3beta3(gammaA22C) and alpha3(betaD390C)3(gammaS90C) subcomplexes, respectively, abolishes cooperative formation of ADP-fluoroaluminate complexes in two catalytic sites. ADP-fluoroaluminate complex formation is restricted to a single catalytic site in the oxidized double mutants. The alpha3beta3delta subcomplex does not form an inhibitory ADP-fluoroaluminate complex under any of the conditions examined for the alpha3beta3gamma subcomplexes.

Adenosine Diphosphate↗
PubMed1997

Direct observation of the rotation of F1-ATPase.

Cells employ a variety of linear motors, such as myosin, kinesin and RNA polymerase, which move along and exert force on a filamentous structure. But only one rotary motor has been investigated in detail, the bacterial flagellum (a complex of about 100 protein molecules). We now show that a single molecule of F1-ATPase acts as a rotary motor, the smallest known, by direct observation of its motion. A central rotor of radius approximately 1 nm, formed by its gamma-subunit, turns in a stator barrel of radius approximately 5nm formed by three alpha- and three beta-subunits. F1-ATPase, together with the membrane-embedded proton-conducting unit F0, forms the H+-ATP synthase that reversibly couples transmembrane proton flow to ATP synthesis/hydrolysis in respiring and photosynthetic cells. It has been suggested that the gamma-subunit of F1-ATPase rotates within the alphabeta-hexamer, a conjecture supported by structural, biochemical and spectroscopic studies. We attached a fluorescent actin filament to the gamma-subunit as a marker, which enabled us to observe this motion directly. In the presence of ATP, the filament rotated for more than 100 revolutions in an anticlockwise direction when viewed from the 'membrane' side. The rotary torque produced reached more than 40 pN nm(-1) under high load.

Actins↗
PubMed1997

Somatic mutations of the von Hippel-Lindau tumor suppressor gene and loss of heterozygosity on chromosome 3p in human glial tumors.

Molecular genetic analysis of von Hippel-Lindau tumor suppressor gene (VHL gene) was performed on 38 tissues of human glial tumors (ependymoma, 1; astrocytoma, 6; oligodendroglioma, 1; oligoastrocytoma, 2; anaplastic oligoastrocytoma, 3; anaplastic astrocytoma, 14; glioblastoma multiforme, 11). Somatic DNAs extracted from frozen tumor specimens were examined by single-strand conformational polymorphism analysis and direct sequencing. In addition, loss of heterozygosity (LOH) on chromosome 3p in 15 glial tumor cases, lymphocyte DNAs of which were available, was examined by use of 10 microsatellite probes and two polymorphism markers for the VHL gene. Two cases of low-grade gliomas showed somatic sense mutations in exon 3 of the VHL gene, and 6 of 15 cases (40.0%) showed LOH of chromosome 3p. The VHL gene-mutated cases also showed LOH. The retention of heterozygosity and high pathological grade of glial tumors were correlated significantly. In addition, Kaplan-Meier survival analysis for patients with glial tumors showed that patients with LOH had a significantly longer survival time than those without LOH. These results suggest that somatic mutations on 3p, including the VHL gene, may be involved in tumorigenesis of some low-grade glial tumors.

Adult↗
PubMed1997

Short hydrophobic segments in the mature domain of ProOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli.

Based on the finding that a series of engineered proOmpAs containing disulfide-bridged loops of different sizes at different positions exhibits a discontinuous mode of polypeptide transit across the cytoplasmic membrane of Escherichia coli, we suggested previously that the translocation of preproteins takes place at every 30 amino acid residues (Uchida, K., Mori, H., and Mizushima, S. (1995) J. Biol. Chem. 270, 30862-30868). In the present study, we investigated the molecular mechanism underlying this stepwise translocation. Deletion or relocation of hydrophobic segments of the mature domain of proOmpA (H1, residues 233-237; H2, residues 261-265) significantly altered the pattern of the stepwise translocation. The stepwise mode of polypeptide insertion was also observed with reconstituted proteoliposomes comprising purified SecA, SecY, and SecE. Cross-linking experiments involving a photoactivable cross-linker revealed that SecY and SecA are the components which interact with the hydrophobic segment of proOmpA. The present results indicate that the hydrophobic segments of the mature domains of preproteins interact with membrane embedded translocase during polypeptide transit across the membrane, which causes a discontinuous mode of polypeptide movement.

Adenosine Triphosphatases↗
PubMed1997

The fourth component of the sarcoglycan complex.

We found a novel dystrophin-associated protein (DAP) exhibiting almost the same mobility as gamma-sarcoglycan on SDS-PAGE. This novel DAP with basic charge is separated from gamma-sarcoglycan by 2-dimensional PAGE or de-N-glycosylation followed by SDS-PAGE. This DAP is most likely the rabbit homologue of "delta-sarcoglycan", the gamma-sarcoglycan-like protein identified previously [Nigro et al. (1996) Hum. Mol. Genet. 5, 1179-1186], since an internal amino acid sequence from the DAP matched the predicted amino acid sequence of "human delta-sarcoglycan" within the limits of species difference and this DAP was recognized by anti-"delta-sarcoglycan" antibody. The DAP was found to be contained in the sarcoglycan fraction which was prepared by treatment of the dystrophin-DAP complex with n-octyl beta-D-glucoside and crosslinked with beta- and/or gamma-sarcoglycan by a chemical crosslinker, dithiobis(succinimidyl propionate). Therefore, we concluded that the DAP is the fourth component of the sarcoglycan complex.

Amino Acid Sequence↗