Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5′ end of the RNA pregenome. Epsilon contains an apical stem–loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem–loop based on NOE, RDC and ¹H chemical shift NMR data. The ¹H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5′-CUGUGC-3′ folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.     

Prototypical type I E-cadherin and type II cadherin-7 mediate very distinct adhesiveness through their extracellular domains

Using a dual pipette assay that measures the force required to separate adherent cell doublets, we have quantitatively compared intercellular adhesiveness mediated by Type I (E- or N-cadherin) or Type II (cadherin-7 or -11) cadherins. At similar cadherin expression levels, cells expressing Type I cadherins adhered much more rapidly and strongly than cells expressing Type II cadherins. Using chimeric cadherins, we found that the extracellular domain exerts by far the dominant effect on cell adhesivity, that of E-cadherin conferring high adhesivity, and that of cadherin-7 conferring low adhesivity. Type I cadherins were incorporated to a greater extent into detergent-insoluble cytoskeletal complexes, and their cytoplasmic tails were much more effective in disrupting strong adherent junctions, suggesting that Type II cadherins form less stable complexes with beta-catenin. The present study demonstrates compellingly, for the first time, that cadherins are dramatically different in their ability to promote intercellular adhesiveness, a finding that has profound implications for the regulation of tissue morphogenesis.     

Wood anatomy of Rauvolfioideae (Apocynaceae): a search for meaningful non-DNA characters at the tribal level

Wood anatomical studies in the economically important Apocynaceae or dogbane family are fragmentary. This study represents a first attempt to unravel the phylogenetic significance and major evolutionary trends in the wood of the family, using existing and new microscopic wood observations within the large subfamily Rauvolfioideae. On the basis of LM and SEM observations of 91 species representing all 10 currently recognized tribes, we found that most of the tribes are characterized by a unique combination of wood characters, such as vessel grouping, vessel element length, fiber type, frequency of uniseriate rays, and fused multiseriate rays. Climbing rauvolfioid taxa can generally be distinguished from erect species by their wider vessels, tendency to form paratracheal axial parenchyma, presence of tracheids, and occurrence of laticifers in rays. With respect to the entire family, there is a general phylogenetic trend toward shorter vessel elements, a higher proportion of vessels in multiples and more vessels per multiple, higher tracheid abundance, more paratracheal parenchyma, and fewer cells per axial parenchyma strand in the more derived Apocynaceae. Most of these evolutionary trends are likely to be triggered by drier environmental conditions and/or shifts from an erect to a climbing habit.     

Foraging for Work and Age-Based Polyethism: The Roles of Age and Previous Experience on Task Choice in Ants

In social insects, colonies commonly show temporal polyethism in worker behavior, such that a worker follows a predictable pattern of changes between tasks as it ages. This pattern usually leads from workers first doing a safe task like brood care, to ending their lives doing the most dangerous tasks like foraging. Two mechanisms could potentially underlie this pattern: (1) age‐based task allocation, where the aging process itself predisposes workers to switch to more dangerous tasks; and (2) foraging for work, where ants switch to tasks that need doing from tasks which have too many associated workers. We tested the relative influence of these mechanisms by establishing nests of Camponotus floridanus with predetermined combinations of workers of known age and previous task specialization. The results supported both mechanisms. Nests composed of entirely brood‐tending workers had the oldest workers preferentially switching to foraging. However, in nests initially composed entirely of foragers, the final distribution of tenders and foragers was not different from random task‐switching and therefore supportive of foraging for work. Thus, it appears that in C. floridanus there is directionality to the mechanisms of task allocation. Switching to more dangerous tasks is age‐influenced, but switching to less dangerous tasks is age‐independent. The results also suggest that older workers are more flexible in their task choice behavior. Younger workers are more biased towards choosing within‐nest tasks. Finally, there are effects of previous experience that tend to keep ants in familiar tasks. Task allocation based on several mechanisms may balance between: (1) concentrating the most worn workers into the most dangerous tasks; (2) increasing task performance levels; and (3) maintaining behavioral flexibility to respond to demographic perturbations. The degree to which behavior is flexible may correlate to the frequency of such perturbations in a species.     

