Compartmentalization of the redox environment in PC-12 neuronal cells.

Neuronal redox phenomena are involved in numerous biochemical pathways and play a key role in many pathological events and clinical situations. The oxidation/reduction (redox) state present in biological compartments is a major target for possible pharmaceutical intervention and, consequently, the processes associated with its change have attracted increased attention in recent years. Here, we analyze the redox environment and its spatial compartmentalization in differentiated neuronal phenotype of PC-12 cells using a redox-sensitive protein (i.e., a mutant of the Yellow Fluorescent protein), employed ratiometrically. Redox maps of cells were generated with an elevate spatial resolution, and the spatial distributions of highly oxidized and highly reduced regions have been determined. A quantitative analysis of redox maps allows the disclosure of a peculiar spatial organization of the redox environment.

Cl- and F- anions regulate the architecture of protofibrils in fibrin gel.

Ischemic heart disease is the leading cause of serious morbidity and mortality in Western society. One of the therapeutic approaches is based on the use of thrombolitic drugs that promote clot lysis. Even if the mechanisms leading to clot lysis are not completely understood, it is widely accepted that they depend on the complex biochemical reactions that occur among fibrin fibers and fibrinolitic agents, and by their ready diffusion into the fibers. Here we investigate the effects of specific anions on the architecture of protofibrils within fibrin fibers in fibrin gels prepared in a para-physiological solution. The results obtained through small-angle X-ray scattering (SAXS) demonstrate that the characteristic axial and longitudinal repeat distances among protofibrils are strongly affected by the action of Cl(-) and F(-) anions.

Dose measurements and calculations of small radiation fields for 9-MV x rays.

Measurements of dose distribution for square fields with sizes ranging from 1 X 1 to 30 X 30 cm for a 9-MV x-ray beam from a Neptune 10 linear accelerator, manufactured by CGR, are reported. Special attention was paid to field sizes smaller than 4 X 4 cm, used in radiosurgery techniques. To express the dose-monitor units relationship, total, collimator, and phantom scatter correction factors were obtained by experimental measurements. A strong dependence of these factors on the smallest field sizes (less than 4 X 4 cm) was shown. Measurements of the maximum depth dose dmax, plotted as a function of field size, showed a maximum at about 5 X 5 cm, in good agreement with previous results. dmax was also measured for the smallest fields, demonstrating that the contaminating electron component of the x-ray beam was not responsible for the dmax shift. Analysis of the penumbra width of cross dose distributions, as a function of field sizes, allowed us to postulate that the dmax shift could be due to the phantom scattered photons, which in turn were generated by the collimator scattered photons. Newly derived tissue-maximum ratio and scatter-maximum ratio data were used for dose profile calculations of 2 X 2, 4 X 4, and 10 X 10 cm field sizes. The agreement between experimental and calculated data was found to be +/- 2% within the geometrical edges of the fields and +/- 6% outside of them. A dose profile from the isocenter of a 2 X 2 cm square field moving through a 360 degree rotation arc was obtained and compared with that from the center of a 125I shielded source, as measured by Ling. Advantages and problems relating to the use of x-ray beams from linear accelerators in radiosurgery are discussed.

p-type silicon detector for brachytherapy dosimetry.

The sensitivity of a cylindrical p-type silicon detector was studied by means of air and water measurements using different photon beams. A lead filter cap around the diode was used to minimize the dependence of the detector response as a function of the brachytherapy photon energy. The radial dose distribution of a high-activity 192Ir source in a brachytherapy phantom was measured by means of the shielded diode and the agreement of these data with theoretical evaluations confirms the method used to compensate diode response in the intermediate energy range. The diode sensitivity was constant over a wide range of dose rates of clinical interest; this allowed one to have a small detector calibrated in terms of absorbed dose in a medium. Theoretical evaluations showed that a single shielding filter around the p-type diode is sufficient to obtain accurate dosimetry for 192Ir, 137Cs, and 60Co brachytherapy sources.

A study of quality of bremsstrahlung spectra reconstructed from transmission measurements.

A numerical method for the reconstruction of bremsstrahlung spectra has been applied to the analysis of simulated data. The method is found to be reliable in reconstructing x-ray spectra of maximum energy up to 10 MeV. Measurements of aluminum transmission data carried out for five linear accelerators have also been analyzed by this method to determine the fractional energy fluence. The values of the Spencer-Attix water/air stopping power ratio, SS.A. w,air, reported as a function of the ratios TPR20 10, are in good agreement with computed results. Quality index values were correlated to mean incident energies of x-ray beams used in radiotherapy. The experimental setup has also provided information regarding the softening effect on the off-axis beam, which can be used in clinical computer dosimetry to correct conventional zero field size tissue maximum ratio.

Linkage effects in a model for cell survival after radiation.

