A spectroscopic study of the chromatic properties of GafChromicEBT3 films.

PURPOSE: This work provides an interpretation of the chromatic properties of GafChromicEBT3 films based on the chemical nature of the polydiacetylene (PDA) molecules formed upon interaction with ionizing radiation. The EBT3 films become optically less transparent with increasing radiation dose as a result of the radiation-induced polymerization of diacetylene monomers. In contrast to empirical quantification of the chromatic properties, less attention has been given to the underlying molecular mechanism that induces the strong decrease in transparency. METHODS: Unlaminated GafChromicEBT3 films were irradiated with a 6 MV photon beam to dose levels up to 20 Gy. The optical absorption properties of the films were investigated using visible (vis) spectroscopy. The presence of PDA molecules in the active layer of the EBT3 films was investigated using Raman spectroscopy, which probes the vibrational modes of the molecules in the layer. The vibrational modes assigned to PDA's were used in a theoretical vis-absorption model to fit our experimental vis-absorption spectra. From the fit parameters, one can assess the relative contribution of different PDA conformations and the length distribution of PDA's in the film. RESULTS: Vis-spectroscopy shows that the optical density increases with dose in the full region of the visible spectrum. The Raman spectrum is dominated by two vibrational modes, most notably by the ν(C≡C) and the ν(C=C) stretching modes of the PDA backbone. By fitting the vis-absorption model to experimental spectra, it is found that the active layer contains two distinct PDA conformations with different absorption properties and reaction kinetics. Furthermore, the mean PDA conjugation length is found to be 2-3 orders of magnitude smaller than the crystals PDA's are embedded in. CONCLUSIONS: Vis- and Raman spectroscopy provided more insight into the molecular nature of the radiochromic properties of EBT3 films through the identification of the excited states of PDA and the presence of two PDA conformations. The improved knowledge on the molecular composition of EBT3's active layer provides a framework for future fundamental modeling of the dose-response.

Quantitative trait loci influencing hepatic copper in rats.

Significant differences in liver copper content have been observed between rat inbred strains. To define loci controlling this trait, the offspring (n = 190) from an (LEW/OlaHsd x BC/CpbU) F(2)-intercross was genetically analyzed. From each F(2) animal, liver copper content was determined and genomic DNA was screened with polymorphic DNA markers. We found a major quantitative trait locus (QTL) for liver copper content in females on chromosome 2 and in males on chromosome 10. Both QTLs accounted for approximately 20% of the genetic variance. In addition, suggestive linkage for liver copper content was found on rat chromosomes 1, 8, 10, 12, 14, and 19. The regions on these chromosomes contain genes that are responsible for 9.0-15.5% of the genetic variance of liver copper content.

Genetic and correlation analysis of hepatic copper content in the rat.

Thirty recombinant inbred (RI) strains derived from the spontaneous hypertensive rat (SHR/OlaIpcv) and the Brown Norway (BN-Lx/Cub) progenitors were used to search for quantitative trait loci (QTLs) that are responsible for differences in liver copper between these two strains. The heritability of liver copper concentration (expressed as microg/g liver wet wt and microg/g liver dry wt) and liver copper store (microg/whole liver) was estimated to be 57, 57, and 46%, respectively. In a total genome scan of the RI strains, involving over 600 genetic markers, suggestive association was found between liver copper store (microg/whole liver) and the D16Wox9 marker on chromosome 16 (lod score = 2.8), and between liver copper concentration (microg/g dry wt) and the D10Cebrp1016s2 marker on chromosome 10 (lod score = 3.0). These putative QTLs are responsible for nearly 34 and 40% of the additive genetic variability for liver copper store and concentration, respectively.

Microelectrode study of voltage-dependent Ba2+ and Cs+ block of apical K+ channels in the skin of Rana temporaria.

The blockage of the apical K+ channels in frog species Rana temporaria by Ba2+ and Cs+ is strongly voltage-dependent. The interaction of both blockers with the K+ channels was studied by recording relations between the K+ currents (IK) and the transepithelial and intracellular potential. Mucosal Ba2+ and Cs+ depress IK, hyperpolarize the cell and induce pronounced nonlinearities in the current/voltage (I/V) relations. The nonlinearities are caused by the voltage-dependent interaction of Ba2+ and Cs+ with the binding site. Consequently, the apical membrane resistance not only depends on the blocker concentration but also on the apical membrane potential. Also the fractional resistance, fRa, and the voltage divider ratio, fVa, will change with blocker concentration and voltage. Owing to this non-ohmic behaviour, measurements of fVa in the presence of Ba2+ deviate markedly from the expected fRa values. The inhibitory effect of Ba2+ and Cs+ was analysed at different transepithelial and apical membrane voltages. The relation between the Michaelis-Menten constants and the voltage could be fitted with equations based on Eyring rate theory with the assumption of a single binding site. With this model we calculated the relative electrical position of the binding site for the blocker (delta), referred to the extracellular side of the channel. We obtained for Ba2+, delta = 0.34 +/- 0.05 and for Cs+, delta = 0.81 +/- 0.01. Comparison of the results from apical and transepithelial I/V relations demonstrates that the analysis of the transepithelial data provides overestimated values of the Hill coefficient and results in an underestimation of delta.

