Hideo Inoue

[27] ATP citrate lyase (citrate-cleavage enzyme)

Publisher Summary The chapter discusses three different methods by which ATP citrate lyase is assayed by determining the amount of acetyl-CoA or oxaloacetate formed. These methods are: The hydroxamate method: The acetyl-CoA formed is trapped as acetylhydroxamate and the latter is determined by the color produced with FeCl 3 . The spectrophotometric method: The oxaloacetate formed is measured by its reaction with NADH in the presence of malate dehydrogenase. The isotopic method: Citrate-l, 5- 14 C is incubated with ATP, CoA, Mg ++ , and enzyme, and the oxaloacetate- 14 C formed is degraded according to the method of Krebs and Eggleston. The 14 CO 2 evolved is trapped and counted. The enzyme is highly specific for citrate. The activity of this enzyme depends on the presence of Mg ++ . Mn ++ can partially substitute for Mg ++ but Ni ++ Fe ++ , Fe 3++ , Cu ++ , and Zn ++ are all inactive. The enzyme requires sulfhydryl compounds for maximal activity. 2-Mercaptoethanol, cysteine, glutathione, and dithiothreitol are all nearly equally effective. The spectrophotometric method is used in the subsequent steps of purification. Because of its high sensitivity, the isotopic method is employed when the ATP citrate lyase activity in some tissue is low.

Synthesis, characterization and platelet adhesion of segmented polyurethanes grafted phospholipid analogous vinyl monomer on surface.

New segmented polyurethanes (SPUs) grafted phospholipid analogous vinyl monomer, 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) on surface were synthesized. The soft segment of these polyurethanes was hydroxylated poly(isoprene) diol and the hard segments were 4,4-methylenediphenyl diisocyanate (MDI) and 1,4-butanediol (BD). SPUs were hydroxylated by potassium peroxodisulfate and MPC was grafted on the surface of hydroxylated SPUs using di-ammonium cerium (IV) nitrate (ceric ammonium nitrate, CAN) as a radical initiator. The bulk characterization of synthesized SPUs was investigated by infrared spectroscopy (IR) and gel-permeation chromatography (GPC). The existence of phospholipid analogous groups on the surface of these SPUs was revealed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. The surfaces of MPC-grafted SPUs showed decreased water contact angles compared to non-grafted SPU and the presence of phosphorylcholine groups. The blood compatibilities of the new polymers were evaluated by platelet rich plasma (PRP) contact studies and viewed by scanning electron microscopy (SEM) using BioSpan and non-grafted polyurethane as references. We found that fewer platelets adhered to the MPC-grafted surfaces and that they showed less shape variation than the references. These results suggest that these grafted polymers may have the possibility of the usage for biomaterials.

[1] Acetyl coenzyme A carboxylase from rat liver

Publisher Summary This chapter describes the determination of acetyl coenzyme A carboxylase from rat liver. In the course of assay, [ 14 C]bicarbonate is converted into the carboxyl group of malonyl-CoA. As a result an acid-volatile compound is converted into an acid-stable compound. The reaction is stopped by addition of acid, and the reaction mixture is taken to dryness. Unreacted [ 14 C] bicarbonate escapes as 14 CO 2 , while the 14 C fixed into malonyl-CoA remains. The radioactivity of the residue is a measure of the activity of the enzyme. This assay gives linear results only during the initial portion of the reaction because the accumulation of malonyl-CoA causes the reaction to become inhibited. A difficulty is encountered, when this assay is applied to crude extracts because such extracts usually contain the enzymes and substrates for other CO 2 -fixing reactions. Even when all the substrates needed for such reactions are not present, exchange reactions can still cause an unwanted CO 2 fixation. This difficulty is readily overcome by subjecting the crude extracts to gel filtration on Sephadex G-25 prior to assay. In crude extracts, acetyl-CoA carboxylase from rat must be activated by incubation with citrate or, better, with citrate and magnesium ions, before it can be assayed under conditions of maximum activity. This type of activation is inhibited by adenosine triphosphate (ATP).

MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells.

Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-β in rat clonal dental pulp cells

Dental pulps contain sulfated glycosaminoglycans (GAGs), such as chondroitin 4-sulfate (CSA/4CS), dermatan sulfate (CSB/DS), and chondroitin 6-sulfate (CSC/6CS). Sulfated GAGs play important roles in mineralization and collagen fibrillogenesis during primary, secondary, and reparative dentin formations. Transforming growth factor-beta (TGF-beta) is a potent regulator for several extracellular matrix (ECM) components and modulates the proliferation and differentiation. Using rat clonal dental pulp cells (RPC-C2A), we investigated the constituents of GAGs synthesized by the cells and the effect of TGF-beta on their synthesis by measuring the radioactivity of [35S]sulfate incorporated into GAG fractions. Cellulose acetate electrophoresis analysis revealed that RPC-C2A cells synthesized CSA and CSB but not CSC and that 10 ng/ml of TGF-beta increased the production of CSA and CSB in the cell/ECM fraction. Measurement of [35S]sulfate incorporation showed a significant increase in the amount of GAGs by TGF-beta, 1.3-fold CSA, and 1.2-fold CSB in the cell/ECM fraction. In the medium fraction the most secreted GAG was CSA, whereas CSB was stored in the cell/ECM fraction. Secreted CSA in the medium was markedly increased by 10 ng/ml of TGF-beta (1.7-fold). These findings indicate that CSA and CSB are major sulfated GAGs synthesized by RPC-C2A cells and that TGF-beta acts as a stimulator of sulfated GAG synthesis in dental pulp cells.