A thermodynamic treatment for the effects of radiation on cell survival is proposed. The treatment is an extension of the linear-quadratic model (K.H. Chadwick and H.P. Leenhouts, Phys. Med. Biol. 13 (1973) 78) following the principles of linkage thermodynamics (E. Di Cera, S.J. Gill and J. Wyman, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 5077). Linkage effects between chemical binding to DNA and radiation action are considered, along with the synergism between different types of radiations. A simple mathematical condition is found for the additivity of radiation doses that result in an isoeffect. The resolvability of the model parameter is investigated by simulations and statistical analysis of the distributions obtained.

Point perturbation analysis of experimental data.

A new method of data analysis is proposed. The method is based on discrete perturbation of experimental data points, which is used to probe the metric of the parameter hyperspace. Perturbation-induced fluctuations in the residual values are analysed by discrete Fourier transform to yield the autocorrelation function and a relaxation length for each experimental point. This parameter provides a quantitative measure of correlation and hence nonrandomness of residuals. The method is applied to the analysis of measurements of the shear viscosity of a 2,6-lutidine/water mixture near the critical point, and to the oxygen and carbon monoxide binding reactions to human hemoglobin. Relaxation profiles are constructed for several experimental data sets. Departure from random behavior in the residuals is discussed in connection with the theoretical interpretations of the phenomenon under consideration.

Point perturbation analysis of experimental data: II. The statistics of relaxation length values of pseudo-random errors.

The statistics of relaxation lengths for pseudo-random deviates as determined by point perturbation analysis (E. Di Cera, F. Andreasi Bassi and G. Arcovito Biophys. Chem. 34 (1989)239),has been constructed by a Monte Carlo study. The values of the relaxation length, L, approximately follow a Gamma distribution. The results allow for a statistical estimation of relaxation profiles and provide a test for randonmess of residuals which is more accurate than other standard procedures.

Dynamic light scattering study of fine semiflexible fibrin networks.

Fine fibrin networks have been investigated using the dynamic light scattering (DLS) technique. At the shortest delay times, t, the dynamic structure factor s(q,t) is found to depend on time according to an exponential function and, at intermediate delay times (up to 1 ms), to a stretched exponential. At longer times (t > 1 ms), a progressively increasing deviation from the stretched exponential behaviour has been observed. These results are in agreement with the theoretical predictions of a recently forwarded model for semiflexible polymers in semidilute solutions [K. Kroy and E. Frey, Physical Review E 55 (1996) p. 3092.], despite the fact that fibrin networks are made up of crosslinked branched polymers. The model, moreover, allows the calculation from the initial decay rate gamma q(0) of the average diameter of the fibrin fibres, a. The value of a = 30 +/- 2 nm, at fibrinogen concentration c(f) = 1676 nM and ionic strength 0.5, fits well into the data reported in electron microscopy studies. A concentration dependence of the average diameter of the fibrin fibres has been observed which saturates at the highest concentrations. The diameter of fibrin fibres is an important component in determining the physical properties of the fibrin networks, since the radial growth of fibrin fibres is limited by twisting during protofibrils aggregation. Our results indicate the importance of taking into account intrinsic semiflexibility in studying the physical properties of 'real' polymers and emphasize the high sensitivity of the DLS technique to investigate biological polymers also at the lowest concentrations where the systems are very fragile.

Ion-recombination correction factor ksat for spherical ion chambers irradiated by continuous photon beams.

The large range of reference air kerma rates of brachytherapy sources involves the use of large-volume ionization chambers. When such ionization chambers are used the ion-recombination correction factor ksat has to be determined. In this paper three spherical ion chambers with volumes ranging from 30 to 10(4) cm3 have been irradiated by photons of a 192Ir source to determine the ksat factors. The ionization currents of the ion chambers as a function of the applied voltage and the air kerma rate have been analysed to determine the contribution of the initial and general ion recombination. The ksat values for large-volume ionization chambers obtained by considering the general ion recombination as predominant (Almond's approach) are in disagreement with the results obtained using methods that consider both initial and general ion-recombination contributions (Niatel's approach). Such disagreement can reach 0.7% when high currents are measured for a high-activity source calibration in terms of reference air kerma rate. In this study a new 'two-voltage' method, independent of the voltage ratio given by a dosimetry system, is proposed for practical dosimetry of continuous x- and gamma-radiation beams. In the case where the Almond approach is utilized, the voltage ratio V1/V2 should be less than 2 instead of Almond's limit of V1/V2 < 5.

Dosimetry of 169 Yb seed model X1267.

