Micromechanical measurement of AChBP binding for label-free drug discovery.

A potential binding assay based on binding-driven micromechanical motion is described. Acetylcholine binding protein (AChBP) was used to modify a microcantilever. The modified microcantilever was found to bend on application of the naturally occurring agonist (acetylcholine) or the antagonist (nicotine and d-tubocurarine). Control experiments show that microcantilevers modified without AChBP do not respond to acetylcholine, nicotine, and d-tubocurarine. K(d) values obtained for acetylcholine, nicotine, and d-tubocurarine are similar to those obtained from radio-ligand binding assays. These results suggest that the microcantilever system has potential for use in label free, drug screening applications.

Laser spray: electric field-assisted matrix-assisted laser desorption/ionization.

A new liquid chromatography/mass spectrometry interface, the laser spray, has been developed. Explosive vaporization and mist formation occur when an aqueous solution effusing out from the tip of the stainless-steel capillary is irradiated from the opposite side of the capillary by a 10.6 microm infrared laser. Weak ion signals could be detected when the plume was sampled through the ion sampling orifice. When a high voltage (3-4 kV) was applied to the stainless-steel capillary, strong ion signals appeared. The ion abundances were found to be orders of magnitude greater than those obtained by conventional electrospray ionization in the case of aqueous solutions. The present method is regarded as an electric-field assisted form of matrix-assisted laser desorption/ionization in which the liquid chromatographic solvent (water, etc.) acts as a liquid matrix. Laser spray ionization is expected to become a versatile method for biological mass spectrometry because this method is compatible with the natural solvent, water.

Persistence of allergy to anaesthetic drugs.

Intradermal testing and RIA testing for specific IgE antibodies to neuromuscular blocking drugs (NMBDs) were performed in patients referred to an Anaesthetic Allergy Clinic. Six patients were initially investigated four to 29 years after clinical anaphylaxis during anaesthesia and two of these patients and sixteen others were investigated by intradermal testing on two occasions at least four years apart. Seven patients had RIA tests for NMBD-specific IgE antibodies on two occasions at the time of skin testing. In all but two patients the evidence for drug-specific antibodies persisted 4-29 years after the reactions. In one patient all tests became negative and in another the skin test became negative but the positive RIA persisted. Evidence of antibodies to NMBDs persisted in 21 of 22 patients who had had anaphylactic reactions to these drugs during anaesthesia. In the absence of evidence of allergy diminishing with time in the majority of patients it would seem wise to avoid drugs responsible for reactions for the rest of the patient's life.

Detection of nicotinic receptor ligands with a light addressable potentiometric sensor.

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.

Gastrocnemius acetylcholinesterase activity in response to burn trauma in the mouse.

In this study we examined the effect of increasing size of body surface area (BSA, 20, 30 and 50 per cent) burn on distally located gastrocnemius acetylcholinesterase (AChE) activity. Burn injury was applied to predefined areas of the dorsal and ventral skin surfaces of mice. AChE activity at 3 weeks postburn was measured by differential extraction and velocity sedimentation on sucrose gradients. Analysis of variance was used for all statistical evaluation. Within the sham-treated controls most of the AChE activity consisted entirely (92 per cent) of the globular forms (S1 and S2). Asymmetric (S3 and S4) and non-extractable (H5) forms were 2 and 5 per cent, respectively. The total globular, asymmetric and non-extractable forms were not significantly different in gastrocnemius among the burn groups. However, individual forms (6.5S, 10S) of the soluble globular group (S1) were decreased (P less than 0.05) by half for all burn groups. Some molecular forms (4S, 10S) of residual globular group (S2) were decreased (P less than 0.05) in the 20 per cent and 50 per cent burn groups. In the asymmetric group (S3) the molecular forms (4S, 10S, 12S and 16S) were increased (P less than 0.001) for the 20 and 30 per cent groups. Within and between extraction groups interrelationships of the molecular forms were apparent. These data provide evidence that in response to burn trauma AChE activity of the gastrocnemius showed a decrease in soluble globular and an increase in asymmetric forms with multimeric dependence.

The medical and scientific evidence in alleged tubocurarine poisonings. A review of the so-called Dr. X case.

