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T Ohshiro

Publications and source records attributed to T Ohshiro.

At least 19 recordsLinked to original sources
PubMed2002

Flavin reductase coupling with two monooxygenases involved in dibenzothiophene desulfurization: purification and characterization from a non-desulfurizing bacterium, Paenibacillus polymyxa A-1.

The dibenzothiophene (DBT) desulfurizing bacterium metabolizes DBT to form 2-hydroxybiphenyl without breaking the carbon skeleton. Of the DBT desulfurization enzymes, DszC and DszA catalyze monooxygenation reactions, both requiring flavin reductase. We searched for non-DBT-desulfurizing microorganisms producing a flavin reductase that couples more efficiently with DszC than that produced by the DBT desulfurizing bacterium Rhodococcus erythropolis D-1, and found Paenibacillus polymyxa A-1 to be a promising strain. The enzyme was purified to complete homogeneity. K(m) values for FMN and NADH were 2.1 microM and 0.57 mM, respectively. Flavin compounds were good substrates, some nitroaromatic compounds were also active, and regarding the electron donor, the activity for NADPH was about 1.5 times that for NADH. In the coupling assay with DszC, only FMN or riboflavin acted as the electron acceptor. The coupling reactions of P. polymyxa A-1 flavin reductase with DszC and DszA proceeded more efficiently (3.5- and 5-fold, respectively) than those of R. erythropolis D-1 flavin reductase when identical enzyme activities of each flavin reductase were added to the reaction mixture. The result of the coupling reaction suggested that, in the microbial DBT desulfurization, flavin reductase from the non-DBT-desulfurizing bacterium was superior to that from the DBT-desulfurizing bacterium.

Amino Acid Sequence↗
PubMed2001

Influence of natural, electrically neutral lipids on the potentiometric responses of cation-selective polymeric membrane electrodes.

Ionophore-free ion exchanger electrodes were found to exhibit quite a high selectivity for the creatininium ion; however, measurements in diluted urine samples revealed large emf drifts. Potentiometric, chromatographic, NMR, and mass spectrometric evidence did not reveal any major cationic interfering agents, and anionic interfering agents cannot trivially explain the consistently positive emf drifts. Ultrafiltration of urine samples showed that the interfering agents have molecular weights below 1000 u. The drifts are apparently caused by electrically neutral lipophilic compounds of low molecular weight that are easily extracted into organic phases. Follow-up experiments showed that p-cresol and cholesterol cause no significant emf responses but that coproporphyrin, phosphatidylserine, taurocholic acid, cholic acid, phosphatidylethanolamine, and octanoic acid cause positive emf drifts of the type that was observed with the urine samples. The extent of the responses and the response time depend not only on the specific compound but also on the cation in the sample solution. These results suggest that the emf drifts are due to extraction of such natural lipids into the organic membrane phase where they interact in an ionophore-like fashion with the analyte and interfering ions. Changes in the potentiometric selectivities after contact with natural lipids support this interpretation. The same effect of natural lipids is also expected for ionophore-based electrodes. Indeed, exposure of a valinomycin-based electrode to a methylene chloride extract of urine resulted in a significant reduction of the Na+ discrimination, increasing log Kpot(K,Na) from -3.9 to -3.1.

Cations↗
PubMed2001

Initial velocity, spectral, and pH studies of the serine-glyoxylate aminotransferase from Hyphomicrobiuim methylovorum.

Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The initial velocity and dead-end inhibition patterns are consistent with a ping-pong kinetic mechanism. The Km values for L-serine and the alternative substrate ketomalonate are 0.28 +/- 0.02 and 1.13 +/- 0.08 mM, respectively. The spectrum of SGAT at pH 7.5 shows an absorbance maximum at 413 nm and a shoulder centered at 330 nm corresponding to the ketoenamine and enolimine forms of the protonated Schiff's base with the enolimine tautomer predominating. As determined by the changes in the enzyme absorbance spectrum the enzyme can be converted from the E-PLP to the E-pyridoxamine 5'-phosphate (E-PMP) form on addition of L-serine. The enzyme can subsequently be converted back to E-PLP by addition of glyoxylate or hydroxypyruvate. The enzyme displays a pH-dependent spectral change with a pK of about 8.2 which is ascribed to the ionization of an enzymatic residue that effects the tautomeric equilibrium between the ketoenamine and enolimine tautomers of the protonated aldimine. The V/K(L-serine) pH profile displays two pK values at pH 7.5 and 8.5 with limiting slopes of 1 and -1. The V/K(ketomalonate) pH profile displays one pK at 8.2 on the basic side with a limiting slope of 1 and the log K(I oxalate) pH profile shows one pK on the basic side at pH 7.2. The data suggest the active enzyme is the protonated aldimine and an enzymatic base with a pK of 7.5 accepts a proton from the alpha-amine of substrate to initiate catalysis.

