96 research outputs found
Investigation of the effects of sample preparation on gluten quantitation in rye and barley flours
Proper gluten quantitation is essential for providing safe gluten-free food for patients living with celiac disease (CD). However, gluten quantitation faces several challenges: the lack of a reference method and certified reference materials, the variability of methods and the effects of genetic and environmental factors on gluten. Among all these challenges our research group focuses on gluten reference material development. Gluten content is determined by enzyme linked immunosorbent assay (ELISA) methods to obtain comparable data for the selection of cultivars used in our reference material development efforts. As ELISA methods are developed for determining low gluten concentrations, application for these special research purposes requires a 10,000-fold dilution. The formerly performed process was a post-extraction liquid dilution that proved to be sufficient for wheat samples. However, gluten contents of rye and barley samples were found to be overestimated by ELISA methods. One of the suggested reasons is the structural and solubility changes of gluten proteins during the dilution process. Therefore, our present study focuses on the comparison of the original dilution method and a revised version using solid-phase dilution in a gluten-free matrix
Myosin and tropomyosin stabilize the conformation of formin-nucleated actin filaments
The conformational elasticity of the actin cytoskeleton is
essential for its versatile biological functions. Increasing
evidence supports that the interplay between the structural and
functional properties of actin filaments is finely regulated by
actin-binding proteins, however, the underlying mechanisms and
biological consequences are not completely understood. Previous
studies showed that the binding of formins to the barbed end
induces conformational transitions in actin filaments by making
them more flexible through long-range allosteric interactions.
These conformational changes are accompanied by altered
functional properties of the filaments. To get insight into the
conformational regulation of formin-nucleated actin structures,
in the present work we investigated in detail how binding
partners of formin-generated actin structures, myosin and
tropomyosin, affect the conformation of the formin-nucleated
actin filaments, using fluorescence spectroscopic approaches.
Time-dependent fluorescence anisotropy and temperature-dependent
Forster-type resonance energy transfer measurements revealed
that heavy meromyosin, similarly to tropomyosin, restores the
formin-induced effects and stabilizes the conformation of actin
filaments. The stabilizing effect of heavy meromyosin is
cooperative. The kinetic analysis revealed that despite the
qualitatively similar effects of heavy meromyosin and
tropomyosin on the conformational dynamics of actin filaments,
the mechanisms of the conformational transition is different for
the two proteins. Heavy meromyosin stabilizes the formin-
nucleated actin filaments in an apparently single-step reaction
upon binding, while the stabilization by tropomyosin occurs
after complex formation. These observations support the idea
that actin-binding proteins are key elements of the molecular
mechanisms that regulate the conformational and functional
diversity of actin filaments in living cells
Investigation of the effects of food processing and matrix components on the analytical results of ELISA using an incurred gliadin reference material candidate
Disorders induced by cereal proteins (e.g. wheat allergy, celiac disease) are widespread in human population. Since their only effective treatment is the avoidance of the problematic proteins, patients have to be familiar with the composition of food products. For checking special foods produced for them, proper analytical methods are necessary. At the moment, in gluten analysis there are no reference methods and reference materials which model real food matrices. During the production and experimental utilisation of our previously developed reference material candidate, numerous questions emerged. As our model product is a real food matrix, interactions can be present between gluten proteins and other macro and micro components. Fat content of the baked cookies is almost 20%, which might affect the results of ELISA measurements. The detectable gluten content is significantly increasing after the defatting procedure, as a pre-treatment of samples. Moreover, baking is a common food processing step that might modify the structure of gluten proteins leading to denaturation and aggregation. In the soluble protein fraction the amount of low molecular weight proteins increases, while that of high molecular weight proteins decreases during the baking procedure
DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.
During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin
Individual Actin Filaments in a Microfluidic Flow Reveal the Mechanism of ATP Hydrolysis and Give Insight Into the Properties of Profilin
A novel microfluidic approach allows the analysis of the dynamics of individual actin filaments, revealing both their local ADP/ADP-Pi-actin composition and that Pi release is a random mechanism
DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.
During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin
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