Genes involved in <Emphasis Type="Italic">Drosophila</Emphasis>glutamate receptor expression and localization

Background  

A clear picture of the mechanisms controlling glutamate receptor expression, localization, and stability remains elusive, possibly due to an incomplete understanding of the proteins involved. We screened transposon mutants generated by the ongoing Drosophila Gene Disruption Project in an effort to identify the different types of genes required for glutamate receptor cluster development.     

Characterization of the system functions of ultrasonic linear phased array inspection systems

This work characterizes the electrical and electromechanical aspects of an ultrasonic linear phased array inspection system, using a matrix of system functions that are obtained from the measured response of individual array elements in a simple reference experiment. It is shown that for the arrays tested all these system functions are essentially identical, allowing one to use a single system function to characterize the entire array, as done for an ordinary single element transducer. The variation of this single system function with the number of elements firing in the array or with changes of the delay law used is examined. It is also demonstrated that once such a single system function is obtained for an array, it can be used in a complete ultrasonic measurement model to accurately predict the array response measured from a reference reflector in an immersion setup.     

Capillary electrophoresis of the scrapie prion protein from sheep brain

Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrP^sc) that has a molecular mass of ca. 27 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrP^sc. The overall concentration of PrP^sc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrP^sc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrP^sc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrP^sc. After treatment with proteinase K and non-ionic detergents, PrP^sc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep. Capillary electrophoresis can be used to show immunocomplex formation when PrP^sc is present in sheep brain.     

Detection of prion protein using a capillary electrophoresis-based competitive immunoassay with laser-induced fluorescence detection and cyclodextrin-aided separation

The development of capillary electrophoresis (CE)-based competitive immunoassay for prion protein (PrP) using carboxymethyl beta-cyclodextrin (CM-beta-CD) as a buffer additive is described here. The assay was based on the competitive binding of PrP and a fluorescein-labeled peptide from the prion protein with a limiting amount of specific antibody. The amount of both free and fluorescein-labeled peptide bound to antibody (immunocomplex) were determined by CE with laser-induced fluorescence detection. In the presence of PrP, the peak height ratio of the immunocomplex and the free peptide was altered compared to the control. These changes were directly proportional to the amount of PrP present. The fluorescently labeled peptide spanning amino acid positions 140-158 of the PrP and its corresponding monoclonal antibody is reported here. The reaction times of the antibody with either the peptide or the recombinant PrP was less than 1 min and is a large improvement over the 16-18 h required to achieve equilibrium for polyclonal antibodies. CM-beta-CD was explored as a buffer additive to suppress analyte adsorption and enhance separation selectivity in the CE analysis. A fast (1.1 min), selective (resolution 4.7), and reproducible (relative standard deviations of migration time for free and bound fluorescein isothiocyanate (FITC)-peptide 0.56% and 0.64%, respectively) separation was obtained with 0.6% CM-beta-CD in 25 mM N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) at pH 8.8. The concentration detection limit of the assay for recombinant PrP was determined to be 80 ng/mL (or mass detection limit 1 pg). When blood samples from scrapie-infected sheep and from normal sheep were tested, the results of the blood assay were consistent with scrapie status of the sheep as determined post mortem by Western blot analysis. Development of this assay will lead to a potentially robust, rapid, and specific preclinical diagnosis for transmissible spongiform encephalopathies (TSEs) in animals and humans.     

K-space Prony’s method for generating the basis functions of multi-Gaussian beam models

The expansion coefficients of a multi-Gaussian ultrasonic beam model are obtained by a new approach that applies Prony´s method in a K-space domain. This method allows the fitting of the Gaussian beam directly at the face of the transducer with very high computational efficiency. It is demonstrated that the K-space Prony’s method can be used to accurately model the transducer field of planar and focused piston transducers, as well as probes that do not act as pistons. The choice of parameters appearing in the method and their influence on performance are discussed.     

Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis

An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.     

Improving the Born approximation for the scattering of ultrasound in elastic solids

It has recently been demonstrated that the Born approximation for predicting the scattering response of flaws can be improved through the use of simple modifications called the "doubly distorted Born approximation". In this paper the doubly distorted Born approximation itself is modified with phase and amplitude corrections that further improve the Born scattering results for isotropic elastic media. The reliability of this new modification of the Born approximation has been evaluated by comparison with the exact solution for spherical inclusions obtained with the method of separation of variables. Unlike the ordinary Born approximation which works well only for very weak scattering inclusions, our modification of the doubly distorted Born approximation gives improved scattering results for both weak and strong scattering inclusions.     

Complete ultrasonic transducer characterization and its use for models and measurements

Transmitting and receiving properties of ultrasonic piezoelectric crystal transducers that directly affect the measured output voltage in an ultrasonic measurement system are described. These transducer properties are the transducer's electrical impedance and sensitivity, the transducer's radiation impedance, and the transducer's effective parameters (effective radius and focal length). It is shown that all these properties can be obtained with a series of calibration measurements, most of them purely electrical in nature. This series of measurements is described, including a newly developed method that makes the determination of the transducer sensitivity simpler than with previous methods. It is demonstrated that by combining these transducer properties with knowledge of the electrical properties of the pulser/receiver and cabling and the acoustic/elastic processes present in an ultrasonic measurement system, it is possible to accurately simulate the output voltage of the system.     

Quasi pulse-echo ultrasonic technique for flaw classification

One of the important flaw characterization tasks in the field of ultrasonic non-destructive evaluation (NDE) is to provide flaw type information by analysing the flaw responses acquired during an inspection. Here a new quasi pulse-echo ultrasonic classification technique is presented which utilizes the time separation and amplitude difference of mode-converted diffracted signals to distinguish between smooth and sharp-edged flaw geometries. Experiments with cylindrical cavities, surface-breaking fatigue cracks and slag inclusions have been used to test the practicality of this approach. All results of these tests show good consistency in the separation of smooth and sharp-edged flaws, provided that the signal-to-noise ratio is sufficient. Furthermore, the scattering feature used for classification in this method is also verified by detailed elastodynamic scattering calculations.