Structural features of an anti-diabetic polysaccharide (TAP) from Tremella aurantia

The structure of an anti-diabetic polysaccharide (TAP) obtained from the fruiting bodies of Tremella aurantia was investigated by methylation analysis, Smith degradation, partial acid hydrolysis, 13C-NMR spectrometry, and enzymatic digestion. The results suggested that TAP was composed of (1-->3)-linked alpha-D-mannopyranosyl residues as a backbone, some of which were substituted at position 2 with (1-->3)-linked beta-D-xylopyranose side chains and with beta-D-glucopyransyluronic acid at position 4 linked to terminal alpha-D-mannopyranose.     

Solid-phase luminescent catalyst immunoassay for human serum albumin with hemin as labeling catalyst

A solid-phase luminescent catalyst immunoassay is described for the determination of human serum albumin (HSA) in solution; hemin is used as a label which catalytically amplifies the sensitivity. The method is essentially a non-radioactive and non-enzymatic sandwich immunoassay. Anti-HSA antibody is covalently bound to a transparent plate, which then undergoes the immunochemical reaction with HSA in the test solution, and with the fixed amount of hemin-labeled anti-HSA antibody. After the two-step immunoreaction, the immunochemically-adsorbed hemin-antibody conjugate is quantified by means of the luminescence produced in a solution containing luminol and hydrogen peroxide. The luminescence intensity is correlated with the amount of HSA. The limit of detection for HSA is 1 ng ml^-1.     

The effect of the anion fraction on the physicochemical properties of EMIm(HF)nF (n = 1.0-2.6)

A series of molten salts EMIm(HF)nF's with different n values has been synthesized by the reaction of EMImHF(2) and anhydrous hydrogen fluoride. The salts contain EMIm cation and some oligomeric fluorohydrogenate anions, (HF)nF-, of which the fraction changes with the change of n. A phase diagram of EMIm(HF)nF's (n = 1.0-2.6) has been constructed which suggests the presence of the stoichiometric compounds, EMIm(HF)1.5F and EMIm(HF)2F, in this range. Compared to the EMIm(HF)2.3F previously reported, EMIm(HF)nF's (n = 1.8-2.0) possess wider liquid temperature ranges because of their similar melting points and superior thermal stabilities at elevated temperatures. The electrochemical windows of EMIm(HF)nF's (n = 1.0-2.6) falls in the range of 2.9-3.4 V. The conductivity of EMIm(HF)nF's (n = 1.0-2.6) increases with the increase of n.     

Photoelectrochemical reaction of chlorophyll immobilized with liquid crystal on metal surface

Chlorophyll was immobilized with liquid crystal on a platinum surface to prepare a photoexcitable electrode. Liquid crystals such as N-(p-methoxybenzylidene)-p-butylanil-ine were found to be effective on the photoexcitation of the immobilized chlorophyll. Such a chlorophyll-liquid crystal electrode produced photocurrent when it was coupled with a solution of nicotinamide adenine dinucleotide and exposed to light. The electron transfer accompanied by the photoelectrochemical reaction is discussed.     

Design and construction of glutamine binding proteins with a self-adhering capability to unmodified hydrophobic surfaces as reagentless fluorescence sensing devices

