Generation of a zinc and rhodium containing metallomacrocycle by rearrangement of a six-coordinate precursor complex.

Two pentadentate N3,P2 ligands coordinate zinc(ii) by their N3 pocket. Four free phosphine donors allow the coordination of four AuCl moieties leading to a pentanuclear ZnAu4 complex. In contrast, the attempt to use the phosphines for chelating coordination of two Rh(CO)Cl units results in a well-organized rearrangement that ends up with the formation of a metallomacrocycle in high yields.

Torsional rigidity of positively and negatively supercoiled DNA.

Time-correlated single-photon counting of intercalated ethidium bromide was used to measure the torsion constants of positively supercoiled, relaxed, and negatively supercoiled pBR322 DNA, which range in superhelix density from +0.042 to -0.123. DNA behaves as coupled, nonlinear torsional pendulums under superhelical stress, and the anharmonic term in the Hamiltonian is approximately 15 percent for root-mean-square fluctuations in twist at room temperature. At the level of secondary structure, positively supercoiled DNA is significantly more flexible than negatively supercoiled DNA. These results exclude certain models that account for differential binding affinity of proteins to positively and negatively supercoiled DNA.

Where plants make oxygen: a structural model for the photosynthetic oxygen-evolving manganese cluster.

In the photosynthetic evolution of oxygen, water oxidation occurs at a catalytic site that includes four manganese atoms together with the essential cofactors, the calcium and chlorine ions. A structural model and a determination of the manganese oxidation states based on x-ray absorption spectroscopy are presented. The salient features, in both higher plants and cyanobacteria, are a pair of di-mu-oxo bridged manganese binuclear clusters linked by a mono-mu-oxo bridge, one proximal calcium atom, and one halide. In dark-adapted samples, manganese occurs in oxidation states (III) and (IV). Data from oriented membranes display distinct dichroism, precluding highly symmetrical structures for the manganese complex.

Chemical bonding information from photoelectron spectroscopy.

High-resolution measurements of photoelectrons produced by x-rays in compounds of iodine and europium have revealed chemical shifts in the core-level energies, from which chemical bonding information can be obtained. The observed shifts, 0.8 electron volt per unit change in oxidation number in iodine and 9.6 electron volts in europium, are discussed in terms of two theoretical models.

Modification of optical responses associated with the action potential of lobster giant axons.

The sources of optical retardation changes and light scattering changes occurring during the action potential propagation of lobster giant axons have been investigated. A technique has been developed for resolving the total transmitted-light intensity change into a retardation change component, dI-r, and a forward direction light scattering change, dI-s. Trypsin, pronase, neuraminidase and hyaluronidase all reduce the magnitude of dI-r without diminishing the action potential, probably by cleaving charged saccharides. Dithiothreitol has no effect. This suggests that glycoproteins and hyaluronic acid polymers at the surface of the axon are involved in the optical responses, either by being passively realigned or by contributing to compression and expansion forces as the membrane electric field changes. Large dI-s responses are generated by trypsin and pronase treatment. The modifying effects of these proteases may be due to modification of the membrane or to increases in the refractive index of the medium surrounding the axon, since similar large dI-s, responses are produced by increasing the refractive index with sucrose. Since large reductions in dIr can be produced without concurrent reductions in the action potential, a significant portion of the optical retardation responses cannot be attributable to structural changes that are causally related to membrane ionic permeability changes during the action potential.

The use of fluorescence correlations spectroscopy to probe chromatin in the cell nucleus.

All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small system, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well-characterized DNA preparations, including calif thymus DNa, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and it decreases as the cells leave the resting state and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.

Intracellular dye heterogeneity determined by fluorescence lifetimes.

The cellular localization of a fluorescent probe molecule depends on both the chemical structure of the dye and the cellular environment. To study the number and types of environments in an epithelial cell line, we have measured in Madin-Darby canine kidney (MDCK) cells the fluorescence lifetimes of three structurally distinct fluorescent dyes--rhodamine-B, 3,3'-dihexadecylindocarbocyanine-(C3) (diI), and Collarein-incorporated into these cells. The latter is a rhodamine-cardiolipin conjugate that we designed and synthesized for the property of exclusive localization in the plasma membrane. The former two dyes required at least two exponential components to fit their fluorescence decay curves, while the decay of Collarein was characterized by a single exponential. These data are consistent with fluorescence microscopic observations, in which diI and rhodamine-B exhibit heterogeneous spatial distributions, while Collarein appears to be located on the cell surface.

The g = 2 multiline EPR signal of the S2 state of the photosynthetic oxygen-evolving complex originates from a ground spin state.

The amplitude of the g = 2 Mn 'multiline' EPR signal of the S2 state of the photosynthetic oxygen-evolving complex varies inversely with temperature, indicating that this signal arises from a ground spin state. Electron spin echo experiments at temperatures of 4.2 K and 1.4 K show such Curie-law behavior of the g = 2 multiline EPR signal, as do continuous-wave EPR experiments performed at a non-saturating microwave power in the range from 15.0 K to 4.2 K.

