873 resultados para Hybrid Floor Plate System


Relevância:

30.00% 30.00%

Publicador:

Resumo:

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein–protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein–protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These “nanodroplets” contain defined media, cells, and one or more beads containing ≈100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered Saccharomyces cerevisiae cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein–protein interactions in living cells or organisms.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Protein–protein interactions play crucial roles in the execution of various biological functions. Accordingly, their comprehensive description would contribute considerably to the functional interpretation of fully sequenced genomes, which are flooded with novel genes of unpredictable functions. We previously developed a system to examine two-hybrid interactions in all possible combinations between the ≈6,000 proteins of the budding yeast Saccharomyces cerevisiae. Here we have completed the comprehensive analysis using this system to identify 4,549 two-hybrid interactions among 3,278 proteins. Unexpectedly, these data do not largely overlap with those obtained by the other project [Uetz, P., et al. (2000) Nature (London) 403, 623–627] and hence have substantially expanded our knowledge on the protein interaction space or interactome of the yeast. Cumulative connection of these binary interactions generates a single huge network linking the vast majority of the proteins. Bioinformatics-aided selection of biologically relevant interactions highlights various intriguing subnetworks. They include, for instance, the one that had successfully foreseen the involvement of a novel protein in spindle pole body function as well as the one that may uncover a hitherto unidentified multiprotein complex potentially participating in the process of vesicular transport. Our data would thus significantly expand and improve the protein interaction map for the exploration of genome functions that eventually leads to thorough understanding of the cell as a molecular system.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

We used a genetic method, the yeast substrate-trapping system, to identify substrates for protein tyrosine phosphatases ζ (PTPζ/RPTPβ). This method is based on the yeast two-hybrid system, with two essential modifications: conditional expression of protein tyrosine kinase v-src (active src) to tyrosine-phosphorylate the prey proteins and screening by using a substrate-trap mutant of PTPζ (PTPζ-D1902A) as bait. By using this system, several substrate candidates for PTPζ were isolated. Among them, GIT1/Cat-1 (G protein-coupled receptor kinase-interactor 1/Cool-associated, tyrosine-phosphorylated 1) was examined further. GIT1/Cat-1 bound to PTPζ-D1902A dependent on the substrate tyrosine phosphorylation. Tyrosine-phosphorylated GIT1/Cat-1 was dephosphorylated by PTPζ in vitro. Immunoprecipitation experiments indicated that PTPζ-D1902A and GIT1/Cat-1 form a stable complex also in mammalian cells. Immunohistochemical analyses revealed that PTPζ and GIT1/Cat-1 were colocalized in the processes of pyramidal cells in the hippocampus and neocortex in rat brain. Subcellular colocalization was further verified in the growth cones of mossy fibers from pontine explants and in the ruffling membranes and processes of B103 neuroblastoma cells. Moreover, pleiotrophin, a ligand for PTPζ, increased tyrosine phosphorylation of GIT1/Cat-1 in B103 cells. All these results indicate that GIT1/Cat-1 is a substrate molecule of PTPζ.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

The enzyme 4-coumarate:coenzyme A ligase (4CL) is important in providing activated thioester substrates for phenylpropanoid natural product biosynthesis. We tested different hybrid poplar (Populus trichocarpa × Populus deltoides) tissues for the presence of 4CL isoforms by fast-protein liquid chromatography and detected a minimum of three 4CL isoforms. These isoforms shared similar hydroxycinnamic acid substrate-utilization profiles and were all inactive against sinapic acid, but instability of the native forms precluded extensive further analysis. 4CL cDNA clones were isolated and grouped into two major classes, the predicted amino acid sequences of which were 86% identical. Genomic Southern blots showed that the cDNA classes represent two poplar 4CL genes, and northern blots provided evidence for their differential expression. Recombinant enzymes corresponding to the two genes were expressed using a baculovirus system. The two recombinant proteins had substrate utilization profiles similar to each other and to the native poplar 4CL isoforms (4-coumaric acid > ferulic acid > caffeic acid; there was no conversion of sinapic acid), except that both had relatively high activity toward cinnamic acid. These results are discussed with respect to the role of 4CL in the partitioning of carbon in phenylpropanoid metabolism.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

