Simple additive effects are rare: a quantitative review of plant biomass and soil process responses to combined manipulations of CO2 and temperature

In recent years, increased awareness of the potential interactions between rising atmospheric CO₂ concentrations ([ CO₂ ]) and temperature has illustrated the importance of multifactorial ecosystem manipulation experiments for validating Earth System models. To address the urgent need for increased understanding of responses in multifactorial experiments, this article synthesizes how ecosystem productivity and soil processes respond to combined warming and [ CO₂ ] manipulation, and compares it with those obtained in single factor [ CO₂ ] and temperature manipulation experiments. Across all combined elevated [ CO₂ ] and warming experiments, biomass production and soil respiration were typically enhanced. Responses to the combined treatment were more similar to those in the [ CO₂ ]‐only treatment than to those in the warming‐only treatment. In contrast to warming‐only experiments, both the combined and the [ CO₂ ]‐only treatments elicited larger stimulation of fine root biomass than of aboveground biomass, consistently stimulated soil respiration, and decreased foliar nitrogen (N) concentration. Nonetheless, mineral N availability declined less in the combined treatment than in the [ CO₂ ]‐only treatment, possibly due to the warming‐induced acceleration of decomposition, implying that progressive nitrogen limitation (PNL) may not occur as commonly as anticipated from single factor [ CO₂ ] treatment studies. Responses of total plant biomass, especially of aboveground biomass, revealed antagonistic interactions between elevated [ CO₂ ] and warming, i.e. the response to the combined treatment was usually less‐than‐additive. This implies that productivity projections might be overestimated when models are parameterized based on single factor responses. Our results highlight the need for more (and especially more long‐term) multifactor manipulation experiments. Because single factor CO₂ responses often dominated over warming responses in the combined treatments, our results also suggest that projected responses to future global warming in Earth System models should not be parameterized using single factor warming experiments.     

Simultaneous quantitative determination of the HIV protease inhibitors indinavir,amprenavir, ritonavir,lopinavir, saquinavir,nelfinavir and the nelfinavir active metabolite M8 in plasma by liquid chromatography

A simple HPLC method that quantitates all six currently available protease inhibitors and the nelfinavir active metabolite M8 in one assay is presented. A 500-microliter plasma sample was treated by liquid-liquid extraction with a mixture of heptane and ethyl acetate. After evaporation, the residue was redissolved in sodium dihydrogenphosphate and acetonitrile and washed twice with heptane. Chromatography was performed with an analytical C(18) column. Ultraviolet detection at 210 and 239 nm was used. The present method is associated with high accuracy and low imprecision in the concentration range of 25-5000 ng/ml of all six protease inhibitors and M8. This makes it suitable for monitoring purposes.     

Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronectin but otherwise normal

Vitronectin (VN) and plasminogen activator inhibitor-1 (PAI-1) have important functional interactions: VN stabilises the protease inhibitory activity of PAI-1 and PAI-1 inhibits binding of adhesion receptors to VN. Having previously mapped the PAI-1 binding area for VN, we have now constructed a PAI-1 variant, R103A-M112A-Q125A, without measurable affinity to VN, but with full protease inhibitory activity and endocytosis receptor binding. As a tool for evaluating the physiological and pathophysiological functions of the PAI-1-VN interaction, our new variant is far superior to the previously widely used PAI-1 variant Q125K, which we have found possesses an only about 10-fold reduced affinity to VN.     

Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein

Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro‐chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin‐resistant under the acidic cleavage conditions (pH 4.5) used in this system. Here, we have modified the pro‐chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both at the level of small and large‐scale preparations. Using short peptide substrates, we further examined the influence of P1′ amino acid (the N‐terminus of the native target protein) and found that chymosin accepts many different, although not all, amino acids. We conclude that chymosin has several appealing characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins.     

Inhibition of 2'-5' oligoadenylate synthetase by divalent metal ions.

OAS1 is the small form and OAS2 is the medium form of the human interferon-induced 2'-5' oligoadenylate synthetases. The p42 isoform of OAS1 and the p69 isoform of OAS2 have been expressed in insect cells and purified to give pure, highly active 2'-5' oligoadenylate synthetase. The catalysis of 2'-5' oligoadenylate synthesis is strictly dependent on double-stranded RNA and magnesium ions. We have examined the effect of a series of divalent metal ions: copper, iron and zinc ions strongly inhibited the enzymatic activity, cobalt and nickel ions were partly inhibitory whereas calcium and manganese ions were without effect. However, manganese ions can replace magnesium ions as activator. The inhibitory effect of zinc ions was characterised in detail. The inhibitory constants of Zn(2+) were estimated to be 0.10 mM for OAS1p42 and to 0.02 mM for OAS2p69. Cross-linking experiments showed that zinc ions can control the oligomerisation by enhancing the formation of tetrameric forms of OAS1p42     

Origin,Migration Routes and Worldwide Population Genetic Structure of the Wheat Yellow Rust Pathogen Puccinia striiformis f.sp. tritici

Analyses of large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. This is particularly important for long-distance migrating fungal pathogens such as Puccinia striiformis f.sp. tritici (PST), capable of rapid spread to new regions and crop varieties. Although a range of recent PST invasions at continental scales are well documented, the worldwide population structure and the center of origin of the pathogen were still unknown. In this study, we used multilocus microsatellite genotyping to infer worldwide population structure of PST and the origin of new invasions based on 409 isolates representative of distribution of the fungus on six continents. Bayesian and multivariate clustering methods partitioned the set of multilocus genotypes into six distinct genetic groups associated with their geographical origin. Analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan regions (China) and a predominant clonal population structure in other regions. The higher genotypic diversity, recombinant population structure and high sexual reproduction ability in the Himalayan and neighboring regions suggests this area as the putative center of origin of PST. We used clustering methods and approximate Bayesian computation (ABC) to compare different competing scenarios describing ancestral relationship among ancestral populations and more recently founded populations. Our analyses confirmed the Middle East-East Africa as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. Although most geographic populations are not markedly affected by recent dispersal events, this study emphasizes the influence of human activities on recent long-distance spread of the pathogen.     

Suppression of eukaryotic translation termination by selected RNAs

Using selection-amplification, we have isolated RNAs with affinity for translation termination factors eRF1 and eRF1.eRF3 complex. Individual RNAs not only bind, but inhibit eRF1-mediated release of a model nascent chain from eukaryotic ribosomes. There is also significant but weaker inhibition of eRF1-stimulated eRF3 GTPase and eRF3 stimulation of eRF1 release activity. These latter selected RNAs therefore hinder eRF1.eRF3 interactions. Finally, four RNA inhibitors of release suppress a UAG stop codon in mammalian extracts dependent for termination on eRF1 from several metazoan species. These RNAs are therefore new specific inhibitors for the analysis of eukaryotic termination, and potentially a new class of omnipotent termination suppressors with possible therapeutic significance.