An approach for analysis of protein toxins based on thin films of lipid mixtures in an optical biosensor.

Biological membrane-like lipid films were deposited on the sensing surface in an optical biosensor instrument. The membranes were mixtures of biologically occurring lipids. Eight surfaces were prepared, some of which contained various glycolipids as minor components. One was supplemented with membrane proteins. The binding of six protein toxins (cholera toxin, cholera toxin B subunit, diphtheria toxin, ricin, ricin B subunit, staphylococcal enterotoxin B) and of bovine serum albumin at pH 7.4 and pH 5.2 to each of the sensor surfaces was studied. Each of the seven proteins gave a distinct binding pattern. The assay is rapid and simple, with no need for reagents. The lipid sensor surface is readily regenerated after binding and very stable. The concept with mixed lipid layers and assays at different pHs gives numerous combinations and could be applicable for developing a sensor for protein toxins.

From liposomes to supported, planar bilayer structures on hydrophilic and hydrophobic surfaces: an atomic force microscopy study.

The sequence of events involved in the transition from attached liposomes to bilayer patches on hydrophilic and hydrophobic solid supports were visualized in situ by Tapping Mode atomic force microscopy in liquid. In a smooth manner, the attached liposomes spread and flattened from the outer edges toward the center until the two membrane bilayers were stacked on top of each other. The top bilayer then either rolls or slides over the bottom bilayer, and the adjacent edges join to form a larger membrane patch. This is clearly visible from the apparent height of 6.0-7.5 nm of the single bilayer, measured in situ. The addition of calcium appeared to increase the rate of the processes preventing the visualization of the intermediate stages. The same intermediate steps appeared to be present on hydrophobic surfaces, although the attached liposomes seemed to be distorted and the resultant membrane edges were uneven. This work has provided visual and detailed information on liposome coalescence (fusion) onto solid supports and demonstrated how the atomic force microscope can be used to study the process.

Distribution and stability of membrane proteins in lipid membranes on solid supports.

Bacteriorhodopsin and the nicotinic acetylcholine receptor were biotinylated and reconstituted in lipidic membranes on silicon supports by fusion with proteoliposomes. The presence and distribution of the proteins were studied by binding with streptavidin. Radio-labelled streptavidin was employed for quantifying the amounts of protein remaining in the supported membranes after storage in buffer. The proteins within the membranes remained bound to the surface for weeks. The biological activity of reconstituted unlabelled receptor upon storage showed stability in membranes formed on silicon supports and a reduced stability when formed onto lipid monolayer covered supports. Atomic force microscopy studies on preparations in liquid showed bilayer structures but also attached, partly fused liposomes and membrane particles. In air, the surface was smoother and contained less of liposomes and more of stacked lipid layers. Preparations labelled with streptavidin conjugated to colloidal gold and imaged in air showed the proteins individually distributed, with no protein-rich patches or protein aggregates.

Helicobacter pylori interactions with human gastric mucin studied with a resonant mirror biosensor.

Helicobacter pylori, a human pathogen colonizing the gastrointestinal tract, is first interacting with mucus glycoproteins to penetrate the gastric mucus layer and then attach to specific epithelial cell targets. An optical biosensor technique based on the resonant mirror was used to study H. pylori interactions with human gastric mucin. The mucin preparation was immobilized on the sensor surface to set up the experimental model, close to the in vivo situation. Both sialylated and sulphated oligosaccharides interfered with the H. pylori binding to the immobilized mucin by reducing or abolishing the binding of the bacteria. Furthermore, the displacement of the bacteria from immobilized mucin by highly sulphated glycosaminoglycans was observed.

Characterization of Helicobacter pylori interactions with sialylglycoconjugates using a resonant mirror biosensor.

