Two hydrophobic protein fractions of ovine pulmonary surfactant: isolation, characterization, and biophysical activity.

Pulmonary surfactant contains two extremely hydrophobic proteins, SP-B and SP-C. We present a novel HPLC method for the preparation of these hydrophobic proteins. It is based on size-exclusion chromatography using the apolar stationary-phase butyl silica gel and isocratic elution with acidified chloroform/methanol. Samples for HPLC were prepared from sheep lung lavage fluid by centrifugation and extraction with chloroform/methanol. Amino acid analyses of the two protein fractions revealed sequences that are consistent with SP-B and SP-C, respectively. MALDI-TOF-MS analyses of the SP-B fraction showed one major peak of dimeric SP-B with m/z 17,361, and additional peaks of monomeric and oligomeric forms, which are predominantly even numbered. The SP-C fraction showed a peak at m/z 4200, consistent with the theoretical mass of the dipalmitoylated form of this protein. The biophysical activity of pure sheep SP-B and SP-C was evaluated by measuring the surface tension using axisymmetric drop shape analysis for captive bubbles. We found distinct surface pressure versus surface area isotherms of SP-B and SP-C indicating different biophysical activities for these surfactant proteins. The new preparative HPLC method is able to replace the established, time-consuming low-pressure liquid chromatography method for the isolation of SP-B and SP-C from lipids.

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the avian inner ear.

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells.

The influence of the subsurface cisterna on the electrical properties of the outer hair cell.

A distributed-parameter computational model of the outer hair cell was used to predict the potentials resulting from current injection. A good fit was found to the length-dependent whole cell conductance and capacitance. The fit required the presence of the subsurface cisterna and a low (0.001 mS/cm2) specific conductivity for the plasma membrane of the lateral wall. The lateral wall could not be space clamped as significant longitudinal currents pass through the extracisternal space vs the axial core. A reduction in the width of the extracisternal space decreased the whole-cell conductance and capacitance. Position-dependent phase shifts were noted during injection of sinusoidal currents (0.001575 rad/micron). These predictions support a role for extracisternal longitudinal potential gradients in experimentally observed non-linear capacitance and electromotility.

Microradiographs of laser fusion targets: 2-D modeling and analysis.

A new contact microradiographic system for analyzing laser fusion targets with 2-D modeling and image analysis techniques is described. This system, which uses a monochromatic x-ray source and Kodak highresolution plate emulsion, is sensitive to spherical wall thickness variations (eccentricities) as small as +/-200 A in hollow shells with a mean wall thickness of 1 microm. Measurements of wall thickness and of local and spherical wall thickness variations by radiographic techniques, using 2-D video, digital image analysis, and optical interferometry, are compared.

Hybrid optical-digital radiography-based system for lung disease detection.

A hybrid optical-digital system for automated measurement and disease classification of chest radiographs has been constructed. Initial results from the use of this system for classification of opacity textural patterns associated with a specific diffuse lung disease (coal workers pneumoconiosis) are presented. This system consists of three subsystems which include a digital image scanner for lung field detection, a computercontrolled X - Y transport for film positioning, and a Fraunhofer diffraction pattern sampling unit for opacity detection and measurement. A minicomputer is used to integrate these subsystems as well as render a film quality and disease classification through the use of discriminant functions and probability estimates.

Automated computer screening of chest radiographs for pneumoconiosis.

The results of two complementary approaches for performing diagnostic screening for the presence of coal workers' pneumoconiosis (CWP) from the routine posterior-anterior chest radiograph are presented. The first is a digital approach utilizing the measurement of image texture, while the second uses hybrid optical-digital methods involving the optical Fourier transform. Both approaches yield classification results comparable to experienced radiologists.