Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca²⁺-dependent manner. In order to study the Ca²⁺-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [¹⁹F]- and [¹H]-NMR and UV-difference spectroscopy. It was estimated from [¹⁹F]-NMR that in the absence of Ca²⁺, thek _1/2 value of TFP was 130 µM, while itsk _1/2 value decreased to 28 µM in the presence of Ca²⁺. The addition of KCl was not antagonistic to the Ca²⁺-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca²⁺-bound S100b was estimated to be 9×10² sec^?1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca²⁺ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg²⁺ was added to a S100b-TFP solution. Thus, we suggest that Ca²⁺ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.     

Clinical and biochemical studies in three patients with severe early infantile Cockayne syndrome

Summary We present clinical and biochemical data from three patients with severe Cockayne syndrome (CS) of very early onset. Unlike in classic CS, signs became evident in the first weeks of life and led to unusually early death. Fibroblasts from two of the patients showed a complete defect of the repair of UV-induced thymine dimer lesions. They were unable to remove thymine dimer lesions from their DNA, had a severe reduction of the RNA synthesis rates after UV irradiation, and showed no reactivation of an UV-inactivated indicator gene and no DNA recondensation after UV irradiation. DNA repair investigated in these two fibroblast cell strains resembled that of xeroderma pigmentosum cells of complementation group A. In contrast, fibroblasts from the third patient showed the same in vitro repair characteristics as classic CS cells.     

"Caged calcium" in Aplysia pacemaker neurons. Characterization of calcium-activated potassium and nonspecific cation currents

We have studied calcium-activated potassium current, IK(Ca), and calcium-activated nonspecific cation current, INS(Ca), in Aplysia bursting pacemaker neurons, using photolysis of a calcium chelator (nitr-5 or nitr-7) to release "caged calcium" intracellularly. A computer model of nitr photolysis, multiple buffer equilibration, and active calcium extrusion was developed to predict volume-average and front-surface calcium concentration transients. Changes in arsenazo III absorbance were used to measure calcium concentration changes caused by nitr photolysis in microcuvettes. Our model predicted the calcium increments caused by successive flashes, and their dependence on calcium loading, nitr concentration, and light intensity. Flashes also triggered the predicted calcium concentration jumps in neurons filled with nitr-arsenazo III mixtures. In physiological experiments, calcium-activated currents were recorded under voltage clamp in response to flashes of different intensity. Both IK(Ca) and INS(Ca) depended linearly without saturation upon calcium concentration jumps of 0.1-20 microM. Peak membrane currents in neurons exposed to repeated flashes first increased and then declined much like the arsenazo III absorbance changes in vitro, which also indicates a first-order calcium activation. Each flash-evoked current rose rapidly to a peak and decayed to half in 3-12 s. Our model mimicked this behavior when it included diffusion of calcium and nitr perpendicular to the surface of the neuron facing the flashlamp. Na/Ca exchange extruding about 1 pmol of calcium per square centimeter per second per micromolar free calcium appeared to speed the decline of calcium-activated membrane currents. Over a range of different membrane potentials, IK(Ca) and INS(Ca) decayed at similar rates, indicating similar calcium stoichiometries independent of voltage. IK(Ca), but not INS(Ca), relaxes exponentially to a different level when the voltage is suddenly changed. We have estimated voltage-dependent rate constants for a one-step first-order reaction scheme of the activation of IK(Ca) by calcium. After a depolarizing pulse, INS(Ca) decays at a rate that is well predicted by a model of diffusion of calcium away from the inner membrane surface after it has entered the cell, with active extrusion by surface pumps and uptake into organelles. IK(Ca) decays somewhat faster than INS(Ca) after a depolarization, because of its voltage-dependent relaxation combined with the decay of submembrane calcium. The interplay of these two currents accounts for the calcium-dependent outward-inward tail current sequence after a depolarization, and the corresponding afterpotentials after a burst     

The role of secreted acid hydrolases in the utilization of complex nutrients byTetrahymena

In an attempt to extend our knowledge of the biology of feeding of the ciliateTetrahymena thermophila, this organism was grown axenically on complex organic material. The nutrient substrate was based on autoclaved wheat grains and adjusted to either pH 5.5 or 7.5. In wild type cultures the cells grew and multiplied only under acidic conditions. In cultures of a mutant cell line blocked in the secretion of acid hydrolases the cells did not grow at either pH value. Thus released acid hydrolases may play a key role in the utilization of complex nutrients in combination with uptake of small organic molecules. Mechanisms in the feeding biology ofTetrahymena thermophila andParamecium tetraurelia are compared.     

Free radical-mediated platelet activation by hemoglobin released from red blood cells.

It is known that the rate of thrombus formation depends on interaction between platelets and erythrocytes, but the mechanism of this process has remained obscure. We here show that nanomolar levels of hemoglobin released from damaged red blood cells can induce platelet aggregation. The molecular mechanism is not receptor-based, but involves oxidation of oxyhemoglobin by platelet-derived hydrogen peroxide, with subsequent generation of a small unknown free radical species, detected by ESR spectroscopy. Methemoglobin and carbon monoxide-treated hemoglobin are unable to cause platelet activation or radical formation. The aggregation of platelets induced by hemoglobin is completely blocked by catalase or radical scavengers. These findings indicate a role for a novel extracellular free radical second messenger in the activation of platelets.     

Phenology of Festuca pallescens in relation to topography in north-western Patagonia

Abstract. The phenological changes in populations of Festuca pallescens (St. Yves) Parodi at different topographic positions and exposure along an altitudinal gradient (600 - 1100 m) were investigated during two growing seasons in northwestern Patagonia. Stepwise multiple regression analysis was used to describe the relationship between phenology and environment during the entire growing season. Analysis of variance was also performed at each sample date to detect significant environmental factors influencing phenology at different sites. The sum of maximum air temperatures was identified as the environmental variable best correlated with the seasonal variation of phenological events of Festuca pallescens over the period of two growing seasons, explaining 93.2 % of the total variance. Significant differences between sites were observed at each sample date. Main effects of altitude and topographic position and two-way interactions between altitude and topographic position, and topographic position and exposure were also detected as significant. Phenology was delayed at increased altitude. Differences in phenology between topographic sites at the same altitude were not detected during the entire growing season and were only observed in the reproductive phase. At this time, the phenology was significantly delayed at high topographic positions on the slopes as compared with low and mid positions. At high altitudes in the valley (950 m a. s. 1.), where steep slopes and humid conditions prevail, phenology was delayed on western exposures and low positions. The results adequately summarize and quantify the effect of spatial and temporal environmental variation on the phenological development of Festuca pallescens in northwestern Patagonia.