The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM +/- VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM - VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM +/- VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM - VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM +/- VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM - VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM - VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a significant factor in the reduced axonation capabilities of mature neurons.

We report here a unique variant of alpha spectrin in a kindred with hereditary elliptocytosis. This novel red blood cell-membrane protein migrated to a position between the normal alphaand beta-spectrin subunits in SDS polyacrylamide gel electrophoresis. It was identified as an alpha spectrin by its binding to anti-alpha spectrin antibodies, by the absence of a phosphorylation site, and by the normal 1:1 stoichiometry between total alpha-and beta-spectrin molecules. The quantity of the alphaspectrin mutant, expressed as a percentage of the total alpha spectrin, varied from 9.9-45.2% among six affected individuals. Two-dimensional electrophoretic analysis of spectrin tryptic digests was qualitatively normal but showed a decreased quantity of a normal alpha IV fragment. The variable quantity of alphaspectrin mutant among family members correlated directly with the increased percentage of spectrin dimers in cold low ionic strength spectrin extracts (r = 0.92) and inversely with red blood cell ghost mechanical stability (r = -0.98). The data suggest that this new alpha-spectrin mutant is responsible for decreased spectrin dimer-dimer association and for red cell instability in affected individuals.

ABSTRACT. The life span of neonatal erythrocytes (60-80 days) is shorter than that of adult erythrocytes (120 days). We studied neonatal red blood cells separated on stractan density gradients to further characterize the aging process and to explore the possibility that senescence antigens play a role in the destruction of neonatal erythrocytes. Quantitation of membrane proteins 4.la and 4.lb served as a marker for cell age and confirmed an enrichment for senescent red cells in the most dense layers of the gradients. Despite the shorter life span of neonatal erythrocytes, cord blood contained a larger percentage of very dense, K+-depleted red cells than did adult blood. ATP levels in dense neonatal and adult cells were decreased to 50-80% of normal values for unseparated red cells. Levels of reduced glutathione did not fall with increasing cell density. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of red cell membrane proteins showed increased membrane-associated globin in senescent neonatal cells, but such gels run without reducing agents did not show oxidative protein cross-linking. Membrane bound immunoglobulins were detected on senescent neonatal and adult red cells by the rosetting antiglobulin test. We conclude that senescence antigens are revealed during the aging process of neonatal erythrocytes, thereby labeling them for antibody-mediated destruction in the reticuloendothelial system. (Pediatr Res 23: 288-292, 1988) Abbreviations GSH, reduced glutathione SDS, sodium dodecyl sulfate PAGE, polyacrylamide gel electrophoresis BSKG, buffered saline with potassium and glucose EA, erythroctye antibody DTT, dithiothreitol RBC, red blood cells Normal human red cells undergo a nonrandom, age-dependent process of removal from the circulation; the most aged or senescent cells are selectively destroyed (1). The sequence of events that targets the senescent erythrocyte for destruction is not completely understood, but recent work has suggested that aging of adult red cells is associated with the exposure of cryptic, senescence antigens on the exterior surface of the red cell membrane (2-6). Such antigens are then recognized by autologous immunoglobulins, thus facilitating the removal of aged cells by the macrophages of the reticuloendothelial system (2-6).The life span of neonatal red cells is significantly shorter than that of adult red cells. While adult erythrocytes survive in the circulation for 120 days, fetal cells survive only 60-80 days (7-9). The mechanisms underlying the shorter survival are unknown, but cross-transfusion experiments have indicated that the decreased life span is intrinsic to the fetal cell, rather than a feature of the fetal circulation (8).Recently, Matovcik et al. (10) studied density-separated red cells and showed that the aging process of human neonatal erythrocytes is associated with a more rapid loss of membrane surface area than is the case with adult red cells. The loss of surface area was demonstrated to have a significant detrimental effect on cell deformability. ...