Genetic identification of units for conservation in tomato frogs, genus Dyscophus

Dyscophus antongilii and D. guineti are two morphologically very similar microhylid frogs from Madagascar of uncertain taxonomy. D. antongilii is currently included in Appendix I of the Convention on the International Trade in Endangered Species (CITES) and its exportation is banned completely. In contrast, D. guineti does not receive any legal protection and it is regularly exported. Field data on ecology and behaviour are to a large extent lacking. Here we report on a genetic survey of D. antongilii and D. guineti using nuclear and mitochondrial DNA markers. Sequences of a fragment of 501 bp of the mitochondrial cytochrome b gene from one population of D. antongilii and two populations of D. guineti resulted in a single haplotype network, without haplotype sharing among the populations. However, haplotypes of D.␣guineti were only 1–4 mutational steps from those of D. antongilii, and did not form a clade. The analysis of eight microsatellites newly developed and standardized for D. antongilii revealed an excess of homozygotes and the absence of Hardy–Weinberg equilibrium. The microsatellite data clearly distinguished between D. antongilii and D. guineti, and fixed differences were observed at one locus. Although confirmation of the status of Dyscophus antongilii and D. guineti as separate species requires further data, our study supports the definition of these two taxa as different evolutionary significant units under the adaptive evolutionary conservation concept.     

Injury of mitochondrial respiration and membrane potential during iron/ascorbate-induced peroxidation

First functional events during peroxidation in mitochondria consisted in a progressive inhibition of the phosphorylating and uncoupled respiration with succinate and glutamate/malate as substrates, whereas the resting state respiration during the same period was virtually not influenced. The membrane potential registered at a time with the respiration rates was capable of being built up for a relatively long time interval with only minor decreases, and broke down rather promptly when the active respiration was highly diminished. Inhibition of respiration proceeded mainly during the initiation phase of peroxidation. Lag phases of varied length, of malondialdehyde formation which were predominantly attributed to the iron/protein ratios correlated closely with different time intervals needed to attain maximal inhibition of respiration and decrease in glutathione. Hence, the lessening of respiration, drop of membrane potential and loss of the antioxidant, glutathione, represent early stages in the causal chain of events which precede the onset of intensive lipid peroxidation.     

Within-host Competition Does Not Select for Virulence in Malaria Parasites; Studies with Plasmodium yoelii

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.     

Long-term effects on the nitrogen budget of a short-rotation grey alder (Alnus incana (L.) Moench) forest on abandoned agricultural land

Short-rotation energy forestry is one of the potential ways for management of abandoned agricultural areas. It helps sequestrate carbon and mitigate human-induced climate changes. Owing to symbiotic dinitrogen (N₂) fixation by actinomycetes and the soil fertilizing capacity and fast biomass growth of grey alders, the latter can be suitable species for short-rotation forestry. In our study of a young grey alder stand (Alnus incana (L.) Moench) on abandoned arable land in Estonia we tested the following hypotheses: (1) afforestation of abandoned agricultural land by grey alder significantly affects the soil nitrogen (N) status already during the first rotation period; (2) input of symbiotic fixation covers an essential part of the plant annual N demand of the stand; (3) despite a considerable N input into the ecosystem of a young alder stand, there will occur no significant environmental hazards (N leaching or N₂O emissions). The first two hypotheses can be accepted: there was a significant increase in N and C content in the topsoil (from 0.11 to 0.14%, and from 1.4 to 1.7%, respectively), and N fixation (151.5 kg N ha⁻¹ yr⁻¹) covered about 74% of the annual N demand of the stand. The third hypothesis met support as well: N₂O emissions (0.5 kg N ha⁻¹ yr⁻¹) were low, while most of the annual gaseous N losses were in the form of N₂ (73.8 kg N ha⁻¹ yr⁻¹). Annual average NO₃-N leaching was 15 kg N ha⁻¹ yr⁻¹ but the N that leached from topsoil accumulated in deeper soil layers. The soil acidifying effect of alders was clearly evident; during the 14-year period soil acidity increased 1.3 units in the upper 0-10 cm topsoil layer.     

