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

Application of support vector machines on the basis of the first Hungarian bankruptcy model

In our study we rely on a data mining procedure known as support vector machine (SVM) on the database of the first Hungarian bankruptcy model. The models constructed are then contrasted with the results of earlier bankruptcy models with the use of classification accuracy and the area under the ROC curve. In using the SVM technique, in addition to conventional kernel functions, we also examine the possibilities of applying the ANOVA kernel function and take a detailed look at data preparation tasks recommended in using the SVM method (handling of outliers). The results of the models assembled suggest that a significant improvement of classification accuracy can be achieved on the database of the first Hungarian bankruptcy model when using the SVM method as opposed to neural networks

Acquisition Correction and Reconstruction for a Clinical SPECT/MRI Insert

The development of the first clinical simultaneous Single Photon Emission Computed Tomography (SPECT) and Magnetic Resonance Imaging (MRI) system was carried out within the INSERT project. The INSERT scanner was constructed under the initial project, but its performance was not fully evaluated; here we have reconstructed the first images on the SPECT system. Calibration and acquisition protocols were developed and used to establish the clinical feasibility of the system. The image reconstruction procedures were implemented on the first phantom images in order to assess the system's imaging capabilities. This study solved issues involving incomplete data sets and pixel failure in the prototype detector system. The final images determined a measure of trans-axial image resolution, giving average values of 9.14 mm and 6.75 mm in the radial and tangential directions respectively. The work carried out on the complete system produced several clinical phantom images which utilized the capabilities of both SPECT and MRI

Development of clinical simultaneous SPECT/MRI

There is increasing clinical use of combined positron emission tomography (PET) and magnetic resonance imaging (MRI) but to date there has been no clinical system developed capable of simultaneous single photon emission computed tomography (SPECT) and MRI. There has been development of preclinical systems, but there are several challenges faced by researchers who are developing a clinical prototype including the need for the system to be compact and stationary with MRI-compatible components. The limited work in this area is described with specific reference to the Integrated SPECT/MRI for Enhanced stratification in Radio-chemo Therapy (INSERT) project, which is at an advanced stage of developing a clinical prototype. Issues of SPECT/MRI compatibility are outlined and the clinical appeal of such a system is discussed, especially in the management of brain tumour treatment

INSERT: A Novel Clinical Scanner for Simultaneous SPECT/MRI Brain Studies

A clinical SPECT insert for a commercial MRI scanner has been developed within the INSERT project, allowing simultaneous SPECT/MRI studies of the human brain. Here we present preliminary experimental results. The reconstructed resolution was 6-11 mm and the sensitivity 280-440 s-1/MBq. The INSERT is the first clinical SPECT prototype for simultaneous SPECT/MRI

Preconditioning-induced ischemic tolerance: a window into endogenous gearing for cerebroprotection

Ischemic tolerance defines transient resistance to lethal ischemia gained by a prior sublethal noxious stimulus (i.e., preconditioning). This adaptive response is thought to be an evolutionarily conserved defense mechanism, observed in a wide variety of species. Preconditioning confers ischemic tolerance if not in all, in most organ systems, including the heart, kidney, liver, and small intestine. Since the first landmark experimental demonstration of ischemic tolerance in the gerbil brain in early 1990's, basic scientific knowledge on the mechanisms of cerebral ischemic tolerance increased substantially. Various noxious stimuli can precondition the brain, presumably through a common mechanism, genomic reprogramming. Ischemic tolerance occurs in two temporally distinct windows. Early tolerance can be achieved within minutes, but wanes also rapidly, within hours. Delayed tolerance develops in hours and lasts for days. The main mechanism involved in early tolerance is adaptation of membrane receptors, whereas gene activation with subsequent de novo protein synthesis dominates delayed tolerance. Ischemic preconditioning is associated with robust cerebroprotection in animals. In humans, transient ischemic attacks may be the clinical correlate of preconditioning leading to ischemic tolerance. Mimicking the mechanisms of this unique endogenous protection process is therefore a potential strategy for stroke prevention. Perhaps new remedies for stroke are very close, right in our cells

Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin.

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin

In vivo direct interaction of the antibiotic primycin on a Candida albicans clinical isolate and its ergosterol-less mutant

Interaction of primycin antibiotic with plasma membrane, and its indirect biological effects were investigated in this study. The antifungal activity of primycin against 13 human pathogenic Candida ATCC and CBS reference species and 74 other Candida albicans clinical isolates was investigated with a microdilution technique. No primycin-resistant strain was detected. Direct interaction of primycin with the plasma membrane was demonstrated for the first time by using an ergosterol-producing strain 33erg+ and its ergosterol-less mutant erg-2. In growth inhibition tests, the 33erg+ strain proved to be more sensitive to primycin than its erg-2 mutant, indicating the importance of the plasma membrane composition in primycin-induced processes. The 64 μg ml−1 (56.8 nM) primycin treatment induced an enhanced membrane fluidity and altered plasma membrane dynamics, as measured by steady-state fluorescence anisotropy applying a trimethylammonium-diphenylhexatriene (TMA-DPH) fluorescence polarization probe. The following consequences were detected. The plasma membrane of the cells lost its barrier function, and the efflux of 260-nm-absorbing materials from treated cells of both strains was 1.5–1.8 times more than that for the control. Depending on the primycin concentration, the cells exhibited unipolar budding, pseudohyphae formation, and a rough cell surface visualized by scanning electron microscopy

INSERT Project: First results of a MR compatible preclinical SPECT based on SiPM photodetectors

We report on the results achieved with the first SPECT insert for general purpose MR scanners, developed within the INSERT project (INtegrated SPECT/MRI for Enhanced stratification of brain tumors in Radio-chemoTherapy). The present paper focuses on the implementation and performance assessment of a preclinical version of the SPECT. A clinical configuration is currently under development and will be optimized accordingly to the results from the preclinical system. The SPECT is composed by a full ring of gamma detection modules based on compact and MR compatible Silicon PhotoMultipliers (SiPMs). The intrinsic spatial resolution of the detection modules has been measured as 1.0 mm FWHM over 40 mm × 40 mm planar field of view (FOV), at 140 keV and at 4 °C. The energy resolution of the detector is 13.7 % for Tc-99m (140 keV, at 4 °C) and has been optimized to permit the simultaneous acquisition of multiple radiotracers. The main components, namely the detection modules, the collimator and custom RF coil, have been realized with a compact design to fit inside common MR bores. Moreover, mutual compatibility between SPECT system and MR has been studied and optimized for all the SPECT components. First tomographic images have been acquired from phantoms and in vivo animal models. The gamma detection modules have been tested in a 7 T MRI (MAGNETOM by Siemens) to measure induced disturbances on the electronic chain. The detection module negligibly affects MRI imaging. The outcomes of mutual compatibility tests provide enough confidence to proceed with the first synchronous SPECT/MR imaging experiments