98 research outputs found

    Synthetic peptide arrays for investigating protein interaction domains

    Get PDF
    AbstractSynthetic peptide array technology was first developed in the early 1990s by Ronald Frank. Since then the technique has become a powerful tool for high throughput approaches in biology and biochemistry. Here, we focus on peptide arrays applied to investigate the binding specificity of protein interaction domains such as WW, SH3, and PDZ domains. We describe array-based methods used to reveal domain networks in yeast, and briefly review rules as well as ideas about the synthesis and application of peptide arrays. We also provide initial results of a study designed to investigate the nature and evolution of SH3 domain interaction networks in eukaryotes

    Региоселективный синтез и свойства ацетильных производных фенолгликозидов

    Get PDF
    Региоселективный синтез и свойства ацетильных производных фенолгликозидов. Работа посвящена выявлению реакционной способности при кислотном дезацетилировании с использованием HCl / EtOH в CHCl3, которая приводит к дезацетилированию на O-3, O-4 и O-6. Описанный реагент оказался общим и уникальным, и была получена серия 2-О-ацетиларилгликозидов. Вообще, частично ацетилированные арилгликозиды широко встречаются в природе. В частности, можно найти множество примеров 2-O-ацетиларилгликозидов.Regioselective synthesis and properties of acetyl derivatives of phenol glycosides. The thesis is devoted to the detection of reactivity during acid deacetylation using HCl / EtOH in CHCl3, which leads to deacetylation at O-3, O-4 and O-6. The described reagent proved to be general and unique and the series of 2-О-acetyl aryl glycosides were prepared. Generally, partially acetylated aryl glycosides are widely found in nature. Particularly, many examples of 2-O-acetyl aryl glycosides can be found

    NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

    Get PDF
    Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes

    Bimodal antagonism of PKA signalling by ARHGAP36

    Get PDF
    Protein kinase A is a key mediator of cAMP signalling downstream of G-protein-coupled receptors, a signalling pathway conserved in all eukaryotes. cAMP binding to the regulatory subunits (PKAR) relieves their inhibition of the catalytic subunits (PKAC). Here we report that ARHGAP36 combines two distinct inhibitory mechanisms to antagonise PKA signalling. First, it blocks PKAC activity via a pseudosubstrate motif, akin to the mechanism employed by the protein kinase inhibitor proteins. Second, it targets PKAC for rapid ubiquitin-mediated lysosomal degradation, a pathway usually reserved for transmembrane receptors. ARHGAP36 thus dampens the sensitivity of cells to cAMP. We show that PKA inhibition by ARHGAP36 promotes derepression of the Hedgehog signalling pathway, thereby providing a simple rationale for the upregulation of ARHGAP36 in medulloblastoma. Our work reveals a new layer of PKA regulation that may play an important role in development and disease

    NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

    Get PDF
    Xenarthrans – anteaters, sloths, and armadillos – have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with 24 domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, ten anteaters, and six sloths. Our dataset includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data-paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the south of the USA, Mexico, and Caribbean countries at the northern portion of the Neotropics, to its austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n=5,941), and Cyclopes sp. has the fewest (n=240). The armadillo species with the most data is Dasypus novemcinctus (n=11,588), and the least recorded for Calyptophractus retusus (n=33). With regards to sloth species, Bradypus variegatus has the most records (n=962), and Bradypus pygmaeus has the fewest (n=12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other datasets of Neotropical Series which will become available very soon (i.e. Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans dataset

    Investigation of the network of preferred interactions in an artificial coiled-coil association using the peptide array technique

    No full text
    We screened a randomized library and identified natural peptides that bound selectively to a chimeric peptide containing α-, β- and γ-amino acids. The SPOT arrays provide a means for the systematic study of the possible interaction space accessible to the αβγ-chimera. The mutational analysis reveals the dependence of the binding affinities of α-peptides to the αβγ-chimera, on the hydrophobicity and bulkiness of the side chains at the corresponding hydrophobic interface. The stability of the resulting heteroassemblies was further confirmed in solution by CD and thermal denaturation

    Detection systems cross reacting with peptides - Analysis at the amino acid level

    No full text
    There are several methods commonly used to measure protein-protein interactions and binding affinities. Quite contrary to most of these methods, protein- and peptide arrays on cellulose membranes or glass slides are suitable for high-throughput measurement, as they provide a higher density of probes and a multitude of peptide-protein interactions can be measured in parallel [1]. The most important application of the SPOT synthesis technique is to simultaneously detect a high number of peptides that have a strong binding affinity to defined targets. The validity of the results, however, depends on the ability of the detection system to indicate binding events whilst not interfering with the experiment itself through cross reaction. We tested three common peptide detection systems (TAMRA, FITC, Biotin/ Streptavidine) for their ability to interact with cellulose bound peptides at the amino acid level. 

Peptides with different amino acid cores were synthesized and tested for interaction with common dyes and detection systems. Our goal was to discover the potential of 5-(and 6)-carboxytetramethylrhodamine [5(6)-TAMRA], fluoresceinisothiocyanate [FITC], biotin and streptavidine to crossreact with individual amino acids. 

To this end we designed 20 peptides of the sequence XXX[aa]5XXX, where [aa]5 denotes five repeats of one of the 20 amino acids, and prepared them via SPOT synthesis [2]. Glycine was chosen as the flanking residue X to act as a spacer molecule. As analytes small peptides (gly-gly-gly) were solid phase synthesized and afterwards labelled with the detection compound of interest. The resulting amino acid library was then incubated with the glycine labelled detection system and evaluated via optical and fluorescent methods.

Our approach identified several amino acids interacting with different detection systems. These results will strengthen the reliability of the analysis of SPOT synthesis generated data in the future.

References

1.Andresen, H. et al. Functional peptide microarrays for specific and sensitive antibody diagnostics. _Proteomics_, 6(5):1376– 1384, Mar 2006.

2.Frank, R. The spot-synthesis technique. Synthetic peptide arrays on membrane supports – principles and applications. _J Immunol Methods_, 267(1):13–26, 2002.
&#xa
    corecore