Automated mode-of-action detection by metabolic profiling.

Rapid classification and identification of the mode-of-action of bioactive compounds applied to plants can be achieved by a robust and easy-to-use metabolic-profiling method. This method uses artificial neural network analysis of one-dimensional proton NMR spectra of aqueous plant extracts to rapidly classify changes in the total metabolic profile caused by application of crop protection chemicals.

Method performance and validation for quantitative analysis by (1)h and (31)p NMR spectroscopy. Applications to analytical standards and agricultural chemicals.

Nuclear magnetic resonance (NMR) can be used to provide an independent and intrinsically reliable determination of chemical purity. Unlike chromatography, it is possible to employ a universal reference standard as an internal standard for the majority of chemical products assayed by quantitative NMR (QNMR). This is possible because the NMR response can be made the same for all chemical components, including the internal standard, by optimizing certain instrumental parameters. Experiments were performed to validate the quantitative NMR method described in this paper for the analysis of organic chemicals. Experimental precision, accuracy, specificity, linearity, limits of detection and quantitation, and ruggedness were systematically addressed, and system suitability criteria were established. The level of the major chemical ingredient can be determined with accuracy and precision significantly better than 1%, and impurities may be quantified at the 0.1% level or below. Thus, QNMR rivals chromatography in sensitivity, speed, precision, and accuracy, while avoiding the need for a reference standard for each analyte. Examples are given of (1)H and (31)P NMR used for quantitative analysis of agricultural chemicals, and a method for characterization of analytical standards is presented.

Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase.

Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes. Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex. The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide. Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes. Expression of recombinant proteins in tobacco plants conferred resistance in vivo. The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.

A deuterium nuclear magnetic resonance study of the condensing effect of cholesterol on egg phosphatidylcholine bilayer membranes. I. Perdeuterated fatty acid probes.

Deuterium nuclear magnetic resonance quadrupole splittings Dq and the related acyl chain segmental order parameters Smol have been determined for perdeuteriostearic acid intercalated as a molecular probe in the lamellar liquid crystalline phase of egg yolk phosphatidylcholine-cholesterol-water mixtures. The 2H NMR data show that cholesterol induces a high degree of order in the acyl chains of the phospholipid while maintaining the general profile of high order near the head group and relatively low order in the middle of the bilayer. This results in a pronounced thickening of the bilayer and concomitant decrease in the average molecular area of the fatty acyl chains. The giometrical changes in the bilayer due to cholesterol are discussed in terms of trans-gauche isomerization in the fatty acyl chains. The picture of the condensing effect revealed by 2H NMR is consistent with that from previous X-ray diffraction experiments. No evidence for a specific complex between cholesterol and phosphatidylcholine is apparent. The condensing effect of cholesterol is attributed to interaction between the fatty acyl chains and the rigid steroid nucleus, and to solvation of the 3beta-hydroxyl group at the aqueous interface.

Molecular motion and order in single-bilayer vesicles and multilamellar dispersions of egg lecithin and lecithin-cholesterol mixtures. A deuterium nuclear magnetic resonance study of specifically labeled lipids.

Deuterium (2H) nuclear magnetic resonance (NMR) quadrupole splittings and relaxation times have been measured for a variety of specifically deuterated lipids intercalated in lamellar-multibilayer dispersions and single-bilayer vesicles of egg lecithin and lecithin-cholesterol mixtures. The deduced order parameters and relaxation times vary with position of deuteration, acyl chain length, unsaturation, and temperature. The order parameters and spinlattice relaxation times T1 indicate rapid intramolecular motions of restricted amplitude in both the choline head group and hydrocarbon chains. The ordering profile for the acyl chains is similar to that predicted by statistical-mechanical theory. The order parameters yield estimates of the bilayer thickness and linear coefficient of expansion in close agreement with the x-ray determinations. A comparison of the deuterium and electron spin resonance spinprobe order parameters demonstrates the perturbation of the bilayer by the bulky nitroxide probe. The transverse relaxation time T2 for single-bilayer vesicles is quantitatively accounted for by a simple modification of classical relaxation theory which takes into account the modulation of the static quadrupole interaction by rapid local molecular motions and the modulation of the residual quadrupole interaction by the slower overall tumbling of the vesicle. It is unambiguously demonstrated that molecular motion and order in single-bilayer vesicles are very similar to those in lamellar multibilayers. Significant differences occur only for a few segments near the terminal methyl groups of the acyl chains, where the order parameters for vesicles are 10-30% smaller than those found for lamellae. The incorporation of cholesterol in lecithin bilayers is shown to increase the degree of orientational order in vesicles and lamellae, and to increase the hydrodynamic radius of vesicles. Thus, single-bilayer vesicles and multilamellar dispersions of phospholipids are equally useful models for biological membranes. They yield equivalent information about the internal organization and mobility of lipid bilayers, when the spectral manifestations of overall vesicle motion are correctly taken into account.

Molecular order in Acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids.

The first application of deuterium magentic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37 degrees C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.