Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.     

Bromination by means of sodium monobromoisocyanurate (SMBI)

A variety of aromatic compounds with both activating and deactivating substituents were brominated with sodium monobromoisocyanurate (SMBI) 1, diethyl ether, diethyl ether-methanesulfonic acid, trifluoroacetic acid, or sulfuric acid were employed as solvents. Thus nitrobenzene was conveniently brominated in sulfuric acid, benzene was readily monobrominated in diethyl ether-methanesulfonic acid, and phenol was selectively brominated at the ortho position under mild conditions in refluxing diethyl ether. With substituents that are easily protonated, trifluoroacetic acid may be employed as solvent in the reaction with 1, in contrast NBS was ineffective in trifluoroacetic acid. This renders 1 a superior reagent relative to NBS. In addition to aromatics, alkenes, ketones and esters were also brominated with 1. Diethyl malonate was brominated with 1 and then subjected to a Bingel reaction with NaH to afford the desired methanofullerene in reasonable yield.     

Immobilization of ultra-thin layer of monoclonal antibody on glass surface

When preparing an affinity column and a biosensor, it is desirable to immobilize a unimolecular layer of pure protein on a matrix. In this work, we tried to immobilize a monoclonal antibody on a surface of a glass test-tube as a model, to confirm the stability of this ultra-thin layer by an enzyme immunoassay, and to estimate the thickness of the layer on a slide glass by Fourier transform infrared reflection spectrometry. A new test-tube was washed and dried. The tube was filled with 5% 3-aminopropyltriethoxysilane. The 3-aminopropylsilylated surface was treated with glutaraldehyde and 5.6.10(-2) mg/ml solution of a normal mouse monoclonal antibody. The Schiff base between glutaraldehyde and the antibody was further reduced with 7.9.10(-3)% NaBH4. The tube was washed with 0.05% Tween 20 to block non-specific binding. The antibody immobilized on the surface was measured by an enzyme immunoassay based on a reaction of anti-mouse immunoglobulin G labelled with alkaline phosphatase, with which p-nitrophenol was produced from p-nitrophenylphosphate as a substrate. Meanwhile, various amounts of the antibody were immobilized on slide glasses in the same manner. The antibody on each surface was measured by Fourier transform infrared reflection spectrometry. The antibody immobilized under the final conditions was detectable by the enzyme immunoassay, and stable at 4 degrees C for ten days. The antibody on the slide glass was a unimolecular layer, as judged from the Fourier transform infrared spectra referred to -CONH- band semiquantitatively. Thus, we found the optimal conditions for immobilizing an ultra-thin layer of the monoclonal antibody on the glass surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of the indole ring in palladium(II) complexes of 2N1O-donor ligands: cyclopalladation and pi-cation radical formation

The Pd(II) complexes of new 2N1O-donor ligands containing a pendent indole, 3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]ethylindole (Htbu-iepp), 1-methyl-3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]ethylindole (Htbu-miepp), 3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]methylindole (Htbu-impp), and 3-(N-2-pyridylmethyl-N-4-hydroxybenzylamino)ethylindole (Hp-iepp) (H denotes a dissociable proton), were synthesized, and the structures of [Pd(tbu-iepp)Cl] (1a), [Pd(tbu-iepp-c)Cl] (1b), [Pd(tbu-miepp)Cl] (3), and [Pd(p-iepp-c)Cl] (4) (tbu-iepp-c and p-iepp-c denote tbu-iepp and p-iepp bound to Pd(II) through a carbon atom, respectively) were determined by X-ray analysis. Complexes 1a prepared in CH(2)Cl(2)/CH(3)CN and 3 prepared in CH(3)CN have a pyridine nitrogen, an amine nitrogen, a phenolate oxygen, and a chloride ion in the coordination plane. Complex 1b prepared in CH(3)CN has the same composition as 1a and was revealed to have the C2 atom of the indole ring bound to Pd(II) with the Pd(II)-C2 distance of 1.973(2) A. The same Pd(II)-indole C2 bonding was revealed for 4. Interconversion between 1a and 1b was observed for their solutions, the equilibrium being dependent on the solvent used. Reaction of 1b and 4 with 1 equiv of Ce(IV) in DMF gave the corresponding one-electron-oxidized species, which exhibited an ESR signal at g = 2.004 and an absorption peak at approximately 550 nm, indicating the formation of the Pd(II)-indole pi-cation radical species. The half-life, t(1/2), of the indole radical species at room temperature was calculated to be 20 s (k(obs) = 3.5 x 10(-)(2) s(-)(1)) for 1b. The cyclic voltammogram for 1b in DMF gave two irreversible oxidation peaks at E(pa) = 0.68 and 0.80 V (vs Ag/AgCl), which were ascribed to the oxidation processes of the coordinated indole and phenolate moieties, respectively.     

