Search

A3-APO, a de novo designed branched dimeric proline-rich antimicrobial peptide (PrAMP), is highly effective against a variety of in vivo bacterial infections. We undertook a selective examination of the mechanism for the Gram-negative Escherichia coli bacterial membrane interaction of the monomer (Chex-Arg20), dimer (A3-APO), and tetramer (A3-APO disulfide-linked dimer). All three synthetic peptides were effective at killing E. coli. However, the tetramer was 30-fold more membrane disruptive than the dimer while the monomer showed no membrane activity. Using flow cytometry and high-resolution fluorescent microscopy, it was observed that dimerization and tetramerization of the Chex-Arg20 monomer led to an alteration in the mechanism of action from non-lytic/membrane hyperpolarization to membrane disruption/depolarization. Our findings show that the membrane interaction and permeability of Chex-Arg20 was altered by multimerization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Introduction: Anthrax toxin comprises a protective antigen (PA) of MW 83 kDa, a lethal factor (LF) and an edema factor (EF). Upon binding to its receptor on cell surfaces, PA(83) is enzymatically cleaved to a 63 kDa product (PA(63)), followed by binding of LF or EF, receptor-mediated internalisation of these factors, and production of their toxic effects. The high-affinity binding of PA(83) to its receptor is essential for the intoxication process. To study the interaction between the PA and its receptor, and inhibition of the binding, an enzyme-linked immunosorbent assay (ELISA) was developed., Methods: One of the two known anthrax toxin receptors (capillary morphogenesis factor 2; CMG2) was adsorbed onto wells of a 96-well plate. Either PA(83) or PA(63) was then added to the receptor-coated wells, followed by sequential addition of anti-PA antibody, anti-species antibody-enzyme conjugate, and enzyme substrate at appropriate time intervals., Results: Best results were obtained by overnight incubation of CMG2 in PBS at 4 degrees C. CMG2 was used at 1 microg/ml because of the cost of the commercial product. The rate of change of absorbance was low, and was measured over 3 h to obtain accurate results. The assay results increased almost linearly with CMG2 concentration to 10 microg/ml. PA(83) was also used at 1 microg/ml, but the assay values reached a plateau at approx. 10 microg/ml. Binding was divalent cation-dependent, almost irreversible, and free CMG2 was a potent inhibitor of binding (I(50) in the nM range). Binding of PA(63) was similar to that of PA(83)., Discussion: The high-affinity binding and divalent cation dependence confirm the validity of the assay as a model for toxin-receptor binding in vivo and as a means of evaluating toxin-receptor binding and inhibitors of the binding. Attempts to use crude lysates of J774A.1 cells or von Willebrand factor as an alternative source of anthrax toxin receptor were not successful.

Enzyme-catalysed degradation of the nerve agents tabun, sarin, ethyl sarin and soman by three variants of an organophosphate-degrading enzyme was studied at low concentrations of nerve agent. The concentration of nerve agent at a given time was determined by its ability to inhibit the enzyme acetylcholinesterase. Experiments were conducted in 96-well microtitre plates. Values of the ratio of k(cat) (turnover number) to K(m) (Michaelis-Menten constant) were calculated. For tabun, this value (for the most effective OpdA variant) exceeded any value published to date for other enzymes. The value was within an order of magnitude for the highest value reported for sarin, but there appears to be no published value for ethyl sarin for comparison. The OpdA enzymes were relatively inefficient in degrading soman.

Many phospholipases of the A or C types show a very limited ability to hydrolyse phospholipids such as phosphatidylcholine or phosphatidylinositol which adopt a bilayer form when hydrated. The addition of unsaturated 1,2 or 1,3 diacylglycerols to such substances produces a marked stimulation of the attack. In contrast, the hydrolysis of phosphatidylethanolamine, which normally exists in the hexagonal II structure, is not stimulated by diacylglycerol. It is suggested that the liberation of diacylglycerol which often occurs during cell stimulation, may play a part in activating the associated cascade of phospholipid reactions.

A commercial controlled-pore glass medium for exclusion chromatography which is substituted with glycerol to eliminate non-specific adsorption (Glyceryl-CPG) was examined. Experiments on the elution of acetylcholinesterase and ganglioside micelles at varied ionic strength and pH showed that a slight anionic character still persisted on the glass matrix. At ionic strengths above 0.1, this had no effect on the elution of proteins. The material was found to have no tendency to adsorb proteins and other compounds, and was judged an excellent medium for exclusion chromatography. As a support for affinity chromatography, Glyceryl-CPG could be activated by periodate to form a virtually neutral matrix-ligand system.

