Responses in pigmentation and anti-oxidant expression in Arctic Daphnia along gradients of DOC and UV exposure

Responses in carapace melanization and expression of the majoranti-oxidant catalase (CAT) and glutathione transferase (GST)in Arctic Daphnia were assessed in enclosures along a gradientof dissolved organic carbon (DOC). This gradient was createdby adding freeze-dried humic matter to 2 m³ UV-transparent enclosures,yielding final nominal concentrations of 1, 2.5, 5 and 10 mgC l^-1. The UV attenuation was strongly affected by additionsof DOC, and attenuation coefficients at 320 nm increased from3.0 in the control to approximately 3.5 and 11.0 m^-1 in the1 and 10 mg DOC treatments respectively. Most Daphnia showedpronounced carapace melanization, and the absorbance of short-waveradiation through the carapace was strongly related to the degreeof melanization. Nevertheless, the different UV climate in theenclosures did not cause any short-term adaptation in Daphniapigmentation over a 3 week period. The levels of CAT and GSTwere assessed over time in the control and at 10 mg DOC. Theseenzymes displayed opposite patterns, with somewhat lower activitiesof CAT at low DOC (control) relative to 10 mg DOC, while theopposite was found for GST. There was also a significant negativecorrelation between CAT and solar irradiation for GST in bothbags, while no effects were found for GST.     

The RAINFOR database: monitoring forest biomass and dynamics

Problem: Data from over 100 permanent sample plots which have been studied for 10–20 years need a suitable system for storage which allows simple data manipulation and retrieval for analysis. Methods: A relational database linking tree records, taxonomic nomenclature and corresponding environmental data has been built in MS Access as part of the RAINFOR project. Conclusion: The database allows flexible and long‐term use of a large amount of data: more than 100 tree plots across Amazonia, incorporating over 80 000 records of individual trees and over 300 000 total records of tree diameter measurements from successive censuses. The database is designed to enable linkages to existing soil, floristic or plant‐trait databases. This database will be a useful tool for exploring the impact of environmental factors on forest structure and dynamics at local to continental scales, and long term changes in forest ecology. As an early example of its potential, we explore the impact of different methodological assumptions on estimates of tropical forest biomass and carbon storage.     

Tyrant flycatchers coming out in the open: phylogeny and ecological radiation of Tyrannidae (Aves, Passeriformes)

Tyrant flycatchers constitute a substantial component of the land bird fauna in all South American habitats. Past interpretations of the morphological and ecological evolution in the group have been hampered by the lack of a well‐resolved hypothesis of their phylogenetic interrelationships. Here, we present a well‐resolved phylogeny based on DNA sequences from three nuclear introns for 128 taxa. Our results confirm much of the overall picture of Tyrannidae relationships, and also identify several novel relationships. The genera Onychorhynchus, Myiobius and Terenotriccus are placed outside Tyrannidae and may be more closely related to Tityridae. Tyrannidae consists of two main lineages. An expanded pipromorphine clade includes flatbills, tody‐tyrants and antpipits, and also Phylloscartes and Pogonotriccus. The spadebills, Neopipo and Tachuris are their closest relatives. The remainder of the tyrant flycatchers forms a well‐supported clade, subdivided in two large subclades, which differ consistently in foraging behaviour, the perch‐gleaning elaeniines and the sallying myiarchines, tyrannines and fluvicolines. A third clade is formed by the genera Myiotriccus, Pyrrhomyias, Hirundinea and three species currently placed in Myiophobus. Ancestral habitat reconstruction and divergence date estimation suggest that early divergence events in Tyrannida took place in a humid forest environment during the Oligocene. Large‐scale diversification in open habitats is confined to the clade consisting of the elaeniines, myiarchines, tyrannines and fluvicolines. This radiation correlates in time to the expansion of semi‐open and open habitats from the mid‐Miocene (c. 15 Mya) onwards. The pipromorphine, elaeniine and myiarchine–tyrannine–fluvicoline clades each employ distinct foraging strategies (upward striking, perch‐gleaning and sallying, respectively), but the degree of diversity in morphology and microhabitat exploitation is markedly different between these clades. While the pipromorphines and elaeniines each are remarkably homogenous in morphology and exploit a restricted range of microhabitats, the myiarchine–tyrannine–fluvicoline clade is more diverse in these respects. This greater ecological diversity, especially as manifested in their success in colonizing a wider spectrum of open habitats, appears to be connected to a greater adaptive flexibility of the search‐and‐sally foraging behaviour.     

Three phenotypes of glucosephosphate isomerase in sheep: improved staining recipe

Contrary to results published recently, we observe three, rather than two, phenotypes for the enzyme glucosephosphate isomerase (EC 5.3.1.9) from sheep. The phenotypic electrophoretic patterns conform to the patterns observed for this dimeric enzyme in other species. Genotype frequencies in a flock of Southdowns do not deviate significantly from those predicted under the assumption of the Hardy-Weinberg equilibrium. A remarkable observation is that the electrophoretically distinct phenotypes of GPI are largely or entirely obliterated by the addition of 1-10 mmol/l MgCl2 to the electrophoretic buffers. Modification of the usual staining recipe for GPI result in greater resolution and shorter staining times.     

Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli.

For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.     

Selection for High Oridonin Yield in the Chinese Medicinal Plant Isodon (Lamiaceae) Using a Combined Phylogenetics and Population Genetics Approach

Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin.     

Effect of high pH on the spectral and catalytic properties of beef heart cytochrome oxidase

Incubation of cytochrome oxidase at high pH induces changes in several spectral properties. The optical Soret maximum shifts to longer wavelength, and there is an apparent loss in intensity of the 655-nm band, effects that are normally assigned either to a spin-state transition in cytochrome a3 or to a reduction of heme a. However, magnetic circular dichroism spectra show that cytochrome a3 remains high spin and that both cytochrome a and cytochrome a3 are oxidized. At the same time, there is the appearance of a low-spin signal indicative of hydroxide-imidazole coordination which we assign as arising from a structural transition at cytochrome a, rather than at cytochrome a3, as has been proposed previously. With longer incubation times, a new copper signal appears with electron paramagnetic resonance parameters markedly different from those obtained from copper centers which have undergone denaturation. Spin quantitation establishes that this new resonance does not arise from CuA and suggests that high pH breaks the magnetic coupling present at the cytochrome a3-CuB center. A significant proportion of cytochrome a3 may be converted to a low-spin thiolate during this process.