Fluorescence lifetime imaging microscopy of Chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient.

Fluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthin-accumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25-35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a 'dimmer switch'. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, the efficient PSI electron acceptor methyl viologen and the protonophore nigericin and. Lower fluorescence intensities and lifetimes were observed for all npq2 mutant samples at the P-level and during the slow fluorescence transient, compared to wild type and the npq1 mutant. The fluorescence lifetime-resolved measurements during the slow fluorescence changes after the P level up to 250 s for the wild type and the two mutants, in the presence and absence of the above inhibitors, were analyzed with a graphical procedure (polar plots) to determine lifetime compositions. At higher illumination intensity, wild type and npq1 cells show a rise in fluorescence intensity and corresponding rise in the species concentration of the slow lifetime component after the initial decrease following the P level. This reversal is absent in the npq2 mutant, and for all samples in the presence of the inhibitors. Lifetime heterogeneities were observed in experiments averaged over multiple cells as well as within single cells, and these were followed over time. Cells in the resting state (induced by several hours of darkness), instead of the normal swimming state, show shortened lifetimes. The above results are discussed in terms of a superposition of effects on electron transfer and protonation rates, on the so-called 'State Transitions', and on non-photochemical quenching. Our data indicate two major populations of chlorophyll a molecules, defined by two 'lifetime pools' centred on slower and faster fluorescence lifetimes.

Fluorescence characteristics of 5-carboxytetramethylrhodamine linked covalently to the 5' end of oligonucleotides: multiple conformers of single-stranded and double-stranded dye-DNA complexes.

Fluorescence steady-state and lifetime experiments have been carried out on duplex and single-stranded DNA molecules labeled at the 5' ends with 5-carboxytetramethylrhodamine (TMRh). The temperature and ionic strength of the solutions were varied over large ranges. The results reveal at least three well-defined states of the TMRh-DNA molecules for the single-stranded as well as for the double-stranded DNA molecules. Two states are fluorescent, with lifetimes in the range of 0.5-1 ns and 2.5-3 ns. A third state of TMRh-DNA does not fluoresce (a dark species of TMRh-DNA). The distribution of the TMRh-DNA molecules among these three states is strongly temperature and ionic strength dependent. Estimates are made of some reaction parameters of the multistate model. The results are discussed in terms of the photophysics of TMRh, and consequences of the multiple conformers of TMRh-DNA for studies involving fluorescence studies with TMRh-labeled DNA are considered.

A three-dimensional model for the hammerhead ribozyme based on fluorescence measurements.

For the understanding of the catalytic function of the RNA hammerhead ribozyme, a three-dimensional model is essential but neither a crystal nor a solution structure has been available. Fluorescence resonance energy transfer (FRET) was used to study the structure of the ribozyme in solution in order to establish the relative spatial orientation of the three constituent Watson-Crick base-paired helical segments. Synthetic constructs were labeled with the fluorescence donor (5-carboxyfluorescein) and acceptor (5-carboxytetramethylrhodamine) located at the ends of the strands constituting the ribozyme molecule. The acceptor helix in helix pairs I and III and in II and III was varied in length from 5 to 11 and 5 to 9 base pairs, respectively, and the FRET efficiencies were determined and correlated with a reference set of labeled RNA duplexes. The FRET efficiencies were predicted on the basis of vector algebra analysis, as a function of the relative helical orientations in the ribozyme constructs, and compared with experimental values. The data were consistent with a Y-shaped arrangement of the ribozyme with helices I and II in close proximity and helix III pointing away. These orientational constraints were used for molecular modeling of a three-dimensional structure of the complete ribozyme.

Kinking of DNA and RNA helices by bulged nucleotides observed by fluorescence resonance energy transfer.

Fluorescence resonance energy transfer (FRET) has been used to demonstrate the bending of DNA and RNA helices for three series of double-stranded molecules containing bulge loops of unopposed adenosine nucleotides (An, n = 0-9). Fluorescein and rhodamine were covalently attached to the 5' termini of the two component strands. Three different methods were applied to measure the FRET efficiencies. The extent of energy transfer within each series increases as the number of bulged nucleotides varies from 1 to 7, indicating a shortening of the end-to-end distance. This is consistent with a bending of DNA and RNA helices that is greater for larger bulges. The FRET efficiency for DNA molecules with A9 bulges is lower than the efficiency for the corresponding A7 bulged molecules, although the A9 molecules exhibit increased electrophoretic retardation. Ranges of bending angles can be estimated from the FRET results.

Incidence and prognostic importance of silent ischaemia after PTCA: a prospective study.

To evaluate the influence of PTCA on symptomatic and asymptomatic ischaemic episodes in 94 patients, 24-h ambulatory, electrocardiographic Holter recordings were obtained before and after successful PTCA. Sixty-four per cent of patients had one-vessel disease, 28% two-vessel disease and 8% had three-vessel disease. Ischaemic episodes were present in 36% of patients before PTCA, of which 71% were silent; after PTCA, 23% of patients had ischaemic episodes, of which 98% were silent; thus silent ischaemic episodes were improved by PTCA to a lesser degree than symptomatic ischaemic episodes. Successful PTCA lead to a significant reduction in total number and duration of ischaemic episodes but not to a complete abolition. Patients with silent ischaemic episodes after PTCA had also a higher incidence of silent ischaemic episodes before PTCA. Functional and haemodynamic improvement was comparable in patients with and without silent ischaemic episodes. No specific cause for the persistent or newly appearing silent ischaemic episodes after PTCA could be identified; they are not indicative of an inadequate dilatation and cannot be considered as a risk factor for early restenosis. A possible explanation could be a traumatically induced, increased vascular tone in susceptible patients.

Comparative efficacy and safety of bumetanide and furosemide in long-term treatment of edema due to congestive heart failure.

Bumetanide and furosemide were compared for efficacy in reducing edema due to congestive heart failure in 28 patients (21 receiving bumetanide and seven receiving furosemide) in a long-term study for periods from one week to 18 months. In both groups the patients showed decreases in body weight, abdominal girth, edema, hepatomegaly, blood pressure, and heart rate. Commonly observed decreases frequently achieved statistical significance, more often with bumetanide, but the differences between treatments were rarely statistically significant. Both drugs were generally well tolerated. A breast nodule and gynecomastia were each reported once in the bumetanide group as was gynecomastia in one patient who had been on furosemide, all remotely related to test drugs. Soft stools, flatulence, mild constipation, and diminished vision each reported once in the bumetanide group were judged to be unrelated or remotely related to the drug therapy. Tendencies toward hypokalemia, hypochloremia, alkalosis, and hyperuricemia without clinical gout were deemed the result of the pharmacologic action of the diuretics. Others were attributable to the underlying disease state of these patients. Both diuretics proved to be effective in the treatment of cardiac edema and other manifestations of heart failure. Bumetanide treatment beyond six months in 11 patients indicated continued safety as well as efficacy.