A novel alpha-D-galactosynthase from Thermotoga maritima converts beta-D-galactopyranosyl azide to alpha-galacto-oligosaccharides

The large-scale production of oligosaccharides is a daunting task, hampering the study of the role of glycans in vivo and the testing of the efficacy of novel glycan-based drugs. Glycosynthases, mutated glycosidases that synthesize oligosaccharides in high yields, are becoming important chemo-enzymatic tools for the production of oligosaccharides. However, while β-glycosynthase can be produced with a rather well-established technology, examples of α-glycosynthases are thus far limited only to enzymes from glycoside hydrolase 29 (GH29), GH31 and GH95 families. α-L-Fucosynthases from GH29 use convenient glycosyl azide derivatives as a strategic alternative to glycosyl fluoride donors. However, the general applicability of this method to other α-glycosynthases is not trivial and remains to be confirmed. Here, β-D-galactopyranosyl azide was converted to α-galacto-oligosaccharides with good yields and high regioselectivity, catalyzed by a novel α-galactosynthase based on the GH36 α-galactosidase from the hyperthermophilic bacterium Thermotoga maritima. These results open a new avenue to the practical synthesis of biologically interesting α-galacto-oligosaccharides and demonstrate more widespread use of β-glycosyl-azide as donors, confirming their utility to expand the repertoire of glycosynthases.     

Seed to seedling transitions in successional habitats across a tropical landscape

Recognition that tree recruitment depends on the balance between seed arrival and seedling survival has led to a surge of interest in seed‐dispersal limitation and seedling‐establishment limitation in primary forests. Virtually unaddressed are comparisons of this balance in mature and early successional habitats. We assessed seed rain and seedling recruitment dynamics of tree species in primary forest, secondary forest and pasture released from grazing in a tropical agricultural landscape. Seed to seedling ratios (seed effectiveness; Φ_i) for 43 species in southern Mexico determined differences in the extent to which seeds produced seedlings by habitat, life history, and dispersal mode. Reproductive potential as estimated by the transition from seed rain to seedling recruitment, differed by habitats, and varied dramatically by life history and dispersal mode. Expected recruit densities (E_it) were higher for animal‐dispersed than wind‐dispersed species, and for non‐pioneer than pioneer species. Non‐pioneers and animal‐dispersed species had higher expected relative recruit abundance (ε_it) in primary forest (median of 4 seeds recruit^?1) whereas in secondary forest wind‐dispersed pioneers had the highest expected relative recruit abundance (median of 16 seeds per recruit). In pastures, wind‐dispersed pioneer species were most successful with many more seeds per recruit (median of 291) than both forest habitats. Seeds per recruit (Φ_i) appeared to decrease with increase in seed mass for 43 species for which data were available (r = –0.55, P < 0.001). This was associated with a negative correlation of Φ_i with seed size in primary forest (r = –0.50, P = 0.08 for 13 species); Φ_i was not correlated with seed size in secondary forest (n = 16) or pasture (n = 14). Metrics of seeds per recruit, expected recruit density and expected relative recruit abundance dramatically illustrate differences in barriers to recruitment in successional habitats.     

King penguin demography since the last glaciation inferred from genome-wide data

How natural climate cycles, such as past glacial/interglacial patterns, have shaped species distributions at the high-latitude regions of the Southern Hemisphere is still largely unclear. Here, we show how the post-glacial warming following the Last Glacial Maximum (ca 18 000 years ago), allowed the (re)colonization of the fragmented sub-Antarctic habitat by an upper-level marine predator, the king penguin Aptenodytes patagonicus. Using restriction site-associated DNA sequencing and standard mitochondrial data, we tested the behaviour of subsets of anonymous nuclear loci in inferring past demography through coalescent-based and allele frequency spectrum analyses. Our results show that the king penguin population breeding on Crozet archipelago steeply increased in size, closely following the Holocene warming recorded in the Epica Dome C ice core. The following population growth can be explained by a threshold model in which the ecological requirements of this species (year-round ice-free habitat for breeding and access to a major source of food such as the Antarctic Polar Front) were met on Crozet soon after the Pleistocene/Holocene climatic transition.     

Alkylation of amide linkages and cleavage of the C chain in the enzyme-activated-substrate inhibition of alpha-chymotrypsin with N-nitrosamides

