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Abstract  

The effect of phalloidin on the thermal stability of skeletal actin filaments polymerized from ADP-binding monomers was investigated with the method of differential scanning calorimetry. Phalloidin shifted the melting temperature of the ADP-F-actin from 59.1±1.0 to 80.0±1.2°C. The stabilizing effect of phalloidin propagated cooperatively along the filament. The cooperativity factor according to the applied model was 1.07±0.11. With these measurements it was possible to demonstrate that the binding of phalloidin has lower influence on the adjacent protomers in ADP- (k=1) than in ATP-actin filaments (k=3).

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Abstract  

The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential scanning calorimetry. Heart muscle contains α-skeletal and α-cardiac actin isoforms. In the absence of phalloidin the melting temperature was 55°C for the α-cardiac actin isoform and 58°C for the α-skeletal one when the filaments were generated from ADP-actin monomers. After the binding of phalloidin the melting temperature was isoform independent (85.5°C). We concluded that phalloidin stabilized the actin filaments of α-skeletal and α-cardiac actin isoforms to the same extent when they were polymerized from ADP-actin monomers.

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Abstract

Rickettsiae are able to spread within infected cell mono-layers by modifying intra-cellular actin formations. The study analyzes whether a visualization of actin modifications in addition to specific immuno-fluorescence staining of rickettsiae might facilitate the proof of rickettsial growth in cell culture.

Cell mono-layers of Vero E6 und BGM cells were infected with Rickettsia honei. Intra-cellular actin was fluorescence stained with TRITC-(tetra-methyl-5,6-isothiocyanate)-labeled phalloidin in addition to specific immuno-fluorescence staining of rickettsiae with FITC-(fluorescein-isothiocyanate)-labeled antibodies. DNA of bacteria and cells was counter-stained with DAPI (4′,6- diamino-2-phenyl-indole). Cell cultures infected with Vaccinia virus were used as positive controls, cell cultures infected with Coxiella burnetii as negative controls.

High concentrations of R. honei are necessary to demonstrate characteristic modifications of the intra-cellular actin. This effect is more pronounced in Vero E6 cells than in BGM cells.

Actin staining with phalloidin is not suited for an early proof of rickettsial growth in cell culture but may confirm unclear findings in specific immuno-fluorescence staining in case of sufficient bacterial density.

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Summary  

In our present study we performed the detailed characterisation of jasplakinolide and phalloidin on the thermal stability of actin filaments. The heat absorption curves were analysed by using the model established by Sanchez-Ruiz et al. [1]. The analysis provided the activation energies attributed to the heat denaturation of actin filaments in the absence and in the presence of toxins. The results indicated that there are kinetic differences between the toxin-mediated stabilization of the Ca2+-and Mg2+-actin filaments. The effect of toxins appeared to be cation dependent.

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The development of the new pharynx in anterior body fragments of G. tigrina was followed for 7 days by immunocytochemical (ICC) study using antiserum to neuropeptide F (NPF) and muscle staining with phalloidin. ICC investigation revealed the presence of NPF in pharyngeal nervous system, peripheral nerve plexuses, in central nervous system of intact planarians. NPF-immunoreactive (IR) nerve fibres were found at the site of regeneration surrounding pharyngeal rudiment. Restoration of the pharynx function during regeneration was analysed by appearance of food response in anterior fragments. Stimulating effects of NPF and FMRF on the pharyngeal regeneration has been observed. The data indicates an important role of neuropeptides in morphogenetic processes.

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Bivalve larvae use catch muscles for rapid shell closure and maintenance of the closed condition. We used specific antibodies against the muscle proteins together with phalloidin and neuronal markers, FMRFamide and serotonin (5-HT), to analyze mutual distribution of muscle and neuronal elements in larvae of the mussel, Mytilus trossulus, and the oyster, Crassostrea gigas. At trochophore and early veliger stages no anatomical connections between muscular and nervous system were detected. By the pediveliger stage the 5-HT innervation of the anterior adductor developed in oyster only, while rich FMRFa innervation of the adductor muscles developed in both species. Possible roles and mechanisms of FMRFamide and serotonin in the regulation of the catch state are discussed.

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. Pónya, Z., Barnabás, B. 2001: Microinjected fluorescent phalloidin in vivo reveals F-actin dynamics in isolated egg cells of wheat ( Triticum aestivum L.) developed in situ and fertilised in vitro. J. Plant Physiol. , 158 , 1527

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.1016/0040-6031(94)87052-7 . 8. Vig , Andrea , Dudás , Réka , Kupi , Tünde , Orbán , J , Hild , G , Lőrinczy , D , Nyitrai , M . Effect of phalloidin on filaments polymerized from heart muscle adp-actin monomers . J Therm Anal Calorim

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. Cooper , J. A. : Effects of cytochalasin and phalloidin on actin . J Cell Biol 105 , 1473 – 1478 ( 1987 ). 10.1083/jcb.105.4.1473 119. Van Goietsenoven , G

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