Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery

OBJECTIVE: To assess whether hypertension is a risk factor for hysterectomy performed for benign diseases. METHODS: Self-report questionnaires were collected from 77% of 2301 Danish women aged 30, 40, 50, or 60 years selected at random in 1982 for a prevalence study. Information about cardiovascular diseases, hypertension, use of medicine, weight and dieting history, life-styles, psychologic factors, gynecologic history (including history of hysterectomy), and social background were recorded. Weight, height, and blood pressure were measured. In an incidence study, the cohort was followed during 1982-1990 via central registers to assess the incidence of hysterectomy. Logistic and Cox regressions were used to analyze data. RESULTS: In the prevalence study, history of hypertension partly explained the relation between hysterectomy and cardiovascular diseases. In the incidence study, history of hypertension and use of diuretics were significant risk factors for hysterectomy. After confounder control, use of diuretics was explained by weight-related variables, and hypertension was a risk factor for hysterectomy in educated women (adjusted relative risk [RR] 2.88, 95% confidence interval [CI] 1.07, 7.76) and in women with weight fluctuations (adjusted RR 3.31, 95% CI 1.35, 8.14). Weight cycling and lack of education remained significant risk factors for hysterectomy in women with and without hypertension, respectively. CONCLUSION: History of hypertension, weight cycling, and lack of education are closely related risk factors for premenopausal hysterectomy. These three risk factors contribute to women undergoing hysterectomy having an increased risk for cardiovascular diseases. We proposed that hypertension might be a plausible biological cause of menorrhagia and an indication for hysterectomy.     

Mathematical description of photobleaching in vivo describing the influence of tissue optics on measured fluorescence signals

The observed decrease in the fluorescence signal during photodynamic therapy (PDT) may contain dosimetric information as this photobleaching provides direct information on the photodynamic processes occurring in the tissue. A correct interpretation of the photobleaching signal, however, is crucial for its use in dosimetry. In this study the influence of scattering and absorption phenomena in tissue on the emitted fluorescence signal are described mathematically. Analytical solutions of the resulting expression show a difference from the single-decaying-exponential function generally used for describing photobleaching signals. The solutions are a function of the fluence rate at the inner side of tissue boundary psi(0*), the photobleaching dose constant beta, the incident irradiation power I0 and time. The accuracy of the results was investigated by comparison of the analytic solutions with numerical calculations using fluence rate profiles and escape functions obtained by Monte Carlo (MC) simulations. Good resemblance is observed when the value for psi(0*) calculated by the MC simulations is used in the analytical solutions. Experimental results in this study indicate the photobleaching dose constant of ALA-induced PpIX to be 33 +/- 3 J cm-2. Determination of beta for different types of photosensitizer and the development of an accurate method to determine psi(0*) can make monitoring of photobleaching during PDT valuable for dosimetry.     

Heterogeneous photobleaching in confocal microscopy caused by differences in refractive index and excitation mode

Changes in brain 5-HT turnover which have been associated with portal-systemic encephalopathy (PSE) in man were studied in rats with experimental PSE for intervals up to 15 weeks following the surgical construction of end-to-side portacaval shunts (PCS). These were compared to changes measured in portacaval transposed rats (PCT) which, show little hepatic dysfunction or cerebral abnormalities but, in common with the PCS rat, sustain total portal-systemic diversion. Thus any differences between these two groups were indicative of hepatic dysfunction and not the systemic diversion of portal blood. After 15 weeks, sustained increases were measured in brainstem and cerebral concentrations of the catabolite of 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), from 0.25+/-0.01 to 0.68+/-0.01*** microg g(-1) brain and from 0.18+/-0.01 to 0.31+/-0.03*** microg g(-1) brain respectively in PCS rats and were statistically greater to those measured in the brainstem and cerebrum of PCT and control rats. Sustained increases in cerebral concentrations alone of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, from 0.17+/-0.01 to 0.23+/-0.02 microg g(-1) brain were measured in PCS rats and were significantly*** greater than in PCT control rats after 15 weeks. Some early increases in 5-HTP were measured in PCS above control rats but these were not significant after 15 weeks. No sustained significant differences between the 3 groups were measured in 5-HT after 15 weeks. These data confirm previous evidence that the elevations in 5-HTP and 5-HIAA concentrations observed in experimental chronic liver failure and PSE are due to liver dysfunction and not portal-systemic diversion and may contribute additional information regarding the role of derangements in central 5-HT turnover as one of the causes of PSE. ***p<0.001, Newman-Keuls ANOVAR followed by Student's unpaired t-test for individual comparisons, (data shown are mean +/- SEM).     

Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics

We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved.     

