Intercellular adhesion and membrane polarity in rat hepatocytes: Localization of cell -CAM 105 and other domain-specific membrane proteins {\it in situ\/} and {\it in vitro\/} by electron microscopy

Cell-CAM 105 has been identified as a cell adhesion molecule (CAM) based on the ability of monospecific and monovalent anti-cell-CAM 105 antibodies to inhibit the reaggregation of rat hepatocytes. Although one would expect to find CAMs concentrated in the lateral membrane domain where adhesive interactions predominate, immunofluorescence analysis of rat liver frozen sections revealed that cell-CAM 105 was present exclusively in the bile canalicular (BC) domain of the hepatocyte. To more precisely define the in situ localization of cell-CAM 105, immunoperoxidase and electron microscopy were used to analyze intact and mechanically dissociated fixed liver tissue. Results indicate that although cell-CAM 105 is apparently restricted to the BC domain in situ, it can be detected in the pericanalicular region of the lateral membranes when accessibility to lateral membranes is provided by mechanical dissociation. In contrast, when hepatocytes were labeled following incubation in vitro under conditions used during adhesion assays, cell-CAM 105 had redistributed to all areas of the plasma membrane. Immunofluorescence analysis of primary hepatocyte cultures revealed that cell-CAM 105 and two other BC proteins were localized in discrete domains reminscent of BC while cell-CAM 105 was also present in regions of intercellular contact. These results indicate that the distribution of cell-CAM 105 under the experimental conditions used for cell adhesion assays differs from that in situ and raises the possibility that its adhesive function may be modulated by its cell surface distribution. The implications of these and other findings are discussed with regard to a model for BC formation.^ Analysis of molecular events involved in BC formation would be accelerated if an in vitro model system were available. Although BC formation in culture has previously been observed, repolarization of cell-CAM 105 and two other domain-specific membrane proteins was incomplete. Since DMSO had been used by Isom et al. to maintain liver-specific gene expression in vitro, the effect of this differentiation system on the polarity of these membrane proteins was examined. Based on findings presented here, DMSO apparently prolongs the expression and facilitates polarization of hepatocyte membrane proteins in vitro. ^

Pencil -beam redefinition algorithm dose calculations for electron therapy treatment planning

The electron pencil-beam redefinition algorithm (PBRA) of Shiu and Hogstrom has been developed for use in radiotherapy treatment planning (RTP). Earlier studies of Boyd and Hogstrom showed that the PBRA lacked an adequate incident beam model, that PBRA might require improved electron physics, and that no data existed which allowed adequate assessment of the PBRA-calculated dose accuracy in a heterogeneous medium such as one presented by patient anatomy. The hypothesis of this research was that by addressing the above issues the PBRA-calculated dose would be accurate to within 4% or 2 mm in regions of high dose gradients. A secondary electron source was added to the PBRA to account for collimation-scattered electrons in the incident beam. Parameters of the dual-source model were determined from a minimal data set to allow ease of beam commissioning. Comparisons with measured data showed 3% or better dose accuracy in water within the field for cases where 4% accuracy was not previously achievable. A measured data set was developed that allowed an evaluation of PBRA in regions distal to localized heterogeneities. Geometries in the data set included irregular surfaces and high- and low-density internal heterogeneities. The data was estimated to have 1% precision and 2% agreement with accurate, benchmarked Monte Carlo (MC) code. PBRA electron transport was enhanced by modeling local pencil beam divergence. This required fundamental changes to the mathematics of electron transport (divPBRA). Evaluation of divPBRA with the measured data set showed marginal improvement in dose accuracy when compared to PBRA; however, 4% or 2mm accuracy was not achieved by either PBRA version for all data points. Finally, PBRA was evaluated clinically by comparing PBRA- and MC-calculated dose distributions using site-specific patient RTP data. Results show PBRA did not agree with MC to within 4% or 2mm in a small fraction (<3%) of the irradiated volume. Although the hypothesis of the research was shown to be false, the minor dose inaccuracies should have little or no impact on RTP decisions or patient outcome. Therefore, given ease of beam commissioning, documentation of accuracy, and calculational speed, the PBRA should be considered a practical tool for clinical use. ^

Structural conservations and diversities of dsRNA viruses: Structural studies of the cytoplasmic polyhedrosis virus by electron cryomicroscopy and 3D reconstruction

The Reoviridae virus family is a group of economically and pathologically important viruses that have either single-, double-, or triple-shelled protein layers enclosing a segmented double stranded RNA genome. Each virus particle in this family has its own viral RNA dependent RNA polymerase and the enzymatic activities necessary for the mature RNA synthesis. Based on the structure of the inner most cores of the viruses, the Reoviridae viruses can be divided into two major groups. One group of viruses has a smooth surfaced inner core, surrounded by complete outer shells of one or two protein layers. The other group has an inner core decorated with turrets on the five-fold vertices, and could either completely lack or have incomplete outer protein layers. The structural difference is one of the determinant factors for their biological differences during the infection. ^ Cytoplasmic polyhedrosis virus (CPV) is a single-shelled, turreted virus and the structurally simplest member in Reoviridae. It causes specific chronic infections in the insect gut epithelial cells. Due to its wide range of insect hosts, CPV has been engineered as a potential insecticide for use in fruit and vegetable farming. Its unique structural simplicity, unparalleled capsid stability and ease of purification make CPV an ideal model system for studying the structural basis of dsRNA virus assembly at the highest possible resolution by electron cryomicroscopy (cryoEM) and three-dimensional (3D) reconstruction. ^ In this thesis work, I determined the first 3D structure of CPV capsids using 100 kV cryoEM. At an effective resolution of 17 Å, the full capsid reveals a 600-Å diameter, T = 1 icosahedral shell decorated with A and B spikes at the 5-fold vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities that are attributed to the transcriptional enzyme complexes. The inside of the full capsid is packed with icosahedrally-ordered viral genomic RNA. The interactions of viral RNA with the transcriptional enzyme complexes and other capsid proteins suggest a mechanism for RNA transcription and subsequent release. ^ Second, the interactions between the turret proteins (TPs) and the major capsid shell protein (CSPs) have been identified through 3D structural comparisons of the intact CPV capsids with the spikeless CPV capsids, which were generated by chemical treatments. The differential effects of these chemical treatment experiments also indicated that CPV has a significantly stronger structural integrity than other dsRNA viruses, such as the orthoreovirus subcores, which are normally enclosed within outer protein shells. ^ Finally, we have reconstructed the intact CPV to an unprecendented 8 Å resolution from several thousand of 400kV cryoEM images. The 8 Å structure reveals interactions among the 120 molecules of each of the capsid shell protein (CSP), the large protrusion protein (LPP), and 60 molecules of the turret protein (TP). A total of 1980 α-helices and 720 β-sheets have been identified in these capsid proteins. The CSP structure is largely conserved, with the majority of the secondary structures homologous to those observed in the x-ray structures of corresponding proteins of other reoviruses, such as orthoreovirus and bluetongue virus. The three domains of TP are well positioned to play multifunctional roles during viral transcription. The completely non-equivalent interactions between LPP and CSP and those between the anchoring domain of TP and CSP account for the unparalleled stability of this structurally simplest member of the Reoviridae. ^