Characterization of the Asialofetuin microtitre plate-binding assay for evaluating inhibitors of ricin lectin activity.

Optimum conditions for the binding of ricin to the glycoprotein asialofetuin immobilized on microtitre plates were investigated for the purpose of evaluating inhibitors of ricin B-chain lectin activity. Such inhibitors are of potential value in the use of immunotoxins based on ricin. This assay was first reported in 1986, but has not been characterized fully. Maximum binding of asialofetuin to the plate was observed at a concentration of ca. 4 microg ml(-1). Binding increased with time of incubation (1-24 h), pH (7.4-9.9) and temperature (2-37 degrees C). The pH effects were more marked at lower temperatures. Saturable binding of ricin to immobilized asialofetuin was observed, and at least 80% of maximum binding was observed by 10 min of incubation time. The binding was found to be very tight, such that an appreciable proportion of ricin added to the wells was bound at low concentrations, and binding was only partially reversible by addition of free galactose. Consequently, only estimates of the ricin-asialofetuin and ricin-galactose dissociation constants could be determined: 1.9 nM and 83 microM, respectively. Binding of ricin A- and B-chains was found to be 47% (at a 200-fold higher concentration) and 26% (at a twofold higher concentration) of that of the whole ricin molecule, respectively. The assay permits qualitative comparison of inhibitors of ricin B-chain lectin activity.

Blockade of cardiac nicotinic responses by anticholinesterases.

1. Tacrine (10 microM) and physostigmine (10 microM) completely inhibited the positive chronotropic and inotropic actions of acetylcholine (ACh) or nicotine in the atropinized guinea pig right atria. 2. Edrophonium (6 microM) and soman (0.1 microM) completely inhibited these nicotinic responses, as well as the associated increase in pyridine nucleotide fluorescence and vasodilation induced by ACh in the atropinized guinea pig perfused heart. 3. The 200-fold increase in noradrenaline release induced by ACh in the perfused heart was blocked by 10 microM tacrine and 6 microM edrophonium. 4. Tacrine (10 microM) significantly (16-32%) reduced the basal heart rate of both preparations. 5. Edrophonium (6 microM) induced a five- to sixfold increase in basal 3,4-dihydroxyphenyl-(ethylene) glycol (DOPEG) release. 6. The inhibition of nicotinic receptor activation in the atria by the anticholinesterases appears mainly non-competitive. IC50 values range from 0.1 to 10 microM in the perfused heart to 1 to 100 microM in atria (in either case tacrine about 2 microM). 7. The possibility that these compounds have a direct action at nicotinic receptors is discussed.

Biosensors for chemical and biological agents of defence interest.

This review discusses current developments in biosensors for toxic materials of defence interest with particular emphasis on the biological element of such devices. A wide variety of synthetic chemicals, toxins of plant or animal origin and biological materials--including various disease micro-organisms as well as some bacterial exotoxins--have either been used as warfare agents or are perceived as having the potential to be used for that purpose. Although an enormous effort is being put into developing biosensors, relatively few analytes, especially toxic materials, can yet be measured by commercially available devices. The factors which currently mitigate against the use of enzyme, natural receptor or antibody based biosensors for unattended continuous environmental monitoring of toxic materials include the inherent instability and availability of suitable proteins and--for receptors and antibodies--the essentially irreversible nature of the binding event, which necessitates a continuous supply of reagents for sequential measurements. Assays involving antibody or DNA based biosensors are time consuming when working in a hazardous environment. Nevertheless, biosensors are capable of being used for extremely sensitive and specific on-site measurements of contamination by specific toxic materials. Methods for improving the stability, extending the range and altering the binding characteristics of sensing molecules are discussed.

Simple high-performance liquid chromatographic method for assessing the deterioration of atropine-oxime mixtures employed as antidotes in the treatment of nerve agent poisoning.

A set of reversed-phase HPLC conditions for determining the degradation of atropine and the oxime (pralidoxime, obidoxime, or HI-6) in autoinjectors designed for use against nerve agent poisoning is described. The assay conditions for atropine do not require its prior separation from the large molar excess of oxime since both the atropine and tropic acid peaks elute well clear of the oxime and its degradation products and the phenolic preservatives. Further dilution of the sample and simple changes to the mobile phase then provide conditions for the oxime and its major degradation products to be quantitated.