A new optical biosensor technique based on the resonant mirror was used to characterize Helicobacter pylori strains according to their sialic acid binding, demonstrating the suitability of using intact bacteria in real-time measurements and classifying strains based on their binding abilities. Results obtained from both competition and displacement assays using different glycoconjugates confirmed that several, but not all, H.pylori strains express sialic acid-binding adhesin(s), specific for alpha-2,3-sialyllactose. The adhesin, removable from the bacterial surface by water extraction, is not related to other reported H.pylori cell surface proteins with binding ability to sialylated compounds such as sialylglycoceramides.

The neuropeptide TRH has a minor effect on the enzymatic activity of acetylcholinesterase in vitro.

The neuropeptide thyrotropin-releasing hormone (TRH) elicits a variety of physiological effects of which some are due to cholinergic mechanisms. TRH modulates in vivo the effects of compounds affecting acetylcholinesterase (AChE). In the present study the in vitro effects of TRH on the activity of AChE were explored. TRH has no effect at physiologically relevant concentrations. At unphysiologically high concentrations (>5 mM) a slight inhibition was found. This was noticed also when the enzyme was exposed to the amide-free tripeptide analog p-Glu-His-Pro. We conclude that any cholinergic effect of TRH observed in vivo is unlikely to be due to a direct interaction of the peptide with AChE.

Planar lipid bilayers on solid supports from liposomes--factors of importance for kinetics and stability.

One method to create planar lipid bilayers on solid substrates involves the transfer of lipids from liposomes to the support. We have varied the composition of liposomes systematically using factorial experimental designs and analyzed the adsorption behaviour of lipids from these liposomes onto solid supports. The hydrophilic supports were either used plain or modified with a monolayer of a lipid mixture, exposing hydrophobic groups. The monolayer-covered supports were used to identify factors important for adhesion and stability. Lipid adsorption kinetics was primarily studied on plain silicon supports in an ellipsometric cell or on a silicon nitride surface in a resonant mirror system (IAsys), using the systematic approach. Saturated phospholipids were essential for the required stability. Mixtures of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylethanolamine and cholesterol in combination with proteins were investigated in further detail as regards kinetics. The propensity to form a supported planar bilayer could be manipulated by the presence of calcium ions.

Retained activities of some membrane proteins in stable lipid bilayers on a solid support.

Highly stable lipid bilayers, composed of biologically relevant lipids such as phosphatidylcholine, phosphatidylethanolamine and cholesterol, were formed on platinum surfaces. Bacteriorhodopsin isolated from purple membrane (PM) from Halobacterium halobium, cytochrome oxidase from bovine heart, acetylcholinesterase from bovine brain and the nicotinic acetylcholine receptor from Torpedo electric organ were also incorporated into these reconstituted membranes. The proteins retained their biological activities. Some of them were active several weeks after the reconstitution and after several cycles of assay, washing and storage.

Protection of guinea pigs against soman poisoning with ferrocene carbamate.

The protective effect of ferrocene carbamate pretreatment against soman poisoning was studied in guinea pigs. At doses corresponding to 1/20 x and 1/10 x LD50 of this carbamate a 20% and 45% decrease of the acetylcholinesterase in blood and brain, respectively, was obtained. In combination with additional pretreatment, diazepam, and therapy, HI-6 and atropine, the protective ratios (LD50 of soman in treated animals/LD50 of soman in untreated animals) were around 20 and 40, respectively. Animals pretreated with the high dose of the ferrocene carbamate that survived 10 x and 15 x LD50s of soman showed no remaining signs of poisoning after 24 h. Thus, the ferrocene carbamate afforded a better protection against soman than physostigmine. The explanation for this could be due to the properties of the ferrocene carbamate, not correlated to its cholinesterase inhibiting activity. This hypothesis is discussed.

Ketamine enantiomers and acetylcholinesterase.

(-)-ketamine was found to be more potent than (+)-ketamine in all the studied reactions with acetylcholinesterase. In most cases the difference was small but for two rate constants the (-)-form was unique in having effects. Thus, the stereoselectivity of ketamine in this system is the opposite of most other systems studied.

Comparison of kinetic parameters for acetylthiocholine, soman, ketamine and fasciculin towards acetylcholinesterase in liposomes and in solution.