Unlike previous brachytherapy sources a number of published studies have been addressed to the dosimetry of 169 Yb seeds, manufactured in several prototypes, before widespread clinical use has been made. Discrepancies seen in the dosimetry obtained for ytterbium seed prototypes appear to be related to inconsistency and non-reproducibility in the vendor's calibration procedure to determine contained activity. Av. The comparison of 169 Yb seed dosimetries demonstrates a need for more accurate implementation of calibration procedures to determine the air kerma rate for the definitive 169 Yb seed design. This paper reports an experimental procedure to determine the reference air kerma rate, Kr (mu Gy h-1), defined as the kerma rate at 1 m along the source transverse axis in free space for the new 169 Yb seed, model X1267. A mean value of the ratio Kr/Av = 1.53 mu Gy h-1 mCi-1 was obtained from determining the Kr value of eleven seeds. Since this ratio is only 3% less than the air kerma rate constant for the 169 Yb point source, (gamma delta)k = 1.58 mu Gy h-1 m2 mCi-1, this means that the Av is closer to an apparent activity than a contained activity, Ac. A Monte Carlo simulation to determine the ratio between reference air kerma rate and the contained activity gave Kr/Ac = 1.33 mu Gy h-1 mCi-1. For the dose rate constant in water we obtained DKr (1, pi/2) = 1.20 +/- 0.05 cGy h-1 (mu Gy h-1)-1, using calibrated thermoluminescent dosimeters (TLDs) and DKr (1, pi/2) = 1.21 +/- 0.03 cGy h-1 (mu Gy h-1)-1 by Monte Carlo simulation. TLDs were used both to determine the radial dose distribution along the seed transverse axis and to calibrate GAFChromic films to obtain the two-dimensional dose distribution around the seed.

Intracavitary dosimetry of a high-activity remote loading device with oscillating source.

Dosimetric experiments have been carried out in order to obtain the dose distribution in water around a Fletcher applicator loaded by a Buchler system containing two 137Cs 148 GBq (4 Ci) sources and one 192Ir 740 GBq (20 Ci) source. The mechanical system which controls the movement of the 192Ir source and the resulting motion of the source are described. The dose distribution around the sources was measured photographically and by a PWT Normal 0.22 cm3 ionisation chamber. The absolute dose rate was measured along the lateral axes of the sources. The measurements of exposure in water near the sources were corrected for the effect due to the finite volume of the chamber. The "quantisation method" described by Cassell (1983) was utilised to calculate the variation of the dose rate along the lateral axes of the sources. The dose distribution around both 192Ir and 137Cs sources was found to be spherical for angles greater than 40 degrees from the longitudinal axes of the sources. A simple algorithm fitting the data for the moving 192Ir source is proposed. A program written in FORTRAN IV and run on a Univac 1100/80 computer has been used to plot dose distributions on anatomical data obtained from CT images. The uncertainties of the measurements and calculations have been examined and the greatest error has been found to be 5.5%. The clinical significance of the treatment method is discussed.

Growth kinetics and structure of fibrin gels.

The structure and kinetics of fibrin gels grown from fibrinogen solutions under quasiphysiological conditions, but in absence of Ca++, were investigated by means of elastic light scattering. By combining classical light scattering and low-angle elastic light scattering, an overall wave-vector range of about three decades was spanned, from q approximately 3 x 10(2) to q approximately 3 x 10(5) cm(-1). The scattered intensity distribution of the gels was measured in absolute units and fitted to a single function, which was able to reproduce accurately the data over the entire wave-vector range. From the fitting, it was possible to estimate the average diameter d of the fibrin fibers, the average crossover length xi of the gel, and establish the fractal nature of the gel structure, with a measure of its fractal dimension D(m). The measure of the intensity in absolute units also allowed the estimate of the density rho of the fibrin fibers and provided an independent measure of their size. The kinetics of formation of the gel was described in terms of a simple growth model: the scaffold of the network is formed very early in the course of the gelation process, at a "networking time," t(n), which is much smaller than the time required to form the final gel. At times t>t(n), the gel structure remains substantially unchanged and the successive growth consists only in a thickening of the gel fibers. Gels prepared under the same physical-chemical conditions, but at different fibrinogen concentrations, exhibited rather similar structures and kinetics, showing that the modalities of the gelation process are mainly governed by the solution conditions, and only secondarily by the fibrinogen concentration. For gels at fibrinogen concentration of approximately 0.24 mg/ml, the gel parameters were d approximately 130 nm, xi approximately 27 microm, D(m) approximately 1.3, and rho approximately 0.4 g/cm(3). Our d and rho values are in very good agreement with electron microscopy- and turbidity-derived literature data, respectively, while xi seems to be related to the mesh size of the initial scaffold formed at t(n), rather than to the mesh size of the final aged gel.

Quality assurance in planning and implementing radiation therapy.

Quality Assurance (QA) programs in radiotherapy are based on an interdisciplinary team work with the participation of physicists and physicians in treatment decision-making and in the determination of accuracy in dosimetry. In this short article, quality control (QC) procedures for computer-assisted treatment planning and implementation, are reported. Particular attention is paid to the technological developments which could make QC programs feasible at Radiotherapy centers.

Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy.

The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.