CaV1.3 as pacemaker channels in adrenal chromaffin cells: specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca (2+) -dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3 (-/-) KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

Comparative landscape genetics of pond‐breeding amphibians in Mediterranean temporal wetlands: The positive role of structural heterogeneity in promoting gene flow

Comparative landscape genetics studies can provide key information to implement cost‐effective conservation measures favouring a broad set of taxa. These studies are scarce, particularly in Mediterranean areas, which include diverse but threatened biological communities. Here, we focus on Mediterranean wetlands in central Iberia and perform a multi‐level, comparative study of two endemic pond‐breeding amphibians, a salamander (Pleurodeles waltl) and a toad (Pelobates cultripes). We genotyped 411 salamanders from 20 populations and 306 toads from 16 populations at 18 and 16 microsatellite loci, respectively, and identified major factors associated with population connectivity through the analysis of three sets of variables potentially affecting gene flow at increasingly finer levels of spatial resolution. Topographic, land use/cover, and remotely sensed vegetation/moisture indices were used to derive optimized resistance surfaces for the two species. We found contrasting patterns of genetic structure, with stronger, finer scale genetic differentiation in Pleurodeles waltl, and notable differences in the role of fine‐scale patterns of heterogeneity in vegetation cover and water content in shaping patterns of regional genetic structure in the two species. Overall, our results suggest a positive role of structural heterogeneity in population connectivity in pond‐breeding amphibians, with habitat patches of Mediterranean scrubland and open oak woodlands (“dehesas”) facilitating gene flow. Our study highlights the usefulness of remotely sensed continuous variables of land cover, vegetation and water content (e.g., NDVI, NDMI) in conservation‐oriented studies aimed at identifying major drivers of population connectivity.     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Site-directed mutagenesis study of the microenvironment characteristics of Lys213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn(2+) as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. Nature Struct. Biol. 4 (1997) 990-994] shows that Lys(213) is one of the ligands to Mn(2+) at the enzyme active site. Coordination of Mn(2+) to a lysyl residue is infrequent and suggests a low pK(a) value for the epsilon-NH(2) group of Lys(213). In this work, we evaluate the role of neighboring Phe(416) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe416Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn(2+), suggesting an increase of the pK(a) of Lys(213). A study of the effect of pH on K(m) for Mn(2+) indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK(a) of 7.1+/-0.2, compatible with the low pK(a) expected for Lys(213). This pK(a) value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys(213) microenvironment. Theoretical calculations of the pK(a) of Lys(213) indicate a value of 6.5+/-0.9, and it increases to 8.2+/-1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol(-1) in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe(416) to Arg(70), a residue previously shown to be important for PEP binding.     

Adaptive foraging of leaf‐cutter ants to spatiotemporal changes in resource availability in Neotropical savannas

1. Generalist herbivores feed on a wide and diverse set of species, but fine‐scale foraging patterns may be affected by the interplay between the quality, quantity and spatial distribution of host plants. 2. The foraging patterns of a prevalent Neotropical herbivore, the leaf‐cutter ant Atta laevigata, in the Brazilian Cerrado savannas were examined in order to determine if patterns observed are in concert with central‐place foraging predictions. 3. The results showed that A. laevigata acts as a polyphagous but highly selective herbivore, with ant attacks often resulting in partial defoliation of less‐preferred species and full defoliation of preferred ones. It was found, for the first time, that there is a strong and positive relationship between the relative attack frequency on plants from preferred species and foraging distance to the nest. This suggests a balance between the quality of plant resources harvested and costs involved in their transportation. It was also observed that colonies focused their harvest on preferred species in months with low availability of young leaves. Consequently, high herbivory rate was more frequent in plants attacked far away from the nest and in dry months. 4. These assessments highlight the fact that Atta colonies may become more selective as foraging distance to the nest increases and in response to fluctuations in the availability of palatable resources throughout the year. The results also show some dissimilarities in the foraging behaviours of A. laevigata when compared with other locations, suggesting that widely distributed herbivores may modify foraging strategies across their geographic range.     

Water permeability and lipid composition of toad urinary bladder: The influence of temperature

Summary The water diffusional permeability, its activation energy and the lipid composition were studied in urinary bladders from toads adapted to different temperatures. It was observed that the unidirectional water flux greatly depends on the temperature at which the experiments are performed. This dependence is greater in the animals adapted to higher temperatures. Toads adapted to cold show strong reduction in the activation energy for water diffusion permeability (from 11.4±1.9 kcal·mol^–1 to 4.4±1.1 kcal·mol^–1) and an increase of 30% in the amount of total lipids from bladder epithelial cells. There were no significant changes in the phospholipid/cholesterol ratio, composition of the paraffinic chains or protein concentration between toads adapted to both temperatures. The possibility that water translocates through the mucosal border of the toad bladder by partitioning in the polar zone and diffusioning between the hydrocarbon chains of the membrane lipids and that cold adaptation would induce a stronger packing of lipids in the membrane is discussed.