Active-site-directed N-nitrosamides inhibit alpha-chymotrypsin through an enzyme-activated-substrate mechanism. In this work, the activation results in the release--in the active site--of benzyl carbonium ions, which alkylate and inhibit the enzyme. The final ratio of benzyl groups to enzyme molecules is 1.0, but the alkyl groups are scattered over a number of sites. Reduction and alkylation of the inhibited enzyme generate peptides insoluble in most media. Guanidine hydrochloride at 6 M proved a good solvent, and its use as an eluant on G-75 Sephadex permitted separation of the peptides. In the case of 14C-labeled enzyme, such an approach has shown that all of the alkylation occurs on the C chain of the enzyme, the chain of which the active site is constructed. Chemical modification of the peptides with ethylenediamine and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide rendered them soluble in dilute acid, permitting high-performance liquid chromatographic separation. Model studies have shown that the benzyl carbonium ions are highly reactive, alkylating amide linkages at both oxygen and nitrogen. Alkylation at oxygen produces imidate esters, which are labile centers. Hydrolysis of protein imidates results in a cleavage of the chain at that point, and separation of the peptides formed (followed by analysis) permits their identification. In our inhibition of alpha-chymotrypsin, a major site of O-alkylation has been identified as the carbonyl oxygen of Ser-214. Alkylation at the nitrogen atom of amide linkages generates stable labels; full hydrolysis with 6 N HC1 then leads to N-benzyl amino acids characteristic of those sites. Chromatography of this mixture and also 13C NMR spectroscopy of the intact inhibited enzyme have shown that three major N-alkylations have occurred. Tryptic digestion of the C chain of chymotrypsin, which contains all of the alkylation sites, provides evidence that the stable N sites are principally located between residue 216 and residue 230. These locations are consistent with predictions of alkylation sites based on inspection of a molecular model of chymotrypsin, with special reference to the aromatic binding pocket.     

Modelling the distribution and compositional variation of plant communities at the continental scale

Aim

We investigate whether (1) environmental predictors allow to delineate the distribution of discrete community types at the continental scale and (2) how data completeness influences model generalization in relation to the compositional variation of the modelled entities.

Location

Europe.

Methods

We used comprehensive datasets of two community types of conservation concern in Europe: acidophilous beech forests and base‐rich fens. We computed community distribution models (CDMs) calibrated with environmental predictors to predict the occurrence of both community types, evaluating geographical transferability, interpolation and extrapolation under different scenarios of sampling bias. We used generalized dissimilarity modelling (GDM) to assess the role of geographical and environmental drivers in compositional variation within the predicted distributions.

Results

For the two community types, CDMs computed for the whole study area provided good performance when evaluated by random cross‐validation and external validation. Geographical transferability provided lower but relatively good performance, while model extrapolation performed poorly when compared with interpolation. Generalized dissimilarity modelling showed a predominant effect of geographical distance on compositional variation, complemented with the environmental predictors that also influenced habitat suitability.

Main conclusions

Correlative approaches typically used for modelling the distribution of individual species are also useful for delineating the potential area of occupancy of community types at the continental scale, when using consistent definitions of the modelled entity and high data completeness. The combination of CDMs with GDM further improves the understanding of diversity patterns of plant communities, providing spatially explicit information for mapping vegetation diversity and related habitat types at large scales.

     

Structural and physiological responses of <Emphasis Type="Italic">Halodule wrightii</Emphasis> to ocean acidification

Coastal areas face high variability of seawater pH. Ocean acidification (OA) and local stressors are enhancing this variability, which poses a threat to marine life. However, these organisms present potential phenotypic plasticity that can offer physiological and structural tools to survive in these extreme conditions. In this study, we evaluated the effects of elevated CO₂ levels and consequent pH reduction on the physiology, anatomy and ultrastructure of the seagrass Halodule wrightii. A mesocosm study was conducted in an open system during a 30-day experiment, where different concentrations of CO₂ were simulated following the natural variability observed in coastal reef systems. This resulted in four experimental conditions simulating the (i) environmental pH (control condition, without CO₂ addition) and (ii) reduced pH by ? 0.3 units, (iii) ? 0.6 units and (iv) ? 0.9 units, in relation to the field condition. The evaluated population only suffered reduced optimum quantum yield (Y(II)), leaf width and cross-section area under the lowest CO₂ addition (? 0.3 pH units) after 30 days of experiment. This fitness commitment should be related to carbon concentration mechanisms present in the evaluated species. For the highest CO₂ level, H. wrightii demonstrated a capacity to compensate any negative effect of the lowest pH. Our results suggest that the physiological behaviour of this primary producer is driven by the interactions among OA and environmental factors, like irradiance and nutrient availability. The observed behaviour highlights that high-frequency pH variability and multifactorial approaches should be applied, and when investigating the impact of OA, factors like irradiance, nutrient availability and temperature must be considered as well.     

Global distribution and status of introduced Siberian chipmunks Eutamias sibiricus

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The effect of cover plants management on soil invertebrate fauna in vineyard in Northern Italy

Cover crops are largely used in vineyard management to improve ecological services such as pest and weed control, soil protection from erosion and soil water balance. Few data are available about the effects of cover crops on soil fauna and, in particular, on important generalist predators such as Carabidae and Araneae. We compare the effects of five different cover crops treatments: Sweet Alyssum, Phacelia, Buckwheat, Faba bean and a mixture of Vetch and Oat. A three years experimental trial was developed in Northern Italy. Soil fauna was investigated using pitfall traps. Cover crops seriously affected soil fauna of most taxa. Higher abundance of predators, in particular Carabidae and Staphylinidae, was present in treatments with legumes (Faba bean and Vetch and Oat) while soil Araneae seemed not to be affected. No vineyard pests seemed to be positively affected by cover crops. We confirm the importance of cover crops in increasing soil predator abundance and consequently improving pest control.