Use of OsiriX in developing a digital radiology teaching library.

Widespread adoption of digital imaging in clinical practice and for the image-based examinations of the Royal College of Radiologists has created a desire to provide a digital radiology teaching library in many hospital departments around the UK. This article describes our experience of using OsiriX software in developing digital radiology teaching libraries.

Calcium and the mechanism of light adaptation in vertebrate photoreceptors.

Vertebrate photoreceptors transduce the absorption of light into a hyperpolarizing change in membrane potential. The mechanism of transduction is becoming fairly well understood and has been shown to occur via a G protein-coupled decrease in cyclic GMP. Attention is now turning to the way the enzymatic machinery in the outer segment of the photoreceptor cell is modulated during light adaptation. Recent studies show that light adaptation cannot occur if changes in the concentration of cytoplasmic free calcium in the outer segment are prevented, suggesting that calcium functions as a second messenger in sensitivity regulation.

Dark adaptation in vertebrate photoreceptors.

Exposure of the eye to bright light bleaches a significant fraction of the photopigment in rods and cones and produces a prolonged decrease in the sensitivity of vision, which recovers slowly as the photopigment is regenerated. This sensitivity decrease is larger than would be expected merely from the decrease in the concentration of the pigment. Recent experiments have shown that the decrease in sensitivity is produced largely by an excitation of the phototransduction cascade by bleached pigment; even in darkness, it produces an equivalent background similar to that produced by real steady background illumination. Thus, excitation produced by a form of rhodopsin thought previously to be inactive has a profound effect on the physiology of the photoreceptor. This raises the possibility that forms of other G protein-coupled receptors thought to be inactive might also play an important role in signal transduction and disease.

Removal of phosphate from phosphohistidine in proteins.

Kinetic constants of KM = 0.8 microM, 3 microM and 1.6 microM, and kcat = 9 s-1, 7 s-1 or 9 s-1 were determined for histidine dephosphorylation by protein phosphatases 1, 2A and 2C respectively. IC50 values were determined for the inhibition of protein phosphatase 1 by inhibitor 1 (IC50 = 1 nM), inhibitor-2 (IC50 = 3 nM) and okadaic acid (IC50 = 30 nM) and for the inhibition of protein phosphatase 2A by okadaic acid (IC50 = 0.02 nM) and microcystin-LR (IC50 = 1 nM). Inhibitor-1 (Ki = 0.7 nM) and okadaic acid (Ki = 32 nM) are noncompetitive with protein phosphatase 1. Some of the IC50 values were low enough to violate the assumptions of the usual inhibition equations and a more general approach to the analysis of the data was used. On the basis of these kinetic parameters and the presence of phosphohistidine, the major cellular protein serine/threonine phosphatases are likely to act as protein histidine phosphatases in the cell.

Cat satellite DNA. Isolation using netropsin with CsCl gradients.

The absence of centromeric bands in the karyotype of Felis catus is confirmed. It is also confirmed that no satellite band is visible in CsCl density gradients. However, a satellite is observed both by recentrifuging the fraction of the DNA that bands at high density in CsCl and by using netropsin to enhance the resolution of a CsCl gradient containing total F. catus DNA. The satellite, about 0.5% of total DNA, was isolated by repeated centrifugation in CsCl alone and in CsCl with netropsin. Netropsin was removed and a pure satellite DNA obtained. The reassociation kinetics (C0t1/2 less than 10(-3) M . s) show that the satellite is of the simple sequence type and hence a candidate for centromeric heterochromatin. Its cytological localisation awaits in situ hybridisation experiments.

Selectively deuterated amino acid analogues. Synthesis, incorporation into proteins and NMR properties.

Selectively deuterated analogues of histidine, tyrosine, phenylalanine and tryptophan have been synthesized by chemical exchange. These analogues have been characterized by NMR spectrometry and used for growth of bacteria. Active lactose repressor protein has been isolated from cells grown on the deuterated amino acids, and denatured 1H and 2H NMR spectra have been determined for the protein.

Actions of Ca2+ on an early stage in phototransduction revealed by the dynamic fall in Ca2+ concentration during the bright flash response.

To study the actions of Ca2+ on "early" stages of the transduction cascade, changes in cytoplasmic calcium concentration (Ca2+i) were opposed by manipulating Ca2+ fluxes across the rod outer segment membrane immediately following a bright flash. If the outer segment was exposed to 0 Ca2+/0 Na+ solution for a brief period immediately after the flash, then the period of response saturation was prolonged in comparison with that in Ringer solution. But if the exposure to 0 Ca2+/0Na+ solution instead came before or was delayed until 1 s after the flash then it had little effect. The degree of response prolongation increased with the duration of the exposure to 0 Ca2+/0 Na+ solution, revealing a time constant of 0.49 +/- 0.03 s. By the time the response begins to recover from saturation, Ca2+i seems likely to have fallen to a similar level in each case. Therefore the prolongation of the response when Ca2+i was prevented from changing immediately after the flash seems likely to reflect the abolition of actions of the usual dynamic fall in Ca2+i on an early stage in the transduction cascade at a site which is available for only a brief period after the flash. One possibility is that the observed time constant corresponds to the phosphorylation of photoisomerized rhodopsin.

