An analysis of the variations in potency of grayanotoxin analogs in modifying frog sodium channels of differing subtypes.

Responses of tetrodotoxin-sensitive (TTX-s) and insensitive (TTX-i) Na(+) channels, in frog dorsal root ganglion (DRG) cells and frog heart Na(+) channels, to two grayanotoxin (GTX) analogs, GTX-I and alpha-dihydro-GTX-II, were examined using the patch clamp method. GTX-evoked modification occurred only when repetitive depolarizing pulses preceded a single test depolarization; modification, during the test pulse, was manifested by a decrease in peak Na(+) current accompanied by a sustained Na(+) current. GTX-evoked modification of whole-cell Na(+) currents was quantified by normalizing the conductance for sustained currents through GTX-modified Na(+) channels to that for the peak current through unmodified Na(+) channels. The dose-response relation for GTX-modified Na(+) channels was constructed by plotting the normalized slope conductance against GTX concentration. With respect to DRG TTX-i Na(+) channels, the EC(50) and maximal normalized slope conductance were estimated to be 31 microM and 0.23, respectively, for GTX-I, and 54 microM and 0.37, respectively, for alpha-dihydro-GTX-II. By contrast, TTX-s Na(+) channels in DRG cells and Na(+) channels in ventricular myocytes were found to have a much lower sensitivity to both GTX analogs. In single-channel recording on DRG cells and ventricular myocytes, Na(+) channels modified by the two GTX analogs (both at 100 microM), had similar relative conductances (range, 0.25-0.42) and open channel probabilities (range, 0.5-0.71). From these observations, we conclude that the differences in responsiveness of DRG TTX-i, and ventricular whole cell Na(+) currents to the GTX analogs studied are related to the number of Na(+) channels modified.

Characteristics of two slow inactivation mechanisms and their influence on the sodium channel activity of frog ventricular myocytes.

Inactivation of the fast Na+ current of heart muscle occurs in two kinetically distinct phases: a fast process operating on a millisecond time scale and a considerably slower process, the kinetic properties of which have not been explored fully. In this study, we analysed the slow inactivation process in isolated frog ventricular myocytes using the whole-cell variation of the patch-clamp method. Slow inactivation of the Na+ current followed a double-exponential time course, corresponding to slow and ultraslow components of Na+ channel inactivation. The individual time constants were 2-7 s (slow component) and 40-560 s (ultraslow component). Recovery from these slow inactivation processes also followed a double-exponential time course, but was characterized by significantly briefer time constants than those for the inactivation process. The relationship between transmembrane potential and steady-state slow or ultraslow inactivation was well described by the Boltzmann equation. The membrane potential at which half the Na+ channels are inactivated (V1/2) and the slope factor were estimated to be -48.1 and 13.6 mV, respectively, for the slow component alone. Under conditions in which the slow and ultraslow inactivation components were both present, these parameters were -53.1 and 8.7 mV respectively. When the fast and the two slow inactivation processes occurred concomitantly, the resultant steady-state inactivation curves were shifted to more negative potentials and the slope factor was decreased. Treatment with 1 mM Cd2+ externally did not affect the time course of slow inactivation, but produced a 3-7 mV depolarizing shift in its steady-state voltage dependency by virtue of cadmium's known effect on the cell surface potential. This study has thus identified two components of slow Na+ inactivation in heart muscle, operating on a time scale of seconds (slow inactivation) and minutes (ultraslow inactivation).

Differential effects of lipid-soluble toxins on sodium channels and L-type calcium channels in frog ventricular cells.

The effect of grayanotoxin I (GTX I), veratridine and aconitine with either an external or internal concentration of 100 microM on L-type calcium (Ca) channels was studied using the whole cell patch clamp and internal dialysis methods. The experimental conditions for the modification of sodium (Na) channels induced by the internal application of these toxins was determined by showing sustained inward currents with depolarizing repetitive pulses. These toxins failed to generate any change in Ca channels under the same experimental protocol as for Na channels. However, external application of these toxins caused a moderate block of the Ca channels without changing the kinetics.

A photographic technique for the restoration of damaged radiographs.

Occasionally during identification efforts, the forensic science team will encounter radiographs needed for comparisons that are damaged and seemingly useless. The simplified darkroom technique presented will facilitate the recovery of useful information from such compromised radiographs on film.

Photographic superimposition in dental identification. Is a picture worth a thousand words?

This paper reports a controversial case involving the dental identification of the skeletal remains of an Air Force pilot whose F-105D aircraft crashed in North Vietnam. Only a portion of the maxilla less the teeth was recovered and used in the dental comparison and positive identification. A statement was made to the brother of the victim that the dental comparison removed any doubt as to the identification. This was interpreted by the brother as the following: "Without the maxilla there was no positive ID." The brother was not familiar with dental terminology and anatomy and was disoriented when trying to interpret the odontological narrative. The principles of photographic superimposition were used for general information and orientation to clarify the odontological narrative after the internment of the skeletal remains.