The rice low-temperature-induced lip19 gene encodes a 148-amino-acid basic region/leucine zipper (bZIP) protein, termed LIP19. In this study we characterized LIP19 and showed that it lacks the usual ability of bZIP proteins to homodimerize and to bind DNA, as does the Fos protein in mammals. Using a yeast two-hybrid system, the cDNA clones whose products interact with LIP19 were screened. This search revealed a clone termed OsOBF1 (Oryza sativa OBF1) that encodes a new bZIP protein (OsOBF1). This protein forms a homodimer and binds to the hexamer motif sequence (5'-ACGTCA-3'). The protein-protein interaction in homo- and hetero-combinations between LIP19 and OsOBF1 was confirmed in vitro and in planta. LIP19 and OsOBF1 most likely interact with each other more strongly than OsOBF1 interacts with itself, and the resulting heterodimer binds to the C/G hybrid sequence but not to the hexamer sequence. Whereas the expression patterns of lip19 and OsOBF1 in response to low temperatures were totally opposite, the locations of their expression were almost identical. Based upon the presented data, we propose a model describing the low-temperature signal switching mediated by LIP19 in rice.

Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs. The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C 2 (delta-) and C 3 (delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C 2 (delta-) accepts a proton from Cys130 and C 3 (delta+) a hydride (or a hydride equivalent) from reduced FMN N 5 in TcDHOD.

We measured levels of platelet-derived microparticles (PMP), which have coagulative activity and are produced by platelet activation or physical stimulation, and CD62P/CD63-positive platelets in patients with diabetes mellitus to determine their clinical significance and effects on complications of diabetes including diabetic nephropathy. We also compared these levels before and after administration of the antiplatelet drug cilostazol. Plasma PMP and CD62P/CD63-positive platelet levels were significantly higher in patients with diabetes mellitus than normal controls. CD62P-positive platelet levels were significantly higher in patients with nephropathy than in patients without complications. After administration of cilostazol, PMP and CD62P/CD63-positive platelet levels were significantly decreased. The increases in platelet activity and its related procoagulant activity appear to account in part for the hypercoagulability observed in diabetes mellitus. Our findings suggest that activated platelets might play a role in the development of diabetic nephropathy. Furthermore, antiplatelet therapy with cilostazol for diabetic patients may be useful as antithrombin therapy including antiplatelet therapy, since it suppresses the production of intrinsic coagulants produced by platelet activation.

Die Chemisorption von H2 an reinen sowie Cu‐bedeckten Ru(0001)‐Oberflächen wird mit Hilfe von LEED, Austrittsarbeitsmessungen und der thermischen Desorptionsspektroskopie untersucht.

To explore the feasibility of alginate gel as a vehicle for liposomes, we investigated the effects of various factors associated with the loading of drug-containing liposomes into the gel beads. The loading process includes (I) mixing of liposomes and alginate solution, (2) calcium induced gelation of alginates, (3) the time-dependent contraction of a gel body squeezing out interior water, and (4) possible leakage or release of a drug entrapped in liposomes in a series of each of theses processes. These effects were examined in terms of the leakage of a marker 5(6)-carboxyfluorescein (CF) from liposomes of egg phosphatidylcholine (EPC) and EPC/cholesterol (EPC/Cho) and liposome (phosphorus) release from curing and fully-cured gel beads whose initial polymer concentrations were 4 and 2%. Major findings were: (1) Alginate induced the leakage of a water-soluble drug incorporated in the liposomes as a function of the polymer concentration and the mixing time. (2) Calcium ions also stimulated the leakage of the drug. EPC/Cho liposomes were several times more resistant to the leakage of CF than were EPC liposomes. (3) The liposomes were well loaded without any loss in the gel bead despite the squeezing outflow of water and the bead contraction during gel curing. (4) Such curing caused leakage of the drug from the EPC liposomes in the very early stage while no effect was observed in the EPC/Cho liposomes. (5) In the gel-eroding medium (pH 7.4 Tris-HCl, 37 degrees C), the total drug release was controlled by the erosion rate of the bead body. Immediately after the bead erosion, EPC liposomes retained about 60% of the drug in the 2% bead and only about 20% in the 4% bead, whereas EPC/Cho liposomes retained more than 85% regardless of the initial alginate concentration. The results provide valuable information for the design and applicability of the gel-loaded liposome delivery system.

No abstract

Intravenous (IV) edaravone is approved as an amyotrophic lateral sclerosis (ALS) treatment. Because IV administration places a burden on patients, development of orally administered ALS treatments is needed. Therefore, 2 phase 1 studies of oral formulations of edaravone in healthy subjects examined the pharmacokinetics (PK), safety, racial differences, and drug‐drug interactions (DDIs) and investigated the dose of the oral formulation considered to be bioequivalent to the approved dose of the IV formulation. Study 1 was a placebo‐controlled, randomized, single‐blind study of single‐ascending‐dose oral edaravone with the dose range of 30 to 300 mg (n = 56). Study 2 was conducted in 2 cohorts (n = 84); the first assessed DDIs with multiple‐dose edaravone 120 mg/day given over 5 or 8 days (coadministered with single‐dose rosuvastatin, sildenafil, or furosemide), and the second evaluated PK and racial (Japanese/White) differences in PK parameters with doses of 100‐mg edaravone. The oral formulation of edaravone was well absorbed, and plasma concentrations of unchanged edaravone increased more than dose proportionally within the dose range of 30 to 300 mg. No effect of race on oral edaravone PK and no notable DDI effects possibly caused by orally administered edaravone were observed. The oral edaravone formulations were safe and tolerable under the assessed conditions. Mathematical modeling determined that equivalent exposures in plasma with the approved dose of the IV edaravone formulation, as reported previously, could be achieved when the oral edaravone formulation was administered at a dose of  ≈100 mg, with an absolute bioavailability of ≈60%.

In this study, we analyzed the activity of a bacterial luciferase (LuxAB of Vibrio fischeri) expressed under the control of a consensus-type promoter, lacUV5, in Escherichia coli, and found that activity declines abruptly upon entry into the stationary growth phase. Since this decline was reproducibly observed in strains cultured in various growth media, we refer to this phenomenon as ADLA (Abrupt Decline of Luciferase Activity) and define the time point when activity begins to decline as T (0). Because the levels of luciferase proteins (LuxA and LuxB) remained constant before and after T (0), ADLA cannot be due to the repression of luciferase gene expression. Further analyses suggested that a decline in the supply of intracellular reducing power for luciferase was responsible for ADLA. We also found that ADLA was alleviated or did not occur in several mutants deficient in nucleoid proteins, suggesting that ADLA is a genetically controlled process involved in intracellular redox flow.