Forskolin activates gated Cl- channels in frog skin.

We investigated the effect of forskolin on Cl- movements across the isolated epithelium of frog skin. With Cl- on both sides, forskolin (50 mumol/l) increased the transepithelial conductance considerably and elicited significant Cl- secretion. Establishing transepithelial Cl- gradients markedly increased the Cl- currents (ICl). During forskolin treatment, the power density spectra (PDS) of the fluctuation in transepithelial current contained a Lorentzian component that depended on the presence of Cl- in the bathing solutions. Mucosal as well as serosal diphenylamine-2-carboxylic acid (DPC; 1 mmol/l) partially depressed ICl as well as the Lorentzian noise component. Microelectrode recordings from cells involved in transepithelial Na+ absorption showed that forskolin activates gated Cl- channels in a cellular pathway in parallel with the Na+-transporting granulosum cells of the frog skin. The activation of the Cl- -dependent currents and Lorentzian noise was rather variable, and adaptation of the animals to solutions that contained 40 or 60 mmol/l NaCl increased the sensitivity to forskolin. In skins of salt-adapted animals, oxytocin (0.1 U/ml) also slightly activated the Cl- pathway. On the other hand, oxytocin and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; 1 mmol/l) were without effect in control skins.

Current-voltage relations of Cs+-inhibited K+ currents through the apical membrane of frog skin.

The voltage-dependence of the inhibitory effect of mucosal Cs+ on the inward K+ current through the apical membrane of frog skin (Rana temporaria) was studied by recording transepithelial current-voltage relations. Experiments were performed with skins exposed to NaCl and KCl Ringer solutions on the serosal and mucosal side respectively (control skins), as well as with tissues incubated with K2SO4 Ringer solutions on both sides (depolarized skins). Studies of the dose-dependence of the Cs+ block showed that under both experimental conditions the apparent affinity of Cs+ increased as the transepithelial potential was clamped at higher mucosal positive voltages. Under control conditions, the concentration of Cs+ required to block 50% of the K+ current (KCs) recorded while the transepithelial voltage was clamped at zero mV was 16 mmol/l. KCs decreased exponentially with mucosal positive voltages. The dependence of KCs on the membrane potential was analyzed with Eyring rate theory in which Cs+ was assumed to block the K+ transport by binding to a site within the channel. The analysis showed that this site is located at a relative electrical distance delta = 0.32 of the voltage drop across the apical membrane, measured from the cytosolic side. The Hill coefficient obtained from this analysis was n = 3.1. Experiments with K+-depolarized tissues showed that only inward K+ currents recorded with positive transepithelial voltages were depressed by external Cs+. Also under these conditions KCs showed an exponential dependence on the transepithelial potential. The analysis of these data with the rate theory revealed delta = 0.09 and n = 1.7.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin stimulates the apical K+ conductance in frog skin.

We measured the effects of oxytocin (0.1 U/ml) on the current (Isc) recorded through skins of Rana temporaria incubated with an isotonic K+ solution on the apical side while the transepithelial potential was clamped to zero. Under these conditions, Isc is carried by inward K+ movements. Oxytocin markedly stimulated this inward K+ current. When the spontaneous fluctuations were analyzed we found that oxytocin increased the plateau (S(1)o) of the spontaneous Lorentzian component without modifying the corner frequency (f(1)c). Addition of Ba2+ to the mucosal solution blocked Isc both in the presence and absence of oxytocin. Moreover, with mucosal Ba2+ a characteristic blocker-induced Lorentzian component appeared in the power spectrum. Analysis of this blocker-induced noise showed that oxytocin increased the number of active K+ channels in the apical membrane, while the changes in single channel current were in agreement with the expected alterations of the electrochemical driving force.

Voltage-dependent Ba2+ block of K+ channels in apical membrane of frog skin.

The characteristics of the blockage of apical K+ channels in the frog skin (Rana temporaria) by Ba2+ were investigated with current-voltage measurements and current-fluctuation analysis. Inward K+ currents were recorded with a transepithelial K+ concentration gradient oriented from mucosa to serosa. When Ba2+ (5-10,000 microM) was added to the mucosal solution, the inward K+ current was progressively depressed. This blockage was strongly voltage dependent. The apparent macroscopic Michaelis-Menten constant decreased exponentially with increasing transepithelial voltage (Vt) (mucosa positive). According to Eyring rate theory, the binding site for the Ba2+ ion in the K+ channel was found to be located at a relative electrical distance of 0.72 of the apical membrane potential field, measured from the cytosolic side. From current-voltage (I-V) measurements and Michaelis-Menten kinetics, the Ba2+ dissociation constant was calculated: KBa = 57 microM (Vt = 0 mV). The voltage dependence of the on- and off-rate of the Ba2+-receptor interaction was obtained from the analysis of current fluctuations induced by Ba2+. An exponential relationship between the rates and Vt was obtained, as was found for KBa from the I-V measurements. KBa (at Vt = 0 mV) calculated from the noise experiments was 66 microM.