Assignment of the g = 4.1 EPR signal to manganese in the S2 state of the photosynthetic oxygen-evolving complex: an X-ray absorption edge spectroscopy study.

X-ray absorption spectroscopy at the Mn K-edge has been utilized to study the origin of the g = 4.1 EPR signal associated with the Mn-containing photosynthetic O2-evolving complex. Formation of the g = 4.1 signal by illumination of Photosystem II preparations at 140 K is associated with a shift of the Mn edge inflection point to higher energy. This shift is similar to that observed upon formation of the S2 multiline EPR signal by 190 K illumination. The g = 4.1 signal is assigned to the Mn complex in the S2 state.

A polarized photobleaching study of chromatin reorientation in intact nuclei.

Polarized fluorescence recovery after photobleaching (pFRAP) was used to monitor the effects that condensation, i.e. compaction and aggregation, have on the (microseconds and ms) internal dynamics of chromatin in intact nuclei. When divalent cations were present with physiological (approximately 90 mM) monovalent salt the chromatin was found to exist in a compact and aggregated state which was characterized by rotational immobilization over timescales that range from 10 microseconds to 40 milliseconds. This immobilization is attributed to suppression of internal dynamics by intermolecular interactions. When the divalent cations were removed, the compact fibers no longer aggregated and were free to reorient with a characteristic decay time of about 1.2 milliseconds. It is shown that this millisecond relaxation could represent rigid rotation of topologically independent structural domains. Dilution of the monovalent salt induced a gradual change in the structural state of the chromatin that was manifest as a dramatic increase in internal flexibility. At the lowest salt concentration studied (11 mM-monovalent salt) the chromatin reorients in fewer than ten microseconds. These changes in flexibility are continuous with salt concentration, indicating that there are no well-defined endpoints to structural transitions and that the microsecond-millisecond internal dynamics of chromatin are a sensitive measure of structure. Measurements made on nuclei from cells that are either transcriptionally quiescent or active indicate that the dynamics mirrors biological activity.

Fiber transformations in multifidus muscle of young patients with idiopathic scoliosis.

STUDY DESIGN: In this study, the authors investigated the superficial multifidus muscle in patients with idiopathic scoliosis. During spinal fusion, biopsies were taken bilaterally at the apex of the curve, and at the upper and lower end vertebrae. OBJECTIVES: To analyze the muscular reactions in response to bracing in patients with idiopathic scoliosis. SUMMARY OF BACKGROUND DATA: The extent to which intervertebral mobility is restricted by an orthosis is still controversial. In addition, the effect of bracing on the erector spinae has not been investigated. METHODS: Of a total 30 patients, 11 had been treated with a corset for a year or more before surgery. Biopsies were investigated histochemically and the muscle fibers classified as Type I, IIA, IIB, or IIC (transitional fibers). The relative distribution of the fibers was calculated and their diameter was measured. RESULTS: In unbraced patients, a shift in the fiber distribution (from "slow" to "fast") was observed exclusively at the concave side of the apex. This shift was paralleled by an increased percentage of the intermediate Type IIC fiber (indicative of fiber transformation). In patients who always wore a corset, the relative amount of Type IIC fibers was increased, without preference for a specific location. CONCLUSIONS: Corset treatment elicits muscle fiber transformation processes at different levels along the scoliosis. This general reaction of the paraspinal muscles provides strong evidence against the existence of muscular disorders that are restricted to the area of the apex and are thus causing the scoliosis. As such, it must be assumed that the muscular changes in the apical region are secondary.

Use of patient ethnography to support quality improvement in benign prostatic hyperplasia.

BACKGROUND: Patient-centeredness is a primary aim of quality improvement (QI) but optimal strategies to achieve that goal remain elusive. Benign prostatic hyperplasia (BPH) is one of the commonest urologic diagnoses and significantly affects quality of life. Patient ethnography is an emerging qualitative method of observation and dynamic interviews to understand the context through which the patient experiences care. We implemented patient ethnography to support our QI infrastructure and improve patient-centeredness in BPH. PROBLEM: Little is known about how to measure whether processes of care are patient-centered. We did not know whether the care processes our patients experienced provided value from their perspective. GOALS: We sought to discover previously unrecognized components of care that patients perceived to be of low value. Our primary goal was to develop QI initiatives that targeted low-value themes identified in the ethnography. Our secondary goal was a rapid rollout of three targeted initiatives. STRATEGY: We used a 4-step patient ethnography: (1) created detailed process maps to define phases of care, (2) interviewed patients, (3) synthesized transcript data in focus groups using the Crawford Slip method, and (4) targeted undesirable components of care for QI. Semi-structured interviews with seven representative patients identified low-value themes. Focus groups, comprised of primary care physicians, case coordinators, nurses, and urologists, evaluated the interview transcripts and generated improvement opportunities prioritized based on feasibility, patient value, scalability, and innovation. We used affinity mapping and priority matrix techniques to prioritize QI opportunities. RESULTS: We identified five low-value themes from the patient interviews and developed corresponding QI opportunities. These included issues surrounding the referral and consultation process as well as postoperative care, especially home urinary catheter maintenance. Six months after completing the ethnography three of five targeted improvement opportunities had been implemented.