Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signalling

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

Perennial legumes native to Australia: a preliminary investigation of nutritive value and response to cutting

Six Australian native herbaceous perennial legumes (Lotus australis, Swainsona colutoides, Swainsona swainsonioides, Cullen tenax, Glycine tabacina and Kennedia prorepens) were assessed in the glasshouse for nutritive value, soluble condensed tannins and production of herbage in response to three cutting treatments (regrowth harvested every 4 and 6 weeks and plants left uncut for 12 weeks). The Mediterranean perennial legumes Medicago sativa and Lotus corniculatus were also included. Dry matter (DM) yield of some native legumes was comparable to L. corniculatus, but M. sativa produced more DM than all species except S. swainsonioides after 12 weeks of regrowth. Dry matter yield of all native legumes decreased with increased cutting frequency, indicating a susceptibility to frequent defoliation. Shoot in vitro dry matter digestibility (DMD) was high (>70%) in most native legumes, except G. tabacina (65%) and K. prorepens (55%). Crude protein ranged from 21-28% for all legumes except K. prorepens (12%). More frequent cutting resulted in higher DMD and crude protein in all species, except for the DMD of C. tenax and L. australis, which did not change. Concentrations of soluble condensed tannins were 2-9 g/kg DM in the Lotus spp., 10-18 g/kg DM in K. prorepens and negligible (<1 g/kg) in the other legumes. Of the native species, C. tenax, S. swainsonioides and L. australis showed the most promise for use as forage plants and further evaluation under field conditions is now warranted.

Calcium Flux in Neuroblastoma Cells Is a Coupling Mechanism between Non-Genomic and Genomic Modes of Estrogens

Estrogens have been demonstrated to rapidly modulate calcium levels in a variety of cell types. However, the significance of estrogen-mediated calcium flux in neuronal cells is largely unknown. The relative importance of intra- and extracellular sources of calcium in estrogenic effects on neurons is also not well understood. Previously, we have demonstrated that membrane-limited estrogens, such as E-BSA given before an administration of a 2-hour pulse of 17beta-estradiol (E(2)), can potentiate the transcription mediated by E(2) from a consensus estrogen response element (ERE)-driven reporter gene. Inhibitors to signal transduction cascades given along with E-BSA or E(2) demonstrated that calcium flux is important for E-BSA-mediated potentiation of transcription in a transiently transfected neuroblastoma cell line. In this report, we have used inhibitors to different voltage-gated calcium channels (VGCCs) and to intracellular store receptors along with E-BSA in the first pulse or with E(2) in the second pulse to investigate the relative importance of these channels to estrogen-mediated transcription. Neither L- nor P-type VGCCs seem to play a role in estrogen action in these cells; while N-type VGCCs are important in both the non-genomic and genomic modes of estrogen action. Specific inhibitors also showed that the ryanodine receptor and the inositol trisphosphate receptor are important to E-BSA-mediated transcriptional potentiation. This report provides evidence that while intracellular stores of calcium are required to couple non-genomic actions of estrogen initiated at the membrane to transcription in the nucleus, extracellular sources of calcium are also important in both non-genomic and genomic actions of estrogens. Copyright (c) 2005 S. Karger AG, Basel.

Alfven waves as a driving mechanism in stellar winds

Alfven waves have been invoked as an important mechanism of particle acceleration in stellar winds of cool stars. After their identification in the solar wind they started to be studied in winds of stars located in different regions of the FIR diagram. We discuss here some characteristics of these waves and we present a direct application in the acceleration of late-type stellar winds. (C) 2009 COSPAR. Published by Elsevier Ltd. All rights reserved.

A mechanism for the formation of oxygen and iron bimodal radial distribution in the disc of our Galaxy

Recently, it has been proposed that there are two type Ia supernova progenitors: short-lived and long-lived. On the basis of this idea, we develop a theory of a unified mechanism for the formation of the bimodal radial distribution of iron and oxygen in the Galactic disc. The underlying cause for the formation of the fine structure of the radial abundance pattern is the influence of the spiral arms, specifically the combined effect of the corotation resonance and turbulent diffusion. From our modelling, we conclude that in order to explain the bimodal radial distributions simultaneously for oxygen and iron and to obtain approximately equal total iron output from different types of supernovae, the mean ejected iron mass per supernova event should be the same as quoted in the literature if the maximum mass of stars, which eject heavy elements, is 50 M(circle dot). For the upper mass limit of 70 M(circle dot), the production of iron by a type II supernova explosion should increase by about 1.5 times.

