Spectro-chemical probes for protein conformation and function.
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1. The bursting pace-maker R-15 cell of Aplysia was injected with the Ca2+ sensitive dye arsenazo III. Changes in absorbance were measured with a differential spectrophotometer to monitor changes in free intracellular Ca2+, [Ca-a], during activity. 2. Dye absorbance increased during each pace-maker-induced burst of action potentials and decreased during the hyperpolarizing phase of the pace-maker cycle. 3. The increase in dye absorbance was, at least in part, dependent upon action potential discharge and was greater when action potential duration was prolonged by treatment with tetraethylammonium chloride. 4. Changes in dye absorbance occurred under voltage clamp conditions when the membrance was depolarized 5-15 mV from a holding potential near the resting potential and were larger with greater step depolarizations. 5. These changes were completely blocked by the addition of 3mM-La3+ to normal ASW. 6. The ratio of the absorbance change between two pairs of wave-lengths during the pace-maker cycle was compared with the ratio observed following injection of Ca2+, Mg2+ and H+ ions. The ratio for the pace-maker cycle was well matched by that for Ca2+ injection, but not by that for injection of Mg2+ or H+. 7. Intracellular Ca2+ injections which increased [Ca]1 to the same amount as occurred during the pace-maker cycle also produced an outward current of sufficient magnitude to account for the post-burst hyperpolarization. 8. Depolarization of the cell membrane by extrinsic current during the burst increased and prolonged the change in dye absorbance as well as the post-burst hyperpolarization. 9 It is suggested that Ca2+ enters during the pace-maker cycle, thereby increasing [Ca]i, and that this increase is sufficient to activate an outward current carried by K+ ions which causes or contributes to the post-burst hyperpolarization.
1. The metallochromic indicator dye, arsenazo III, was injected into somata of molluscan neurones from Archidoris monteryensis. Membrane current and dye absorbance change were simultaneously monitored under voltage clamp. 2. Absorbance measured at 660 nm increased during positive-going voltage steps large enough to activate membrane conductances. In situ idifference spectra were qualitatively similar to dye, dye-calcium difference spectra recorded in vitro. The absorbance change was abolished by either a thorough removal of external calcium or internal chelation of calcium by EGTA. It was concluded that the absorbance increase primarily reflected changing internal calcium concentration and that the calcium entered from the outside. 3. Dye absorbance increased in a nearly linear fashion during voltage clamp pulses of 100--300 msec duration. This is in qualitative agreement with electrical stuidies which demonstrated only fractional inactivation of calcium conductance during such periods. Plots of absorbance change vs. Vm peaked at +30 to +40 mV and fell off sharply until approximately +70 mV where the slope became less steep. A null or reversal of the absorbance change was generally observed aroung +110 mV. Evidence is presented that calcium influx was in some cases sufficient to cause sizeable changes in its equilibrium potential. 4. During multisecond voltage clamps the slope of the absorbance change showed a large decline. Where barium substituted for calcium as the influx species in identical clamps, the absorbance at 660 nm also increased but in a much more linear fashion. Except for a slight effect on the initial few pulses, the absorbance signal did not recover after a period of barium influx. These results suggest that part of the slope decline might result from processes related to calcium uptake and not to membrane conductance decrease. 5. Dye absorbance changes during normal and TEA action potentials were measured. Comparison of these changes with voltage clamp records indicated that calcium influx during a spike was capable of raising concentration by roughly 2 X 10(-7) M if the cell were considered to be a uniform sphere with no buffering capacity. Calcium influx during action potentials was increased dramatically by TEA, primarily as a result of a prolonged plateau phase. The existence and duration of the plateau was controlled mainly by potassium conductance systems, however. There was no evidence found for facilitation of the calcium conductance. 6. Following a moderate influx of calcium it required 20--60 sec, depending on the neurone, for the dye absorbance to return to base line (at 9 degrees C). The recovery time course showed a marked difference when examined at different wavelengths. For lambda = 660 nm there was an initial period in which the absorbance decreased rapidly, followed by a slower phase which generally carried the absorbance below the initial (prepulse) value...
Arsenazo III was used to measure changes in the free intracellular calcium concentration during spontaneous bursting pacemaker activity in the Aplysia R1K neuron. Intracellular calcium increased during the burst, and this increase was sufficient to cause the hyperpolarization that followed. The results suggest that the interval between bursts is determined by the rate of subsequent decline of free intracellular calcium.
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The recurrence of allergic inflammation, as examined by exudate accumulation, infiltration of polymorphonuclear leukocytes into the exudate, edema formation, vascular permeability and prostaglandin E2 levels in the exudate, was induced by injecting the antigen, azobenzene arsonate-conjugated acetyl bovine serum albumin, into the capsule of the proliferative granulation tissue which had been formed by the first-time antigenic challenge injection into an air pouch in the dorsum of the sensitized rat. The recurrence of the allergic inflammation 4 and 24 h after the antigenic challenge was inhibited dose-dependently by treatment with cyclooxygenase inhibitors, suggesting the possible participation of cyclooxygenase products, especially prostaglandin E2. The difference in the allergic inflammatory responses induced by the first-time antigenic challenge and the second-time antigenic challenge was discussed from the viewpoint of chemical mediators.
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Eczematous lesions in VM mice were invariably infected with Staphylococcus pyogenes aureus. A variety of preparations was used in treatment and although most were ineffective, one combining steroid and antibiotic therapy achieved remarkable results.
Inorganic arsenic-the more acutely toxic form of this metalloid element-contaminates drinking water supplies around the world. In the United States, the most serious arsenic contamination occurs in the West, Midwest, Southwest, and Northeast; as many as 20 million people-many getting their water from unregulated private wells-may be exposed to excess arsenic in their drinking water. In Bangladesh, it's estimated that as many as 40 million people may be suffering from arsenic poisoning; contaminated drinking water is also a problem in many other countries, including Argentina, China, Chile, Ghana, Hungary, India, and Mexico.
Gallium arsenide (GaAs), a chemical compound of gallium and arsenic, causes various toxic effects including pulmonary diseases in animals. Since the toxicity is not completely investigated, GaAs has been used in workplaces as the material of various semiconductor products. The present study was conducted to clarify the toxicity of GaAs particles in the alveolar macrophages of hamsters using magnetometry, enzyme release assays and morphological examinations. Alveolar macrophages obtained from hamsters by tracheobronchial lavage and adhered to the disks in the bottom of wells were exposed to ferrosoferric oxide and GaAs particles. Ferrosoferric oxide particles were magnetized externally and the remanent magnetic field was measured. Relaxation, a fast decline of the remanent magnetic fields radiated from the alveolar macrophages, was delayed and decay constants were decreased dose-dependently due to exposure to GaAs. Because the relaxation is thought to be associated with cytoskeleton, the exposure of GaAs may have impaired the motor function of them. Enzyme release assay and morphological findings indicated the damage to the macrophages. Thus the cytotoxicity causes cytostructural changes and cell death. According to DNA electrophoresis and the TUNEL method, necrotic changes occur more frequently than apoptotic changes.