Bovine serum albumin, acetylcholinesterase and two non-protein markers were chromatographed on columns of controlled-pore glasses in buffer solutions of varied ionic strengths. Adsorption of protein to the glass and ionic effects due to repulsion of the negatively-charged solutes from the glass matrix were observed. When glasses were treated with Carbowax 20M, the adsorption disappeared but ion exclusion effects persisted. The ionic behavior of Carbowax-treated glasses was similar to that observed by one of the authors when using agarose columns.

Simulated experimental data were generated from error-free data following the equation y = A - Be-kt where A, B, and k are constants and were analyzed by iterative nonlinear regression using one of two basic published computer programs. The effect of the simulated experimental error in y on the precision of the computed constants A, B, and k was evaluated. The errors were either independent of y (simple errors) or proportional to y (relative errors) and outliers were sometimes introduced. Other factors investigated were the number of data points per regression, the range of values of y, and the effect of weighting the data. The results show that the errors in the computed constants, and particularly the rate constant k, may be considerably magnified with respect to the errors in the experimental data. The quantitative relationships that are presented are useful aids in the design of biochemical experiments in which the above equation is applicable.

An improved method for assaying carbamylated acetylcholinesterase is described which has substantial benefits over current methods. Acetylcholinesterase was carbamylated with neostigmine and diluted extensively into buffer to allow decarbamylation to occur. At various times, phenyldichlorophosphate was added to the mixture of free and carbamylated enzyme, whereupon two very rapid, simultaneous reactions occurred: near total, and permanent, inactivation of free acetylcholinesterase by the organophosphate, and inactivation of phenyldichlorophosphate by hydrolysis. The carbamylated acetylcholinesterase was allowed to reactivate fully and then assayed for enzyme activity. The assay provided a measure of the amount of carbamylated enzyme present at the time of addition of phenyldichlorophosphate, thereby enabling the first-order rate constant for decarbamylation to be calculated. This new method of studying decarbamylation was applied to two systems of soluble acetylcholinesterase, where the half-life for decarbamylation was approximately 1/2 h or 4 min, respectively, and to membrane-bound acetylcholinesterase. The results agreed well with those determined by a conventional method; moreover, the standard error of the mean was lower for the new method. The advantages of the method using phenyldichlorophosphate over conventional methods are particularly evident when decarbamylation is rapid or when in vivo studies are being performed and it is not practical or desirable to run assays immediately on isolation of the tissue. The new method also has advantages over a published related technique using the organophosphate anticholinesterase soman.

The optimum conditions for measuring radioactivity in the filtration assay of muscarinic cholinoceptors with tritiated quinuclidinyl benzilate are to use Whatman GF/B filters and to add a simple toluene scintillant to them while they are still damp. Practically all the radioactive material is then slowly extracted into the scintillant and high counting efficiencies are obtained after 24 h. Dried filters, or dry filters in control experiments in the absence of receptor, adsorb much of the radioactivity with a 30% reduction in counting efficiency. Other scintillants were able to extract the radioactive material from dry filters, but were generally not preferable to toluene. The GF/B filters performed better than other glass microfiber and cellulose filters in terms of retention of receptor-bound ligand, rapid filtration rates, and low filter blanks. Toluene is unsuitable as a scintillant with GF/B filters for some other radioligands examined.

Intracellular phospholipases (A2 and C types) can hydrolyse bilayer-forming phospholipids much more rapidly when diacylglycerol is added. Unsaturated long-chain diacylglycerols are much more effective than short-chain saturated diacylglycerols or 1-oleoyl,2-acetylglycerol. Diacylglycerol does not change the electrokinetic properties of the phospholipid water interface, nor does it enhance enzymic digestion of monolayers. 31P-NMR of phosphatidylcholine indicates that diacylglycerol causes an isotropic component to develop in the spectrum of the bilayers which correlates approximately with the enhancement of phospholipase A2 attack. Addition of further diacylglycerol converts this transitional stage of unknown structural origin to the hexagonal II phase with a total loss of enzyme activity.