Trophallaxis in Dehydrated Leaf Cutting Colonies of Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Trophallaxis, the transfer of liquid among individuals by oral regurgitation or anal deposition, occurs in many insect groups including ants. The first indication that trophallaxis could occur in leaf cutting ants (Atta sexdens rubropilosa) was made by Autuori in 1942. He reported water collection by this ant species, and highlighted what in those days was an undescribed behavior for this species. In 2005, Da-Silva and Ribeiro presented preliminary results suggesting the existence of trophallaxis in A. sexdens rubropilosa. Here we report on a formal test of the hypothesis of trophallaxis in that species. Our approach was to test ant pairs in which only one individual (Group I) had access to blue-dyed water and the other individual (Group II), a nest-mate, came from a colony dehydrated by offering dry crushed corn for fungal growth. Positive results for trophallaxis were obtained in ants from four colonies and accounted for 33%-46% of all tests in which ants from Group I drank dyed water. These results indicate that trophallaxis occurs in this species.

Colony dehydration and water collection by specialized caste in the leaf-cutting ant Atta sexdens rubropilosa

Social organization enables leaf-cutting ants to keep appropriate micro-ecological nest conditions for the fungus garden (their main food), eggs, larvae and adults. To maintain stability while facing changing conditions, individual ants must perceive destabilising factors and produce a proper behavioral response. We investigated behavioral responses to experimental dehydration in leaf-cutting ants to verify if task specialization exists, and to quantify the ability of ant sub-colonies for water management. Our setup consisted of fourteen sub-colonies, ten of which were randomly assigned to different levels of experimental dehydration with silica gel, whereas the remaining four were controls. The ten experimental sub-colonies were split into two groups, so that five of them had access to water. Diverse ant morphs searched for water in dehydrated colonies, but mainly a caste of small ants collected water after sources had been discovered. Size specialization for water collection was replicable in shorter experiments with three additional colonies. Ants of dehydrated colonies accumulated leaf-fragments on the nest entrance, and covering the fungus garden. Behaviors that may enhance humidity within the nests were common to all dehydration treatments. Water availability increased the life span of dehydrated colonies.

Structural and Biochemical Characterization of Peroxiredoxin Q beta from Xylella fastidiosa CATALYTIC MECHANISM AND HIGH REACTIVITY

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 107 and 106 M(-1) s(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie similar to 12.3 angstrom apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro

Muscarinic (mAChRs) and nicotinic acetylcholine receptors (nAChRs) are involved in various physiological processes, including neuronal development. We provide evidence for expression of functional nicotinic and muscarinic receptors during differentiation of P19 carcinoma embryonic cells, as an in vitro model of early neurogenesis. We have detected expression and activity alpha(2)-alpha(7), beta(2), beta(4) nAChR and M1-M5 mAChR subtypes during neuronal differentiation. Nicotinic alpha(3) and beta(2) mRNA transcription was induced by addition of retinoic acid to P19 cells. Gene expression Of alpha(2), alpha(4)-alpha(7), beta(4) nAChR subunits decreased during initial differentiation and increased again when P19 cells underwent final maturation. Receptor response in terms of nicotinic agonist-evoked Ca2+, flux was observed in embryonic and neuronal-differentiated cells. Muscarinic receptor response, merely present in undifferentiated P19 cells, increased during neuronal differentiation. The nAChR-induced elevation of intracellular calcium ([Ca2+](i)) response in undifferentiated cells was due to Ca2+ influx. In differentiated P19 neurons the nAChR-induced [Ca2+](i) response was reduced following pretreatment with ryanodine, while the mAChR-induced response was unaffected indicating the contribution of Ca2+ release from ryanodine-sensitive stores to nAChR- but not mAChR-mediated Ca2+ responses. The presence of functional nAChRs in embryonic cells suggests that these receptors are involved in triggering Ca2+ waves during initial neuronal differentiation. (C) 2007 Elsevier Ltd. All rights reserved.

