Enantiomer-specific selection of amino acids

Dietary intake of l-amino acids impacts on several physiological functions, including the control of gastrointestinal motility, pancreatic secretion, and appetite. However, the biological mechanisms regulating behavioral predilections for certain amino acid types remain poorly understood. We tested the hypothesis that, in mice, the potency with which a given glucogenic amino acid increases glucose utilization reflects its rewarding properties. We have found that: (1) during long-, but not short-, term preference tests, l-alanine and l-serine were preferred over their d-enantiomer counterparts, while no such effect was observed for l-threonine vs. d-threonine; (2) these behavioral patterns were closely associated with the ability of l-amino acids to promote increases in respiratory exchange ratios such that those, and only those, l-amino acids able to promote increases in respiratory exchange ratios were preferred over their d-isomers; (3) these behavioral preferences were independent of gustatory influences, since taste-deficient Trpm5 knockout mice displayed ingestive responses very similar to those of their wild-type counterparts. We conclude that the ability to promote increases in respiratory exchange ratios enhances the reward value of nutritionally relevant amino acids and suggest a mechanistic link between substrate utilization and amino acid preferences.     

Tolerance to deer herbivory and resistance to insect herbivores in the common evening primrose (Oenothera biennis)

The evolution of plant defence in response to herbivory will depend on the fitness effects of damage, availability of genetic variation and potential ecological and genetic constraints on defence. Here, we examine the potential for evolution of tolerance to deer herbivory in Oenothera biennis while simultaneously considering resistance to natural insect herbivores. We examined (i) the effects of deer damage on fitness, (ii) the presence of genetic variation in tolerance and resistance, (iii) selection on tolerance, (iv) genetic correlations with resistance that could constrain evolution of tolerance and (v) plant traits that might predict defence. In a field experiment, we simulated deer damage occurring early and late in the season, recorded arthropod abundances, flowering phenology and measured growth rate and lifetime reproduction. Our study showed that deer herbivory has a negative effect on fitness, with effects being more pronounced for late‐season damage. Selection acted to increase tolerance to deer damage, yet there was low and nonsignificant genetic variation in this trait. In contrast, there was substantial genetic variation in resistance to insect herbivores. Resistance was genetically uncorrelated with tolerance, whereas positive genetic correlations in resistance to insect herbivores suggest there exists diffuse selection on resistance traits. In addition, growth rate and flowering time did not predict variation in tolerance, but flowering phenology was genetically correlated with resistance. Our results suggest that deer damage has the potential to exert selection because browsing reduces plant fitness, but limited standing genetic variation in tolerance is expected to constrain adaptive evolution in O. biennis.     

Method for the characterization of size-exclusion chromatography media for preparative purification of DNA restriction fragments

A protocol has been developed to characterize the potential of size exclusion chromatography media for the separation of DNA fragments at a preparative scale. A standard DNA mixture composed of -DNA cut with the restriction enzymes, AluI and HaeIII, was chromatographed on a column packed with the gel of interest. Fractions were collected and examined by PAGE. A digitized image of the gel was analyzed with the aid of a computer software program to determine the DNA fractionation range and selectivity curve for each chromatography gel. Four gels were characterized using this protocol. The effect of the elution buffer ionic strength on the fractionation of DNA was also investigated.The US Government right to retain a non-exclusive royalty-freelicense in and to any copyright is acknowledged.     

Phytotoxicity and volatile constituents from leaves of Callicarpa japonica Thunb

The essential oil from the leaves of Callicarpa japonica was analyzed by GC-MS, and 84 compounds were identified. The main constituents of the essential oil were spathulenol (18.1%), germacrene B (13.0%), bicyclogermacrene (11.0%), globulol (3.3%), viridiflorol (2.6%), alpha-guaiene (2.3%), and gamma-elemene (2.0%). The essential oil constituents of C. japonica were significantly different from those found in our previous work on Callicarpa americana. The oil of C. japonica was selectively phytotoxic to bentgrass compared to lettuce seeds, with 80-100% growth reduction observed at 0.3 mg/ml.     

The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase

The inhibitory activity of 34 natural products of various structural classes on hydroxyphenylpyruvate dioxygenase (HPPD), the target site for triketone herbicides, and the mode of interaction of selected natural products were investigated. Recombinant HPPD from arabidopsis is sensitive to several classes of natural compounds including, in decreasing order of sensitivity, triketones, benzoquinones, naphthoquinones and anthraquinones. The triketone natural products acted as competitive tight-binding inhibitors, whereas the benzoquinones and naphthoquinones did not appear to bind tightly to HPPD. While these natural products may not have optimal structural features required for in vivo herbicidal activity, the differences in their kinetic behavior suggest that novel classes of HPPD inhibitors may be developed based on their structural backbones.     

Separation of large circular DNA by electrophoresis in agarose gels

The electrophoresis of circular DNA, ranging in size from 4.4 kilobase pairs (kbp) to 220 kbp, was studied in agarose gels. Bacterial artificial chromosome (BAC) DNA was used as a source of large supercoiled and open circular (relaxed) forms. The open circles above approximately 50 kbp were trapped at the sample wells of 1% agarose gels during electrophoresis at 3 V/cm. Field inversion gel electrophoresis (FIGE) was used to relieve the trapping of the open circles in the gels. Using FIGE (30 s forward pulse time), open circles with sizes of 115 and 220 kbp required reverse pulse times of 3 and 6 s, respectively, to free the circles from open-ended gel fibers. A minimum in the gel velocity of the open circles was measured at approximately 20 kbp. Open circles below approximately 20 kbp migrated slower than the supercoiled forms, and above 20 kbp the order was reversed. These results indicate that when the size of the open circles exceeded the average pore size of a gel and it was forced to span multiple pores, the open circles gained a mobility advantage. Decreasing the ionic strength of the electrophoresis buffer significantly decreased the mobility of the smaller circles and slightly increased the mobility of the larger circles.