Combined observational and experimental data provide limited support for facilitation in lichens

It is increasingly recognized that facilitative interactions can shape communities. One of the mechanisms through which facilitation may operate is when one species facilitates the colonization of another through the exchange of shared symbionts. Lichens are symbiotic associations composed of a mycobiont (lichenised‐fungus) and one or two photobionts (algae or cyanobacteria). Different lichen species may have overlapping specificity for photobionts, creating the possibility that facilitation drives lichen community assembly. To investigate whether facilitation occurs in lichens, we combined an observational study (a) with a manipulative field experiment (b). For (a), we quantified the effect of local patch conditions, facilitation and the size of the surrounding metapopulation on colonizations of an epixylic lichen species (Cladonia botrytes) in an area of managed boreal forest. This was done by twice surveying lichens on 293 stumps, located in stands of three age classes. For (b), we treated unoccupied surfaces of 56 cut stumps with algal mixtures of an Asterochloris photobiont and recorded C. botrytes colonizations over three years. In (a), colonization rates of C. botrytes increased with increasing abundance of other lichen species with specificity for Asterochloris photobionts, consistent with an effect of facilitation. However, in the field experiment (b), colonizations of the focal species did not provide support for facilitation. We conclude that our study provides limited support for facilitation in green‐algal lichens, underscoring the importance of combining observational studies with experiments when studying species interactions.     

Regulation of the muscle-specific AMP-activated protein kinase alpha2beta2gamma3 complexes by AMP and implications of the mutations in the gamma3-subunit for the AMP dependence of the enzyme

The AMP-activated protein kinase is an evolutionarily conserved heterotrimer that is important for metabolic sensing in all eukaryotes. The muscle-specific isoform of the regulatory gamma-subunit of the kinase, AMP-activated protein kinase gamma3, has a key role in glucose and fat metabolism in skeletal muscle, as suggested by metabolic characterization of humans, pigs and mice harboring substitutions in the AMP-binding Bateman domains of gamma3. We demonstrate that AMP-activated protein kinase alpha2beta2gamma3 trimers are allosterically activated approximately three-fold by AMP with a half-maximal stimulation (A(0.5)) at 1.9 +/- 0.5 or 2.6 +/- 0.3 microm, as measured for complexes expressed in Escherichia coli or mammalian cells, respectively. We show that mutations in the N-terminal Bateman domain of gamma3 (R225Q, H306R and R307G) increased the A(0.5) values for AMP, whereas the fold activation of the enzyme by 200 microm AMP remained unchanged in comparison to the wild-type complex. The mutations in the C-terminal Bateman domain of gamma3 (H453R and R454G), on the other hand, substantially reduced the fold stimulation of the complex by 200 microm AMP, and resulted in AMP dependence curves similar to those of the double mutant, R225Q/R454G. A V224I mutation in gamma3, known to result in a reduced glycogen content in pigs, did not affect the fold activation or the A(0.5) values for AMP. Importantly, we did not detect any increase in phosphorylation of Thr172 of alpha2 by the upstream kinases in the presence of increasing concentrations of AMP. Taken together, the data show that different mutations in gamma3 exert different effects on the allosteric regulation of the alpha2beta2gamma3 complex by AMP, whereas we find no evidence for their role in regulating the level of phosphorylation of alpha2 by upstream kinases.     

The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity

Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.     

Long‐term rearing affects pheromone‐mediated flight behaviour of the Indian meal moth,Plodia interpunctella

The pest Plodia interpunctella (Hübner) is reared in many research laboratories. In a culture established in 1996, attraction of males to the female‐produced sex pheromone in flight tunnel assays gradually decreased after ≈15 years of rearing. A new culture was established to enable comparison with the old culture regarding traits associated with mate finding. Female calling activity, pheromone titre and male antennal response to pheromone components did not differ between cultures. In contrast, very few males from the old culture reached the pheromone source in flight tunnel assays compared with 61%–81% of males from the other culture. Our results highlight the importance of maintaining viable insect cultures for research purposes and suggest frequent evaluation of traits involved in chemical communication in such cultures to ensure reliable results in experiments.     

School performance in Danish children exposed to maternal type 1 diabetes in utero: A nationwide retrospective cohort study

BackgroundConflicting results have been reported concerning possible adverse effects on the cognitive function of offspring of mothers with type 1 diabetes (O-mT1D). Previous studies have included offspring of parents from the background population (O-BP), but not offspring of fathers with type 1 diabetes (O-fT1D) as the unexposed reference group.Methods and findingsThis is a population-based retrospective cohort study from 2010 to 2016. Nationally standardized school test scores (range, 1 to 100) were obtained for public school grades 2, 3, 4, 6, and 8 in O-mT1D and compared with those in O-fT1D and O-BP. Of the 622,073 included children, 2,144 were O-mT1D, and 3,474 were O-fT1D. Multiple linear regression models were used to compare outcomes, including the covariates offspring with type 1 diabetes, parity, number of siblings, offspring sex, smoking during pregnancy, parental age, and socioeconomic factors. Mean test scores were 54.2 (standard deviation, SD 24.8) in O-mT1D, 54.4 (SD 24.8) in O-fT1D, and 56.4 (SD 24.7) in O-BP. In adjusted analyses, the mean differences in test scores were −1.59 (95% CI −2.48 to −0.71, p < 0.001) between O-mT1D and O-BP and −0.78 (95% CI −1.48 to −0.08, p = 0.03) between O-fT1D and O-BP. No significant difference in the adjusted mean test scores was found between O-mT1D and O-fT1D (p = 0.16). The study’s limitation was no access to measures of glycemic control during pregnancy.ConclusionsO-mT1D achieved lower test scores than O-BP but similar test scores compared with O-fT1D. Glycemic control during pregnancy is essential to prevent various adverse pregnancy outcomes in women with type 1 diabetes. However, the present study reduces previous concerns regarding adverse effects of in utero hyperglycemia on offspring cognitive function.

Anne Lærke Spangmose and colleagues examine the association between school performance and exposure to maternal or paternal type 1 diabetes in utero in Denmark.     

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k)

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.