Purified acetylcholinesterase from bovine brain was reconstituted by a detergent depletion technique into liposomes, prepared from soybean lecithin. The kinetics for the substrate acetylthiocholine and for three inhibitors with very different binding properties was studied. The results were compared with results from corresponding experiments with solubilized enzyme in detergent solution. The reconstituted enzyme showed a higher affinity for acetylthiocholine, ketamine and fasciculin. Parameters unaffected by the reconstitution were: turnover number for the substrate; the non-competitive component in ketamine inhibition and the kinetics for the active site-directed irreversible inhibitor soman.

Monoamine metabolite concentrations and cholinesterase activities in cerebrospinal fluid of progressive dementia patients: relation to clinical parameters.

Forty-five well clinically characterized patients with progressive dementia were investigated for lumbar cerebrospinal fluid (CSF) monoamine metabolites and cholinesterase activities. Monoamine concentrations were determined by reverse phase liquid chromatography with electrochemical detection and the cholinergic enzymes were measured photometrically. Firstly, all clinical and CSF parameters were studied in statistical cluster analyses to detect groups of variables which demonstrated a high correlation with respect to each other. The CSF transmitter markers were then used in multiple regression models to explain the variance of clinical variables as chosen from the cluster analyses. The degree of dementia, as assessed by global deterioration score (GDS) and activity in daily life (ADL) status, as well as the Alzheimer-related symptoms dyspraxia and dysphasia, were associated with low AChE activities in CSF. A presumed subgroup of dementia patients clinically characterized by asymmetry of neurological signs, increased unilateral tonus, stepwise progression, and high Hachinski score, showed low HVA concentrations in CSF. These data suggest a coupling of clinical/neurological parameters to different CSF transmitter profils and, thus, that CSF biochemical parameters are of use as antemortem markers in dementia conditions.

Increased monoamine metabolite concentrations and cholinesterase activities in cerebrospinal fluid of patients with acute stroke.

Twenty-one patients with acute brain infarction, 8 with transient ischemic attack and 20 controls were investigated for lumbar cerebrospinal fluid (CSF) monoamine metabolites and cholinesterases. The diseased patients were lumbar punctured on 2 occasions, mean Days 1 (0-3) and 5 (3-9) after debut of symptoms. Monoamine concentrations were determined by reverse phase liquid chromatography with electrochemical detection and the cholinergic enzymes were measured photometrically. Increased concentrations of 3-methoxytyramine (3-MT), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HI-AA) and increased activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in lumbar cerebrospinal fluid was found in patients with acute brain infarction when compared to control values, while the levels of 3-methoxy-4-hydroxyphenylglycol (MHPG) were not altered. No change of any neurotransmitter metabolite concentration/enzyme activity were found between Day 1 and Day 5 in the diseased patients. These data suggest an increased release of these neurotransmitter markers from necrotic brain areas into the cerebrospinal fluid and/or altered barriers between blood, brain and CSF and/or a dysfunction of the arachnoid villi to clear substances from the CSF. We therefore concluded that CSF neurotransmitters may be useful as specific brain markers in acute stroke.

Reactivation of nerve agent inhibited human acetylcholinesterases by HI-6 and obidoxime.

Acetylcholinesterase was purified from human caudate nucleus and skeletal muscle. The enzyme preparations were used to study aging and reactivation by HI-6 and obidoxime after inhibition by soman and its isomers. HI-6 was found to be the most potent reactivator. For both enzyme preparations a higher reactivatability and a higher rate of aging were observed after inhibition by C+-soman than after inhibition by C(-)-soman. Aging was retarded by propidium diiodide. Reactivation by the two oximes was also studied after inhibition by tabun, sarin and VX. Tissue homogenates were used for this part of the work. Our conclusion is that HI-6 is superior to obidoxime for human acetylcholinesterases inhibited by soman and sarin, while obidoxime is better towards tabun-inhibited enzyme.

