The co-occurrence of ecdysones with bufadienolides and steroidal saponins in the genus Helleborus

A method is described for the detection of ecdysones in small quantities of plant material. When applied to 14 taxa of Helleborus L., ecdysterone and 5 β-hydroxyecdysterone were detected and quantified in 11; the other 3, which are morphologically distinct, gave a negative result. The co-occurrence of ecdysones with bufadienolides and steroidal saponins is discussed. The isolation of the ecdysones from the aerial parts of H. orientalis hybrids is described.     

Improved separation of high-molecular-weight proteins by preparative sodium dodecyl sulfate-gel electrophoresis: Application to apolipoprotein B

We have described modifications of apparatus and technique of preparative sodium dodecyl sulfate-gel electrophoresis which improve the resolution of very large proteins. These modifications permit the resolution of several components of apolipoprotein B from human blood plasma.     

Photosensitized oxidation of elastase. Selective modification of the tryptophyl residues

Spectral studies demonstrated that acidic pH values induce a two-step denaturation of porcine elastase, the first conformational transition occuring over the pH range 4.2–3.8, the second between pH 3.3 and 2.9. The proflavine-sensitized photooxidation of elastase in its native state, as well as in its denatured conformations, allowed us to isolate elastase derivatives selectively modified at given tryptophyl residues, hence to draw reliable conclusions about their degree of burial inside the protein matrix and their functional and conformational role. In particular, tryptophan-26 and -164 are located at the surface of the protein molecule, and their oxidation to N-formylkynurenine has no appreciable effect on the elastolytic activity and three-dimensional geometry of elastase. Tryptophan-83 is partially shielded from the aqueous environment; its modification affects only slightly the enzymic efficiency, while the tertiary structure of the protein perhaps increases its rigidity. Tryptophan-12 must be largely buried in internal regions, since its photooxidation is possible only after the native elastase structure has been extensively randomized; its indole ring appears to be of critical importance for the enzymic activity and the conformational stability of elastase. Finally, our data suggest that tryptophan-39, -132, and -232 are deeply buried; consequently, we failed to achieve the specific or preferential modification of these residues.     

Molecular systematics and Holarctic phylogeography of cestodes of the genus Anoplocephaloides Baer, 1923 s. s. (Cyclophyllidea,Anoplocephalidae) in lemmings (Lemmus,Synaptomys)

The present molecular systematic and phylogeographic analysis is based on sequences of cytochrome c oxidase subunit 1 (cox1) (mtDNA) and 28S ribosomal DNA and includes 59 isolates of cestodes of the genus Anoplocephaloides Baer, 1923 s. s. (Cyclophyllidea, Anoplocephalidae) from arvicoline rodents (lemmings and voles) in the Holarctic region. The emphasis is on Anoplocephaloides lemmi (Rausch 1952) parasitizing Lemmus trimucronatus and Lemmus sibiricus in the northern parts of North America and Arctic coast of Siberia, and Anoplocephaloides kontrimavichusi (Rausch 1976) parasitizing Synaptomys borealis in Alaska and British Columbia. The cox1 data, 28S data and their concatenated data all suggest that A. lemmi and A. kontrimavichusi are both non‐monophyletic, each consisting of two separate, well‐defined clades, that is independent species. As an example, the sister group of the clade 1 of A. lemmi, evidently representing the ‘type clade’ of this species, is the clade 1 of A. kontrimavichusi. For A. kontrimavichusi, it is not known which one is the type clade. There is also fairly strong evidence for the non‐monophyly of Anoplocephaloides dentata (Galli‐Valerio, 1905)‐like species, although an earlier phylogeny suggested that this multispecies assemblage may be monophyletic. The results suggest a deep phylogenetic codivergence of Lemmus spp. and A. lemmi, primarily separating the two largely allopatric host and parasite species at the Kolyma River in east Siberia. There are also two allopatric sublineages within each main clade/species of A. lemmi and Lemmus, but the present distributions of the sublineages within the eastern L. trimucronatus and clade 1 of A. lemmi are not concordant. This discrepancy may be most parsimoniously explained by an extensive westward distributional shift of the easternmost parasite subclade. The results further suggest that the clade 1 of A. kontrimavichusi has diverged through a host shift from the precursor of L. trimucronatus to S. borealis.     

