Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.     

Welwitschiaceae from the Lower Cretaceous of northeastern Brazil

Welwitschiaceae, a family in the Gnetales, is known today from only one extant species, Welwitschia mirabilis. This species is distributed in the Namibian desert, along the western coast of southern Africa, about 10 km inland from the coast. Very little is known about the fossil record of this family. Lower Cretaceous megafossils of various organs, assigned to Welwitschiaceae, are presented here. These fossils include young stems with paired cotyledons attached (Welwitschiella austroamericana n. gen. et sp.), isolated leaves (Welwitschiophyllum brasiliense n. gen. et sp.), and axes bearing male cones (Welwitschiostrobus murili n. gen. et sp.). They were collected in the Crato Formation, which is dated by palynomorphs and ostracods as Late Aptian (114 to 112 million years ago). These sediments are exposed in the Araripe Basin of northeastern Brazil. This study brings together new information of the megafossil record of Welwitschia-like plants and also reports of pollen said to be similar to that of Welwitschia from Lower Cretaceous sediments.     

Certainly can't live without this: SIRT6

Cellular metabolic rates might regulate aging by impinging on genomic stability through the DNA repair pathways. A new study published in Cell (Mostoslavsky et al., 2006) reports that deficiency in one of the mammalian Sir2 homologs, SIRT6, results in genome instability through the DNA base excision repair pathway and leads to aging-associated degenerative phenotypes.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Enzymatic antioxidant response of a labrid fish (Coris julis) liver to environmental caulerpenyne

Exposure of marine animals to certain toxic compounds can enhance reactive oxygen species production with subsequent damage to macromolecules and alterations in oxidant defenses levels. Caulerpenyne is the major metabolite synthesized by Caulerpa species, used as chemical defense affecting several cellular and molecular targets. We assessed the changes produced by the presence of Caulerpa spp. in the activities of antioxidant enzymes as well as lipid peroxidation levels in liver of the teleost Coris julis. Fish were captured at two stations with Caulerpa species-Caulerpa taxifolia and Caulerpa prolifera-and at a region with the seagrass Posidonia oceanica as negative control. Caulerpenyne concentration was significantly higher in C. prolifera than in C. taxifolia (p<0.05). Glutathione S-transferase, glutathione peroxidase and glutathione reductase activities were significantly higher in both Caulerpa stations compared to the P. oceanica (p<0.05). No statistical difference (p>0.05) existed in catalase activity between groups. Glutathione reductase activity is significantly higher in C. prolifera station than in C. taxifolia (p<0.05). Despite the variations in the antioxidant enzyme activities, there was no significant difference in malondialdehyde concentration. In conclusion, the production of caulerpenyne by Caulerpa species could induce an antioxidant adaptation in the liver of C. julis in order to prevent oxidative damage.     

Structural Characterization of Protein-Protein Complexes by Integrating Computational Docking with Small-angle Scattering Data

X-ray crystallography and NMR can provide detailed structural information of protein-protein complexes, but technical problems make their application challenging in the high-throughput regime. Other methods such as small-angle X-ray scattering (SAXS) are more promising for large-scale application, but at the cost of lower resolution, which is a problem that can be solved by complementing SAXS data with theoretical simulations. Here, we propose a novel strategy that combines SAXS data and accurate protein-protein docking simulations. The approach has been benchmarked on a large pool of known structures with synthetic SAXS data, and on three experimental examples. The combined approach (pyDockSAXS) provided a significantly better success rate (43% for the top 10 predictions) than either of the two methods alone. Further analysis of the influence of different docking parameters made it possible to increase the success rates for specific cases, and to define guidelines for improving the data-driven protein-protein docking protocols.     

IL-1 beta breaks tolerance through expansion of CD25+ effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.     

Exclusive frugivory and seed dispersal of Rhamnus alaternus in the bird breeding season

We studied avian frugivory and seed dispersal in a dioecious shrub, Rhamnus alaternus, focusing on the quantitative and qualitative components of effectiveness. The study took place at three locations in the northeast of the Iberian Peninsula, and examined bird behaviour, intensity of feeding, and the consequences for seedling emergence. The coincidence between the bird breeding season and fruit ripening of R. alaternus in the absence of other ripe fruit, generates a monospecific interaction. The extant frugivorous species were mainly legitimate seed dispersers and their abundance was low. Sylvia melanocephala and S. undata were the most important at one site whereas S. atricapilla, Erithacus rubecula and Turdus merula predominated at the other two sites. Fruit handling took place directly on the branches. Bird species used microhabitats differently as first post-feeding perch, which usually was a short distance away. The low density of frugivorous birds in all localities, among others factors, resulted in satiation of the disperser community and many mature fruits unconsumed. Both adults and juveniles feed upon the plants and their foraging patterns are similar. Adults of S. melanocephala were observed to feed fruit to nestlings and consequently a second phase of dispersal potentially arises from the transport of fecal sacs. Pulp removal and passage through the digestive tract increased the probability of seedling emergence. This plant-dispersal interaction has important consequences, both positive and negative for the plant. Positively, the fruiting of R. alaternus at a time when other ripe fruits are not available avoids interspecific competition for seed dispersers. In addition, a low density of seed rain may reduce intraspecific competition. Negatively, the low density and small size of the breeding frugivorous bird community limit fruit handling and removal away from the parent plants, while the territorial behaviour of birds at that time of the year reduces the potential distances of seed dispersal.     

Norepinephrine induces calcium spikes and proinflammatory actions in human hepatic stellate cells

Catecholamines participate in the pathogenesis of portal hypertension and liver fibrosis through alpha1-adrenoceptors. However, the underlying cellular and molecular mechanisms are largely unknown. Here, we investigated the effects of norepinephrine (NE) on human hepatic stellate cells (HSC), which exert vasoactive, inflammatory, and fibrogenic actions in the injured liver. Adrenoceptor expression was assessed in human HSC by RT-PCR and immunocytochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was studied in fura-2-loaded cells. Cell contraction was studied by assessing wrinkle formation and myosin light chain II (MLC II) phosphorylation. Cell proliferation and collagen-alpha1(I) expression were assessed by [3H]thymidine incorporation and quantitative PCR, respectively. NF-kappaB activation was assessed by luciferase reporter gene and p65 nuclear translocation. Chemokine secretion was assessed by ELISA. Normal human livers expressed alpha(1A)-adrenoceptors, which were markedly upregulated in livers with advanced fibrosis. Activated human HSC expressed alpha(1A)-adrenoceptors. NE induced multiple rapid [Ca2+]i oscillations (Ca2+ spikes). Prazosin (alpha1-blocker) completely prevented NE-induced Ca2+ spikes, whereas propranolol (nonspecific beta-blocker) partially attenuated this effect. NE caused phosphorylation of MLC II and cell contraction. In contrast, NE did not affect cell proliferation or collagen-alpha1(I) expression. Importantly, NE stimulated the secretion of inflammatory chemokines (RANTES and interleukin-8) in a dose-dependent manner. Prazosin blocked NE-induced chemokine secretion. NE stimulated NF-kappaB activation. BAY 11-7082, a specific NF-kappaB inhibitor, blocked NE-induced chemokine secretion. We conclude that NE stimulates NF-kappaB and induces cell contraction and proinflammatory effects in human HSC. Catecholamines may participate in the pathogenesis of portal hypertension and liver fibrosis by targeting HSC.