A calmodulin-stimulated Ca-ATPase from plant vacuolar membranes with a putative regulatory domain at its N-terminus

A cDNA, BCA1, encoding a calmodulin-stimulated Ca²⁺-ATPase in the vacuolar membrane of cauliflower (Brassica oleracea) was isolated based on the sequence of tryptic peptides derived from the purified protein. The BCA1 cDNA shares sequence identity with animal plasma membrane Ca²⁺-ATPases and Arabidopsis thaliana ACA1, that encodes a putative Ca²⁺ pump in the chloroplast envelope. In contrast to the plasma membrane Ca²⁺-ATPases of animal cells, which have a calmodulin-binding domain situated in the carboxy-terminal end of the molecule, the calmodulin-binding domain of BCA1 is situated at the amino terminus of the enzyme.     

Phase-polarisation contrast for surface plasmon resonance biosensors

A technique of phase-polarisation contrast (PPC) for the enhancement of the contrast of a surface plasmon resonance (SPR) intensity profile is proposed and experimentally realised. The technique exploits the peculiarities of light phase and polarisation behaviour under SPR. It applies to non-optimum SPR coupling conditions and enables one to lower the resonant minimum of reflected intensity nearly to zero, and hence to increase substantially the ratio of the intensity from the resonance to that at the minimum. We observed the contrast enhancement by more than one order of magnitude when we applied the PPC scheme. The PPC can be efficiently employed in commercial SPR sensors, as it significantly reduces restrictions on allowable parameters of SPR-supporting metal films and biomolecular layers immobilised on them, facilitates SPR observation, and increases the accuracy of SPR shift measurements.     

Fucoxanthin metabolites in egg yolks of laying hens

Feeding experiments were conducted with White leghorn laying hens fed a carotenoid depleted control diet (containing some zeaxanthin and lutein) or this diet supplemented with 15% seaweed meal of established carotenoid composition. Egg yolk colour was estimated by use of a Roche Yolk Colour Fan and by detailed quantitative and qualitative carotenoid analysis of individual eggs of three laying hens during 4 weeks. Identification of the carotenoids included HPLC, VIS, MS, ¹H NMR data and partial synthesis. The results confirmed that fucoxanthin, the major carotenoid in seaweed meal, is not transferred to the yolk. However, fucoxanthin gave rise to the metabolites fucoxanthinol, fucoxanthinol 3'-sulphate and paracentrone, that are ascribed to enzymatic modifications occurring in the hens. The difuranoid auroxanthin encountered in the egg yolk was ascribed to violaxanthin and/or its furanoid derivatives present in the seaweed meal. Colour of individual yolks varied considerably. The pigmentation level is discussed.     

Salt-independent adsorption of human serum proteins on cyanocarbon gels

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene–divinyl sulfone–agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Determination and pharmacokinetics of a furosemide–amiloride drug combination

The study presents an accurate and precise HPLC assay for the determination of furosemide and amiloride in human specimens. Both drugs were extracted from human plasma with ethyl acetate; furosemide was extracted at pH 1 and amiloride at pH 12. While chromatographic separation conditions, i.e., column, mobile phase and flow-rate were the same for both investigated drugs, furosemide was detected using a UV absorbance detector, whereas amiloride, because of its very low therapeutic range, was detected with a spectrofluorimetric detector. The linearity of the furosemide and amiloride assays were confirmed over the range of 30–3000 ng/ml and 0.5–30 ng/ml, respectively. These concentrations correspond well with the therapeutic ranges of both drugs. The extraction recoveries, depending on concentration, exceed 80% for furosemide and 74% for amiloride. The reported methods were applied to pharmacokinetic investigations of the two compounds taken in form of a drug combination.     

cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca²⁺-ATPases is discussed.     

A new interference-free lysine biosensor using a non-conducting polymer film

An electrochemical biosensor for the determination of lysine to be used for rapid evaluation of food quality has been developed. Platinum electrodes have been coated by electropolymerisation with 1,2-diaminobenzene (1.2-DAB) using cyclic voltammetry. The reduction in the oxidation of interferents compared with the bare platinum electrode was 100% for ascorbic acid, 99% for acetaminophen and 99% for cysteine. The enzyme L-lysine-α-oxidase was then immobilised onto the polymer layer by passive adsorption and a calibration curve for lysine constructed. This gave a linear range of 1×10⁻⁵ mol/l to 1×10⁻³ mol/l and a limit of detection of 2×10⁻⁷ mol/l.     

Volume conduction models for surface EMG; confrontation with measurements

Volume conduction models are used to describe and explain recorded motor unit potentials (MUPs). So far it has remained unclear which factors have to be taken into account in a volume conduction model. In the present study, five different models are confronted with measured MUP distributions over the skin surface above the m. biceps brachii generated by MUs at different depths and recorded by small surface electrodes. All model simulations include fibres of finite length. The models differ in the size of the volume conductor (finite/infinite), the number of different layers (1, 2 or 3) and the conductivities of these layers (representing muscle, subcutaneous fat and skin). All measured and simulated MUPs contain a mainly negative propagating wave followed by a positive wave simultaneously present at all electrode positions. The magnitude of the different MUP components relative to each other and as a function of motor unit (MU) and electrode position differ between the models studied and the measurements. All simulated MUPs changed faster with observation distance than the measured MUPs. The three-layer model, in which muscle tissue was surrounded by a subcutaneous fat layer and by a layer of skin resulted in MUPs closest to the measured MUPs.     

Quercetin metabolites inhibit copper ion-induced lipid peroxidation in rat plasma

The oxidative susceptibility of plasma obtained from rats after intragastric administration of quercetin was studied to know whether or not quercetin acts as an in vivo antioxidant after metabolic conversion. Quercetin was raised in the rat blood plasma essentially as glucuronide and/or sulfate conjugates. The plasma obtained from rats after quercetin administration was more resistant against copper sulfate-induced lipid peroxidation than the control plasma on the basis of the accumulation of cholesteryl ester hydroperoxides and the consumption of α-tocopherol. The results strongly suggest that some conjugated metabolites of quercetin act as effective antioxidants when plasma is subject to metal ion-induced lipid peroxidation.