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At least 19 recordsLinked to original sources
PubMedDate not supplied

Singlet oxygen in plants--its significance and possible detection with double (fluorescent and spin) indicator reagents.

Direct detection of reactive oxygen species (ROS), especially singlet oxygen, in plants under stress conditions is of special importance, not only to identify primary events of oxidative damage, but also in studies exploring the potential role of ROS as signal molecules. Due to short life-times and diffusion distances of ROS, these tasks require highly reactive and selective indicator reagents, localized at the presumed site of production. In the present study, we compared four double sensors: ROS indicator reagents in which partial fluorescence quenching of a dansyl moiety occurs as a result of nitroxide radical formation from a sterically hindered amine constituent. Our experiments support the idea that shorter donor-acceptor distances within these molecules result in higher reactivity to ROS. The presence of a diethylaminoethyl side chain resulted in better selectivity to singlet oxygen: reagents lacking such substituent had an additional reactivity to superoxide anions, probably as a result of the formation of zwitterionic structures. Fluorescence localization studies of the indicator reagents in tobacco leaves and in Chlamydomonas cells show promising perspectives of their applications to plant stress studies.

Biology↗
PubMed1983

False positive results in class-specific rheumatoid factor (RF) assays due to interaction between RF and Fc fragments of anti-immunoglobulin indicator reagents.

A recently developed, simple immunological method, diffusion-in-gel ELISA, has been adapted for class-specific determination of rheumatoid factor (RF). Evidence was obtained of an analytical error due to interaction between RF and antigenic determinants on the Fc part of the indicator immunoglobulin (Ig) molecules used. This was eliminated by replacing the usual indicator reagent, complete Ig molecules, by conjugated Fab or F(ab')2 fragments. The findings imply that unless the RF-Fc interaction mentioned is avoided in the assaying technique, false positive results may be obtained for, e.g. IgG RF and IgA RF in sera containing IgM RF.

Antibodies, Anti-Idiotypic↗
PubMed2003

Evaluation of FLIPR Calcium 3 Assay Kit--a new no-wash fluorescence calcium indicator reagent.

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT(2C)), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT(2C)) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT(2C) receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC(50) values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compelling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format.

Aniline Compounds↗
PubMed2001

A portable fiber-optic chemical device for the quantitative determination of carbon monoxide from automobile exhaust emissions.

A colorimetric method for the quantitative determination of CO by diffuse reflectance is described. This method is based on the reduction by CO of Mo (VI) from the indicator reagent molybdosilicic acid (H8Si[Mo2O7]6). The reduction yielded a change of color from clear yellow to dark green on white disk filter chart paper wetted with reagent indicator solution. The gaseous mixture containing CO was forced to pass through this chart paper, initiating the reaction. The intensity of the color produced, measured by diffuse reflectance, was proportional to the CO concentration present in exhaust gases in the range from 0.02 to 12% volume/volume (v/v). A 650-nm light-emitting diode was used as a light source. A two-fiber-optic system carried the light from the source to the detection system, which was composed of a photodiode, an amplification circuit, and a digital display. The method was applied with success in field measurements for automobiles in the Otto cycle. In a previous paper, this method was used for the quantitative determination of exhaust emissions from diesel-fueled vehicles.

Carbon Monoxide↗
PubMed1982

[Effectiveness of erythrocytic diagnostic reagents in indicating Shigellae].

The possibility of the preparation of dysentery antibody diagnostic reagents on the basis of adsorbed sera is shown. Such diagnostic reagents permit to determine 200000-600000 microbial bodies or 0.0008-0.0016 microgram of the antigen per ml. The use of antigenic erythrocytic diagnostic reagents for the indication of shigellae is expedient in the second variant of the passive hemagglutination inhibition test in the following modification: immune sera should be diluted not 2-fold, but only 1.5-fold; the material to be tested for the presence of shigellae should be added to these dilutions in a double volume.