The chemically and genetically remodeling of proteins with ligand binding specificities can be utilized to synthesize various protein-based microsensors for detecting single biomolecules. Here, we describe the construction and characterization of fluorophore-labeled glutamine binding proteins (QBP) and derivatives coupled to the independently designed hydrophobic polypeptide (E12) that can adhere onto solid surfaces via hydrophobic interactions. The single cysteine mutant (N160C QBP) modified with the three environmentally sensitive fluorescent dyes (IAANS, acrylodan, and IANBD ester) showed increased changes in fluorescence intensity induced by glutamine binding. The use of these conjugates as reagentless fluorescence sensors enables us to determine the glutamine concentrations (0.1-50 microM) in homogeneous solution. The fusion of N160C QBP with E12, (Gly4-Ser)n spacers (GSn), and IANBD resulted in the novel fluorescence sensing elements having an adhering capability to hydrophobic surfaces of unmodified microplates. In ELISA and fluorescence experiments for the microplates treated with a series of the conjugates, IANBD-labeled N160C QBP-GS1-E12 displayed the best reproducibility in adhesion onto the hydrophobic surfaces and the precise correlation between fluorescence changes and glutamine concentrations. The performance of the biosensor-attached microplate for glutamine titrations demonstrated that the hydrophobic interaction of E12 with solid surfaces is useful for effective immobilization of proteins that need specific conformational movements in recognizing particular biomolecules. Therefore, the technique using E12 as a surface-linking domain for protein adhesion onto unmodified substrates could be applied effectively to prepare microplates/arrays for a wide variety of high-throughput assays on chemical and biological samples.     

Effects of chemical modifications of membranes on transmembrane potential

The role of membrane surface substances on the generation of transmembrane potential was studied. Several functional groups such as amino, epoxy, and carboxyl groups were covalently introduced to a bromoacetyl cellulose membrane. These functional groups caused a marked change in the surface potential of the membrane. The transmembrane potential shift caused by the chemical modification was attributed to the charge of the functional groups. Several proteins were covalently immobilized to the modified membrane. The modification process was followed through the transmembrane potential. The transmembrane potential of the protein-binding membranes showed that lysozyme and egg albumin at the membrane surface produced a positive and a negative charge, respectively. It was concluded that attachment of protein to the surface of the membrane affects a change in the charge density of the membrane surface with a resulting change in transmembrane potential.     

HEMATOPORPHYRIN DERIVATIVE UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS: THE SPECTRA OF FLUORESCENCE FROM HEMATOPORPHYRIN DERIVATIVE DEMONSTRATED BY AN EXCIMER DYE LASER

Abstract A new diagnostic and therapeutic endoscopic system consisting of an excimer pulse dye laser is presented. This report demonstrates the accumulation of hematoporphyrin derivative (HpD) in atheroma as shown by the fluorescence of HpD using this equipment. Atheroma was induced in the aorta of WHHL (Watanabe heritable hyperlipidemic) rabbits, 5 mg kg⁻¹ HpD was injected intravenously and the rabbits were sacrificed 24 h later. The aorta was dissected and the localization of HpD was examined. Characteristic peaks of the fluorescence of HpD at 630, 665 and 690 nm wavelength were detected in the atheromatous lesion. However, in the fatty plaque, the emission peak at 630 nm was lower and the 665 nm peak faded away. No fluorescence with peaks was detected in the normal area. The ratio of fluorescence intensity in atheroma, border zones and normal areas was 10.4 : 5.0 : 1.0. On normal rabbits made atherosclerotic by diet and balloon damage, an ultra thin endoscopic catheter was inserted from the descending aorta of atherosclerotic rabbits under anesthesia. Essentially the same data was obtained by these studies in vivo as was obtained in the in vitro studies. The above data suggests the possibility of future applications of this equipment for diagnosis of atheroma.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.     

Organelle sensor: amperometric determination of nadh by immobilized mitochondrial electron transport particles

A new sensor for NADH was developed by making use of an immobilized subcellular organelle. Mitochondria was used as a model system for assembling an organelle sensor. Mitochondrial electron transport particles (ETP) were prepared from beef heart muscle and entrapped in the membrane formed of agar gel. The membrane-bound ETP was found capable of NADH oxidation: $$NADH + \tfrac{1}{2}O_2 + H^ + \xrightarrow{{ETP}}NAD^ - + H_2 O$$ The membrane was tightly attached to the surface of an oxygen electrode capable of amperometric detection of O2. The sensor responded to NADH in solution with a resulting electric output. The response was enhanced by the addition of 2,4-dinitrophenol (DNP). NADH was determined in the concentration range 1–300 µM. NADH was alternatively determined for 2 weeks without replacing the ETP-bound membrane.