Conformational stability, folding, and ligand-binding affinity of single-chain Fv immunoglobulin fragments expressed in Escherichia coli

A fluorescein-binding single-chain Fv (scFv) was chosen as a model for the study of the physicochemical parameters associated with synthetic IgG fragments. Three such scFv proteins were designed from the primary sequences of one anti-fluorescyl monoclonal antibody (Mab 4.4.20). These were constructed with varying-length interdomain peptide linkers of between 12 and 25 residues, expressed in Escherichia coli, and the protein folding, stability, and antigen-binding characteristics were assessed. Efficient renaturation could be accomplished in vitro to yield approximately 26 mg of active scFv/L of fermentation. Scatchard analysis for fluorescein ligand binding revealed that the scFv designs come within 2-fold of the Ka = 1.99 (+/- 0.18) x 10(9) observed for the parental 4.4.20 Fab and have identical stoichiometries (n approximately 0.99). Reversible solvent denaturation studies demonstrated that the unfolding/refolding equilibria for the scFv proteins can be fit to a simple two-state model and that two of the scFv designs were found to be slightly more stable than single IgG domains (VL and CL) when assessed in terms of the free energy of unfolding, delta Gon-u, or nearly identical to other multiple domain immunoglobulin proteins such as light chains and Fab's when relative transition midpoints, Cm, are compared. Linkers which conferred conformational flexibility beyond the minimally required length of 12 residues were found to have a stabilizing effect. By these criteria of ligand-binding function and protein stability, the scFv proteins were found to be bona fide minimal replicas of their parental IgG molecules.     

The resolution of 25α- and 25β-spirosta-3,5-dienes from sapogenin yielding plants

The separation of the artefacts, 25α- and 25β-spirosta-3,5-dienes, is described and their individual characters are recorded. Following acid hydrolysis of plant material the ratios of diene to sapogenin and of 25α- to 25β-diene varied with different species and morphological parts. The 25α-diene commonly predominated as did diosgenin over yamogenin. Diene production is reduced when, before undergoing acid hydrolysis, the plant material is incubated with water for increased sapogenin yield. The label from acetate-2-¹⁴C was found predominantly in the 25α-diene from such an incubation-acid hydrolysis experiment.     

Effect of the width of the herbicide strip on mite dynamics in apple orchards

Herbicide strips are used in apple orchards to promote tree growth and survival, to increase yield and to reduce the risk of rodent damage to tree bark. However, herbicide strips, particularly wider ones, may cause problems including soil erosion, reduced organic matter, leaching of nitrates into ground water and increased incidence of plant diseases and pests, including two-spotted spider mites, Tetranychus urticae Koch. In this 2 year study we monitored mite dynamics in apple trees and used sticky bands on tree trunks to determine rates of T. urticae immigration into Nova Spy apple trees in plots with wide (2 m) or narrow (0.5 m) herbicide strips. Use of wider herbicide strips promoted two risk factors that could trigger outbreaks of tetranychid mites. First, concentrations of leaf N in apple trees were higher and those of P and K were lower with the wide strips. Such changes in nutritional quality of leaves would increase the potential for more rapid population growth of T. urticae, and to a lesser extent, the European red mite, Panonychus ulmi (Koch). Second, there were higher rates of T. urticae immigration from the ground cover vegetation into the trees. In 2006, and for most of 2007, densities of T. urticae were higher with wide herbicide strips, whereas densities of P. ulmi were not enhanced. However, by late August to early September in 2007, densities of both tetranychids were lower with wide herbicide strips. This is because both risk factors were counterbalanced, and eventually negated, by the enhanced action of phytoseiid predators, mostly Typhlodromus pyri Scheuten. From July through September 2006, ratios of phytoseiids to tetranychids were always several-fold lower with wide herbicide strips but in 2007, from mid-July onwards, predator–prey ratios were usually several-fold higher with wide strips. However, this numerical response of phytoseiids to prey density can only occur where the pesticide program in orchards is not too harsh on phytoseiids. Hence the impact of width of herbicide strip is contingent on the composition and size of the phytoseiid complex and the impact of pesticides on predation.