A scanning fluorometer for imaging ischaemic areas in traumatized muscle.

The criteria used to evaluate the state of muscle surrounding a bullet wound are: a lack of contractility, a lack of capillary bleeding, and changes in colour and consistency. Muscle with all these properties may be assumed to be irreversibly damaged. However, the boundary between such tissue and tissue with potentially reversible levels of damage may not be clear cut. In general, oxygen lack due to blood vessel damage may be sufficient to cause irreversible damage or may result in a site of anaerobic infection. Such areas may occur at some distance from the missile track. Tissue responses to oxygen lack can be monitored by observing changes in intrinsic cellular fluorescence. The blue autofluorescence of intracellular pyridine nucleotides increases with anoxia, whereas the green autofluorescence of intracellular flavoprotein has been found to decrease with anoxia. We have developed a scanning fluorometer which rapidly provides an image of the distribution of anoxic areas in soft tissues. In experiments with anaesthetized rabbits, occlusion of the blood vessels to the gracilis muscle caused an increase in its pyridine nucleotide fluorescence and a decrease in its flavoprotein fluorescence. The changes were hastened by stimulation of the muscle to fatigue. Arterial infusion of colloidal carbon confirmed that adjacent muscles still had a blood flow. The apparatus has potential for the identification of hypoxic zones peripheral to the permanent wound cavity. Conversely, it may also help to indicate whether vascular repair after trauma of large blood vessels has led to improvement of the metabolic status of tissue being reperfused.

Scanning fluorometer for the rapid assessment of pyridine nucleotide and flavoprotein fluorescence changes in tissues in vivo.

This paper describes a scanning fluorometer which produces images in real time of the distribution of pyridine nucleotide or flavoprotein fluorescence at the surface of tissues in vivo. The basic difference between this device and others reported in the literature is that fluorescence changes at any selected point within the image can be quantified as they occur. We suggest that the apparatus has potential application in those areas of surgery where vascular replacement or repair is required and where it would be advantageous to have an immediate measure of the cellular response to a return of blood flow.

A cytoplasmic component of pyridine nucleotide fluorescence in rat diaphragm: evidence from comparisons with flavoprotein fluorescence.

Pyridine nucleotide (PN) and flavoprotein (Fp) fluorescence were monitored in the isolated intact rat diaphragm. A substantial increase in PN fluorescence occurred when N2 replaced O2 in glucose medium. This response was much reduced in pyruvate medium and/or by pretreatment with iodoacetic acid (IAA). The anaerobic levels of Fp fluorescence were less affected by substrate and IAA. Substitution of glucose by pyruvate did not alter the PN fluorescence of the resting aerobic tissue, but increased Fp fluorescence. After a tetanus with glucose present the PN of the anaerobic muscle, but not the Fp underwent a substantial transient oxidation. This oxidation was absent in pyruvate medium. It is concluded that a cytoplasmic component of the PN fluorescence is present in skeletal muscle. The levels of Fp fluorescence in the resting and contracting aerobic tissue supplied with pyruvate suggest that the resting tissue respiration was ADP limited. On this basis the level of PN fluorescence in the aerobic resting state was less than expected; the source of the PN fluorescence was both mitochondrial and cytoplasmic.

Fine-structure studies of experimental skeletal muscle trauma.

A study was made of damage to skeletal muscle caused by a high-velocity rifle bullet. Such damage extends peripherally from the permanent wound cavity and is focal in nature. A fine-structure investigation of this region suggests that some components of the muscle are more susceptible to the wounding process than others. The sarcoplasmic reticulum appeared most sensitive and areas as far as 3 cm from the wound cavity frequently showed gross vacuolization. Mitochrondrial damage was seen, but only in areas where there was also damage to myofibrils and the microvasculature. Focal capillary leakage up to 3 cm from the wound cavity was demonstrated in an earlier study by the use of a fluorescein-labelled dextran (Paddle and Freeman, 1979). This finding was confirmed. A possible correlate at the fine structural level was swelling of te capillary endothelial cells, which occurred in the absence of other signs of microvascular damage. Damage to the endothelial junctions was not observed, even in severely damaged tissue. Intravascular colloidal carbon escaped into the extravascular space only when the microvasculature was fractured. The relationship of these findings to macroscopic damage is discussed.