The Adaptor 3BP2 Is Required for Early and Late Events in Fc{varepsilon}RI Signaling in Human Mast Cells

Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.     

Foliar Paclobutrazol Application Suppresses Olive Tree Growth While Promoting Fruit Set

In some agricultural practices, application of plant growth regulators is very useful as an efficient means to modulate olive tree vigour to optimize fruit production. However, the physiological mechanisms underlying these effects are still poorly understood. Here, we evaluated the effectiveness of paclobutrazol, chlormequat and prohexadione on the modulation of vegetative growth and fruit set, with a special emphasis on the underlying effects exerted by phytohormones, in one-year-old olive trees (Olea europea cv. Arbosana). Paclobutrazol and chlormequat were tested using both foliar spraying and soil irrigation, while prohexadione was applied by foliar spraying only, and all treatments were compared to a control. Among treatments, paclobutrazol and chlormequat were the most effective in promoting vegetative growth retardation, when applied through foliar spray. Growth retardation by foliar application of paclobutrazol was associated with proline accumulation and reduced gibberellin contents in leaves. Furthermore, hormonal profiling revealed that not only all treatments were effective in reducing the endogenous contents of gibberellins, but also that the entire hormonal profiling was altered upon treatments. It is concluded that (i) various plant growth regulators can be effectively used to control olive tree growth, and (ii) paclobutrazol seems to be more efficient on vegetative growth retardation than the other tested treatments, being foliar application more effective than soil treatment.

     

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background

Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E₂ (PGE₂) are probably also released and could explain the refractory period observed in patients with EIB.

Objective

This study aimed to evaluate the protective effect of PGE₂ on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.

Methods

We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE₂ and prostanoid receptor (EP) antagonists for EP_1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.

Results

Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE₂ significantly reduced mannitol-induced degranulation through EP₂ and EP₄ receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.

Conclusions

Our data show a protective role for the PGE₂ receptors EP₂ and EP₄ following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.     

Aclacinomycin 10-hydroxylase is a novel substrate-assisted hydroxylase requiring S-adenosyl-L-methionine as cofactor

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-L-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-epsilon-rhodomycin, a step in the biosynthesis of the polyketide antibiotic beta-rhodomycin. S-Adenosyl-L-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis. 18O2 experiments show that the hydroxyl group is derived from molecular oxygen. The reaction further requires thiol reagents such as glutathione or dithiothreitol. Incubation of the enzyme with substrate in the absence of reductant leads to the accumulation of an intermediate with a molecular mass consistent with a perhydroxy compound. This intermediate is turned into product upon addition of glutathione. The crystal structure of an abortive enzyme-AdoMet product ternary complex reveals large conformational changes consisting of a domain rotation leading to active site closure upon binding of the anthracycline ligand. The data suggest a mechanism where decarboxylation of the substrate results in the formation of a carbanion intermediate, which is stabilized by resonance through the aromatic ring system of the anthracycline substrate. The delocalization of the electrons is facilitated by the positive charge of the cofactor AdoMet. The activation of oxygen and formation of a hydroxyperoxide intermediate occurs in a manner similar to that observed in flavoenzymes. Aclacinomycin-10-hydroxylase is the first example of a AdoMet-dependent hydroxylation reaction, a novel function for this cofactor. The enzyme lacks methyltransferase activity due to the positioning of the AdoMet methyl group unfavorable for a SN2-type methyl transfer to the substrate.     

Contrasting phenotypic plasticity in the photoprotective strategies of the invasive species Carpobrotus edulis and the coexisting native species Crithmum maritimum

Photoprotective strategies vary greatly within the plant kingdom and reflect a plant's physiological status and capacity to cope with environment variations. The plasticity and intensity of these responses may determine plant success. Invasive species are reported to show increased vigor to displace native species. Describing the mechanisms that confer such vigor is essential to understanding the success of invasive species. We performed an experiment whereby two species were monitored: Carpobrotus edulis, an aggressive invasive species in the Mediterranean basin, and Crithmum maritimum, a coexisting native species in the Cap de Creus Natural Park (NE Spain). We analyzed their photoprotective responses to seasonal environmental dynamics by comparing the capacity of the invader to respond to the local environmental stresses throughout the year. Our study analyses ecophysiological markers and photoprotective strategies to gain an insight into the success of invaders. We found that both species showed completely different but effective photoprotective strategies: in summer, C. edulis took special advantage of the xanthophyll cycle, whereas the success of C. maritimum in summer stemmed from morphological changes and alterations on β‐carotene content. Winter also presented differences between the species, as the native showed reduced F_v/F_m ratios. Our experimental design allowed us to introduce a new approach to compare phenotypic plasticity: the integrated phenotypic plasticity index (PP_int), defined as the maximum Euclidian distance between phenotypes, using a combination of different variables to describe them. This index revealed significantly greater phenotypic plasticity in the invasive species compared to the native species.     

Physiological Responses in Relation to Performance during Competition in Elite Synchronized Swimmers

Purpose

We aimed to characterize the cardiovascular, lactate and perceived exertion responses in relation to performance during competition in junior and senior elite synchronized swimmers.

Methods

34 high level senior (21.4±3.6 years) and junior (15.9±1.0) synchronized swimmers were monitored while performing a total of 96 routines during an official national championship in the technical and free solo, duet and team competitive programs. Heart rate was continuously monitored. Peak blood lactate was obtained from serial capillary samples during recovery. Post-exercise rate of perceived exertion was assessed using the Borg CR-10 scale. Total competition scores were obtained from official records.

Results

Data collection was complete in 54 cases. Pre-exercise mean heart rate (beats·min⁻¹) was 129.1±13.1, and quickly increased during the exercise to attain mean peak values of 191.7±8.7, with interspersed bradycardic events down to 88.8±28.5. Mean peak blood lactate (mmol·L⁻¹) was highest in the free solo (8.5±1.8) and free duet (7.6±1.8) and lowest at the free team (6.2±1.9). Mean RPE (0–10+) was higher in juniors (7.8±0.9) than in seniors (7.1±1.4). Multivariate analysis revealed that heart rate before and minimum heart rate during the routine predicted 26% of variability in final total score.

Conclusions

Cardiovascular responses during competition are characterized by intense anticipatory pre-activation and rapidly developing tachycardia up to maximal levels with interspersed periods of marked bradycardia during the exercise bouts performed in apnea. Moderate blood lactate accumulation suggests an adaptive metabolic response as a result of the specific training adaptations attributed to influence of the diving response in synchronized swimmers. Competitive routines are perceived as very to extremely intense, particularly in the free solo and duets. The magnitude of anticipatory heart rate activation and bradycardic response appear to be related to performance variability.