The development of non-separation time-resolved fluoroimmunoassays for the measurement of urinary metabolites

We describe the principles of a new generation of sequential or simultaneous time-resolved fluoroimmunoassays, namely, simple, rapid, liquid-phase non-separation procedures which may be applied to the measurement of urinary steroid and drug metabolites. As an example, a method for the measurement of estrone-3-glucuronide in undiluted urine is reported. This method has a similar sensitivity, specificity and accuracy to a conventional separation fluoroimmunoassay or radioimmunoassay but in terms of speed, convenience, precision, reliability and clinical utility the new method has many advantages. The labelled antigen is a novel fluorescent europium chelate covalently linked to estrone-3-glucuronide. The antibody-binding reaction involves the incubation of the labelled antigen (2ng) with a limited concentration of polyclonal or monoclonal antibodies to estrone-3-glucuronyl-6-BSA and an aliquot of standard or sample (undiluted urine; 10 μl) in microtitre wells. After a 10 min incubation, the fluorescence which emanates from the antibody-free label is measured in a time-resolved fluorometer and is proportional to the concentration of estrone-3-glucuronide in the standard or sample. The method may be applied for the monitoring of ovarian function in women.     

Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator

Summary To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ·cm², and short circuit current (I _sc=2–20 A/cm²), representing active secretion of Cl, increased >threefold with addition of 10 m isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescencevs. [Cl] (0–90mm) was carried out using high-K buffers containing the ionophores nigericin (5 m) and tributyltin (10 m); SPQ fluorescence was quenched with a Stern-Volmer constant of 13m ^–1. Intracellular Cl activity was 43±4mm. Cl flux was measured in response to addition and removal of 114mm Cl from the bathing solution. Addition of 10 m isoproterenol increased Cl efflux from 0.10 to 0.27mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1mm). In the absence of isoproterenol, removal of external Na or addition of 0.5mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.     

Conformational transitions and charge translocation by the Na,K pump: Comparison of optical and electrical transients elicited by ATP-concentration jumps

Summary The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (8000 m^–2) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive electrode. ATP was released in the medium from a photolabile inactive ATP derivative (caged ATP) by a 40-sec light flash. Electrical signals resulting from transient charge movements in the protein under single-turnover conditions were recorded in the external measuring circuit. In parallel experiments carried out under virtually identical conditions, the fluorescence of membrane fragments containing Na,K-ATPase with covalently-bound 5-iodoacetamido-fluorescein (5-IAF) was monitored after the ATP-concentration jump. When the medium contained Na⁺, but no K⁺, the fluorescence of the 5-IAF-labeled protein decreases monotonously after release of ATP. In the experiments with membrane fragments bound to a planar bilayer, a transient pump current was observed which exhibited virtually the same time behavior as the fluorescence decay. This indicates that optical and electrical transients are governed by the same rate-limiting reaction step. Experiments with chymotrypsin-modified Na,K-ATPase suggest that both the fluorescence change as well as the charge movement are associated with the deocclusion of Na⁺ and release to the extracellular side. In experiments with Na⁺-free K⁺ media, a large inverse fluorescence change is observed after the ATP-concentration jump, but no charge translocation can be detected. This indicates that deocclusion of K⁺ is an electrically silent process.     

Effect of copper deficiency on photosynthetic electron transport in wheat plants

Copper deficiency in wheat ( Triticum aestivum L. cv. Nazareno Stramppeli) markedly affects photosynthetic activity. Flag leaves of copper-deficient plants showed a 50% reduction of the photosynthetic rate expressed as mg CO₂ dm⁻²h⁻¹. The activities of PSI and PSII, determined for isolated chloroplasts, as well as fluorescence measurements on intact leaves of copper-deficient plants, indicated a low activity of photosynthetic electron transport. Ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity was not affected by copper deficiency but copper deficiency affected the chloroplast ultrastructure, especially at the level of grana, where a disorganization of thylakoids is evident.     

Estimation of the effect of photoinhibition on the carbon gain in leaves of a willow canopy

The occurrence of photoinhibition of photosynthesis in leaves of a willow canopy was examined by measuring the chlorophyll-a fluorescence ratio of F _V/F _M (F_M is the maximum fluorescence level of the induction curve, and F_V is the variable fluorescence, F _V=F _M–F ₀, where F₀ is the minimal fluorescence). The majority of the leaves situated on the upper parts of peripheral shoots showed an afternoon inhibition of this ratio on clear days. This was the consequence of both a decrease in F _M and a rise in F _O. In the same leaves the diurnal variation in intercepted photosynthetic photon flux density (PPFD) was monitored using leaf-mounted sensors. Using the multivariate method, partial least squares in latent variables, it is shown that the dose of PPFD, integrated and linearly weighted over the last 6-h period, best predicts photoinhibition. Photoinhibition occurred even among leaves that did not intercept PPFDs above 1000 mol·m^–2·s^–1. Exposure of leaves to a standard photoinhibitory treatment demonstrated that the depression in the F _V/F _M ratio was paralleled by an equal depression in the maximal quantum yield of CO₂ uptake and a nearly equal depression in the rate of bending (convexity) of the light-response curve of CO₂ uptake. As a result, the rate of net photosynthesis is depressed over the whole natural range of PPFD. By simulating the daily course in the rate of net photosynthesis, it is estimated that in the order of one-tenth of the potential carbon gain of peripheral willow shoots is lost on clear days as a result of photoinhibition. This applies to conditions of optimal temperatures. Photoinhibition is even more pronounced at air temperatures below 23° C, as judged from measurements of the F_V/F_M ratio on clear days: the afternoon inhibition of this ratio increased in a curvilinear manner from 15% to 25% with a temperature decrease from 23° to 14° C.Abbreviations and Symbols F_O minimum fluorescence - F_V variable fluorescence - F_M maximum fluorescence - PLS partial least squares in latent variables - PPFD photosynthetic photon flux density - VPD water vapour-pressure deficit This study was supported by the Swedish Natural Science Research Council. We are indebted to Dr. Jerry Leverenz (Department of Plant Physiology, University of Umeå, Sweden) for guidance with the modelling of the photosynthesis data.     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum     

