Linking life history traits in successive phases of a complex life cycle: effects of larval biomass on early juvenile development in an estuarine crab, Chasmagnathus granulata

In marine benthic invertebrates with complex life cycles, recruitment success, juvenile survival, and growth may be affected by variation in both maternal factors and environmental conditions prevailing during preceding embryonic or larval development. In an estuarine crab, Chasmagnathus granulata, previous investigations have shown that initial larval biomass is positively correlated with the biomass of recently extruded eggs, and it depends also on the salinity experienced during embryogenesis. Biomass at hatching has consequences for the subsequent larval development which, in this species, comprises two alternative developmental pathways with four or five zoeal instars (short or long pathway) and a megalopa. Larvae hatching with a lower than average biomass tend to develop through the long pathway and metamorphose to megalopae with higher biomass. In the present study, we show experimentally that the long pathway produces also significantly larger juveniles (crab size measured as carapace width, biomass as dry mass, carbon and nitrogen contents). Compared with juveniles originating from the short pathway, those from the long pathway showed in successive instars longer moulting cycles and larger carapace width, but lower size increments at ecdysis. In consequence, differences in size or biomass of long pathway vs short pathway crabs tended to disappear in later instars (after stage V). Furthermore, we tested in juveniles the tolerance of starvation at three salinities (5‰, 15‰, 32‰). Tolerance of starvation was significantly higher in juveniles originating from the long pathway, indicating higher energy reserves. While salinity played only a minor role for survival, it exerted significant effects on the time of moulting to the second juvenile instar, regardless of the preceding developmental pathway. The biomass of first juveniles obtained from the short pathway showed a significant positive correlation with the biomass of the freshly hatched zoea I, but not in those from the long pathway. In conclusion, the fitness of juvenile C. granulata is linked with previous developmental processes and environmental conditions during the embryonic and larval phase. Hence, a better understanding and prediction of the recruitment success of marine benthic invertebrates with a complex life cycle may require more comprehensive life‐history investigations.     

Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans

His6-tagged xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotope effect when [8-2H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, alphaKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/alphaKG dioxygenase.     

Calcium occlusion in plasma membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.     

The use of an unoccupied aerial vehicle to survey shark species over sand and rocky-reef habitats in a marine protected area

Cabo Pulmo National Park was established in 1995 and has since seen a large increase in fish biomass. An unoccupied aerial vehicle (UAV) was used to survey shallow coastal habitat in which lemon sharks (Negaprion brevirostris), bull sharks (Carcharhinus leucas) and Pacific nurse sharks (Ginglymostoma unami) were recorded. Sharks were more common in the afternoon, potentially using warmer shallow areas to behaviourally thermoregulate. This study highlights UAV surveying to be a viable tool for species identification, a limitation of previous terrestrial surveys conducted in the area.     

The Light Chain 1 Subunit of the Microtubule-Associated Protein 1B (MAP1B) Is Responsible for Tiam1 Binding and Rac1 Activation in Neuronal Cells

Microtubule-associated protein 1B (MAP1B) is a neuronal protein involved in the stabilization of microtubules both in the axon and somatodendritic compartments. Acute, genetic inactivation of MAP1B leads to delayed axonal outgrowth, most likely due to changes in the post-translational modification of tubulin subunits, which enhances microtubule polymerization. Furthermore, MAP1B deficiency is accompanied by abnormal actin microfilament polymerization and dramatic changes in the activity of small GTPases controlling the actin cytoskeleton. In this work, we showed that MAP1B interacts with a guanine exchange factor, termed Tiam1, which specifically activates Rac1. These proteins co-segregated in neurons, and interact in both heterologous expression systems and primary neurons. We dissected the molecular domains involved in the MAP1B-Tiam1 interaction, and demonstrated that pleckstrin homology (PH) domains in Tiam1 are responsible for MAP1B binding. Interestingly, only the light chain 1 (LC1) of MAP1B was able to interact with Tiam1. Moreover, it was able to increase the activity of the small GTPase, Rac1. These results suggest that the interaction between Tiam1 and MAP1B, is produced by the binding of LC1 with PH domains in Tiam1. The formation of such a complex impacts on the activation levels of Rac1 confirming a novel function of MAP1B related with the control of small GTPases. These results also support the idea of cross-talk between cytoskeleton compartments inside neuronal cells.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Implication of new atherosclerotic carotid plaques in the cardiovascular outcome of patients with rheumatoid arthritis

Early detection of atherosclerosis is of major importance to reduce the increased incidence of cardiovascular (CV) complications observed in patients with rheumatoid arthritis (RA). Prospective studies have shown that an abnormally increased carotid intima-media thickness and the presence of plaques assessed by carotid ultrasound are good markers to predict the development of CV events in these patients. Age, classic CV risk factors, and corticosteroid use are also predictors of new plaque formation in patients with RA. Active treatment of the disease may decrease the inflammatory burden, leading to a reduction in the progression of subclinical atherosclerosis in these patients.