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.     

Biosynthetic study of amphidinolide B

The biosynthetic origins of amphidinoide B (1) were investigated on the basis of 13C-NMR data of 13C-enriched samples obtained by feeding experiments with [1-(13)C], [2-(13)C], and [1,2-(13)C2] sodium acetates in cultures of a dinoflagellate Amphidinium sp. These incorporation patterns suggested that 1 was generated from three successive polyketide chains, an isolated C1 unit from C-2 of acetates, six branched C1 units from C-2 of acetates, and an "m-m" and an "m-m-m" unit derived only from C-2 of acetates. The labeling patterns of amphidinolide B (1) were different from those of amphidinolide H (2), a 26-membered macrolide closely related to 1.     

Determination of salicylate in blood serum using an amperometric biosensor based on salicylate hydroxylase immobilized in a polypyrrole-glutaraldehyde matrix

The use of an amperometric biosensor for the salicylate determination in blood serum is described. The biosensor is based on salicylate hydroxylase (EC 1.14.13.1) electropolymerized onto a glassy carbon-working electrode with polypyrrole and glutaraldehyde, to improve the biosensor lifetime. The hexacyanoferrate (II) was also incorporated to work as a redox mediator to minimize possible interferences. The salicylate is enzymatically converted to catechol, which is monitored amperometrically by its electrooxidation at+0.170 V versus SCE (saturated calomel electrode). Salicylate determination was carried out maintaining the ratio between beta-NADH and salicylate at 4:1 (30 degrees C). The amperometric response of the biosensor was linearly proportional to the salicylate concentration between 2.3x10(-6) and 1.4x10(-5) mol l(-1), in 0.1 mol l(-1) phosphate buffer (pH 7.8), containing 0.1 mol l(-1) KCl and 5.0x10(-4) mol l(-1) Na(2)H(2)EDTA, as supporting electrolyte. The recovery studies, in the presence of several interfering compounds, showed recoveries between 96.4 and 104.8%. The useful lifetime of the biosensor in the concentration range evaluated was at least 40 days, in continuous use. Blood serum samples analyzed by this biosensor showed a good correlation compared to the spectrophotometric method (Trinder) used as reference, presenting relative deviations lower than 7.0%.     

Effects of solvent in photo-induced graft copolymerization of vinyl monomers on cellulose

The effects of swelling of the sample and polymerization solvents were studied for photo-induced graft copolymerization of vinyl monomers on cellulose. The graft copolymerization of methyl methacrylate (MMA) was activated by swelling of the sample or organic solvent-water solutions within a certain range of their concentrations. Though each organic solvent gave a maximum in per cent grafting and the number of grafts at about 25 vol-% concentration, the initiation reaction scarcely took place at 100% concentration; thus, the solvent itself is considered to have a negative effect. The solvents used in the experiments were all hydrophilic, such as methanol, acetone, and dioxane. The average molecular weight of the grafted PMMA differed in each solvent, indicating a different characteristic effect of solvent on the growing grafted polymer radicals. The presence of ferric ion as a sensitizer stimulated further the contributions of the sample swelling and the organic solvents to the copolymerization reaction. A similar effect was observed for styrene as for MMA, but not for acrylic acid and methacrylic acid.     

Aromatic polyesters obtained directly from hydroxybenzoic acids by hexachlorocyclotriphosphatriazene (PNC) in pyridine

Polyaryl esters of moderate molecular weights were prepared by the direct polycondensation reaction of hydroxybenzoic acids and its derivatives with hexachlorocyclotriphosphatriazene (also known as phosphonitrilic chloride trimer, PNC) as a condensing agent in pyridine. Copolymerization of p-hydroxybenzoic acid with several hydroxybenzic acid derivatives was carried out to improve the processability of the polymer of p-hydroxybenzoic acid, which yields fusible and soluble copolymers of high inherent viscosities. Polymer solubility and thermal behavior were examined.     

Photo-induced graft copolymerization of methyl methacrylate on cellulose

Graft copolymerization initiated by ultraviolet light irradiation at 40°C in a hard glass vessel under nitrogen was examined. The graft copolymerization was observed to occur easily after some induction period without any use of photosensitizer, though it was found the per cent grafting and the grafting efficiency were markedly affected by the quantities of cellulose and monomer. In the system without cellulose, homopolymerization of methyl methacrylate hardly took place, but the use of cellulose caused the formation of homopolymer too, and a grafting efficiency in the range of 60–80% generally resulted. Ferric chloride or sodium anthraquinone-2,7-disulfonate (AQ) acted on the polymerization reaction as photosensitizers to reduce its induction period. Though ferric chloride acted to develop both the per cent grafting and the number of grafts, not the same effects were observed with AQ. Oxalic acid, which was employed with the object of eliminating very small amount of metals contained in cellulose, was found to act favorably in the formation of grafts, much like ferric chloride.     

Ability of poly(vinyl alcohol) fibers to initiate graft copolymerization

It was noted in a graft copolymerization reaction involving a system of poly(vinyl alcohol) fibers (PVA fibers), methyl methacrylate, and water that fibers oxidized with hydrogen peroxide or sodium hypochlorite or ferric ion-adsorbing fibers effectively initiate the reaction as in the case of cellulose fibers. The initiation reaction of the sample was markedly activated by the presence of a pretreatment with an oxidizing agent at a concentration on the order of 10^?9 mole/l., but the oxidized samples were sharply deactivated by such means as reduction and oximation. Since model initiators for the present system, such as iron(III) acetylacetonate-adsorbing PVA fibers or ferric ion-adsorbing fibres, with the co-existence of acetylacetone were observed to initiate graft copolymerization effectively, it is believed that carbonyl groups in modified PVA fibers could be a major factor for initiating copolymerization through interaction with metallic ions.     

Stabilization of DNA triplexes by dangling aromatic residues

Novel nucleoside analogues containing 2^′-deoxyinosine and aromatic rings, which are connected by short linker groups, were synthesized and incorporated into oligodeoxyribonucleotides (ODNs). ODNs containing the nucleoside analogues formed stable duplexes and triplexes with target nucleic acids. The stacking interaction between base residues in the nucleoside analogues appears to be a major cause of stabilization.     

Temperature stabilized optical waveguide modulator

A low drive-voltage optical modulator using a Ti-diffused UNbO₃ optical waveguide has been fabricated. Stabilization against ambient temperature change was realized by using a miniature halfwave plate. The halfwave voltage, 3 dB bandwidth, optical insertion loss and extinction ratio were 3·8 V (at 1·06m wavelength), 850 MHz, 10 dB and 13 dB, respectively. A reduction scheme for the optical absorption caused by metallic electrodes, and an analysis of the modulator high frequency response are also reported.