Adsorption↗
PubMed2006

Optical biochemical sensor for determining hydroperoxides in nonpolar organic liquids as archetype for sensors consisting of amphiphilic conetworks as immobilisation matrices.

This paper reports the successful design of a prototype of an optical biochemical sensor for the determination of hydroperoxides in nonpolar organic liquids. The sensor consists of a matrix of an amphiphilic polymer conetwork (APCN), a novel class of very promising polymeric materials for easy preparation of biochemical sensor matrices. APCNs are characterised by nanoscopic phase separation between the hydrophilic and the hydrophobic phases. For medium ratios of conetwork composition, the domains of both phases are interconnected both on the surface of the conetworks and throughout the bulk. The APCNs have peculiar swelling properties-the hydrophilic phase swells in hydrophilic media and the hydrophobic phase swells in hydrophobic media. In both types of media dissolved reagents can diffuse from the solution into the swollen phase of the polymeric conetwork. This enables loading of the hydrophilic phase of the APCNs with enzymes and indicator reagents by simple impregnation. Hydrophobic analytes can diffuse into the polymeric conetwork via its hydrophobic phase and react with indicator reagents immobilised in the hydrophilic phase at the huge internal interface between the two opposite phases.To prepare the described hydroperoxide-sensitive biosensors, we used APCN films consisting of 58% (w/w) poly(2-hydroxyethyl acrylate) (PHEA) as hydrophilic chains and 42% (w/w) polydimethylsiloxane (PDMS) as hydrophobic linkers. Horseradish peroxidase (HRP) and diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as indicator reagent were co-immobilised in this optically clear and transparent matrix. In this feasibility study the conditions investigated were principally those relevant to characterisation of the innovative matrix material and the disposable biosensor produced from it; the biosensor was not optimised. Sensitivity toward tert-butylhydroperoxide (tBuOOH) dissolved in n-heptane was acceptable, between approximately 1 and at least 50 mmol L(-1), even in the dry state. The response time was 1.7 to 5.0 min. No leaching of immobilised reagents was observed during a period of at least one hour. Pre-swelling the sensors with water increased the reaction rate and the total turnover number of the enzyme. In a dry atmosphere at 4 degrees C the sensors were found to be stable for at least two weeks.

Biosensing Techniques↗
PubMed2006

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: characterization of TMs IV-VII and IX-XII and their accessibility to the aqueous translocation pathway.

We have determined the accessibility of the Rickettsia prowazekii ATP/ADP translocase transmembrane domains (TMs) IV-VII and IX-XII to the putative, water-filled ATP translocation pathway. A library of 177 independent mutants, each with a single cysteine substitution, was expressed in Escherichia coli, and those with substantial ATP transport activity were assayed for inhibition by thiol-reactive, methanethiosulfonate (MTS) reagents. The MTS reagents used were MTSES (negatively charged), MTSET (positively charged), and MTSEA (amphipathic). Inhibition of ATP transport by a charged MTS reagent indicates the exposure of a TM to the water-filled ATP translocation pathway. The eight TMs characterized in this study had 32 mutants with no assayable transport activity, indicating that cysteine substitution at these positions is not tolerated. ATP transport proficient mutants in TMs IV, V, VII, X, and XI were inhibited by charged MTS reagents, indicating that these TMs are exposed to the aqueous ATP translocation pathway, which is a pattern similar to those of TMs I, II (Alexeyev, M. F. (2004) Biochemistry 43, 6995-7002), and VIII (Winkler, H. H. (2003) Biochemistry 42, 12562-12569). Conversely, ATP-transport-proficient mutants in TMs VI, IX, and XII were not inhibited by charged MTS reagents, indicating that these TMs are sequestered from the aqueous environment, which is a pattern similar to that of TM III (Alexeyev, M. F. (2004) Biochemistry 43, 6995-7002). Preexposure of several MTS-sensitive mutants in TMs V, VII, X, and XI to ATP concentrations 10 times the K(m) resulted in protection from MTS-mediated inhibition; thus, confirming exposure of these TMs to the aqueous ATP translocation pathway, a pattern of protection similar to that observed for TMs I, II, and VIII.