Distribution and elimination of the stereoisomers of soman and their effect on brain acetylcholine.

The four stereoisomers of soman (O-(1,2,2-trimethylpropyl)-methyl-fluorophosphonate) have been analyzed in vivo in mouse blood and tissues after administration of doses corresponding to 0.75 X LD50 of the two diastereoisomeric pairs of soman (Sc- and Rc-soman). The disappearance of the four isomers has been studied in vitro in the presence of enzymes involved in the toxicity and detoxification of soman, e.g., acetyl- and pseudocholinesterase, aliesterase, and phosphorylphosphatase. The effect of Sc- and Rc-soman on brain acetylcholine was studied in the mouse. The analytical methods used are based on gas chromatography-mass spectrometry with deuterated internal standards. Rc-Rp- and ScRp-soman, the two isomers that preferentially react with acetylcholinesterase, were found in blood and liver. In liver the concentration of ScRp was higher than that of RcRp and could be followed for 18 hr. In blood only ScRp could be found. Its presence there could be followed during 18 hr. The levels were, however, lower than in liver. The results indicate that the liver might be a depot for soman and that ScRp might be responsible for the delayed intoxication noted after treatment with antidotes. Rc-soman was found to have a more pronounced effect on the acetylcholine synthesizing system than has Sc-soman, which might explain its higher in vivo toxicity.

Acetylcholinesterase inhibition by sulphur and selenium heterosubstituted isomers of N,N-diethylcarbamyl choline and carbaryl.

Nine sulphur and selenium heterosubstituted isomers of N,N-diethylcarbamylcholine and carbaryl have been prepared and their inhibiting activity towards electric eel acetylcholinesterase (E.C. 3.1.1.7) have been measured. The N,N-diethylcarbamylcholines acted only as reversible inhibitors, i.e. they could not carbamylate the enzyme. The reversible inhibition was of mixed type. Sulphur and selenium substitution had only marginal effects on Ki(0.2-0.3 mM) but reduced the value of K'i. The heterosubstituted carbaryl analogues were, with one exception, found to be irreversible inhibitors, about 100 times less potent than carbaryl.

Aging and reactivatability of plaice cholinesterase inhibited by soman and its stereoisomers.

A simple and rapid method to study aging of soman-inhibited cholinesterases was developed. The method was applied to study the aging characteristics of soman-inhibited cholinesterase from the muscles of the plaice (Pleuronectes platessa). The orientation of the soman molecule in the active site is decisive both for the rate of aging and the degree of reactivation of unaged enzyme, a conclusion reached by using soman stereoisomers. Fluoride ions were found to affect reactivatability as well as aging rate.

Kinetics for the inhibition of acetylcholinesterase from the electric eel by some organophosphates and carbamates.

The inhibition kinetics for some organophosphates (paroxon, diisopropylfluorophosphate, sarin, VX, soman and soman isomers) and carbamates (physostigmine, neostigmine, pyridostigmine and carbaryl) in the reaction with acetylcholinesterase from electric eel have been studied. Dissociation constants and rate constants for the irreversible step were determined. The great differences in inhibitory power of the organophosphates were almost entirely due to differences in affinity. A possible correlation between affinity and bonding rate is discussed.

Ferrocene-carbamate as prophylaxis against soman poisoning.

The effect of a carbamate derivative of ferrocene as a prophylactic agent toward soman poisoning was studied in mice. A sixfold decrease of the acute toxicity (24-hr LD50) of soman was obtained when the carbamate (5.5 mg/kg = 1/30 X LD50) was given intraperitoneally 30 min before soman. In this experiment atropine (20 mg/kg ip) was given 10 min before soman as support. The protection was lower when atropine or atropine plus toxogonin were given as therapy (1 min after soman). At these protective doses of the ferrocene -carbamate, a 30% inhibition of blood acetylcholinesterase activity was seen. Like physostigmine, the ferrocene -carbamate inhibited the brain acetylcholinesterase, suggesting that the compound entered the brain tissue.