We describe a system of hybrid dysgenesis in Drosophila virilis in which at least four unrelated transposable elements are all mobilized following a dysgenic cross. The data are largely consistent with the superposition of at least three different systems of hybrid dysgenesis, each repressing a different transposable element, which break down following the hybrid cross, possibly because they share a common pathway in the host. The data are also consistent with a mechanism in which mobilization of a single element triggers that of others, perhaps through chromosome breakage. The mobilization of multiple, unrelated elements in hybrid dysgenesis is reminiscent of McClintock's evidence [McClintock, B. (1955) Brookhaven Symp. Biol. 8, 58-74] for simultaneous mobilization of different transposable elements in maize.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Nanomedicine is a new branch of medicine, based on the potentiality and intrinsic properties of nanomaterials. Indeed, the nanomaterials ( i.e. the materials with nano and under micron size) can be suitable to different applications in biomedicine. The nanostructures can be used by taking advantage of their properties (for example superparamagnetic nanoparticles) or functionalized to deliver the drug in a specific target, thanks the ability to cross biological barriers. The size and the shape of 1D-nanostructures (nanotubes and nanowires) have an important role on the cell fate: their morphology plays a key role on the interaction between nanostructure and the biological system. For this reason the 1D nanostructure are interesting for their ability to mime the biological system. An implantable material or device must therefore integrate with the surrounding extracellular matrix (ECM), a complex network of proteins with structural and signaling properties. Innovative techniques allow the generation of complex surface patterns that can resemble the structure of the ECM, such as 1D nanostructures. NWs based on cubic silicon carbide (3C-SiC), either bare (3C-SiC NWs) or surrounded by an amorphous shell (3C-SiC/SiO2 core/shell NWs), and silicon oxycarbide nanowires (SiOxCy NWs) can meet the chemical, mechanical and electrical requirements for tissue engineering and have a strong potential to pave the way for the development of a novel generation of implantable nano-devices. Silicon oxycarbide shows promising physical and chemical properties as elastic modulus, bending strength and hardness, chemical durability superior to conventional silicate glasses in aggressive environments and high temperature stability up to 1300 °C. Moreover, it can easily be engineered through functionalization and decoration with macro-molecules and nanoparticles. Silicon carbide has been extensively studied for applications in harsh conditions, as chemical environment, high electric field and high and low temperature, owing to its high hardness, high thermal conductivity, chemical inertness and high electron mobility. Also, its cubic polytype (3C) is highly biocompatible and hemocompatible, and some prototypes of biomedical applications and biomedical devices have been already realized starting from 3C-SiC thin films. Cubic SiC-based NWs can be used as a biomimetic biomaterial, providing a robust and novel biocompatible biological interface . We cultured in vitro A549 human lung adenocarcinoma epithelial cells and L929 murine fibroblast cells over core/shell SiC/SiO2, SiOxCy and bare 3C-SiC nanowire platforms, and analysed the cytotoxicity, by indirect and direct contact tests, the cell adhesion, and the cell proliferation. These studies showed that all the nanowires are biocompatible according to ISO 10993 standards. We evaluated the blood compatibility through the interaction of the nanowires with platelet rich plasma. The adhesion and activation of platelets on the nanowire bundles, assessed via SEM imaging and soluble P-selectin quantification, indicated that a higher platelet activation is induced by the core/shell structures compared to the bare ones. Further, platelet activation is higher with 3C-SiC/SiO2 NWs and SiOxCyNWs, which therefore appear suitable in view of possible tissue regeneration. On the contrary, bare 3C-SiC NWs show a lower platelet activation and are therefore promising in view of implantable bioelectronics devices, as cardiovascular implantable devices. The NWs properties are suitable to allow the design of a novel subretinal Micro Device (MD). This devices is based on Si NWs and PEDOT:PSS, though the well know principle of the hybrid ordered bulk heterojunction (OBHJ). The aim is to develop a device based on a well-established photovoltaic technology and to adapt this know-how to the prosthetic field. The hybrid OBHJ allows to form a radial p–n junction on a nanowire/organic structure. In addition, the nanowires increase the light absorption by means of light scattering effects: a nanowires based p-n junction increases the light absorption up to the 80%, as previously demonstrated, overcoming the Shockley-Queisser limit of 30 % of a bulk p-n junction. Another interesting employment of these NWs is to design of a SiC based epicardial-interacting patch based on teflon that include SiC nanowires. . Such contact patch can bridge the electric conduction across the cardiac infarct as nanowires can ‘sense’ the direction of the wavefront propagation on the survival cardiac tissue and transmit it to the downstream surivived regions without discontinuity. The SiC NWs are tested in terms of toxicology, biocompatibility and conductance among cardiomyocytes and myofibroblasts.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Two new hybrid molybdenum(IV) Mo3S7 cluster complexes derivatized with diimino ligands have been prepared by replacement of the two bromine atoms of [Mo3S7Br6]2− by a substituted bipyridine ligand to afford heteroleptic molybdenum(IV) Mo3S7Br4(diimino) complexes. Adsorption of the Mo3S7 cores from sample solutions on TiO2 was only achieved from the diimino functionalized clusters. The adsorbed Mo3S7 units were reduced on the TiO2 surface to generate an electrocatalyst that reduces the overpotential for the H2 evolution reaction by approximately 0.3 V (for 1 mA cm−2) with a turnover frequency as high as 1.4 s−1. The nature of the actual active molybdenum sulfide species has been investigated by X-ray photoelectron spectroscopy. In agreement with the electrochemical results, the modified TiO2 nanoparticles show a high photocatalytic activity for H2 production in the presence of Na2S/Na2SO3 as a sacrificial electron donor system.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Hybrid magnetic graphitic nanocomposites (MGNC) prepared by inclusion of magnetite nanoparticles (obtained by coprecipitation) into an organic-organic self-assembly system, followed by calcination, revealed high activity for the catalytic wet peroxide oxidation (CWPO) of 4-nitrophenol solutions (5 g L-l), with pollutant removais up to 1245 mg g-' h-l being obtained when considering the mass ratio [pollutant]/[catalyst] =10. The stability of the MGNC catalyst against metal leaching was ascribed to the confinement effect of the carbon based material. These observations, together with the magnetically recoverable characteristics of MGNC, open new prospects for the wide use of this catalyst in highly efficient CWPO applications.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

