Structure of myoglobin-ethyl isocyanide. Histidine as a swinging door for ligand entry

The structure of myoglobin(Fe II)-ethyl isocyanide has been solved at 1.68 A resolution by X-ray crystallography. The isocyano group of the ligand is distorted from the linear conformation observed in solution and in model compounds. Local changes in the protein conformation are also seen. The side-chain of Arg-CD3 moves out into the solvent, and the side-chain of His-E7 swings up and away from the ligand. Both of these side-chains show disorder indicative of dynamic behavior. These outward movements of His-E7 and Arg-CD3 side-chains clear a path from the solvent to the heme iron, suggesting a mechanism for ligand entry.     

A method for the rapid,automated filtration of protein hydrolyzates

A method for the automated filtration of protein hydrolyzates prior to amino acid analysis is described. Minor modification of a Technicon Sampler II enables it to function simultaneously as a sampler and a filtrate collector. Samples are drawn from cups in the sampler tray and are forced through a Teflon filter (pore size, 0.2 μm) in a Millipore Swinnex filter holder by a variable-speed Technicon proportioning pump. The filtrates are collected in cups in the sampler tray opposite those containing unfiltered hydrolyzates. Using this technique, 12 hydrolyzates can be filtered in 25 min compared to the approximately 2 h of technician time required for their manual filtration. Aliquots from each of 48 samples representing different proteins and hydrolysis conditions are filtered manually and by the automated technique. Analysis of variance of the resulting recoveries of each amino acid indicate little likelihood of effects due to filtration method.     

Behavioural responses of an Australian colubrid snake (<Emphasis Type="Italic">Dendrelaphis punctulatus</Emphasis>) to a novel toxic prey item (the Cane Toad <Emphasis Type="Italic">Rhinella marina</Emphasis>)

The invasion of a toxic prey type can differentially affect closely related predator species. In Australia, the invasive Cane Toad (Rhinella marina) kills native anurophagous predators that cannot tolerate the toad’s toxins; but predators that are physiologically resistant (i.e., belong to lineages that entered Australia recently from Asia, where toads of other species are common) have been more resilient. In the current study, we examine the case of an Asian-derived predator lineage that relies on behavioural not physiological adaptations to deal with toads. Despite their Asian origins, Common Tree Snakes (Dendrelaphis punctulatus) are highly sensitive to toad toxins; yet this snake has not declined in abundance due to toads. We exposed captive (field-collected) snakes to toads of different sizes and ontogenetic stages, to quantify feeding responses and outcomes. Tree Snakes were less likely to attack toads than to attack native frogs, and rarely retained their hold on large toads. Tree Snakes ingested frogs of a wide range of body sizes but only ingested very small toads (<?1 g vs. up to 30 g for frogs). Behavioural responses were virtually identical between Tree Snakes from invaded versus yet-to-be-invaded areas, suggesting that preadaptation (from Asia) rather than adaptation (within Australia) is the key to successful utilisation of this novel but potentially toxic prey resource. Nonetheless, a previously-documented shift in relative head sizes of Tree Snakes coincident with toad invasion suggests that the ancestral behavioural tactic may have been reinforced by a recent morphological shift that further reduces maximal prey size, and hence the risk of fatal poisoning.     

Molecular characterisation of gibberellin 20-oxidases. Structure-function studies on recombinant enzymes and chimaeric proteins

Gibberellin (GA) 20-oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C-20 methyl group of a diterpene carboxylic acid precursor (e.g. GA₁₂) to form an alcohol (in our case GA₁₅-open lactone) and an aldehyde (GA₂₄). The aldehyde is either oxidised to a tricarboxylic acid (GA₂₅) or, with loss of carbon-20 and lactonisation, to a C₁₉-GA (GA₉). This branching is interesting to study, because C₁₉-GA derivatives function as plant hormones in different tissues, whereas the C₂₀-GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20-oxidase from immature seeds of Cucurbita maxima L., which produces mainly C-20 carboxylic acids, with a 20-oxidase from Marah macrocarpus immature seeds, which forms predominantly CC₁₉-GAs. The cDNAs encoding these two very similar 20-oxidases were digested with restriction endonucleases Van 911. Bcl 1, and Bsa WI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1 -construct was obtained by exchanging nt 303–809 of the Cucurbita cDNA with the homologous DNA from the March 20-oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810–992, nt 993–end, nt 303–992, nt 810–end, and nt 311–end were exchanged, respectively. All constructs were cloned in a pUC18 vector and functionally expressed in E. coli NM522 cells. GA 20-oxidase activity was detectable in cell-lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA₁₂was found with pCM1 and pCM3, one-tenth of this conversion was observed with pCM5 and pCM6, and one-hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C₁₉-end product, GA₉, nor the C₂₀-end product, GA₂₅-was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6. GA₉accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C₁₉-GAs in this order. Although GA₂₄is the immediate precursor, much less end products were formed by using this substrate.     

Effects of aerial spray volume, coverage, and abamectin on Scirtothrips perseae (Thysanoptera: Thripidae).

Helicopter applications using abamectin in different spray volumes were made against Scirtothrips perseae Nakahara in Ventura County, CA. On small (2.2 m tall) trees, spray coverage on water-sensitive papers was 24-48% and 43-97% for 468 and 935 liter/ha volume treatments, respectively. On large (6.2-8.1 m tall) trees, spray coverage was lower and quite variable, from 1 to 28% and 10 to 70% for 468 and 935 liter/ha treatments, respectively. On small trees, 468, 701, and 935 liter/ha with a high abamectin rate (26 g [AI]/ha) were equally effective against larvae from 13 to 27 d after treatment (DAT). On medium (4.2 m tall) trees, 468 and 935 liter/ha with the high rate were equally effective from 23 to 113 DAT. On large (6.5-8.1 m tall) trees, 468 and 935 liter/ha with a low abamectin rate (13 g [AI]/ha) were ineffective in three tests. In a fourth large (6.8 m tall) tree test, 468 and 935 liter/ha with the high rate were effective at 3 and 37 DAT. In a fifth large (6.2 m tall) tree test, 468-1,403 liter/ha with the high and 935 liter/ha with the low rate were equally effective 2-22 DAT. After all effective treatments, thrips numbers were lower than in controls for 1-3 mo. However, stable and highest reductions in populations were sometimes delayed until 20-23 DAT even when coverage was high. The variability in spray coverage on the lower levels of large trees and the delayed effect may explain inconsistencies in the reporting of or in actual aerial application results.