1,721,016 research outputs found
Optimizing a kinase assay for IKK.beta. on an HTS station
No full textUsing a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)—based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z' factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)—fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z' factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min
Recent applications of retro-inverso peptides
Full text linkNatural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides pos-sess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in im-munology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Structural investigations on the Nodal-Cripto binding: a theoretical and experimental approach
No full textNodal, a member of the TGF-beta superfamily, is a potent embryonic morphogen also implicated in tumor progression. Unfortunately, up to date structural information on the interaction of Nodal with its molecular partners is unknown. In order to deepen our understanding about mechanisms underlying both embryonic development and Nodal/Cripto-dependent tumor progression, we are presenting here a molecular model of ALK4/Cripto/Nodal complex built by homology modeling as well as docking tests aimed at identifying potential binding epitopes. Starting from this model, we have predicted a large interaction surface on Nodal which encompasses residues 43-69 and includes the pre-helix loop and the H3 helix. This hypothesis has been subsequently assessed by Surface Plasmon Resonance binding assays between the full length Cripto and synthetic peptides reproducing the selected Nodal regions. In addition the binding affinity between the full length Nodal and Cripto proteins has been evaluated for the first time
Discovery of Small Peptide Antagonists of PED/PEA15-D4α Interaction from Simplified Combinatorial Libraries
No full textMost biological processes involve permanent and temporary interactions between different proteins: protein complexes often play key roles in human diseases and, as a consequence, molecules that prevent protein-protein interactions can be potential new therapeutic agents to treat diseases. Here, we describe a simplified approach by which small synthetic peptide libraries were screened to identify the inhibitors of the complex between phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes15 (PED/PEA15) and D4α, a functional domain of the phospholipase D1, that is involved in the molecular mechanisms of insulin resistance occurring in type 2 diabetes. By using an enzyme-linked immunosorbent assay (ELISA)-based screening, performed on a fully automated platform, we analyzed two simplified peptide libraries in a positional scanning format. This screening led to the identification of small peptides able to inhibit PED/PEA15-D4α interaction. The selection of inhibitors was carried out employing combined competitive and direct experiments, through ELISA and surface plasmon resonance techniques, providing peptides with IC50 values in the micromolar range. Our results showed that the protein complex PED/PEA15-D4α is susceptible to peptides having H-donor groups and aromatic rings on specific positions. These small sequences can be considered as promising scaffolds that could be converted into higher-affinity inhibitor compounds. By a simplified approach small synthetic peptide libraries were screened to identify inhibitors of the complex between PED/PEA15 and D4α that is involved in molecular mechanisms of insulin resistance in type 2 diabetes. Small peptide sequences having H-donor groups and aromatic rings on specific positions are able to inhibit the complex and are promising scaffolds that could be converted into higher-affinity inhibitor compounds. © 2011 John Wiley & Sons A/S
Molecular basis of the PED/PEA15 interaction with the C-terminal fragment of phospholipase D1 revealed by NMR spectroscopy
No full textPED/PEA15 is a small protein involved in many protein-protein interactions that modulates the function of a number of key cellular effectors involved in major cell functions, including apoptosis, proliferation and glucose metabolism. In particular, PED/PEA15 interacts with the phospholipase D (PLD) isoforms 1 and 2 increasing protein kinase C-α isoform activity and affects both insulin-stimulated glucose transport and glucose-stimulated insulin secretion. The C-terminal portion (residues 712-1074) of PLD1, named D4, is still able to interact with PED/PEA15. In this study we characterized, by means of NMR spectroscopy, the molecular interaction of PED/PEA15 with D4α, a smaller region of D4, encompassing residues 712-818, shown to have the same affinity for PED/PEA15 and to induce the same effects as D4 in PED/PEA15-overexpressing cells. Chemical shift perturbation (CSP) studies allowed to define D4α binding site of PED/PEA15 and to identify a smaller region likely affected by an allosteric effect. Moreover, ELISA-like experiments showed that three 20-mer overlapping synthetic peptides, covering the 762-801 region of D4α, strongly inhibit PED/PEA15-D4α interaction through their binding to PED/PEA15 with KDs in low micromolar range. Finally, molecular details of the interaction of PED/PEA15 with one of the three peptides have been revealed by CSP and saturation transfer difference (STD) analyses. © 2013 Elsevier B.V. All rights reserved
A Comprehensive Analysis of the Intrinsic Visible Fluorescence Emitted by Peptide/Protein Amyloid-like Assemblies
No full textAmyloid aggregation is a widespread process that involves proteins and peptides with different molecular complexity and amino acid composition. The structural motif (cross-β) underlying this supramolecular organization generates aggregates endowed with special mechanical and spectroscopic properties with huge implications in biomedical and technological fields, including emerging precision medicine. The puzzling ability of these assemblies to emit intrinsic and label-free fluorescence in regions of the electromagnetic spectrum, such as visible and even infrared, usually considered to be forbidden in the polypeptide chain, has attracted interest for its many implications in both basic and applied science. Despite the interest in this phenomenon, the physical basis of its origin is still poorly understood. To gain a global view of the available information on this phenomenon, we here provide an exhaustive survey of the current literature in which original data on this fluorescence have been reported. The emitting systems have been classified in terms of their molecular complexity, amino acid composition, and physical state. Information about the wavelength of the radiation used for the excitation as well as the emission range/peak has also been retrieved. The data collected here provide a picture of the complexity of this multifaceted phenomenon that could be helpful for future studies aimed at defining its structural and electronic basis and/or stimulating new applications
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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