Important GABAergic mechanism within the NTS and the control of sympathetic baroreflex in SHR

Inhibitory neurotransmission has an important role in the processing of sensory afferent signals in the nucleus of the solitary tract (NTS), particularly in spontaneously hypertensive rats (SHR). In the present study, we tested the hypothesis that gamma-aminobutyric acid (GABA) mediated neurotransmission within the NTS produces an inhibition of the baroreflex response of splanchnic sympathetic nerve discharge (sSND). In urethane-anesthetized, artificially ventilated and vagotomized male SHR and Wistar Kyoto (WKY) rats we compared baroreflex-response curves evoked after bilateral injections into the NTS of the GABA-A antagonist bicuculline (25 pmol/50 nl) or the GABA-B antagonist CGP 35348 (5 nmol/50 nl). Baseline MAP in SHR was higher than the WKY rats (SHR: 153+/-5, vs. WKY: 112+/-6 mm Hg, p<0.05). Bilateral injection of bicuculline or CGP 35348 into the NTS induced a transient (5 min) reduction in MAP (Delta = -26+/-4 and -41+/-6 mm Hg, respectively vs. saline Delta = +4+/-3 mm Hg, p<0.05) and sSND (Delta = -21+/-13 and -78+/-7%, respectively vs. saline: Delta = +6+/-4% p<0.05). Analysis of the baroreceptor curve revealed a decrease in the lower plateau (43+/-11 and 15+/-5%, respectively vs. saline: 78+/-6%, p<0.05) and an increase in the sympathetic gain of baroreflex (6.3+/-0.3, 7.2+/-0.8% respectively vs. saline: 4.2+/-0.4%, p<0.05). Bicuculline or CGP35348 into the NTS in WKY rats did not change MAP, sSND and sympathetic baroreflex gain. These data indicate that GABAergic mechanisms within the NTS act tonically reducing sympathetic baroreflex gain in SHR. Crown Copyright (C) 2010 Published by Elsevier By. All rights reserved.

Three-dimensional Reconstruction of Ovaries of Leaf-cutting Ant (Atta sexdens rubropilosa) Queens (Hymenoptera: Formicidae)

In this study, the ovary morphology of newly emerged ant queens of Atta sexdens rubropilosa was studied in whole mount preparations by confocal microscopy. The ovaries are composed of approximately 40 ovarioles, showing non-synchronic oocyte maturation. The terminal filament with clusters of undifferentiated cells was found at the distal end of the ovarioles. Next to this region is the germarium, composed of several elongated cystocytes interconnected by cytoplasmic bridges. The nurse cells (23-28 cells) result from asymmetric mitosis. Cytoskeleton analysis showed F-actin concentrated at the muscle cells of the external tunica and in fusomes inside the ovarioles. Microtubules were concentrated around the nuclei of the nurse and follicular cells. In contrast, the oocytes and the external tunica showed faint staining for tubulin.

Celecoxib prevents tumor growth in an animal model by a COX-2 independent mechanism

Nonsteroidal antiinflammatory drugs (NSAIDs) have been shown to reduce cell growth in several tumors. Among these possible antineoplastic drugs are cyclooxygenase-2 (COX-2)-selective drugs, such as celecoxib, in which antitumoral mechanisms were evaluated in rats bearing Walker-256 (W256) tumor. W256 carcinosarcoma cells were inoculated subcutaneously (10(7) cells/rat) in rats submitted to treatment with celecoxib (25 mg kg(-1)) or vehicle for 14 days. Tumor growth, body-weight gain, and survival data were evaluated. The mechanisms, such as COX-2 expression and activity, oxidative stress, by means of enzymes and lipoperoxidation levels, and apoptosis mediators were also investigated. A reduction in tumor growth and an increased weight gain were observed. Celecoxib provided a higher incidence of survival compared with the control group. Cellular effects are probably COX-2 independent, because neither enzyme expression nor its activity, measured by tumoral PGE(2), showed significant difference between groups. It is probable that this antitumor action is dependent on an apoptotic way, which has been evaluated by the expression of the antiapoptotic protein Bcl-xL, in addition to the cellular changes observed by electronic microscopy. Celecoxib has also a possible involvement with redox homeostasis, because its administration caused significant changes in the activity of oxidative enzymes, such as catalase and superoxide dismutase. These results confirm the antitumor effects of celecoxib in W256 cancer model, contributing to elucidating its antitumoral mechanism and corroborating scientific literature about its effect on other types of cancer.

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.