Adenosine Triphosphate↗
PubMed1989

Simple fibre optic spectrophotometric cell for pH determination.

A simple, inexpensive fibre optic spectrophotometric cell has been developed for clinical use. The system may employ one of two indicator reagents for the measurement: phenol red and BDH universal indicator. The spectrophotometer uses two ultrabright LED sources and a PIN diode photodetector. The fibre is of the plastic type with a core diameter of 1.0 mm. Two alternative analytical methods are available: electronic or computer processing. In the case of phenol red the measuring range using computer processing is between 6.0 and 8.0 pH units, with an accuracy of 0.015 pH units. The range for electronic circuit processing is from 6.8 to 8.0 pH units with an accuracy of 0.02 pH units. Using a BDH universal indicator, the range for computer processing is between 5.5 and 8.5 pH units with an accuracy of 0.05 pH units, while with electronic processing the range is between 6.0 and 8.0 pH units with an accuracy of 0.03 pH units. A description of the optoelectronics, an analysis of the indicator reagents and the calibration procedure are presented here, together with some example results.

Calibration↗
PubMed1980

[Effect of certain factors on the quality of the indicator tubules for determining hydrogen sulfide].

Silicagel with size of granules 0.315 - 0.400 mm was used for carrier of indicator reagents in tubes for hydrogen sulphide. A planned experiment was carried out, with the purpose of establishing the influence of seven factors on the qualities of the silicagel-carrier and the tubes. It was found that essential is indicator powder porosity and humidity. An optimal amount of water to be added to keep powder humidity was found. The influence of acid processing of the carrier was also studied. Acid treatment was shown to increase carrier adsorption capacity, which affected both the ability of the carrier to take in indicator reagents and tubes quality. It was shown that tubules, prepared on the basis of acid-nontreated, but only heated at 600 degrees C carrier, are of superior quality, so long as the requirements for indicator powder humidity have strictly been observed.

Gels↗
PubMed1992

Electrochemically modulated optrode for glucose.

One of the problems with fibre-optic sensors is the difficulty of finding reversible indicator reagents. This is a particular problem for fibre-optic glucose sensors. The development of an electrochemically modulated fibre-optic probe or optrode has been proposed as a convenient solution. Here the indicator reagent is regenerated electrochemically. In this work a design is proposed that offers considerable advantages in practical applications. In particular, it can be used in the same way as conventional optrodes. The optimization of working parameters and the application of the optrode to flow analysis under steady-state and flow-injection conditions is described.

Biosensing Techniques↗
PubMed2006

Nanophase-separated amphiphilic conetworks as versatile matrixes for optical chemical and biochemical sensors.

As a novel class of sensor matrixes, nanophase-separated amphiphilic polymeric conetworks (APCNs) open a new dimension for optical chemical and biochemical sensing. These conetworks consist of a hydrophilic phase-we used poly(2-hydroxyethyl acrylate), poly(2-(dimethylamino)ethyl acrylate), or polycationic poly(2-(trimethylammonium)ethyl acrylate)-and of a hydrophobic phase-poly(dimethylsiloxane). Sensors can be prepared by simple impregnation of the matrix. Due to nanophase separation, there is a spatial separation between areas in which the indicator reagents are well immobilized and areas that advantageously take care of the diffusive transport of the analyte, whereby these functionalities of the contrary phases can be exchanged. Thanks to the huge interface between the contrary phases, the accessibility of the indicator reagents is good, which makes it possible to design sensors with high sensitivity. To demonstrate the advantages of APCNs as matrixes, different prototypes of sensors were prepared, e.g., one to determine gaseous chlorine based on its reaction with immobilized o-tolidine and another to determine vaporous acids based on immobilized bromophenol blue dianions. As a breakthrough in biochemical sensing, we are also able to present an easily producible, optically transparent biochemical sensor to determine peroxides in nonpolar organic media-based on coimmobilized horseradish peroxidase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate).

