Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B).

Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.

Numerical analysis of the acoustic signature of a surface-breaking crack.

The boundary element method is used to calculate the acoustic signature, produced by a line focus scanning acoustic microscope, of an elastic object containing a surface-breaking crack. The acoustic signature has a vertical (z) and horizontal (x) dependence. A model of the microscope developed earlier is used and extended to take account of the crack. The mathematical formulation of the scattering problem for the cracked object leads to hypersingular integral equations. A suitable technique is employed to solve such equations by the boundary element method. An electromechanical reciprocity identity is used to relate the received voltage to the acoustic wavefields collected by the lens. The acoustic wavefield scattered from the cracked object is investigated, and curves are presented that display the acoustic signature, as functions of (x ,z), for cracks of various depths and orientations. A method to measure the depth of a surface-breaking crack using the acoustic signature is suggested.

Wave analysis of the acoustic material signature for the line focus microscope.

A model is presented for the computation of the acoustic material signature for a line focus scanning acoustic microscope, based on a boundary element calculation and an electromechanical reciprocity identity. This identity is used to relate the voltage at the terminals of the microscope's transducer to the acoustic wavefields at the interface between the specimen and the coupling fluid. A Gaussian beam, launched in the buffer rod, is tracked through the lens and its matching layer. A model for the matching layer that is convenient for use with the boundary element technique is presented. The wavefields scattered from the surface of the specimen, including the leaky Rayleigh wave, are then calculated. Knowing the wavefields incident on and scattered from the specimen, the acoustic signature is calculated using the reciprocity relation. Results are presented for a defect free halfspace, and are compared with those of an analytical model and an experimental measurement.