CRIPTO and its signaling partner GRP78 drive the metastatic phenotype in human osteotropic prostate cancer.

CRIPTO (CR-1, TDGF1) is a cell surface/secreted oncoprotein actively involved in development and cancer. Here, we report that high expression of CRIPTO correlates with poor survival in stratified risk groups of prostate cancer (PCa) patients. CRIPTO and its signaling partner glucose-regulated protein 78 (GRP78) are highly expressed in PCa metastases and display higher levels in the metastatic ALDHhigh sub-population of PC-3M-Pro4Luc2 PCa cells compared with non-metastatic ALDHlow. Coculture of the osteotropic PC-3M-Pro4Luc2 PCa cells with differentiated primary human osteoblasts induced CRIPTO and GRP78 expression in cancer cells and increases the size of the ALDHhigh sub-population. Additionally, CRIPTO or GRP78 knockdown decreases proliferation, migration, clonogenicity and the size of the metastasis-initiating ALDHhigh sub-population. CRIPTO knockdown reduces the invasion of PC-3M-Pro4Luc2 cells in zebrafish and inhibits bone metastasis in a preclinical mouse model. These results highlight a functional role for CRIPTO and GRP78 in PCa metastasis and suggest that targeting CRIPTO/GRP78 signaling may have significant therapeutic potential.

Ga(+) beam lithography for nanoscale silicon reactive ion etching.

By using a dry etch chemistry which relies on the highly preferential etching of silicon, over that of gallium (Ga), we show resist-free fabrication of precision, high aspect ratio nanostructures and microstructures in silicon using a focused ion beam (FIB) and an inductively coupled plasma reactive ion etcher (ICP-RIE). Silicon etch masks are patterned via Ga(+) ion implantation in a FIB and then anisotropically etched in an ICP-RIE using fluorinated etch chemistries. We determine the critical areal density of the implanted Ga layer in silicon required to achieve a desired etch depth for both a Pseudo Bosch (SF(6)/C(4)F(8)) and cryogenic fluorine (SF(6)/O(2)) silicon etching. High fidelity nanoscale structures down to 30 nm and high aspect ratio structures of 17:1 are demonstrated. Since etch masks may be patterned on uneven surfaces, we utilize this lithography to create multilayer structures in silicon. The linear selectivity versus implanted Ga density enables grayscale lithography. Limits on the ultimate resolution and selectivity of Ga lithography are also discussed.

Alumina etch masks for fabrication of high-aspect-ratio silicon micropillars and nanopillars.

We introduce using sputtered aluminum oxide (alumina) as a resilient etch mask for fluorinated silicon reactive ion etches. Achieving selectivity of 5000:1 for cryogenic silicon etching and 68:1 for SF(6)/C(4)F(8) silicon etching, we employ this mask for fabrication of high-aspect-ratio silicon micropillars and nanopillars. Nanopillars with diameters ranging from below 50 nm up to several hundred nanometers are etched to heights greater than 2 microm. Micropillars of 5, 10, 20, and 50 microm diameters are etched to heights of over 150 microm with aspect ratios greater than 25. Processing and characterization of the sputtered alumina is also discussed.

Reduced expression of dystroglycan in breast and prostate cancer.

Cellular interactions with the extracellular matrix are an important factor in the development and progression of many types of cancer. Dystroglycan is a cell surface receptor for several extracellular matrix proteins and plays a central role in the formation of basement membranes in tissues. Because abnormalities in the structure and function of basement membranes are hallmarks of metastatic disease, we examined the status of dystroglycan expression in prostate and breast tumors. In 15 cases of surgically resected prostate cancer, we noted reduced expression of dystroglycan as judged by intensity of immunohistochemical staining. This reduction was most pronounced in high-grade disease. We found similar results in 6 cases of mammary ductal adenocarcinoma, suggesting that reduced expression of dystroglycan may be a conserved feature of epithelial neoplasia. These data suggest that reduced expression of dystroglycan in prostate and breast cancers may lead to abnormal cell-extracellular matrix interactions and thus contribute to progression to metastatic disease.

Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization.

Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface. Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In beta1 integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells, the formation of complex laminin-1 structures is defective, implicating perlecan in the laminin matrix assembly process. Moreover, laminin and perlecan reciprocally modulate the organization of the other on the cell surface. Taken together, the data support a model whereby DG serves as a receptor essential for the initial binding of laminin on the cell surface, whereas beta1 integrins and perlecan are required for laminin matrix assembly processes after it binds to the cell.

Dystroglycan binding to laminin alpha1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro.

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.

Maturation and maintenance of the neuromuscular synapse: genetic evidence for roles of the dystrophin--glycoprotein complex.

The dystrophin-glycoprotein complex (DGC) links the cytoskeleton of muscle fibers to their extracellular matrix. Using knockout mice, we show that a cytoplasmic DGC component, alpha-dystrobrevin (alpha-DB), is dispensable for formation of the neuromuscular junction (NMJ) but required for maturation of its postsynaptic apparatus. We also analyzed double and triple mutants lacking other cytoskeletal DGC components (utrophin and dystrophin) and myotubes lacking a alpha-DB or a transmembrane DGC component (dystroglycan). Our results suggest that alpha-DB acts via its linkage to the DGC to enhance the stability of postsynaptic specializations following their DGC-independent formation; dystroglycan may play additional roles in assembling synaptic basal lamina. Together, these results demonstrate involvement of distinct protein complexes in the formation and maintenance of the synapse and implicate the DGC in the latter process.