The problem of proving the presence or absence of a poison in a buried cadaver is the central theme of this presentation. Certain general questions are posed which may serve to guide those seeking to determine the cause of death in buried cadavers and allegedly due to a poison. Medicolegal and scientific evidence is presented from the court records of five deaths which were alleged homicides due to intravenous tubocurarine. As to the medical evidence: The prosecution claimed absence of adequate medical causes but full congruence with intravenous tubocurarine as the cause of death. The defense claimed and presented its evidence, including history, clinical picture, gross and microscopic pathological findings--for the deaths having occurred from competent natural causes in all but one case. In that one case the cause was undetermined. In two of the four cases evidence was presented for the mechanism of death and why they died at the time that they did. As to the forensic toxicological evidence: The prosecution claimed qualitative identification but with no particular quantitative detection or identification limits of tubocurarine in the remains based on results of combinations of HPLC followed by RIA and of some selected ion direct inlet mass spectrometry. The defense corroborated--along with a quantitative estimate--the presence of a substantial concentration of tubocurarine in the liver specimen of one case. However the chain of custody of this particular specimen was compromised for a period of several days between post-exhumation autopsy and submission to the prosecution toxicologists. With respect to all the other specimens examined by the defense, direct inlet mass spectrometry failed to show ions which are critical for establishing the identity of tubocurarine. The defense also presented results of experiments which showed that the tissues of the cases in question destroyed tubocurarine at such a rate that no reasonably conceivable administered amount could have survived the 10 years of burial of these cases. In each of the five cases exhumation and re-autopsy would have been found to be neither justified nor even indicated had an objective examination of the available record been made and supplemented by a similarly objective review of the literature and the simple stability experiments used by the defense. After an 8-month trial, the jury brought in a not guilty verdict on all counts after less than 2 h of deliberations.

Preparation of semisynthetic (+)-tubocurarine chloride.

Semisynthetic (+)-tubocurarine chloride (II) was prepared by monoquaternization of (+)-tubocurine. The method involved treating (+)-tubocurine with a 0.5 M equivalent of hydrochloric acid prior to quaternization with methyl iodide, followed by neutralization and iodide-chloride ion-exchange. Column chromatography and crystallization procedures were utilized for pure semisynthetic II preparation. The neuromuscular junction blocking activities of the semisynthetic and commercial II were determined by the in vivo cat hypoglossal nerve-tongue muscle preparation. No delectable differences among physical constants, spectral data, and neuromuscular junction blocking activities were noted between the commercial product and the semisynthetic II. This result substantiates the chemical and biological data for the well-accepted new formula for II. The unexplained M + n14 mass spectral peaks shown by the curare-type bases are characteristic of the molecular species rather than a result of contaminants.

Neuromuscular junction blocking agents: an initial screening bioassay.

A simple, rapid and sensitive in vivo bioassay for the initial screening of neuromuscular junction (NMJ) blocking agents has been accomplished. The i.v. retro-orbital plexus (IVROP) mode of injection was utilized, for the first time, in conjunction with the mouse inclined screen bioassay.

Quantitative analysis of d-tubocurarine chloride in curare by column liquid chromatography.

An improved quantitative analysis of d-tubocurarine chloride in the plant extract curare is presented. Gradient high-performance liquid chromatography on a hydrophobic stationary phase was found to be very suitable for the analysis of quaternary ammonium bases such as the complex mixture of curare alkaloids. Owing to the residual free silanol groups on the modified silica surface, the curare alkaloids are eluted from a reversed-phase column only if an electrolyte is added to the mobile phase. In order to optimize the separation, the effects of pH, the nature of the cation in the buffer and the concentration of the buffer of the retention of the alkaloids were investigated. Using a tetramethylammonium phosphate buffer at pH 4 in a gradient of water-methanol, undesirable retardation effects on the reversed-phase column could be suppressed sufficiently. As a result, an accurate method for the determination of d-tubocurarine chloride in curare was obtained. The coefficient of variation of this analysis is only 1.3%.

On the localisation of d-tubocurarine in rat liver lysosomes in vivo by electron microscopy and subcellular fractionation.

After i.v. injection in the rat, d-tubocurarine is taken up and concentrated by the liver. A method is developed for the visualisation of d-tubocurarine inside the liver cell by electron microscopy. Glutaraldehyde fixed liver blocks were immersed in an ammonium molybdate solution; d-tubocurarine was precipitated at sites of high concentration by molybdate, to form an insoluble d-tubocurarine-molybdate complex. This precipitate was found predominantly at the surface of lysosome-like particles, but also inside these organelles. In subcellular fractionation experiments, d-tubocurarine was found with a high relative specific "activity" in the lysosomal fraction, lending support to a lysosomal localisation of d-tubocurarine.