Synthesis of Ultrafine SiC Powders from Carbon-Silica Hybridized Precursors with Carbothermic Reduction

Inorganic-organic hybrid gels were prepared by simultaneous condensation of the liquid mixture of ethyl silicate, ethyl borate and water soluble phenol resin with low molecular weight. The liquid mixtures were changed into lightly colored transparent gels after a catalyst addition and stirring. The obtained gels were crushed, dried and fired in controlled conditions to yield the inorganic precursors. Since the tailored inorganic precursors mainly consisted of SiO2 and carbon, silicon carbide was formed with the heat treatments in an Ar atmosphere beyond 1773 K with carbothermic reduction. The properties and morphology of the formed silicon carbide powders in terms of the starting precursor compositions and the conditions of the carbothermic reduction were investigated with SEM, XRD and TG-DTA analysis.     

Intramolecularly hydrogen-bonded 3-phenacyl-lH-pyrido[3,4-b]-pyrazin-2-one and 2-Phenacyl-4H-pyrido[3,4-b]pyrazin-3-one

Twelve phenacyl derivatives of 1H-pyrido[3,4-b]pyrazin-2-ones and 4H-pyrido[3,4-b]pyrazin-3-ones have been synthesized. In these compounds, C = N double bonds at the 3 and 4 positions in the former compounds and those at the 1 and 2 positions in the latter compounds migrate onto the side chains to form phenacyli-dene structures. These migrations are facilitated by chelation between side chain carbonyl and the proton attached to the ring nitrogen atom which was generated by the migration. All the hydrogen-bonded structures appear to be stable as shown by their ir spectra in the crystalline state, and by their ¹H nmr spectra in solution.     

Structure of aszonalenin,a new metabolite of aspergillus zonatus

Aszonalenin ($\text{1}$) has been isolated from $\text{Aspergillus zonatus}$ together with LL-S490β ($\text{2}$) and the structure of $\text{1}$ has been established by spectroscopic evidences and chemical transformation.     

Specific heat and neutron diffraction study on quaternary sulfides BaNd2CoS5 and BaNd2ZnS5

Magnetic and electrical properties are investigated for quaternary neodymium sulfides BaNd₂TS₅ (T=Co, Zn) through the specific heat, neutron diffraction, and electrical conductivity measurements. Their electrical conductivities show semiconductive behavior, which follows the Arrhenius temperature dependence with the activation energy of E_a=1.46 eV for BaNd₂ZnS₅ and E_a=1.19 eV for BaNd₂CoS₅. The specific heat of BaNd₂ZnS₅ has a λ-type anomaly at 2.8 K due to the antiferromagnetic ordering of the Nd³⁺ moments and a Schottky-type anomaly at around 60 K, which results from the crystal field splitting of the ⁴I_9/2 ground state of the Nd³⁺ ion. The specific heat of BaNd₂CoS₅ shows two λ-type anomalies at 5.7 K due to the antiferromagnetic ordering of Nd³⁺ and at 58 K due to the antiferromagnetic ordering of Co²⁺. The latter overlaps with the Schottky-type anomaly due to the crystal field splitting of the Nd³⁺ ion. Neutron diffraction measurements for BaNd₂CoS₅ show that a magnetic arrangement of the Co²⁺ moments has a collinear antiferromagnetic structure, while that of the Nd³⁺ moments has a noncollinear one.