From the Introduction. Russia’s annexation of Crimea and its aggression in eastern Ukraine have triggered a debate about the main directions of defence policy in the Nordic and Baltic region. In the Baltic states, but also in the Nordic countries and Poland, much attention is being paid to questions of Territorial Defence Forces (TDF). TDF are viewed as one of the elements in the national defence systems’ response during the early stages of a hybrid conflict. The Baltic states have decided to adapt their Territorial Defence Forces to new threats by making a number of changes to their functioning, depending on the local conditions in each case. Given the growing uncertainty in the region, they have opted not to undertake any in-depth reforms of TDF at this stage, as that could entail a temporary disorganisation in the armed forces. In the coming years Estonia, Latvia and Lithuania will invest in increasing the size and combat readiness of their Territorial Defence Forces, providing them with better training and equipment, and creating a system of incentives to encourage more people to serve in volunteer formations.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Transects of a Remotely Operated Vehicle (ROV) providing sea-bed videos and photographs were carried out during POLARSTERN expedition ANT-XV/3 focussing on the ecology of benthic assemblages on the Antarctic shelf in the South-Eastern Weddell Sea. The ROV-system sprint 103 was equiped with two video- and one still camera, lights, flash-lights, compass, and parallel lasers providing a scale in the images, a tether-management system (TMS), a winch, and the board units. All cameras used the same main lense and could be tilted. Videos were recorded in Betacam-format and (film-)slides were made by decision of the scientific pilot. The latter were mainly made under the aspect to improve the identification of organisms depicted in the videos because the still photographs have a much higher optical resolution than the videos. In the photographs species larger than 3 mm, in the videos larger than 1 cm are recognisable and countable. Under optimum conditions the transects were strait; the speed and direction of the ROV were determined by the drift of the ship in the coastal current, since both, the ship and the ROV were used as a drifting system; the option to operate the vehicle actively was only used to avoide obstacles and to reach at best a distance of only approximately 30 cm to the sea-floor. As a consequence the width of the photographs in the foreground is approximately 50 cm. Deviations from this strategy resulted mainly from difficult ice- and weather conditions but also from high current velocity and local up-welling close to the sea-bed. The sea-bed images provide insights into the general composition of key species, higher systematic groups and ecological guilds. Within interdisciplinary approaches distributions of assemblages can be attributed to environmental conditions such as bathymetry, sediment characteristics, water masses and current regimes. The images also contain valuable information on how benthic species are associated to each other. Along the transects, small- to intermediate-scaled disturbances, e.g. by grounding icebergs were analysed and further impact to the entire benthic system by local succession of recolonisation was studied. This information can be used for models predicting the impact of climate change to benthic life in the Southern Ocean. All these approaches contribute to a better understanding of the fiunctioning of the benthic system and related components of the entire Antarctic marine ecosystem. Despite their scientific value the imaging methods meet concerns about the protection of sensitive Antarctic benthic systems since they are non-invasive and they also provide valuable material for education and outreach purposes.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

"August 1979

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Armorial book-plate of the Marquess of Ely.

Relevância:

30.00% 30.00%

Publicador:

Resumo:

Each plate accompanied by a leaf with descriptive letterpress.