Hydrogen-Ion Concentration↗
PubMed2001

Scanning tunneling microscopy with chemically modified tips: discrimination of porphyrin centers based on metal coordination and hydrogen bond interactions.

STM gold tips chemically modified with 4-mercaptopyridine (4MP) were found capable of discriminating zinc(II) 5,15-bis(4-octadecyloxyphenyl)porphyrin (Por-Zn) from its metal-free porphyrin (Por-2H) and nickel(II) complexes (Por-Ni) in the mixed monolayers of these compounds, spontaneously formed at the solution/graphite interface. The porphyrin centers in STM images observed with 4MP-modified tips exhibited bright spots, while those measured with unmodified tips exhibited the porphyrin centers as dark depressions. The centers of Por-Zn were brighter than those of Por-2H and Por-Ni, thereby allowing the discrimination of Por-Zn from Por-2H or Por-Ni in mixed monolayers. The changes in the contrasts of porphyrin centers of Por-2H and Por-Zn/ Por-Ni were explained by facilitated electron tunneling due to hydrogen bond and metal coordination interactions, respectively, between porphyrin centers and the pyridyl group of 4MP on the tip.

Hydrogen Bonding↗
PubMed2001

Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1.

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.

Escherichia coli↗
PubMed2001

[Treatment outcomes with vinorelbine for metastatic breast cancer patients previously treated with both doxorubicin and docetaxel].

The purpose of this study was to evaluate the efficacy and toxicity of weekly vinorelbine (VNB) in patients with metastatic breast cancer previously treated with both adriamycin (ADM) and docetaxel (TXT). VNB was administered weekly at the dose 20 mg/m2 by i.v. infusion over 20 minutes followed by flushing the vein with 100 ml of normal saline. From June 1999 to August 2000, ten patients were enrolled in this study. Patient characteristics were that the cumulative doses (median) of previous ADM and TXT were 300 mg (range, 120-880 mg), 560 mg (range, 120-960 mg) respectively. The median number of metastatic sites was four, with poor performance status (ECOG 1-2: 40%, 3-4: 60%). The median cycles of weekly VNB were seven (range: 2-12). Two of 10 assessable patients obtained partial response, with an overall response rate of 20%. The main toxicity (NCI grade 4) was leukopenia in 10% of 10 patients. Phlebitis (grade 2) was observed in 4 of 10 patients (40%). VNB is an active agent against metastatic breast cancer pretreated with both ADM and TXT, possessing no severe toxicities.

Adult↗
PubMed2000

Role of cortical tumour-suppressor proteins in asymmetric division of Drosophila neuroblast.

Cellular diversity during development arises in part from asymmetric divisions, which generate two distinct cells by transmitting localized determinants from a progenitor cell into one daughter cell. In Drosophila, neuroblasts undergo typical asymmetric divisions to produce another neuroblast and a ganglion mother cell. At mitosis, neural fate determinants, including Prospero and Numb, localize to the basal cortex, from which the ganglion mother cell buds off; Inscuteable and Bazooka, which regulate spindle orientation, localize apically. Here we show that a tumour-suppressor protein, Lethal giant larvae (Lgl), is essential for asymmetric cortical localization of all basal determinants in mitotic neuroblasts, and is therefore indispensable for neural fate decisions. Lgl, which itself is uniformly cortical, interacts with several types of Myosin to localize the determinants. Another tumour-suppressor protein, Lethal discs large (Dlg), participates in this process by regulating the localization of Lgl. The localization of the apical components is unaffected in lgl or dlg mutants. Thus, Lgl and Dlg act in a common process that differentially mediates cortical protein targeting in mitotic neuroblasts, and that creates intrinsic differences between daughter cells.

Amino Acid Sequence↗
PubMed2000

Purification, characterization and crystallization of enzymes for dibenzothiophene desulfurization.

DszC and DszA, DBT monooxygenase and DBT sulfone monooxygenase, respectively, involved in dibenzothiophene (DBT) desulfurization, were purified to homogeneity from Rhodococcus erythropolis D-1. The two enzymes were crystallized and enzymologically characterized. We found a high activity of flavin reductase in the non-DBT-desulfurizing bacterium, Paenibacillus polymyxa A-1, which is essential for DszC and A activities, and purified to homogeneity and characterized the enzyme.

Biodegradation, Environmental↗
PubMed2000

A novel enzyme, L-tryptophan oxidase, from a basidiomycete, Coprinus sp. SF-1: purification and characterization.