An anionic trypsin-like enzyme from Streptomyces eythreus.

It had been considered that microbial proteases have very wide ranges of side-chain specificity [I] . Recent investigations, however, have shown the existence of a protease possessing a substrate specificity similar to that of bovine pancreatic trypsin [2,3]. The protease was found in Strepromyces fTadiae [2] or in Streptomycesgriseus [3], and has come to be called trypsin-like enzyme [4] . We have subsequently found a third trypsln-like enzyme, from Streptomyces eryrhreus. It is of interest to isolate enzymes responsible for tryptic activity because of possible structural homologies among trypsin-like enzymes, and particularly in view of the study at present in progress on the tertiary structure of trypsin [S] . This report describes the purification and properties of a trypsin-like enzyme from St. erythreus and comparison with the known trypsin-like enzymes and with trypsin.

Bright red light-emitting organic electroluminescent devices based on a novel thiophene-containing europium complex as an emitting layer

A novel europium complex, Eu(DTP)3(dipphen) (DTP = 1,3-di(2-thienyl)propane-1,3-dione, dipphen = 4,7-diphenyl-1,10-phenanthroline), has been synthesized and applied to organic electroluminescent (EL) devices as an emitting and electron-transporting material. The EL device structure can be described as indium tin oxide (ITO)/hole-transporting layer (triphenyldiamine derivative (TPD))/emitting layer (Eu(DTP)3(dipphen))/electron transport layer (Alq3)/AlLi (99∶1). The emitting layers were formed by a codeposition technique. The deposited EL devices were observed to emit a bright red light originating from Eu(DTP)3(dipphen) with a maximum luminance of 450xa0cdxa0m–2 at 15xa0V and 200xa0mAxa0cm–2.

Constitutive expression of thrombospondin 1 in MC3T3‐E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3‐E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18‐ and 24‐mer antisense oligonucleotides caused concentration‐dependent increases in the number of mineralized nodules, acid‐soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide‐treated MC3T3‐E1 cells, thickened extracellular matrix, well‐developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3‐E1 cells. Furthermore, MC3T3‐E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose‐dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322–332, 2006.

Pseudomonas aeruginosa proteinase: II. Molecular weight and molecular dimension

Abstract 1. 1. In order to determine the molecular weight of the crystalline proteinase of Pseudomonas aeruginosa (IFO 3080) sedimentation-velocity experiments have been carried out. 2. 2. The boundary-gradient curves obtained have been analysed according to Fujitas theory. The sedimentation coefficient s 0 and the diffusion coefficient D 0 at infinite dilution are thus calculated to be 3.99·10 −13 sec, and 7.4·10 −7 cm 2 /sec. respectively. 3. 3. Insertion of the above values into the Svedberg equation gives a mol. wt. of 48 400. 4. 4. On the assumption that the proteinase molecule is a rigid ellipsoid of revolution, hydrated to 30%, the molecular dimensions are calculated as follows: a axis 35 A, b axis 68 A for an oblate ellipsoid; a axis 86 A, b axis 43 A for a prolate ellipsoid.

Electroluminescent devices based on polymers forming hole-transporting layers. II. Polyimides containing β-naphthyldiphenylamine units

Two polyimides, PI(C1-BA) and PI(C1-6FDA), based on an aromatic di- amine compound and two aromatic dianhydrides (biphenyltetracarboxylic dianhydride (BA) and 4,49-(hexafluoroisopropylidene)diphthalic anhydride (6FDA)) were newly syn- thesized. They were characterized by viscosity, Fourier transform infrared measure- ments, and X-ray analyses. X-ray results revealed that the crystallinity of PI(C1-BA) was lower than that of PI(C1-6FDA). The synthesized polyimides could be formed into stable and homogeneous thin films on an ITO (indium tin oxide) electrode, functioning as good hole-transport materials for organic electroluminescent (EL) devices. The double-layered EL devices, consisting of the hole-transport layer of these polyimides and an emitting layer of tris(8-quinolinolato)aluminum complex, exhibited a peak emission wavelength in the bright green at 524 -527 nm. Moreover, a maximum luminance of 355 cd/m 2 was achieved at a voltage of 13 V, with a current density of 600 mA/cm 2 for the EL device using PI(C1-BA) as a hole-transport layer. The phenomenon of an extended luminance of these EL devices was also found when the voltage was