MicroRNA-binding is required for recruitment of human Argonaute 2 to stress granules and P-bodies

Argonaute proteins are the core components of the RNA-induced silencing complex, the central effector of the mammalian RNA interference pathway. In the cytoplasm, they associate with at least two types of cytoplasmic RNA granules; processing bodies and stress granules, which function in mRNA degradation and translational repression, respectively. The significance of Argonaute association with these RNA granules is not entirely clear but it is likely related to their activities within the RNAi pathway. Understanding what regulates targeting of Argonautes to RNA granules may provide clues as to their functions at these organelles. To this end, there are a number of conflicting reports that describe the role of small RNAs in targeting Argonaute proteins in mammalian cells. We employed quantitative microscopic analyses of human Argonaute 2 (hAgo2) mutants to study factors that govern localization of this RNA-binding protein to cytoplasmic RNA granules. We report, for the first time, that hAgo2 is recruited to stress granules as a consequence of its interaction with miRNAs. Moreover, loading of small RNAs onto hAgo2 is not required for its stability, suggesting that a pool of unloaded hAgo2 may exist for extended periods of time in the cytoplasm.     

Cytotoxic effects of new trans-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines and their interaction with antitumoral drugs gemcitabine and paclitaxel on cellular lines of human breast cancer

Two tetrahydroquinoline compounds, called DM8 and DM12, from a new series of the cis-2,4-diaryl-r-3-methyl-1,2,3,4-tetrahydroquinolines, were selected for cytotoxic effects studies on cellular lines of human breast cancer. The synergistic, additive and antagonistic effects in combination of these compounds with anticancer drugs, such as paclitaxel and gemcitabine, were studied. The isobolograms and their analysis demonstrated models of synergism, additivity and antagonism of these tetrahydroquinolines in the presence of paclitaxel and gemcitabine. Results showed that compounds DM8 and DM12 individually induced growth inhibition on breast cancer cell lines MCF-7 and SKBR3, and the addition of paclitaxel and gemcitabine intensified their cytotoxic activity on both cell lines at conc. below 1 μg/mL. During these studies the compound DM12 was identified as new, perspective and safe agent for adjuvant therapy.     

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60(c-src) efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60(c-src), only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.     

Crystal structure of cardosin A, a glycosylated and Arg-Gly-Asp-containing aspartic proteinase from the flowers of Cynara cardunculus L.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manbeta4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two beta-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.     

Insights into the activation of brain serine racemase by the multi-PDZ domain glutamate receptor interacting protein, divalent cations and ATP

Brain serine racemase contains pyridoxal phosphate as a prosthetic group and is known to become activated by divalent cations such as Ca(2+) and Mg(2+), as well as by ATP and ADP. In vivo, brain serine racemase is also activated by a multi-PSD-95/discs large/ZO-1 (PDZ) domain glutamate receptor interacting protein (GRIP) that is usually coupled to the GluR2/3 subunits of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid Ca(2+) channel. In the present study, we analysed the mechanisms by which serine racemase becomes activated by GRIP, divalent cations and ATP. We show that binding of PDZ6 of GRIP to serine racemase does not result in increased d-serine production. However, full-length GRIP does augment significantly enzymatic activity. We expressed various GRIP shorter constructs to map down the regions within GRIP that are necessary for serine racemase activation. We observed that, whereas recombinant proteins containing PDZ4-PDZ5-PDZ6 are unable to activate serine racemase, other constructs containing PDZ4-PDZ5-PDZ6-GAP2-PDZ7 significantly augment its activity. Hence, activation of serine racemase by GRIP is not a direct consequence of the translocation towards the calcium channel but rather a likely conformational change induced by GRIP on serine racemase. On the other hand, the observed activation of serine racemase by divalent cations has been assumed to be a side-effect associated with ATP binding, which is known to form a complex with Mg(2+) ions. Because no mammalian serine racemase has yet been crystallized, we used molecular modelling based on yeast and bacterial homologs to demonstrate that the binding sites for Ca(2+), ATP and the PDZ6 domain of GRIP are spatially separated and modulate the enzyme through distinct mechanisms.     

Macroinvertebrate community dynamics in a temperate European Atlantic river. Do they conform to general ecological theory?