A basidiomycete, Coprinus sp. SF-1, was found to produce an L-Trp-oxidizing enzyme by screening from the culture collection of our laboratory. After solubilization by 1 M NaSCN from the particulate fraction of disrupted cells of the strain, the enzyme was purified about 76-fold to essential homogeneity. The enzyme had a molecular mass of about 420 kDa and the subunit molecular mass was 68 kDa. The enzyme contained 1 mol of non-covalently bound FAD per mol of the subunit. It catalyzed the simultaneous reactions of oxidative deamination and oxygenative decarboxylation of L-Trp to form indolepyruvic acid and indole-3-acetamide, the former of which was further oxidized to indole-3-acetic acid. The molar ratio of the respective reaction products was about 9:1. The enzyme specifically oxidized L-Trp, and slightly acted on L-Phe and L-Tyr. The Km for L-Trp was about 0.5 mM in both oxidase and oxygenase reactions. Thus, the enzyme is a novel one and was tentatively designated "L-Trp oxidase (deaminating and decarboxylating)". The optimum pHs of oxidase and oxygenase activities were 7.0 and 9.0, respectively. The optimum temperatures of both activities were 50 degrees C. The enzyme was stable at pH 6.0-10.5 and below 50 degrees C, and at 4 degrees C for 1 year.

Absorption↗
PubMed2000

Identification of the gene encoding a NAD(P)H-flavin oxidoreductase coupling with dibenzothiophene (DBT)-desulfurizing enzymes from the DBT-nondesulfurizing bacterium Paenibacillus polymyxa A-1.

The gene encoding NAD(P)H-flavin oxidoreductase (flavin reductase), which couples efficiently with dibenzothiophene (DBT)-desulfurizing monooxygenases of Rhodococci, was cloned from a DBT-non-desulfurizing bacterium Paenibacillus polymyxa A-1 in Escherichia coli, and designated as flv. Cell-free extracts from the recombinant exhibited a flavin reductase activity about forty times higher than that of the E. coli carrying the vector DNA only. Nucleotide sequence analysis reveals that the gene product consists of 208 amino acids and showed about 27%, 32% and 21% identity in amino acid sequence with FRase I, the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several members of the nitroreductase family, respectively. The coexpression of flv with two kinds of desulfurizing genes, dszABC and tdsABC, in E. coli enhanced the rate of DBT degradation by about 10 and 5 times as high as in the case without flv, respectively.

Journal Article↗
PubMed1999

Purification and characterization of dibenzothiophene (DBT) sulfone monooxygenase, an enzyme involved in DBT desulfurization, from Rhodococcus erythropolis D-1.

Dibenzothiophene (DBT), a model of organic sulfur compound in petroleum, is microbially desulfurized to 2-hydroxybiphenyl by Rhodococcus erythropolis D-1. Three desulfurization (Dsz) enzymes--DszC, A, and B--and flavin reductase are involved in sulfur-specific DBT desulfurization. In this study, DszA was purified, characterized, and crystallized from R. erythropolis D-1. DszA, DBT sulfone monooxygenase, is the second enzyme in microbial DBT desulfurization metabolism and catalyzes the conversion of DBT sulfone to 2'-hydroxybiphenyl 2-sulfinic acid in the presence of flavin reductase with cleavage of the carbon-sulfur bond in the DBT skeleton. Using anion-exchange column chromatography, the four enzyme fractions responsible for DBT desulfurization were separated, and DszA was then purified to homogeneity. Polygonal crystals of DszA were observed within a week. DszA was found to have a molecular mass of 97 kDa and to consist of two subunits with identical masses of 50 kDa. The N-terminal amino acid sequence of the purified DszA completely coincided with the deduced amino acid sequence for dszA of R. erythropolis IGTS8 except for a methionine residue at the latter N-terminal. The optimal temperature and pH for DszA activity were 35 degrees C and about 7.5. The activity of the enzyme was inhibited by Mn2+, Ni2+, 2,2'-bipyridine, and 8-quinolinol, suggesting that a metal might be involved in its activity. DszA acted on not only DBT sulfone but also on dibenz[c,e][1,2]oxathiin 6-oxide and dibenz[c,e][1,2]oxathiin 6,6-dioxide. Dihydroxybiphenyl was formed from the latter two substrates.

Journal Article↗
PubMed1999

Microbial desulfurization of organic sulfur compounds in petroleum.