Biosensing Techniques↗
PubMed1977

Characteristics of binding between reagent-strip indicators and urinary proteins.

We describe the binding characteristics of two indicator dyes that are widely used in commercial "dip-stick" indicators for protein in urine. Binding was studied by both gel filtration chromatography and by spectrophotometric titrations. The results demonstrate that the well-known lack of sensitivity of these strips to proteins other than albumin relates to the number and affinity of binding sites on these proteins as compared to albumin. The data suggest that these test strips can screen more effectively for albuminuria than for other proteinurias.

Chromatography, Gel↗
PubMed1981

Propionic acid-metabisulphite-Schiff reagent for quick Feulgen staining.

This communication presents a method for the preparation of Schiff reagent in which N hydrochloric acid has been replaced by a low concentration of propionic acid. The result of using such a Schiff reagent indicates that the dye-reagent thus prepared is extrafast in action on acid-hydrolysed mammalian tissue sections. The intensity of nuclear colouration is also increased considerably, since the pH of the dye-reagent is 6.0. It is, therefore advocated that this newly developed Schiff reagent be used at 5 degrees C.

Animals↗
PubMed1993

Conformation and thermodynamics of DNA "necks". Models for three-arm branch formation in a duplex.

The properties of three-arm DNA junctions differ from those of four-arm junctions in several respects. Most apparently, bases flanking the branch are reactive to single strand specific agents in three-arm junctions but not four-arm junctions. To determine the basis for this, we have designed and synthesized a series of complexes in which a short duplex, a neck, progressively extends from a parent 16-mer DNA duplex. These structure allow us to investigate how a three-arm branch forms, and how its properties change as the neck extends. Comparison of the properties of a nicked duplex with those of the neck structures using native gel electrophoresis with reporter arms attached reveals progressively greater geometrical perturbation of the complexes as the number of base-pairs in the neck increases. Footprinting by single-strand specific reagents indicates that the reactivity to single-strand reagents near the branch occurs when only a single pair is possible. The branch in each neck interacts tightly with ethidium, as does a nick in the same duplex. The thermodynamics of neck formation have been evaluated by calorimetry and from the concentration dependence of absorbance temperature profiles. Each neck complex is destabilized with respect to duplex DNA or a nicked duplex, and has a lower enthalpy of formation despite the increased number of base-pairs present. A model is proposed to account for these properties in which the bases in the pair adjacent to the duplex interact directly with the duplex, via transient insertion.

Base Sequence↗
PubMed1990

Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5'-diphospho-5'-adenosine and pyridoxal phosphate reactive residue.

Pyridoxal 5'-diphospho-5'-adenosine (AP2PL) inhibits lamb kidney (Na,K)-ATPase and that inhibition and covalent modification is blocked by the presence of ATP. After trypsin digestion of the labeled, purified alpha subunit and subsequent peptide mapping of the fluorescently labeled peptides by means of high performance liquid chromatography, the main labeled peptide was further purified and analyzed by amino acid composition analysis and peptide sequencing. The obtained peptide had the sequence Ile470-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys480-Tyr-Gln-Le u-Ser-Ile-His- Lys487. Lysine 480 is the residue modified by AP2PL in the absence, but not in the presence of ATP. The beta subunit is not differentially labeled by AP2PL in the presence or absence of ATP. Interestingly, the same results were obtained using pyridoxal phosphate as the labeling and inactivation reagent, indicating that the specificity of labeling by these reagents is not due to the presence of the adenosine moiety, but instead that the initial recognition of nucleotides by the ATP-binding site of (Na,K)-ATPase may be due to recognition of the phosphate moiety. The amino acid sequence surrounding this lysine residue labeled by both reagents is highly conserved in (Na,K)-ATPase and the related (H,K)-ATPase sequences thus far obtained, which may signify a functional importance for this region of the putative ATP-binding site in these transport proteins.