Spatial and temporal dynamics of macroinvertebrate communities have usually been linked to several environmental and anthropic factors. The aim of this study is to elucidate how important are these factors in structuring macroinvertebrate communities from temperate regions. Regarding the macroinvertebrate number of taxa, the Habitat Template Model, the Dynamic Equilibrium Hypothesis and the Intermediate Disturbance Hypothesis will be tested in order to know how important the diversity of instream elements and the hydrological disturbance frequency are in defining the macroinvertebrate taxonomic richness. Thus, the structure and composition of macroinvertebrate communities were analysed in nine sites of the Pas River basin, a temperate Atlantic basin in northern Spain, during winter, spring, summer and autumn 2005, together with water physicochemical and environmental characteristics. Macroinvertebrate abundance increased downstream and during summer, probably favoured by lower hydraulic stress and water organic enrichment. As predicts the Habitat Template Model, the macroinvertebrate number of taxa was related to habitat heterogeneity. However, no clear relationship amongst macroinvertebrate richness and water quality was found. The macroinvertebrate taxonomic richness did not correspond exactly with the Dynamic Equilibrium Hypothesis and the Intermediate Disturbance Hypothesis because it was relatively high in the absence of hydrological disturbances (summer). Thus, disturbance events may play a secondary role in determining the seasonal dynamic of the number of taxa. However, hydrological disturbances can be considered the most important factors explaining the seasonal pattern of macroinvertebrate abundance. On the other hand, spatial patterns of macroinvertebrate community structure and composition were mainly determined by resource availability, hydraulic conditions, habitat heterogeneity and human alterations, whilst hydrological predictability and resource availability might play a major role in determining seasonal dynamics.     

Functional and biophysical characterization of a hyperthermostable GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Thermotoga petrophila</Emphasis>

A hyperthermostable glycoside hydrolase family 51 (GH51) α-l-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-l-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.     

Carotenoid supplementation and sex-specific trade-offs between colouration and condition in common tern chicks

Carotenoids, as pigments with antioxidant and immunoregulatory properties, play a crucial role in developing chicks. Carotenoids must be acquired through diet and are relatively scarce, suggesting that their availability is a limiting factor leading to a trade-off between colour displays and physiological functions. However, potential differences in this trade-off between male and female chicks have been little studied. We manipulated carotenoid availability in 9 days old common tern Sterna hirundo chicks by supplementing their fish diet with four carotenoids during 9 days. Our aim was to examine sex-specific responses to the experimental increase of dietary carotenoids on plasma circulation, physiological and condition variables and successful fledging. Furthermore, to explore the functional and evolutionary basis of the trade-off, we studied the relationships among carotenoid concentration, mediated immune response and foot colouration. After treatment, control chicks showed decreasing plasma levels for most carotenoid types, whereas supplemented chicks had strong increases. Colour luminosity and saturation increased in both treatment groups, while hue only changed significantly towards redder feet in supplemented females. Supplemented chicks presented neither different T-cell-mediated immunity nor other differences compared to control chicks. Nevertheless, supplemented females showed tendencies towards decreased immune responses and increased δ¹⁵N signatures, and supplemented males towards greater body mass. Our results indicate colouration may have, in females, a signalling function as to compensate for immunological costs. In males, additional availability of carotenoids may contribute to improve the body condition. This study suggests that trade-off responses to carotenoid availability are sex-specific in tern chicks. Thus, parental carotenoid supply to chicks may be an unrecognised component in sex allocation.     

Neuroinfections in developed versus developing countries

In recent supplement of neuroendocrinology letters, first time the authors from West and East, North and South of EU and the "Third World" present data on neuroinfections in high technology society - on nosocomial meningitis and vice versa in low technology and income countries of sub-Saharan Africa. 14 years survey of 171 cases of nosocomial paediatric meningitis is presented by Rudinsky et al. [1] and subpopulations of Acinetobacter baumannii and Pseudomonas aeruginosa [1,2] within last 20 years are briefly analyzed by Huttova et al. [2] and Ondrusova et al. [3]. All cases were complicating high technology procedures, such as neurosurgery, very low birth weight neonates after shunt implants etc. Current problems of management of nosocomial meningitis are reviewed by Bauer et al. [4] and consequence of inappropriate therapy by Huttova et al. [5]. Another high technology associated infection is septic embolisation followed by brain abscess and meningitis in patients with endocarditis after cardiac surgery (Kovac et al.) [6]. Experience from more than 600 cases is discussed in the article by Karvaj et al. [7] who outlines extremely high mortality in patients with endocarditis embolizing to central nervous system - up to 60%. The rest of papers are in contrary to problems of neuroinfections in EU and US focused on meningitis and cerebral malaria as commonest neuroinfections in the third world: 261 cases of cerebral malaria are discussed in a brief research note by Sudanese team of tropical programme in area of famine and civil war in southern Sudan (Bartkovjak and Ianetti et al.) [8]. Fungal neuroinfections complicating AIDS are of decreasing trend as reported by Njambi et al. from Kenya [9] and data from 497 cases from Uganda, Ethiopia and Burundi are presented by Benca et al. [10]. Finally an outbreak of meningococcal meningitis is reported by Benca et al. [11] from meningitis belt in Darfur and southern Sudan. We hope that the supplement may show difference in etiology, risk factors, therapy and outcome of neuroinfections (which is a burning public health and social problem in tropics) in other third world countries versus developed high-tech medical settings of US, EU and other high income countries, as presented by Benca et al. [12].