Non-photochemical quenching of Fo in leaves is emission wavelength dependent: consequences for quenching analysis and its interpretation

The effects of light-induced non-photochemical quenching on the minimal F_o, and variable F_v, fluorescence emissions at 690 and 730 nm in leaves were determined. Non-photochemical quenching of F_o, but not F_v, was found to be dependent upon the wavelength of emission, and was greater at 690 nm than at 730 nm. For emission at 730, compared to at 690 nm, approx. 30% of F_o was not affected by non-photochemical quenching processes in leaves of C3 plants; in maize leaves this was found to be approx. 50%. The data indicate that a substantial proportion of the pigments contributing to F_o emission at 730 nm are not quenched by light-induced, non-photochemical quenching processes and that there are large differences in the pigment matrices contributing to F_o and F_v emissions at 730 nm, compared to those at 690 nm. These findings have important implications for the accurate estimation and interpretation of non-photochemical quenching of fluorescence parameters and their use in the calculation of photochemical efficiencies in leaves. Measurements of fluorescence emissions at wavelengths above 700 nm are likely to give rise to significant errors when used for determinations of photochemical and non-photochemical quenching parameters.     

Studies on the mechanism of photosystem II photoinhibition I. A two-step degradation of D1-protein

The role of D1-protein in photoinhibition was examined. Photoinhibition of spinach thylakoids at 20°C caused considerable degradation of D1-protein and a parallel loss of variable fluorescence, Q_B-independent electron flow and Q_B-dependent electron flow. The breakdown of D1-protein as well as the loss of variable fluorescence and Q_B-independent electron flow were largely prevented when thylakoids were photoinhibited at 0°C. The Q_B-dependent electron flow markedly decreased under the same conditions. This inactivation may represent the primary event in photoinhibition and could be the result of some modification at the Q_B-site of D1-protein. Evidence for this comes from fluorescence relaxation kinetics following photoinhibition at 0°C which indicate a partial inactivation of Q_A ^--reoxidation. These results support the idea of D1-protein breakdown during photoinhibition as a two step process consisting of an initial inactivation at the Q_B-site of the protein followed by its degradation. The latter is accompanied by the loss of PS II-reaction centre function.Abbreviations Asc ascorbate - p-BQ 1, 4-benzoquinone - DAD diaminodurene - DPC diphenylcarbazide - DQH₂ duroquinole - Fecy ferricyanide - MV methylviologen - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - SiMo silicomolybdate     

Time-resolved spectroscopy of the blue fluorescence of spinach leaves

When excited by ultraviolet radiation, leaves of a great number of species of higher plants exhibit emission of blue fluorescence, comparable in intensity to the red emission of chlorophyll. The fluorescence decay of the blue emission of spinach leaves recorded by single photon counting techniques is decomposed into exponential components and it is shown that at least three different components are present. The lifetime of the three components does not show significant variations with the excitation or emission wavelengths. The excitation and emission spectra of each component were determined. The nature of the chemical compounds which cause this emission is discussed in relation to these spectra.     

State 1-state 2 adaptation in the cyanobacteria Synechocystis PCC 6714 wild type and Synechocystis PCC 6803 wild type and phycocyanin-less mutant

The mechanism of excitation energy distribution between the two photosystems (state transitions) is studied in Synechocystis 6714 wild type and in wild type and a mutant lacking phycocyanin of Synechocystis 6803. (i) Measurements of fluorescence transients and spectra demonstrate that state transitions in these cyanobacteria are controlled by changes in the efficiency of energy transfer from PS II to PS I (spillover) rather than by changes in association of the phycobilisomes to PS II (mobile antenna model). (ii) Ultrastructural study (freeze-fracture) shows that in the mutant the alignment of the PS II associated EF particles is prevalent in state 1 while the conversion to state 2 results in randomization of the EF particle distribution, as already observed in the wild type (Olive et al. 1986). In the mutant, the distance between the EF particle rows is smaller than in the wild type, probably because of the reduced size of the phycobilisomes. Since a parallel increase of spillover is not observed we suggest that the probability of excitation transfer between PS II units and between PS II and PS I depends on the mutual orientation of the photosystems rather than on their distance. (iii) Measurements of the redox state of the plastoquinone pool in state 1 obtained by PS I illumination and in state 2 obtained by various treatments (darkness, anaerobiosis and starvation) show that the plastoquinone pool is oxidized in state 1 and reduced in state 2 except in starved cells where it is still oxidized. In the latter case, no important decrease of ATP was observed. Thus, we propose that in Synechocystis the primary control of the state transitions is the redox state of a component of the cytochrome b ₆/f complex rather than that of the plastoquinone pool.Abbreviations DCCD dicyclohexylcarbodiimide - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - EF exoplasmic face - PQ plasto-quinone - PS photosystem - PBS phycobilisome