Engagement of ICAM-3 activates polymorphonuclear leukocytes: aggregation without degranulation or beta 2 integrin recruitment

ICAM-3 is a preferred counterreceptor for the leukocyte alpha(L)beta2 integrin. It activates T cells through outside-in signaling, but polymorphonuclear leukocytes (PMN) are reported to be refractory to ICAM-3 stimulation. We found that engagement of ICAM-3 by a mAb (CAL3.10), which binds in the region where alpha(L)beta2 integrin binds, activates PMN homotypic aggregation and adhesion to surfaces. These functional changes were due to ICAM-3 outside-in signaling because aggregation and adhesion were beta2 integrin-dependent, tyrosine kinase and protein kinase C activities were activated, and there was a reorganization of the cytoskeleton. This reorganization and kinase activity was required for ICAM-3-, but not FMLP-, induced aggregation. This is not an Fc-mediated event as an appropriate anti-ICAM-3 F(ab')2 fragment still induced aggregation. Another anti-ICAM-3 Ab (HP2/19), which activates T cells, did not activate PMN. Strikingly, anti-ICAM-3 did not induce degranulation or cause an increase in surface beta2 integrin expression, so adhesion and aggregation were due solely to the activation of the constitutively expressed beta2 integrins. Aggregation in response to ICAM-3, but not FMLP, was compromised at lower cell densities, showing that beta2 integrin recruitment enhances aggregation under suboptimal conditions. We conclude that engagement of ICAM-3 stimulates PMN as well as T cells, but that the appropriate epitope varies between these two cells. ICAM-3 outside-in signaling reorganizes the cytoskeleton without causing degranulation, induces serine and tyrosine kinase activation, and activates existing surface beta2 integrins to a proadhesive state.     

Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1

The sphingolipid metabolite sphingosine-1-phosphate (SPP) has been implicated as a second messenger in cell proliferation and survival. However, many of its biological effects are due to binding to unidentified receptors on the cell surface. SPP activated the heterotrimeric guanine nucleotide binding protein (G protein)-coupled orphan receptor EDG-1, originally cloned as Endothelial Differentiation Gene-1. EDG-1 bound SPP with high affinity (dissociation constant = 8.1 nM) and high specificity. Overexpression of EDG-1 induced exaggerated cell-cell aggregation, enhanced expression of cadherins, and formation of well-developed adherens junctions in a manner dependent on SPP and the small guanine nucleotide binding protein Rho.     

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.     

The stretching nonlinearity of the basilar membrane in a cochlear model

Recently it has been suggested that the stretching nonlinearity of the basilar membrane might be responsible for the observed nonlinear behaviour of basilar membrane motion. In the present study this type of nonlinearity is investigated, both by estimating its influence in an analytical manner, and by calculating its effect numerically, using a regular pertubation method. The conclusion reads that the stretching nonlinearity does not explain the observed nonlinear phenomena; not only is stretching negligible at normal sound levels, but it also fails to fit the data qualitatively, because of its typical hard-spring effect. In consequence, the origin of cochlear nonlinearity is not to be sought in the macromechanics of the inner ear, but in the more detailed processes in th organ of Corti-tectorial membrane complex.     

Magnetic resonance imaging of primary cardiomyopathies

Cardiomyopathies are diseases of the myocardium of unknown etiology associated with cardiac dysfunction. On the grounds of their morphology and pathophysiology, primary or idiopathic cardiomyopathies may be classified into a number of disorders; namely, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, and restrictive cardiomyopathy. The term "secondary cardiomyopathies" is reserved to specific heart muscle diseases clinically very similar to primary cardiomyopathies. Cardiac magnetic resonance imaging has long been used to study cardiac morphology and, more recently, to assess blood flow, perfusion, and contractile function. The emerging role of magnetic resonance imaging for the understanding and treatment of primary cardiomyopathies cannot be underestimated. From a clinical point of view, an examination based on a single, efficient, and noninvasive MR study focusing on the clinically relevant features of cardiomyopathies is an objective and reproducible means for diagnosing and monitoring hypertrophic, arrhythmogenic, dilated, and restrictive cardiomyopathies.