Adenosine Diphosphate↗
PubMed1996

Characterization of the 46-kDa intermediates of matrix metalloproteinase 3 (stromelysin 1) obtained by site-directed mutation of phenylalanine 83.

The precursor of matrix metalloproteinase 3 (MMP-3/ stromelysin 1) is activated in vitro by proteinases or mercurial compounds by stepwise processes which include the initial formation of short-lived intermediates and the subsequent intermolecular cleavage of the His82-Phe83 bond to generate the fully activated mature MMP-3 (Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G. (1990) Biochemistry 29, 5783-5789). To study the enzymatic properties of the intermediates we have mutated either His82 or Phe83 to Arg to obtain a stable MMP-3 intermediate. The mutant proteins were expressed in Chinese hamster ovary K-1 cells using a mammalian expression system. The proMMP-3(H82R) mutant was activated by chymotrypsin, elastase, and 4-aminophenylmercuric acetate to the 45-kDa MMP-3 with similar mechanism and kinetics as the wild-type. In contrast, the activation of the proMMP-3(F83R) mutant by proteinases or 4-aminophenylmercuric acetate resulted in 46-kDa forms, which retained 13, 14, or 15 amino acids of the pro-domain depending on the activators. The proteinase-activated MMP-3(F83R) intermediates exhibited little enzymatic activity, but they were partially active after treatment with SH-reacting reagents. These molecules could bind to the tissue inhibitor of metalloproteinases-1 and alpha 2-macroglobulin. However, the SH group of Cys75 of the intermediates was not modified by SH-reagents, indicating that the enzymatic activity generated by SH-reagents resulted from molecular perturbation of the enzyme rather than their interaction with Cys75. When gelatin and transferrin were digested with the 46-kDa intermediates the products were different from those generated by the wild-type MMP-3, suggesting an alteration in substrate specificity. The treatment of proMMP-3 with trypsin resulted in the formation of a 45-kDa MMP-3 with an NH2-terminal Thr85, whose activity and substrate specificity were similar to those of the 46-kDa MMp-3(F83R) obtained from the proMMP-3(F83R) mutant. These observations indicate that the correct processing at the His82-Phe83 bond is critical for expression of the full activity and the specificity of MMP-3.

Amino Acid Sequence↗
PubMed2004

Structure and function of extracellular loop 4 of the serotonin transporter as revealed by cysteine-scanning mutagenesis.

Residues 386-423 of the rat brain serotonin transporter (SERT) are predicted to form a hydrophilic loop connecting transmembrane spans 7 and 8 (extracellular loop 4 or EL4). EL4 has been hypothesized to play a role in conformational changes associated with substrate translocation. To more fully investigate EL4 structure and function, we performed cysteine-scanning mutagenesis and methanethiosulfonate (MTS) accessibility studies on these 38 residues. Four EL4 mutants (M386C, R390C, G402C, and L405C) showed very low transport activities, low cell surface expression, and strong inhibition by MTS reagents, indicating high structural and functional importance. Twelve mutants were sensitive to very low MTS concentrations, indicating positions highly exposed to the aqueous environment. Eleven mutants were MTS-insensitive, indicating positions that were either buried in EL4 structure or functionally unimportant. The patterns of sensitivity to mutation and MTS reagents were used to produce a structural model of EL4. Positions 386-399 and 409-421 are proposed to form alpha-helices, connected by nine consecutive MTS-sensitive positions, within which four positions, 402-405, may form a turn or hinge. The presence of serotonin changed the MTS accessibility of cysteines at nine positions, while cocaine, a non-transportable blocker, did not affect accessibility. Serotonin-induced accessibility changes required both Na(+) and Cl(-), indicating that they were associated with active substrate translocation. With the exception of a single mutant, F407C, neither mutation to cysteine nor treatment with MTS reagents affected SERT affinities for serotonin or the cocaine analog beta-CIT. These studies support the role of EL4 in conformational changes occurring during translocation and show that it does not play a direct role in serotonin binding.

Animals↗