Insights into the catalytic mechanism of human sEH phosphatase by site-directed mutagenesis and LC-MS/MS analysis

We have recently reported that human soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a novel phosphatase enzymatic activity. Based on a structural relationship with other members of the haloacid dehalogenase superfamily, the sEH N-terminal phosphatase domain revealed four conserved sequence motifs, including the proposed catalytic nucleophile D9, and several other residues potentially implicated in substrate turnover and/or Mg²⁺ binding. To enlighten the catalytic mechanism of dephosphorylation, we constructed sEH phosphatase active-site mutants by site-directed mutagenesis. A total of 18 mutants were constructed and recombinantly expressed in Escherichia coli as soluble proteins, purified to homogeneity and subsequently analysed for their kinetic parameters. A replacement of residues D9, K160, D184 or N189 resulted in a complete loss of phosphatase activity, consistent with an essential function for catalysis. In contrast, a substitution of D11, T123, N124 and D185 leads to sEH mutant proteins with altered kinetic properties. We further provide evidence of the formation of an acylphosphate intermediate on D9 by liquid chromatography-tandem mass spectrometry based on the detection of homoserine after NaBH₄ reduction of the phosphorylated enzyme, which identifies D9 as the catalytic nucleophile. Surprisingly, we could only show such homoserine formation using the D11N mutant, which strongly suggests D11 to be involved in the acylphosphate hydrolysis. In the D11 mutant, the second catalytic step becomes rate limiting, which then allows trapping of the labile intermediate. Substrate turnover in the presence of ¹⁸H₂O revealed that the nucleophilic attack during the second reaction step occurs at the acylphosphate phosphorous. Based on these findings, we propose a two-step catalytic mechanism of dephosphorylation that involves the phosphate substrate hydrolysis by nucleophilic attack by the catalytic nucleophile D9 followed by hydrolysis of the acylphosphate enzyme intermediate supported by D11.     

Intrinsic and extrinsic causes of spatial variability across scales in a metacommunity

The relative importance of extrinsic and intrinsic causes of variability is among the oldest unresolved problems in ecology. However, the interaction between large-scale intrinsic variability in species abundance and environmental heterogeneity is still unknown. We use a metacommunity model with disturbance-recovery dynamics to resolve the interaction between scales of environmental heterogeneity, biotic processes and of intrinsic variability. We explain how population density increases with environmental variability only when its scale matches that of intrinsic patterns of abundance, through their ability to develop in heterogeneous environments. Succession dynamics reveals how the strength of local species interactions, through its control of intrinsic variability, can in turn control the scale of metapopulation response to environmental scales. Our results show that the environment and species density might fail to show any correlation despite their strong causal association. They more generally suggest that the spatial scale of ecological processes might not be sufficient to build a predictive framework for spatially heterogeneous habitats, including marine reserve networks.     

Chromatin assembly factor 1 regulates the cell cycle but not cell fate during male gametogenesis in Arabidopsis thaliana

The interdependence of cell cycle control, chromatin remodeling and cell fate determination remains unclear in flowering plants. Pollen development provides an interesting model, as it comprises only two cell types produced by two sequential cell divisions. The first division separates the vegetative cell from the generative cell. The generative cell divides and produces the two sperm cells, transported to the female gametes by the pollen tube produced by the vegetative cell. We show in Arabidopsis thaliana that loss of activity of the Chromatin assembly factor 1 (CAF1) pathway causes delay and arrest of the cell cycle during pollen development. Prevention of the second pollen mitosis generates a fraction of CAF1-deficient pollen grains comprising a vegetative cell and a single sperm cell, which both express correctly cell fate markers. The single sperm is functional and fertilizes indiscriminately either female gamete. Our results thus suggest that pollen cell fate is independent from cell cycle regulation.     

Effects of two types of neuromuscular electrical stimulation training on vertical jump performance

This study examined the effects of different types of neuromuscular electrical stimulation (NMES) programs on vertical jump performance. Twenty seven healthy trained male students in sports-sciences were recruited and randomized into three groups. The control group (C group, n = 8) did not perform NMES training. Two other groups underwent 3 training sessions a week over 5 weeks on the quadriceps femoris muscle [F group (n = 9): stimulation with an 80 Hz current for 15 min for improving muscle strength; E group (n = 10): stimulation with a 25 Hz current for 60 min for improving muscle endurance]. The height of the vertical jump was measured before NMES training (test 1), one week (test 2) and five weeks (test 3) after the end of the programs. The results showed that the height of the vertical jump significantly increased in both the F and E groups between tests 1 and 2 (5 cm and 3 cm respectively). Results of test 3 showed that both groups preserved their gains. A NMES training program destined to improve muscle endurance does not interfere on vertical jump performance. It can even durably enhance it in the same way as a NMES training program destined to improve muscle strength. Thus, to improve muscle endurance without deteriorating muscle power, sportsmen can use electrical stimulation.