Dynamics of gaseous nitrogen and carbon fluxes in riparian alder forests

We studied greenhouse gas (GHG) fluxes in two differently loaded riparian Alnus incana-dominated forests in agricultural landscapes of southern Estonia: a 33-year-old stand in Porijõgi, in which the uphill agricultural activities had been abandoned since the middle of the 1990s, and a 50-year-old stand in Viiratsi, which still receives polluted lateral flow from uphill fields fertilized with pig slurry. In Porijõgi, closed-chamber based sampling lasted from October 2001 to October 2009, whereas in Viiratsi the sampling period was from November 2003 to October 2009. Both temporal and spatial variations in all GHG gas fluxes were remarkable. Local differences in GHG fluxes between micro-sites (“Edge”, “Dry” and “Wet” in Porijõgi, and “Wet”, “Slope” and “Dry” in Viiratsi) were sometimes greater than those between sites. Median values of GHG fluxes from both sites over the whole study period and all microsites did not differ significantly, being 45 and 42 mg CO₂-C m⁻² h⁻¹, 8 and 0.5 μg CH₄-C m⁻² h⁻¹, 1.0 and 2.1 mg N₂-N m⁻² h⁻¹, and 5 and 9 μg N₂O-N m⁻² h⁻¹, in Porijõgi and Viiratsi, respectively. The N₂:N₂O ratio in Viiratsi (40-1200) was lower than in Porijõgi (10-7600). The median values-based estimation of the Global Warming Potential of CH₄ and N₂O was 19 and 185 kg CO₂ equivalents (eq) ha⁻¹ yr⁻¹ in Porijõgi and −14 and 336 kg CO₂ eq ha⁻¹ yr⁻¹ in Viiratsi, respectively. A significant Spearman rank correlation was found between the mean monthly air temperature and CO₂, CH₄ and N₂ fluxes in Porijõgi, and N₂O flux in Viiratsi, and between the monthly precipitation and CH₄ fluxes in both study sites. Higher groundwater level significantly increases CH₄ emission and decreases CO₂ and N₂O emission, whereas higher soil temperature significantly increases N₂O, CH₄ and N₂ emission values. In Porijõgi, GHG emissions did not display any discernable trend, whereas in Viiratsi a significant increase in CO₂, N₂, and N₂O emissions has been found. This may be a result of the age of the grey alder stand, but may also be caused by the long-term nutrient load of this riparian alder stand, which indicates a need for the management of similar heavily loaded riparian alder stands.     

The Effect of Maillard Conjugation of Deamidated Wheat Proteins with Low Molecular Weight Carbohydrates on the Secondary Structure of the Protein

A soluble isolated wheat protein fraction (sIWP) prepared from isolated wheat protein (30–35% deamidation) was incubated alone or in the presence of glucose or maltodextrins of various molecular weights (MW 1, 1.9 and 4.3 kDa) at 60 °C and 75% relative humidity to promote the formation of Maillard conjugates. The formation of Maillard conjugates was confirmed by the loss of available -NH₂ groups on incubation. Approximately 3–4 carbohydrate moieties (glucose or low molecular weight carbohydrates in the commercial maltodextrin) were attached per mole of sIWP after 24 h incubation. Principal component analysis of attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra measured in the dry state showed that there were no major structural changes among non-incubated sIWP, sIWP incubated alone, sIWP–glucose conjugate and sIWP–maltodextrin (MW 1 kDa) conjugate. Structural changes were observed when the protein was incubated with larger molecular weight maltodextrin (MW 1.9 kDa or 4.3 kDa). However, there were no detectable differences in their circular dichroism (CD) spectra suggesting the absence of conformational changes in proteins with or without attached carbohydrates in solution state. The differences between the FTIR and CD results are possibly due to differences in water content of the samples although pressure-induced changes to protein structure induced in the ATR cell and the influence of unreacted maltodextrins cannot be discounted. Attachment of low molecular weight carbohydrate moieties on a relatively large molecular weight protein (i.e. sIWP with average MW of 40.4 kDa) with low lysine content (average three per mole of protein) is not sufficient to have an impact on the secondary structure of the protein.     

Dipeptidyl nitrile inhibitors of Cathepsin L

A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.