Temporal models for mitotic phase labelling.

With the widespread use of time-lapse data to understand cellular function, there is a need for tools which facilitate high-throughput analysis of data. Fluorescence microscopy of genetically engineered cell lines in culture can be used to visualise the progression of these cells through the cell cycle, including distinctly identifiable sequential stages of cell division (mitotic phases). We present a system for automated segmentation and mitotic phase labelling using temporal models. This work takes the novel approach of using temporal features evaluated over the whole of the mitotic phases rather than over single frames, thereby capturing the distinctive behaviour over the phases. We compare and contrast three different temporal models: Dynamic Time Warping, Hidden Markov Models, and Semi Markov Models. A new loss function is proposed for the Semi Markov model to make it more robust to inconsistencies in data annotation near transition boundaries. The models are tested under two different experimental conditions to explore robustness to changes in biological conditions.

Demonstration of literal three-dimensional imaging.

In a recent paper a new technique was proposed for remote ranging and topographical mapping by using a system with a single-photon-counting detector and a low-power pulsed laser [Appl. Opt. 35, 441 (1996)]. We report on the results from the laboratory and the field demonstration of this literal three-dimensional imaging technique. Using a detector system developed at Los Alamos with a commercial pulsed laser and observing from a single remote vantage point, we demonstrate use of this technique in the literal mapping of three-dimensional topography and the probing of a complex scene. With a reasonably short exposure this system can resolve features with height variations as small as 5 cm.

Enhancing patient well-being: advocacy or negotiation?

The United Kingdom Central Council for Nursing, Midwifery and Health Visitors (UKCC) document, Exercising Accountability, states that the role of patient's advocate is an essential aspect of good professional nursing practice (1). The author examines the case for and against the nurse being the best person to act as advocate, and critically evaluates the criteria of advocacy. The problematic moral issues arising are discussed, and a case made for negotiation between the members of the multidisciplinary team and the patient/client (or a significant person to the patient) in order to promote the well-being of the patient and to minimise suffering. She concludes that the health care professional's (including the nurse's) role is to help people to assert control over the factors which affect their lives, that is empowerment, rather than advocacy.

Fluorescence characterization of Trp 21 in rat glutathione S-transferase 1-1: microconformational changes induced by S-hexyl glutathione.

The glutathione S-transferase (GST) isoenzyme A1-1 from rat contains a single tryptophan, Trp 21, which is expected to lie within alpha-helix 1 based on comparison with the X-ray crystal structures of the pi- and mu-class enzymes. Steady-state and multifrequency phase/modulation fluorescence studies have been performed in order to characterize the fluorescence parameters of this tryptophan and to document ligand-induced conformational changes in this region of the protein. Addition of S-hexyl glutathione to GST isoenzyme A1-1 causes an increase in the steady-state fluorescence intensity, whereas addition of the substrate glutathione has no effect. Frequency-domain excited-state lifetime measurements indicate that Trp 21 exhibits three exponential decays in substrate-free GST. In the presence of S-hexyl glutathione, the data are also best described by the sum of three exponential decays, but the recovered lifetime values change. For the substrate-free protein, the short lifetime component contributes 9-16% of the total intensity at four wavelengths spanning the emission. The fractional intensity of this lifetime component is decreased to less than 3% in the presence of S-hexyl glutathione. Steady-state quenching experiments indicate that Trp 21 is insensitive to quenching by iodide, but it is readily quenched by acrylamide. Acrylamide-quenching experiments at several emission wavelengths indicate that the long-wavelength components become quenched more easily in the presence of S-hexyl glutathione. Differential fluorescence polarization measurements also have been performed, and the data describe the sum of two anisotropy decay rates. The recovered rotational correlation times for this model are 26 ns and 0.81 ns, which can be attributed to global motion of the protein dimer, and fast local motion of the tryptophan side chain. These results demonstrate that regions of GST that are not in direct contact with bound substrates are mobile and undergo microconformational rearrangement when the "H-site" is occupied.

The catalytic mechanism of glutathione S-transferase (GST). Spectroscopic determination of the pKa of Tyr-9 in rat alpha 1-1 GST.

The rat alpha 1-1 glutathione S-transferase (GST) contains a single, non-essential tryptophan and only 8 tyrosines in each subunit. One of these tyrosines, Tyr-9, hydrogen bonds to the substrate glutathione and stabilizes the nucleophilic thiolate anion. Two mutant proteins that allow for the spectrocopic determination of the pKa of this catalytic residue have been constructed. The W21F mutant provides a fully active GST with no tryptophans, and the double mutant W21F/Y9F lacks both tryptophan and the active site tyrosine. The intrinsic fluorescence and absorbance properties of these mutants are dominated by tyrosine. Fluorescence emission, fluorescence excitation, and absorbance spectral changes of samples containing the W21F mutant at several pH values in the range 6.8-9.0 reveal a pH-dependent increase in the contribution of tyrosinate. No spectral changes are observed with the W21F/Y9F protein in this pH range. At pH 12.5, both proteins exhibit complete deprotonation of all tyrosines. The pKa of Tyr-9 determined from these spectroscopic changes is 8.3-8.5. The changes in absorbance at 250 and 295 nm correspond to titration of 0.95 +/- 0.29 tyrosines/subunit in the W21F protein between pH 6.9 and 9.3. Moreover, addition of the inhibitor S-hexylglutathione results in an apparent increase in the pKa of Tyr-9. Together, these results indicate that the catalytically active Tyr of GSTs has a pKa value that is 1.8-2.0 pKa units below tyrosine in solution. It is likely that this decrease in the pKa of Tyr-9 contributes to catalysis by altering the equilibrium position of the proton shared between Tyr-9 and GSH, and this active site residue may function as a general base catalyst in addition to a hydrogen bond donor.