Dystroglycan inside and out.

Dystroglycan connects the extracellular matrix and cytoskeleton. Key findings in the past year indicate that dystroglycan interacts with a wider repertoire of extracellular ligands than originally appreciated, that dystroglycan plays a critical role in organizing extracellular matrix molecules on the cell surface and in basement membranes, and that at least two human pathogens utilize dystroglycan to gain access to host cells. Together, these advances begin to help elucidate important biological roles for dystroglycan in development and disease.

Dystrophic phenotype induced in vitro by antibody blockade of muscle alpha-dystroglycan-laminin interaction.

alpha-dystroglycan is a glycoprotein expressed on the surface of skeletal muscle fibres and other cell types. In muscle, alpha-dystroglycan provides a link between the myofibre cytoskeleton through its indirect binding to dystrophin, and the basal lamina through its binding to laminin-2, a protein of the extracellular matrix. The disruption of this linkage between the myofibre cytoskeleton and the extracellular matrix is a common feature of Duchenne and other muscular dystrophies, though the pathogenic mechanisms leading to muscle wasting remain unknown. By treating primary mouse muscle cultures with a monoclonal antibody which blocks alpha-dystroglycan binding to laminin, we show here the induction of a dystrophic phenotype in vitro. The phenotype is inducible in differentiated cultures only, is characterised by reduced myotube size, myofibril disorganisation, loss of contractile activity, reduced spontaneous clustering of acetylcholine receptors and is reversed by addition of excess exogenous laminin-2. Thus, alpha-dystroglycan may be part of a signalling pathway for the maturation and maintenance of skeletal myofibres. Detailed knowledge of this signalling pathway may provide insights into the molecular pathology of the various inherited muscular dystrophies, and identify valuable pharmacological targets and new therapeutic strategies.

Identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus.

A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be alpha-dystroglycan (alpha-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified alpha-DG protein. Soluble alpha-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, alpha-DG is a cellular receptor for both LCMV and LFV.

A role for dystroglycan in basement membrane assembly.

Basement membranes are composed of ordered arrays of characteristic extracellular matrix proteins, but little is known about the assembly of these structures in vivo. We have investigated the function of dystroglycan, a cell-surface laminin receptor expressed by cells contacting basement membranes in developing and adult tissues. We find that dystroglycan is required for the formation of a basement membrane in embryoid bodies. Our results further indicate that dystroglycanlaminin interactions are prerequisite for the deposition of other basement membrane proteins. Dystroglycan may exert its influence on basement membrane assembly by binding soluble laminin and organizing it on the cell surface. These data establish a role for dystroglycan in the assembly of basement membranes and suggest fundamental mechanisms underlying this process.

Dystroglycan in development and disease.

Our understanding of the structure and function of dystroglycan, a cell surface laminin/agrin receptor, has increased dramatically over the past two years. Structural studies, analysis of its binding partners, and targeted gene disruption have all contributed to the elucidation of the biological role of dystroglycan in development and disease. It is now apparent that dystroglycan plays a critical role in the pathogenesis of several muscular dystrophies and serves as a receptor for a human pathogen as well as being involved in early development, organ morphogenesis, and synaptogenesis.

Distribution of dystroglycan in normal adult mouse tissues.

Dystroglycan is a cell surface protein which, in muscle, links the extracellular matrix protein laminin-2 to the intracellular cytoskeleton. Dystroglycan also binds laminin-1 and the binding occurs via the E3 fragment of laminin-1. Recently, it was found that dystroglycan is expressed in developing epithelial cells of the kidney. Moreover, antibodies against dystroglycan can perturb epithelial development in kidney organ culture. Therefore, dystroglycan may be an important receptor for cell-matrix interactions in non-muscle tissues. However, information about the tissue distribution of dystroglycan is limited, especially in adult tissues. Here we show that dystroglycan is present in epithelial cells in several non-muscle organs of adult mice. Dystroglycan is enriched towards the basal side of the epithelial cells that are in close contact with basement membranes. We suggest that dystroglycan is involved in linking basement membranes to epithelial and muscle cells. Dystroglycan may be important for the maintenance of tissue integrity.

Dystroglycan is essential for early embryonic development: disruption of Reichert's membrane in Dag1-null mice.

