Thioredoxin A active-site mutants form mixed disulfide dimers that resemble enzyme-substrate reaction intermediates

Thioredoxin functions in nearly all organisms as the major thiol-disulfide oxidoreductase within the cytosol. Its prime purpose is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds into dithiols in a disulfide exchange reaction. Thioredoxin has been reported to contribute to a wide variety of physiological functions by interacting with specific sets of substrates in different cell types. To investigate the function of the essential thioredoxin A (TrxA) in the low-GC Gram-positive bacterium Bacillus subtilis, we purified wild-type TrxA and three mutant TrxA proteins that lack either one or both of the two cysteine residues in the CxxC active site. The pure proteins were used for substrate-binding studies known as “mixed disulfide fishing” in which covalent disulfide-bonded reaction intermediates can be visualized. An unprecedented finding is that both active-site cysteine residues can form mixed disulfides with substrate proteins when the other active-site cysteine is absent, but only the N-terminal active-site cysteine forms stable interactions. A second novelty is that both single-cysteine mutant TrxA proteins form stable homodimers due to thiol oxidation of the remaining active-site cysteine residue. To investigate whether these dimers resemble mixed enzyme-substrate disulfides, the structure of the most abundant dimer, C32S, was characterized by X-ray crystallography. This yielded a high-resolution (1.5Å) X-ray crystallographic structure of a thioredoxin homodimer from a low-GC Gram-positive bacterium. The C32S TrxA dimer can be regarded as a mixed disulfide reaction intermediate of thioredoxin, which reveals the diversity of thioredoxin/substrate-binding modes.     

Xeromorphy increases in shoots of Pseudotsuga menziesii (Mirb.) Franco seedlings with exposure to elevated temperature but not elevated CO2

Seedling structure influences tree structure and function, ultimately determining the potential productivity of trees and their competitiveness for resources. We investigated changes in shoot structure for seedlings of Pseudotsuga menziesii (Douglas-fir) grown under climate change scenarios of ambient or elevated CO₂ (+180 mol mol^–1) plus ambient or elevated temperature (+3.5°C), for 4 years in outdoor, sunlit chambers. Mass allocation and allometry were measured for buds, leaves, branches, and stems, and anatomy was evaluated for leaves and stems. Seedlings became more xeromorphic with elevated temperature: allocation of total mass to branches over stems and leaves increased, sapwood area to height ratio increased, number of growing points relative to seedling size increased, and stem and branch length and mass decreased for sections initiated during the three full CO₂ and temperature seasons. Neither stem nor leaf anatomy was affected by elevated temperature. Elevated CO₂ increased specific mass of leaves, but had few other effects on mass allocation, allometry, or anatomy for any shoot organ. There were no CO₂ × temperature interactions for any important parameter. Thus, under realistic simulated field environmental conditions representative for in at least some P. menziesii forests (i.e., OR, USA, forests with limited soil nitrogen and summer soil moisture), elevated temperature, but not elevated CO₂, may affect seedling shoot structure and, hence, function.     

Diversity and composition of macrobenthic community associated with sandy shoals of the Louisiana continental shelf

Along the Louisiana, USA continental shelf, sandy shoals are shallow, possibly oxygen-rich “islands” surrounded by deeper muddy deposits prone to hypoxia. Shoals also contain significant quantities of fine sand that may be mined in the future for coastal restoration. The ecological role of shoals remains poorly understood and we hypothesized that shoals provide critical habitat for benthic invertebrates. Using Ship Shoal as a model system, we assessed the diversity and structure of macrobenthic assemblages and how community structure varies with season and environmental parameters. High biomass (averaging 26.7 g m⁻²) and high diversity (161 species) of macrobenthos was found in 2006. Polychaetes (45%—72 species) and crustaceans comprised most of the species (28%—46 species); spionids and amphipods dominated the polychaete and crustacean groups respectively, both in terms of number of species and abundances. Sharp decreases in diversity, abundance and biomass occurred from spring to autumn. Species diversity and total abundance significantly increased with decreasing sediment grain size and increasing bottom water dissolved oxygen. Across seasons, mole crabs Albunea paretii and amphioxus Branchiostoma floridae typified the community and contributed most of the biomass. The polychaetes Nephtys simoni, Neanthes micromma, Dispio uncinata, Mediomastus californiensis and Magelona sp. A, the amphipod Acanthohautorius sp. A and the burrowing shrimp Ogyrides alphaerostris also contributed to variation in community composition. Cluster analyses quantified seasonal variation, mainly based on sharp decreases in abundance, as well as spatial differences in species composition oriented along both east–west and north–south gradients. Variation in benthic assemblages was correlated with water depth and sediment characteristics (mean grain size and percentage of gravel-sized shell debris). We conclude that Ship Shoal is an unrecognized biodiversity hotspot and a hypoxia refuge compared to the immediate surrounding area where the benthic community is affected by seasonal hypoxia events and we discuss how sand-mining may influence this community.     