Liquid titanium solute diffusion measured by pulsed ion-beam melting

The diffusivities of Sn, Mo, Zr, and Hf in liquid Ti were determined by pulsed ion-beam melting of thin liquid layers. Time-resolved optical reflectance and one-dimensional heat-flow simulations were employed to determine the melt duration. The broadening of nearly Gaussian solute concentration-depth profiles was determined ex situ using Rutherford backscattering spectrometry. Solute diffusivities in the range of 5 to 9×10⁻⁵ cm²/s were determined at temperatures in the range of 2200 to 2500 K. Calculations of buoyancy and Marangoni convection indicate that convective contamination is unlikely.     

DNA curvature in solution measured by fluorescence resonance energy transfer

The sequence-induced curvature of DNA fragments free in solution was characterized by measurements of the end-to-end distance using fluorescence resonance energy transfer (FRET). The 31 bp oligonucleotides were labeled at their 5' ends with fluorescein as the donor and rhodamine X as the acceptor. We compared a curved oligonucleotide with three phased A6 blocks and a control containing (AT)3 instead of the A6 blocks. The increased efficiency of energy transfer of the A6-containing DNA indicates the existence of a permanent sequence-induced curvature, the magnitude of which is in good agreement with estimates from theoretical curvature predictions. Energy transfer efficiency and correspondingly curvature increases with NaCl concentration.     

Characterization of cosmids and telomeres in cytogenetic preparations by 3D confocal fluorescence

OBJECTIVE: To assess a new laparoscopic technique of paravaginal repair, adapted from a classic laparotomy procedure, for genuine stress urinary incontinence. STUDY DESIGN: From January 1992 to July 1997, 28 patients in a consecutive, prospective clinical case study were subjected to laparoscopic paravaginal repair. No concomitant surgery was performed. A clinical diagnosis of genuine stress urinary incontinence was documented by cystometry following a positive cough stress test. When indicated, a multichannel urodynamics study was performed. RESULTS: In 16 patients (57%) of 28, the right pelvic side was affected, and in 43% fascia damage was identified and repaired bilaterally. The average operative time was 2 hours, 45 minutes; average blood loss was 1.2 g hemoglobin. No intraoperative, immediate postoperative, delayed postoperative or anesthesia-associated complications were observed. Patients were discharged from the surgical units in an average of 5 hours, 15 minutes. There was no postoperative hospital readmission. CONCLUSION: Laparoscopic paravaginal repair is simple and safe and has a 93% cure rate. It is an attractive alternative to laparotomy.     

Hyperglycemia delays terminal depolarization and enhances repolarization after peri-infarct spreading depression as measured by serial diffusion MR mapping

The absorption, distribution, and excretion of GT31-104, a novel bile acid sequestrant, was studied in rats and dogs after both acute and subchronic oral administration. The polyallylamine backbone of GT31-104 was labeled with tritium and one of the alkyl side chains was labeled with 14C. The mean blood and plasma concentration of [3H, 14C]GT31-104 in rats, in both treatment regimens, was negligible at all time points, with the highest amount observed being 0.69 microgram eq/g blood; in dogs the mean blood and plasma concentration of [3H, 14C]GT31-104 was below the limit of quantitation (< 0.001% total dose) at all time points. In both rats and dogs, the mean total urinary excretion of [3H, 14C]GT31-104 was approximately 0.06% of the total dose. The fecal excretion data indicates that both 3H- and 14C-derived radioactivity was excreted entirely in the feces. Mean total radioactivity excreted in the feces ranged from approximately 95 to 105% in the rats and 92 to 102% in the dogs. Across the different treatment regimens, in both species, tissue concentrations were negligible (< 0.01% total dose) and no differences in tissue profile were noted, indicating that there was no effect of pretreatment on [3H, 14C]GT31-104 absorption. GT31-104 was extracted with water, and the water-soluble portion contained radioactivity that would correlate to approximately 0.19% of the 3H dose and 0.41% of the 14C dose; this portion probably accounted for the negligible radioactivity observed systemically. Analysis of gastrointestinal (GI) tract tissues with contents indicated that GT31-104 is rapidly cleared from the GI tract. These data indicate that GT31-104 is not absorbed from the GI tract in rats and dogs.     

Solute diffusion in liquid nickel measured by pulsed ion beam melting

Measurements of liquid-phase diffusion coefficients for dilute tungsten and molybdenum in molten nickel were made using a pulsed ion-beam melting technique. A high-intensity beam of nitrogen ions is focused on the surface of a nickel substrate that was implanted with known concentration profiles of W and Mo. Melting of the surface to a depth of ∼1 μm allows broadening of the implant profiles while molten. Solute concentration-depth profiles were determined before and after melting using Rutherford backscattering spectrometry. Using a series of numerical simulations to estimate the melt history and diffusional broadening for mean liquid temperatures in the range 1755 to 2022 K, an effective diffusion coefficient is determined in each case by comparison to the measured depth profiles. This is found to be (2.4±0.2)×10⁻⁵ cm²/s for W and (1.6±0.4)×10⁻⁵ cm²/s for Mo, with an additional systematic uncertainty of ±0.5×10⁻⁵ due to instrumental and surface effects.     

Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope

Fluorescence photobleaching methods have been widely used to study diffusion processes in the plasma membrane of single living cells and other membrane systems. Here we describe the application of a new photobleaching technique, scanning microphotolysis. Employing a recently developed extension module to a commercial confocal microscope, an intensive laser beam was switched on and off during scanning according to a user definable image mask. Thereby the location, geometry, and number of photolysed spots could be chosen arbitrarily, their size ranging from tens of micrometers down to the diffraction limit. Therewith we bleached circular areas on the surface of single living 3T3 cells labeled with the fluorescent lipid analog NBD-HPC. Subsequently, the fluorescence recovery process was observed using the attenuated laser beam for excitation. This yielded image stacks representing snapshots of the spatial distribution of fluorescent molecules. From these we computed the radial distribution functions of the photobleached dye molecules. The variance of these distributions is linearly related to the diffusion constant, time, and the mobile fraction of the diffusing species. Furthermore, we compared directly the theoretically expected and measured distribution functions, and could thus determine the diffusion coefficient from each single image. The results of these two new evaluation methods (D = 0.3 +/- 0.1 micron 2/s) agreed well with the outcome of conventional fluorescence recovery measurements. We show that by scanning microphotolysis information on dynamical processes such as diffusion of lipids or proteins can be acquired at the superior spatial resolution of a confocal laser scanning microscope.     

Oligomeric state of human erythrocyte band 3 measured by fluorescence resonance energy homotransfer

The oligomeric state of the erythrocyte anion exchange protein, band 3, has been assayed by resonance energy homotransfer. Homotransfer between oligomeric subunits, labeled with eosin-5-maleimide at Lys430 in the transmembrane domain, has been demonstrated by steady-state and time-resolved fluorescence spectroscopy, and is readily observed by its depolarization of the eosin fluorescence. Polarized fluorescence measurements of HPLC-purified band 3 oligomers indicate that eosin homotransfer increases progressively with increasing species size. This shows that homotransfer also occurs between labeled band 3 dimers as well as within the dimers, making fluorescence anisotropy measurements sensitive to band 3 self-association. Treatment of ghost membranes with either Zn2+ or melittin, agents that cluster band 3, significantly decreases the anisotropy as a result of the increased homotransfer within the band 3 clusters. By comparison with the anisotropy of species of known oligomeric state, the anisotropy of erythrocyte ghost membranes at 37 degrees C is consistent with dimeric and/or tetrameric band 3, and does not require postulation of a fraction of large clusters. Proteolytic removal of the cytoplasmic domain of band 3, which significantly increases the rotational mobility of the transmembrane domain, does not affect its oligomeric state, as reported by eosin homotransfer. These results support a model in which interaction with the membrane skeleton restricts the mobility of band 3 without significantly altering its self-association state.     

Lifetime enhancement of ultrasmall fluorescent liquid polymeric film based optodes by diffusion-induced self-recovery after photobleaching

The major concern with optodes, especially miniaturized ones, has been their photobleaching limited lifetime. Liquid polymer [highly plasticized poly(vinyl chloride)] films are commonly used to prepare fluorescent optical fiber sensors. A major advantage is the ease of their fabrication. It is demonstrated here that, with proper choice of excitation power and illumination time, the sensor will completely recover itself from photobleaching after each measurement. This self-recovery is demonstrated on single-mode optical fibers with 80 microns diameter (3.1 microns active region) and on near-field scanning optical microscope pulled fiber tips with submicrometer diameter (250 nm active region). The single-mode optode can be used for 30,000 measurements with only a 5% signal loss at a signal/noise of > 66. This opens the way for prolonged ratiometric application of such optodes.     

Efficient Monte Carlo simulation of confocal microscopy in biological tissue

A variance-reduction technique is described that greatly improves the efficacy of Monte Carlo simulations of reflection-mode confocal microscopy in anisotropically scattering media. The efficiency gain is large enough that the performance of confocal microscopes probing as deep as 5 scattering lengths can be simulated with a desktop computer. We use the technique to simulate the response of a true confocal microscope probing biological tissue, a problem that has been impractical to undertake by using conventional Monte Carlo methods. Our most important finding is that operation of a confocal microscope in the true confocal mode enables much more effective rejection of undesired scattered light than operation in the partially coherent mode, but the maximum probing depths of microscopes operated in either mode are similar (2-3) scattering lengths) in practice because of sensitivity limitations.     