Dystroglycan is a central component of the dystrophin-glycoprotein complex (DGC), a protein assembly that plays a critical role in a variety of muscular dystrophies. In order to better understand the function of dystroglycan in development and disease, we have generated a null allele of dystroglycan (Dag1neo2) in mice. Heterozygous Dag1neo2 mice are viable and fertile. In contrast, homozygous Dag1neo2 embryos exhibit gross developmental abnormalities beginning around 6.5 days of gestation. Analysis of the mutant phenotype indicates that an early defect in the development of homozygous Dag1neo2 embryos is a disruption of Reichert's membrane, an extra-embryonic basement membrane. Consistent with the functional defects observed in Reichert's membrane, dystroglycan protein is localized in apposition to this structure in normal egg cylinder stage embryos. We also show that the localization of two critical structural elements of Reichert's membrane--laminin and collagen IV--are specifically disrupted in the homozygous Dag1neo2 embryos. Taken together, the data indicate that dystroglycan is required for the development of Reichert's membrane. Furthermore, these results suggest that disruption of basement membrane organization might be a common feature of muscular dystrophies linked to the DGC.

Testing surfactant treatment responses: a comparison of two models.

We have compared treatment responses of two surfactants in two animal models of respiratory distress syndrome. Preterm rabbits at 27 days gestation and premature lambs at 126 days gestation were treated with natural sheep surfactant Survanta (Ross Laboratories), or no surfactant and then were ventilated with the same ventilation style to achieve tidal volumes of 8 ml/kg. Dynamic compliances at 15 min were higher for control and surfactant-treated rabbits than for preterm lambs. Sheep surfactant and Survanta increased compliances in rabbits, but only sheep-surfactant-treated lambs had compliances higher than controls at 15 min of age. Maximal lung volumes (milliliters per kilogram) of the rabbits (after 15 min ventilation) were about twice those of the lambs (after 4 h of ventilation). Lung volumes of sheep-surfactant-treated rabbits were larger than for Survanta-treated rabbits, but no differences in lung volume were evident between the surfactant-treated lambs. The two models for testing surfactants do not give the same results when directly compared.

Overdenture retention and stabilization with ball-and-socket attachments: principles and technique.

Implant attachments of various designs are used to retain, stabilize and sometimes support overdentures. Tissue supported overdentures stabilized and retained by two to four implants are often the restorations of choice due to patient preference, limitations in finances, insufficient available bone to accommodate a greater number of implants, or needed improvements in esthetics, phonetics and oral hygiene. Female retentive sockets in the overdenture base that snap onto male ball abutments offer such advantages as reduction in hydraulic resistance to coupling, lower functional stress on the implants, retention that can be adjusted downward, easy component replacement and only relatively parallel abutments.

Dystroglycan: an extracellular matrix receptor linked to the cytoskeleton.

Dystroglycan provides a crucial linkage between the cytoskeleton and the basement membrane for skeletal muscle cells. Disruption of this linkage leads to various forms of muscular dystrophy. Significant recent advances in understanding the structure and function of dystroglycan include detailed in vitro and in vivo analyses of its binding partners in muscle, an examination of its function at the neuromuscular junction, and emerging evidence of its roles in nonmuscle tissues.

Ultrasonic nebulized in comparison with instilled surfactant treatment of preterm lambs.

To evaluate the efficiency and distribution of ultrasonic nebulized versus instilled surfactant in the treatment of surfactant deficiency at varying degrees of maturation, twin or triplet lamb fetuses were delivered at 125 to 137 d gestational age and received nebulized natural surfactant (Neb Only), instilled surfactant followed by a second instilled dose (Inst/Inst), instilled surfactant followed by nebulized surfactant (Inst/Neb), or no surfactant (Control). The lambs were ventilated for 6 h. Twenty-eight lambs were categorized into two groups (low compliance versus moderate compliance) based on initial physiologic lung characteristics. Efficiency of deposition of nebulized surfactant directly correlated with the compliances and ventilatory efficiency indices measured at 15 min of age. The low-compliance group (Low Comp) had significantly lower efficiency of surfactant deposition (7.6 +/- 1.6%) than did the moderate-compliance group (Mod Comp) (23.4 +/- 2.5%) (p < 0.01). Overall, instilled surfactant had a reasonably homogeneous distribution, whereas nebulized surfactant had a less homogeneous distribution, except for the Low Comp, Inst/Neb group, which had a distribution pattern similar to that for instilled surfactant. The potential for nebulized surfactant therapy for respiratory distress syndrome (RDS) may be limited by the nonhomogeneous nature of ventilation in the preterm lung.

Interdomain interactions of radixin in vitro.

We have assayed the domains of the ERM protein radixin for binding activities in vitro. Affinity columns bearing the amino-terminal domain of radixin selectively bound a small subset of the proteins of the chicken erythrocyte cytoskeleton. Two of those proteins were identified as radixin itself and band 4.1. In contrast, the carboxyl-terminal domain of the molecule bound neither protein, and full-length radixin did not bind band 4.1 (binding of full-length radixin to itself was not evaluated). Columns bearing a mixture of the amino- and carboxyl-terminal domains of radixin also failed to bind radixin and band 4.1. These results suggested that the amino- and carboxyl-terminal sequences can interact with one another either in cis or in trans, and so interfere with radixin's interactions with other ligands. Using affinity co-electrophoresis, we confirmed a direct interaction in solution between the two radixin domains; the data are consistent with the formation of a 1:1 complex with a dissociation constant of approximately 5 x 10(-8) M. Competition between intramolecular and intermolecular interactions may help to explain the provocative and dynamic localization of ERM proteins within cells.