Na+-dependent Ca2+ extrusion governs response recovery in frog olfactory receptor cells.

To study the mechanism by which Ca2+, which enters during the odor response, is extruded during response recovery, recordings were made from isolated frog olfactory receptor cells using the suction pipette technique, while superfusing the olfactory cilia with solutions of modified ionic composition. When external Na+ was substituted with another cation, the response to odor was greatly prolonged. This prolongation of the response was similar irrespective of whether Na+ was replaced with Li+, which permeates the cyclic nucleotide-gated conductance, or choline, which does not. The prolonged current was greatly reduced by exposure to 300 microM niflumic acid, a blocker of the calcium-activated chloride channel, indicating that it is carried by this conductance, and abolished if Ca2+ was omitted from the external solution, demonstrating that Ca2+ influx is required for its generation. When the cilia were exposed to Na+-free solution after odor stimulation, the recovery of the response to a second stimulus from the adaptation induced by the first was greatly reduced. We conclude that a Na+-dependent Ca2+ extrusion mechanism is present in frog olfactory cilia and that it serves as the main mechanism that returns cytoplasmic Ca2+ concentration to basal levels after stimulation and mediates the normally rapid recovery of the odor response and the restoration of sensitivity after adaptation.

Bleached pigment activates transduction in salamander cones.

We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11-cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods.

Persistent activation of transducin by bleached rhodopsin in salamander rods.

The hydrolysis-resistant GTP analogue GTP-gamma-S was introduced into rods isolated from the retina of the salamander Ambystoma tigrinum to study the origin of the persistent excitation induced by intense bleaching illumination. Dialysis of a dark-adapted rod with a whole-cell patch pipette containing 2 mM GTP-gamma-S resulted in a gradual decrease in circulating current. If the rod was first bleached and its sensitivity allowed to stabilize for at least 30 min, then dialysis with GTP-gamma-S produced a much faster current decay. The circulating current could be restored by superfusion with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting that the decay in current originated from persistent excitation of the phosphodiesterase by transducin bound to GTP-gamma-S. We conclude that the persistent excitation which follows bleaching is likely to involve the GTP-binding protein transducin, which mediates the normal photoresponse. This observation suggests that a form of rhodopsin which persists long after bleaching can activate transducin much as does photoisomerized rhodopsin, although with considerably lower gain.

Prolongation of actions of Ca2+ early in phototransduction by 9-demethylretinal.

During adaptation Ca2+ acts on a step early in phototransduction, which is normally available for only a brief period after excitation. To investigate the identity of this step, we studied the effect of the light-induced decline in intracellular Ca2+ concentration on the response to a bright flash in normal rods, and in rods bleached and regenerated with 11-cis 9-demethylretinal, which forms a photopigment with a prolonged photoactivated lifetime. Changes in cytoplasmic Ca2+ were opposed by rapid superfusion of the outer segment with a 0Na+/0Ca2+ solution designed to minimize Ca2+ fluxes across the surface membrane. After regeneration of a bleached rod with 9-demethlyretinal, the response in Ringer's to a 440-nm bright flash was prolonged in comparison with the unbleached control, and the response remained in saturation for 10-15s. If the dynamic fall in Ca2+i induced by the flash was delayed by stepping the outer segment to 0Na+/0Ca2+ solution just before the flash and returning it to Ringer's shortly before recovery, then the response saturation was prolonged further, increasing linearly by 0.41 +/- 0.01 of the time spent in this solution. In contrast, even long exposures to 0Na+/0Ca2+ solution of rods containing native photopigment evoked only a modest response prolongation on the return to Ringer's. Furthermore, if the rod was preexposed to steady subsaturating light, thereby reducing the cytoplasmic calcium concentration, then the prolongation of the bright flash response evoked by 0Na+/0Ca2+ solution was reduced in a graded manner with increasing background intensity. These results indicate that altering the chromophore of rhodopsin prolongs the time course of the Ca2+-dependent step early in the transduction cascade so that it dominates response recovery, and suggest that it is associated with photopigment quenching by phosphorylation.

Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods.

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade.

Phosphohistidine goes undetected in conventional studies of protein phosphorylation, although it may account for 6% of total protein phosphorylation in eukaryotes. Procedures for studying protein N- kinases are described. Genes whose products are putative protein histidine kinases occur in a yeast and a plant. In rat liver plasma membranes, activation of the small G-protein, Ras, causes protein histidine phosphorylation. Cellular phosphatases dephosphorylate phosphohistidine. One eukaryotic protein histidine kinase has been purified, and specific proteins phosphorylated on histidine have been observed. There is a protein arginine kinase in mouse and protein lysine kinases in rat. Protein phosphohistidine may regulate the mitogen-activated protein kinase cascade.

Flecainide exerts paradoxical effects on sodium currents and atrial arrhythmia in murine RyR2-P2328S hearts.