Anomalous diffusion of water in biological tissues

This article deals with the characterization of biological tissues and their pathological alterations. For this purpose, diffusion is measured by NMR in the fringe field of a large superconductor with a field gradient of 50 T/m, which is rather homogenous and stable. It is due to the unprecedented properties of the gradient that we are able not only to determine the usual diffusion coefficient, but also to observe the pronounced Non-Debye feature of the relaxation function due to cellular structure. The dynamics of the probability density follow a stretched exponential or Kohlrausch-Williams-Watts function. In the long time limit the Fourier transform of the probability density follows a long-tail Lévy function, whose asymptotic is related to the fractal dimension of the underlying cellular structure. Some of the properties of Lévy walk statistics are discussed and its potential importance in understanding certain biophysical phenomena like diffusion processes in biological tissues are pointed out. We present and discuss for the first time NMR data giving evidence for Lévy processes that capture the essential features of the observed power law (scaling) dynamics of water diffusion in fresh tissue specimens: carcinomas, fibrous mastopathies, adipose and liver tissues.     

Nucleotide binding to IAF-labelled Na+/K(+)-ATPase measured by steady state fluorescence quenching by TNP-ADP

Nucleotide binding to 5-iodoacetamidofluorescein (IAF) labelled Na+/K(+)-ATPase was measured by steady state fluorescence quenching of the fluorescein label via energy transfer to trinitrophenyl (TNP) labelled nucleotide. TNP-nucleotides are valuable probes of nucleotide binding to ATPases. Interpretation of these and other experiments in our laboratory using TNP-nucleotides with the Na+/K(+)-ATPase rely on having a good model for the interaction of TNP-nucleotide with the enzyme. Sets of fluorescence quenching curves obtained by titrating the enzyme with TNP-ADP in the presence of various concentrations of ADP could not be adequately modelled using a simple model with a single nucleotide binding site. Therefore, we compare various models which allow for additional TNP-nucleotide binding to the enzyme. In the two-site model, the additional binding is to a second specific site for which TNP-nucleotide and unlabelled nucleotide compete. In two other models, the additional binding (in one case saturable, and in the other case non-saturable) of TNP-nucleotide is not blocked by or affected by unlabelled nucleotide, and is, therefore, referred to as non-specific binding of the TNP-nucleotide. The goal of this work is to determine which of the distinctly different physical pictures associated with these models most accurately describes the interaction of TNP-nucleotide with the enzyme. We find that the interaction of TNP-ADP with IAF-labelled Na+/K(+)-ATPase is best described by a model in which there are two classes of binding: TNP-ADP and ADP compete for a specific binding site with dissociation binding constants of 0.13 microM for TNP-ADP and 2.0 microM for ADP; and non-saturable non-specific binding of TNP-ADP.     

Adsorption of complement, cytokines, and proteins by different dialysis membrane materials: evaluation by confocal laser scanning fluorescence microscopy

The membranes tested in the present study were cellulose triacetate (CTA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN). The adsorption by each membrane of albumin, IgG, C3a, interleukin-1beta (IL-1beta), interleukin-6 (IL-6), human neutrophil elastase (HNE), and tumor necrosis factor alpha (TNFalpha) was examined and semiquantitatively graded by confocal laser scanning fluorescence microscopy (CLSFM). After clinical use the dialyzers were treated with antibodies for these proteins and cytokines. Then the samples were incubated with fluorescein isothiocyanate-labeled anti-IgG antibody and observed by CLSFM. The changes in the blood levels of C3a and cytokines were also studied. In the CTA membrane, the adsorption of these substances, except for albumin and HNE, was less than in the synthetic membranes. The PAN membrane revealed the most abundant adsorption, especially for IL-1beta, IL-6, and TNFalpha. Although a marked elevation of C3a in the blood was observed in the CTA membrane, considerable adsorption was evident in the PMMA and the PAN membranes. Because the changes in the blood levels could be affected by membrane adsorption, both the blood levels and the adsorption of the biocompatibility parameters should be evaluated when membrane biocompatibility is discussed.     

Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies

This review demonstrates the large analytical and diagnostic potential of fluorescence correlation spectroscopy applied to freely diffusing biomolecules in solution. All applications discussed here in detail are based on changes in the diffusion characteristics of fluorescenctly labeled complementary strands of nucleic acids when they associate. However, the principle of the measurement can be extended to many different reactions with characteristic association times between several minutes up to several hours. If the reaction significantly affects the diffusion constants of at least one partner, single-color auto-correlation analysis is sufficient to extract kinetic parameters. If the observed binding process has only a moderate effect on diffusion coefficients, the detection selectivity and sensitivity can be improved by dual-color cross-correlation analysis. Finally, we show that diffusional analysis on the single-molecule level even opens up diagnostic applications, such as the detection of minute amounts of infectious agents like HIV-1 viruses in blood.