Long-term social bonds promote cooperation in the iterated Prisoner's Dilemma

Reciprocal altruism, one of the most probable explanations for cooperation among non-kin, has been modelled as a Prisoner''s Dilemma. According to this game, cooperation could evolve when individuals, who expect to play again, use conditional strategies like tit-for-tat or Pavlov. There is evidence that humans use such strategies to achieve mutual cooperation, but most controlled experiments with non-human animals have failed to find cooperation. One reason for this could be that subjects fail to cooperate because they behave as if they were to play only once. To assess this hypothesis, we conducted an experiment with monogamous zebra finches (Taeniopygia guttata) that were tested in a two-choice apparatus, with either their social partner or an experimental opponent of the opposite sex. We found that zebra finches maintained high levels of cooperation in an iterated Prisoner''s Dilemma game only when interacting with their social partner. Although other mechanisms may have contributed to the observed difference between the two treatments, our results support the hypothesis that animals do not systematically give in to the short-term temptation of cheating when long-term benefits exist. Thus, our findings contradict the commonly accepted idea that reciprocal altruism will be rare in non-human animals.     

A multispecies approach reveals hot spots and cold spots of diversity and connectivity in invertebrate species with contrasting dispersal modes

Genetic diversity is crucial for species’ maintenance and persistence, yet is often overlooked in conservation studies. Species diversity is more often reported due to practical constraints, but it is unknown if these measures of diversity are correlated. In marine invertebrates, adults are often sessile or sedentary and populations exchange genes via dispersal of gametes and larvae. Species with a larval period are expected to have more connected populations than those without larval dispersal. We assessed the relationship between measures of species and genetic diversity, and between dispersal ability and connectivity. We compiled data on genetic patterns and life history traits in nine species across five phyla. Sampling sites spanned 600 km in the northwest Mediterranean Sea and focused on a 50‐km area near Marseilles, France. Comparative population genetic approaches yielded three main results. (i) Species without larvae showed higher levels of genetic structure than species with free‐living larvae, but the role of larval type (lecithotrophic or planktotrophic) was negligible. (ii) A narrow area around Marseilles, subject to offshore advection, limited genetic connectivity in most species. (iii) We identified sites with significant positive contributions to overall genetic diversity across all species, corresponding with areas near low human population densities. In contrast, high levels of human activity corresponded with a negative contribution to overall genetic diversity. Genetic diversity within species was positively and significantly linearly related to local species diversity. Our study suggests that local contribution to overall genetic diversity should be taken into account for future conservation strategies.     

Non-synonymous polymorphisms in the human SLCO1B1 gene: an in vitro analysis of SNP c.1929A>C

Polymorphisms in the SLCO1B1 gene encoding the human hepatocellular uptake transporter OATP1B1 can be associated with alterations in transporter properties and may affect the pharmacokinetics of drugs transported by OATP1B1. Therefore, it is of interest to investigate newly identified genetic variations on their impact on the pharmacokinetic of OATP1B1 substrates. We analyzed the allelic frequencies of five variations (c.452A>G, c.1007C>G, c.1454G>T, c.1628T>G, and c.1929A>C) in Caucasians and investigated the influence of SNP c.1929A>C which had an allelic frequency of 4.7%. None of the 285 Caucasian DNA donors were carriers of c.452A>G, c.1007C>G, c.1454G>T, c.1628T>G. Liver samples carrying SNP c.1929A>C were analyzed for OATP1B1 protein expression demonstrating no differences in expression levels compared to wild-type samples. Possible functional consequences were analyzed using HEK cells stably expressing the mutated OATP1B1 protein (OATP1B1-Leu643Phe). Uptake experiments with sulfobromophthalein, estradiol-17ssD-glucuronide, pravastatin, and taurocholic acid showed no significant difference in the uptake kinetics compared to wild-type OATP1B1. We showed that four variations frequent in the Asian population were not detected in Caucasians and demonstrated that the frequent SNP c.1929A>C had no effect on the hepatic OATP1B1 protein expression and on the transport properties. Therefore, it is unlikely that c.1929A>C contributes to interindividual variability in drug disposition.     

Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria

Studies of lactic acid bacteria (LAB) as delivery vehicles have focused mainly on the development of mucosal vaccines, with much effort being devoted to the generation of genetic tools for antigen expression in different bacterial locations. Subsequently, interleukins have been co-expressed with antigens in LAB to enhance the immune response that is raised against the antigen. LAB have also been used as a delivery system for a range of molecules that have different applications, including anti-infectives, therapies for allergic diseases and therapies for gastrointestinal diseases. Now that the first human trial with a Lactococcus strain that expresses recombinant interleukin-10 has been completed, we discuss what we have learnt, what we do not yet understand and what the future holds for therapy and prophylaxis with LAB.     

Crystal structure of a cold-adapted class C beta-lactamase

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.