AIMS: Cardiac ryanodine receptor mutations are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), and some, including RyR2-P2328S, also predispose to atrial fibrillation. Recent work associates reduced atrial Nav 1.5 currents in homozygous RyR2-P2328S (RyR2(S/S) ) mice with slowed conduction and increased arrhythmogenicity. Yet clinically, and in murine models, the Nav 1.5 blocker flecainide reduces ventricular arrhythmogenicity in CPVT. We aimed to determine whether, and how, flecainide influences atrial arrhythmogenicity in RyR2(S/S) mice and their wild-type (WT) littermates. METHODS: We explored effects of 1 µm flecainide on WT and RyR2(S/S) atria. Arrhythmic incidence, action potential (AP) conduction velocity (CV), atrial effective refractory period (AERP) and AP wavelength (λ = CV × AERP) were measured using multi-electrode array recordings in Langendorff-perfused hearts; Na(+) currents (INa ) were recorded using loose patch clamping of superfused atria. RESULTS: RyR2(S/S) showed more frequent atrial arrhythmias, slower CV, reduced INa and unchanged AERP compared to WT. Flecainide was anti-arrhythmic in RyR2(S/S) but pro-arrhythmic in WT. It increased INa in RyR2(S/S) atria, whereas it reduced INa as expected in WT. It increased AERP while sparing CV in RyR2(S/S) , but reduced CV while sparing AERP in WT. Thus, RyR2(S/S) hearts have low λ relative to WT; flecainide then increases λ in RyR2(S/S) but decreases λ in WT. CONCLUSIONS: Flecainide (1 µm) rescues the RyR2-P2328S atrial arrhythmogenic phenotype by restoring compromised INa and λ, changes recently attributed to increased sarcoplasmic reticular Ca(2+) release. This contrasts with the increased arrhythmic incidence and reduced INa and λ with flecainide in WT.

Protein histidine phosphatase activity in rat liver and spinach leaves.

Whole cell extracts from rat liver or spinach leaves contain divalent ion-independent protein histidine phosphatase activity due to phosphatases of the PP1/PP2A family. In the rat liver extract, almost all the activity was found in the PP1, PP2A1 and PP2A2 peaks. In the spinach leaf extract, four phosphorylase phosphatase activity peaks were resolved--three containing PP1 and one containing PP2A--and all showed histidine phosphatase activity. Thus, protein histidine phosphatase activity is expressed in the cytosolic forms of protein phosphatases of the PP1/PP2A family in mammalian and plant cells.

Phosphohistidine is found in basic nuclear proteins of Physarum polycephalum.

Nuclear extracts of the true slime mold, Physarum polycephalum, show protein histidine kinase activity towards exogenous histones [(1985) J. Biol. Chem. 260, 16106-16113]. Physarum microplasmodia were labeled with [32P]phosphate in vivo and two basic proteins containing alkali-stable phosphate were detected. The labeled proteins comigrated with Physarum histones H1 (approximately) and H2A and phosphoamino acid analysis showed that each protein contained [32P]-phosphohistidine. The H2A-like protein was also labeled in isolated nuclei incubated with [35S]thio-ATP. We conclude that some Physarum nuclear proteins contain phosphohistidine.

The effect of pH on the block by L-cis-diltiazem and amiloride of the cyclic GMP-activated conductance of salamander rods.

Block by L-cis-diltiazem and amiloride of the cyclic GMP-activated conductance was studied in inside-out patches excised from the salamander rod outer segment. When cytoplasmic pH was varied, the steady-state level of block by L-cis-diltiazem changed in the way that would be predicted if it were the protonated ammonium group that is responsible for effecting block. This is in contrast to the recent results of Haynes (J. gen. Physiol. 100, 783 (1992)) in catfish cones, where no such change was seen. Amiloride was found to block the conductance with a similar voltage dependence to that of L-cis-diltiazem. The dependence of amiloride block on cytoplasmic pH was found to be shifted relative to that of L-cis-diltiazem, consistent with the 1 pH-unit higher pKa value of amiloride and the idea that it is only the charged form of amiloride which can effect block. This suggests that the results seen with L-cis-diltiazem were indeed due to changes in the proportion of blocker in the protonated form, and not to effects of protons on the channel.

Voltage-dependent block by L-cis-diltiazem of the cyclic GMP-activated conductance of salamander rods.

Block by L-cis-diltiazem of the cyclic GMP-activated conductance was studied in excised inside-out patches from the salamander rod outer segment. When L-cis-diltiazem was applied from the cytoplasmic face of the patch, current suppression increased monotonically with membrane depolarization, the ratio of blocked to unblocked current varying e-fold in 50 mV. This suggests that L-cis-diltiazem interacts with a binding site located about half-way across the membrane field, and is unable to fully traverse the cyclic GMP-activated channel. The kinetics of block were accelerated by increasing L-cis-diltiazem concentration and by depolarization. These results can be fitted by a single barrier model in which the barrier peak is located about a third of the way across the membrane field from the cytoplasmic face. Application of L-cis-diltiazem from the extracellular face of the patch also resulted in an enhancement of block with membrane depolarization. Indirect evidence supports the notion that this block resulted from partition of the unchanged form of the blocker across the membrane, and its subsequent interaction with the cytoplasmic face of the conductance.