Sulfur removal from petroleum is important from the standpoint of the global environment because the combustion of sulfur compounds leads to the production of sulfur oxides, which are the source of acid rain. As the regulations for sulfur in fuels become more stringent, the existing chemical desulfurizations are coming inadequate for the "deeper desulfurization" to produce lower-sulfur fuels without new and innovative processes. Biodesulfurization is rising as one of the candidates. Several microorganisms were found to desulfurize dibenzothiophene (DBT), a representative of the organic sulfur compounds in petroleum, forming a sulfur-free compound, 2-hydroxybiphenyl. They are promising as biocatalysts in the microbial desulfurization of petroleum because without assimilation of the carbon content, they remove only sulfur from the heterocyclic compounds which is refractory to conventional chemical desulfurization. Both enzymological and molecular genetic studies are now in progress for the purpose of obtaining improved desulfurization activity of organisms. The genes involved in the sulfur-specific DBT desulfurization were identified and the corresponding enzymes have been investigated. From the practical point of view, it has been proved that the microbial desulfurization proceeds in the presence of high concentrations of hydrocarbons, and more complicated DBT analogs are also desulfurized by the microorganisms. This review outlines the progress in the studies of the microbial desulfurization from the basic and practical point of view.

Biodegradation, Environmental↗
PubMed1998

Cloning and expression of the gene for a vanadium-dependent bromoperoxidase from a marine macro-alga, Corallina pilulifera.

The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned hpol was introduced into Escherichia coli, and the expressed PO1 was purified from the recombinant strain. The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine.

Amino Acid Sequence↗
PubMed1998

miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis.

When neuroblasts divide, prospero protein and mRNA segregate asymmetrically into the daughter neuroblast and sibling ganglion mother cell. miranda is known to localize prospero protein to the basal cell cortex of neuroblasts while the staufen RNA-binding protein mediates prospero mRNA localization. Here we show that miranda is required for asymmetric staufen localization in neuroblasts. Analyses using miranda mutants reveal that prospero and staufen interact with miranda under the same cell-cycle-dependent control. miranda thus acts to partition both prospero protein and mRNA. Furthermore, miranda localizes prospero and staufen to the basolateral cortex in dividing epithelial cells, which express the three proteins prior to neurogenesis. Our observations suggest that the epithelial cell and neuroblast (both of epithelial origin) share the same molecular machinery for creating cellular asymmetry.

Alleles↗
PubMed1997

Transcriptional regulation of breathless FGF receptor gene by binding of TRACHEALESS/dARNT heterodimers to three central midline elements in Drosophila developing trachea.

The development of Drosophila trachea is under the control of spatially and/or quantitatively regulated activity of BREATHLESS FGF receptor, which is also essential for midline glial migration. Here, we identified the minimum enhancer region of breathless, cloned the Drosophila ARNT gene (dARNT), and showed biochemical and genetic evidence that breathless expression in developing trachea is regulated by direct interactions between TRACHEALESS/dARNT heterodimers and three central midline elements (TACGTGs) situated in the minimum enhancer region. Our results also showed that SINGLE-MINDED/dARNT heterodimers, which are essential for breathless expression in midline precursor cells, share DNA targets in common with TRACHEALESS/dARNT, indicating that two different basic helix-loop-helix-PAS protein complexes act through the same target sites in vivo.

Amino Acid Sequence↗
PubMed1995

Stimulatory factors for enzymatic biotin synthesis from dethiobiotin in cell-free extracts of Escherichia coli.

The activity of biotin synthesis from dethiobiotin was found in cell-free extracts of an Escherichia coli bioB transformant. Among the sulfur compounds tested, only S-adenosyl-L-methionine (AdoMet) had a significant effect, while methionine and cysteine were inert. The activity was linearly stimulated by increasing protein concentration. When the dialyzed cell-free extracts were used for the reaction, NADP+, NADPH, and FAD among the well-known cofactors tested promoted the activity. Furthermore, in the presence of AdoMet, cysteine was apparently effective for biotin synthetic activity.

Bacillus↗
PubMed1995

Effectiveness of an inactivated goose parvovirus vaccine in Muscovy ducks.

With goose parvovirus (GPV) strain IH, an inactivated vaccine was prepared from allantoic fluid of embryonating Muscovy duck eggs inoculated with GPV. The response to vaccination was measured by virus neutralizing antibody titer against GPV. Offsprings from the vaccinated flock were introduced in a farm in which GPV infection had been experienced and examined for resistance to exposure to GPV. The results showed that the intramuscular vaccination to Muscovy ducks at any age stimulated significant virus neutralizing antibody levels, and that more than 90% Muscovy ducklings from the vaccinated parent flock survived after the exposure to GPV. Muscovy ducklings that passively possessed high level virus neutralizing antibodies also could respond to the vaccination and the induced antibodies remained for more than 2 months.

Aging↗