Nectar yeasts of two southern Spanish plants: the roles of immigration and physiological traits in community assembly

Recent studies have shown that dense yeast populations often occurring in floral nectar are numerically dominated by a few species from the flower-insect interface specialized genus Metschnikowia, while generalist yeast species commonly occurring on leaf surfaces, soil, freshwater, and air were rarely isolated from nectar samples. This study was designed to understand the main factors responsible for the assembly of nectar yeast communities, by combining field experiments with laboratory tests characterizing the physiological abilities of all yeast species forming the pool of potential colonizers for two Spanish flowering plants (Digitalis obscura and Atropa baetica). Yeast frequency and species richness were assessed in external sources (bee glossae, air, plant phylloplane) as well as in pollinator rewards (pollen, nectar). Yeasts were most frequent in external sources (air, flower-visiting insects), less so in the proximate floral environment (phylloplane), and least in pollen and nectar. Nectar communities appeared to be considerably impoverished versions of those in insect glossae and phylloplane. Nectar, pollen, and insect yeast assemblages differed in physiological characteristics from those in other substrates. Nectarivorous Metschnikowia were not more resistant than other yeast species to plant secondary compounds and high sugar concentrations typical of nectar, but their higher growth rates may be decisive for their dominance in ephemeral nectar communities.     

A palmitoylation switch mechanism regulates Rac1 function and membrane organization

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.     

Mitochondrial reactive oxygen species and Ca2+ signaling

Mitochondria are an important source of reactive oxygen species (ROS) formed as a side product of oxidative phosphorylation. The main sites of oxidant production are complex I and complex III, where electrons flowing from reduced substrates are occasionally transferred to oxygen to form superoxide anion and derived products. These highly reactive compounds have a well-known role in pathological states and in some cellular responses. However, although their link with Ca²⁺ is well studied in cell death, it has been hardly investigated in normal cytosolic calcium concentration ([Ca²⁺]_i) signals. Several Ca²⁺ transport systems are modulated by oxidation. Oxidation increases the activity of inositol 1,4,5-trisphosphate and ryanodine receptors, the main channels releasing Ca²⁺ from intracellular stores in response to cellular stimulation. On the other hand, mitochondria are known to control [Ca²⁺]_i signals by Ca²⁺ uptake and release during cytosolic calcium mobilization, specially in mitochondria situated close to Ca²⁺ release channels. Mitochondrial inhibitors modify calcium signals in numerous cell types, including oscillations evoked by physiological stimulus. Although these inhibitors reduce mitochondrial Ca²⁺ uptake, they also impair ROS production in several systems. In keeping with this effect, recent reports show that antioxidants or oxidant scavengers also inhibit physiological calcium signals. Furthermore, there is evidence that mitochondria generate ROS in response to cell stimulation, an effect suppressed by mitochondrial inhibitors that simultaneously block [Ca²⁺]_i signals. Together, the data reviewed here indicate that Ca²⁺-mobilizing stimulus generates mitochondrial ROS, which, in turn, facilitate [Ca²⁺]_i signals, a new aspect in the biology of mitochondria. Finally, the potential implications for biological modeling are discussed. mitochondria; calcium     

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/α‐synuclein complex by Hip

The ATP‐dependent protein chaperone heat‐shock protein 70 (Hsp70) displays broad anti‐aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of α‐synuclein (αSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation‐prone polypeptides. We show a nucleotide‐dependent interaction between Hsp70 and αSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with αSyn. Such a co‐aggregation phenomenon can be prevented in vitro by the co‐chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of αSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.     

A Single Mutation at the Sheet Switch Region Results in Conformational Changes Favoring λ6 Light-Chain Fibrillogenesis

Systemic amyloid light-chain (LC) amyloidosis is a disease process characterized by the pathological deposition of monoclonal LCs in tissue. All LC subtypes are capable of fibril formation although λ chains, particularly those belonging to the λ6 type, are overrepresented. Here, we report the thermodynamic and in vitro fibrillogenic properties of several mutants of the λ6 protein 6aJL2 in which Pro7 and/or His8 was substituted by Ser or Pro. The H8P and H8S mutants were almost as stable as the wild-type protein and were poorly fibrillogenic. In contrast, the P7S mutation decreased the thermodynamic stability of 6aJL2 and greatly enhanced its capacity to form amyloid-like fibrils in vitro. The crystal structure of the P7S mutant showed that the substitution induced both local and long-distance effects, such as the rearrangement of the V_L (variable region of the light chain)-V_L interface. This mutant crystallized in two orthorhombic polymorphs, P2₁2₁2₁ and C222₁. In the latter, a monomer that was not arranged in the typical Bence-Jones dimer was observed for the first time. Crystal-packing analysis of the C222₁ lattice showed the establishment of intermolecular β-β interactions that involved the N-terminus and β-strand B and that these could be relevant in the mechanism of LC fibril formation. Our results strongly suggest that Pro7 is a key residue in the conformation of the N-terminal sheet switch motif and, through long-distance interactions, is also critically involved in the contacts that stabilized the V_L interface in λ6 LCs.     

Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D ,L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐L ‐amino acids as well as the known N‐carbamoyl‐L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐L ‐amino acids than for N‐carbamoyl and N‐acetyl‐L ‐derivatives, with a catalytic efficiency (k_cat/K_m) of 8.58 × 10⁵, 1.83 × 10⁴, and 1.78 × 10³ (s^?1 M^?1), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co²⁺, Mn²⁺, and Ni²⁺ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co²⁺‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

How reproductive,vegetative and defensive strategies of Mediterranean grassland species respond to a grazing intensity gradient

We analysed the morpho-functional response of grassland species to a grazing intensity gradient (1–3.5 sheep ha⁻¹) in the Mediterranean-type climate region of Chile. A total of nine morpho-functional traits (with a total of 24 attributes) were determined for 79 herbaceous plant species. Valuation of the traits enabled calculation of the reproductive, vegetative and defensive potentials for each species. A classification analysis for species x potentials identified five groups of plant functional strategies, and we analysed their responses along a grazing intensity gradient both for native and non-native species. The defensive potential of the species was negatively correlated with reproductive but was not significant in relation to vegetative potential. Grazing intensification favoured the presence of species with high defensive potential, to the detriment of those with high reproductive potential. This process affected both native and non-native species, but was more intense in the former presenting higher defensive potential. The functional group with a higher defensive strategy showed an increase in relative frequency with grazing intensity. However, self-defence alone is insufficient. This group also presents a certain reproductive potential that ensures the persistence of its annual or biennial species. The functional group combining high reproductive and vegetative potentials is the one that exhibits the biggest decrease in relative frequency due to intensified grazing. This group, however, is dominant in all the stocking treatments. All the functional groups identified include both native and non-native species, although the former dominate in those with greater defensive potential.