Sialylated Form of the Neural Cell Adhesion Molecule ... - Diabetes

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Université Paris 7, Tour 23-33, 1° étage, case 7126, 2 Place Jussieu, 75251 ..... Kiss et al. (14) demonstrated that the insulin-secreting cell line INS-1 and ...
Sialylated Form of the Neural Cell Adhesion Molecule (NCAM) A New Tool for the Identification and Sorting of -Cell Subpopulations With Different Functional Activity Catherine Bernard-Kargar, Nadim Kassis, Marie-France Berthault, William Pralong, and Alain Ktorza

To clarify the relationship between variations in -cell mass and pancreatic function, we investigated the possibility to analyze, quantify, and sort -cell subpopulations with different functional maturity. To this aim, we tested the reliability of the sialylated form of neural cell adhesion molecule (NCAM) (PSA-NCAM) as a marker of -cell functional activity. Islet cells isolated from adult rats were analyzed for their PSA-NCAM abundance using an anti–PSA-NCAM antibody. We found that PSA-NCAM is expressed only in -cells. The PSANCAM labeling was also studied with a fluorescenceactivated cell sorter. We showed that the -cell population is heterogeneous for PSA-NCAM labeling. To directly determine the relationship between PSANCAM labeling and -cell activity, in vitro insulin secretion studies were performed on sorted -cell subpopulations using a perifusion technique. Two -cell subpopulations were analyzed: one that was highly labeled for PSA-NCAM and another that was poorly labeled. Insulin secretion from high PSA-NCAM– labeled -cells was significantly higher than that in low PSA-NCAM–labeled -cells. This differential expression in the -cell population was well correlated with differences in glucose responsiveness. PSA-NCAM seems thus suitable for use as a tool to identify -cell subpopulations according to their glucose responsiveness. Diabetes 50 (Suppl. 1):S125–S130, 2001

From the Laboratoire de Physiopathologie de la Nutrition, Université Paris, Paris, France. Address correspondence and reprint requests to Catherine BernardKargar, Laboratoire de Physiopathologie de la Nutrition, CNRS ESA 7059, Université Paris 7, Tour 23-33, 1° étage, case 7126, 2 Place Jussieu, 75251 Paris cedex 05, France. E-mail: [email protected]. Received for publication 21 May 2000 and accepted 14 August 2000. This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educational grant from Les Laboratoires Servier. BSA, bovine serum albumin; FACS, fluorescence-activated cell sorter; FAD, flavin adenine dinucleotide; FFA, free fatty acid; FSC, forward scatter; KRBH, Krebs-Ringer bicarbonate-HEPES buffer; NCAM, neural cell adhesion molecule; PBS, phosphate-buffered saline; PSA-NCAM, polysialic-NCAM. DIABETES, VOL. 50, SUPPLEMENT 1, FEBRUARY 2001

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here is increasing evidence that, in adult mammals, the mass of pancreatic endocrine -cells is submitted to long-term dynamic changes (endocrine pancreas plasticity). In certain physiological situations such as pregnancy (1) and in physiopathological situations such as obesity (2,3), -cell increase correlates with increased -cell responsiveness to glucose, suggesting that -cell maturity is maintained despite -cell expansion. This finding is of crucial importance in type 2 diabetes, in which impairment of insulin secretion is believed to be related in part to a reduction in the number of fully functional -cells. Whereas new therapeutic approaches aim to preserve the pancreatic -cell mass, in several experimental models in which sustained -cell regeneration could be induced (2–4), the degree of improvement of pancreatic function did not correlate with the magnitude of the regenerative process. This result suggests that all the regenerating -cells did not achieve full functional maturity. To clarify the relationship between the variations in -cell mass and pancreatic function, it is necessary to identify -cell subpopulations with different functional maturity. It has been postulated that at least two -cell populations may be distinguished in the pancreas of normal adult rats: one that has high redox potential and is highly glucose-responsive and another that has low redox potential and is poorly sensitive to glucose (concept of -cell heterogeneity) (5). According to this concept, we investigated the possibility to analyze, quantify, and sort -cell subpopulations more directly on the basis of their ability to secrete insulin. For this purpose, we tested the reliability of the sialylated form of neural cell adhesion molecule (NCAM) (PSA-NCAM) as a marker of -cell functional activity. NCAM is a member of the immunoglobulin superfamily that promotes cell adhesion (6). However, the attachment of carbohydrate sialic acid polymers (PSA) linked through -2,8 binding to NCAM modulates the adhesive property of NCAM and decreases cell adhesion (7,8). In mammals, PSA-NCAM is involved in the development of the nervous system and in tissue remodeling (9,10). After birth, PSA-NCAM persists in the adult, mainly in brain regions that display potential for plasticity and morphological changes such as axonal fasciculation and glial cell migration (11–13). In the adult, PSAS125

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NCAM is also expressed in pancreatic -cells (14). Moreover, in the INS-1 cell line, PSA-NCAM is present in the membrane of the insulin secretory granules and exposed at the cell surface when insulin secretion is stimulated by glucose (14). Overall, these data suggest that PSA-NCAM could be a good candidate to identify -cell subpopulations according to their functional activity. We attempted to isolate and sort different -cell subpopulations with a fluorescence-activated cell sorter (FACS) using the abundance of PSA-NCAM as a marker of -cell activity. To validate this tool, we also performed insulin secretion studies on sorted -cell subpopulations using a perifusion technique. RESEARCH DESIGN AND METHODS Chemicals. Type V collagenase, Histopaque 1077, polyornithine, gentamycin, and forskolin were obtained from Sigma (St. Louis, MO). Trypsin 1:250 was from Difco (Detroit, MI). Culture medium RPMI-1640 and fetal calf serum were from ICN Biochemicals (Aurora, OH). Free fatty acid (FFA) bovine serum albumin (BSA) was from Boehringer Mannheim (Mannheim, Germany). Animals. Three-month-old male Wistar rats weighing 270–300 g were used. They had free access to water and standard laboratory diet (N° 113; Usine d’Alimentation Rationelle, Villemoisson-sur-Orge, France). Islet isolation. Rats were anesthetized with pentobarbital (Sanofi Santé Animale, Libourne, France) (4 mg/100 g body wt intraperitoneally). Islets of Langerhans were isolated after collagenase digestion of the pancreas as previously described (15). Islet cell preparation. The isolated islets were pooled in 15 ml chelation saline buffer (phosphate-buffered saline [PBS] with 5 mmol/l EGTA) and then resuspended in the same buffer supplemented with 0.1 mg/ml trypsin. The suspension was incubated for 5–6 min at 37°C with occasional mixing through a Pasteur pipette. The digestion was stopped by adding cold Krebs-Ringer bicarbonate-HEPES buffer (KRBH) containing 140 mmol/l NaCl, 3.6 mmol/l KCl, 0.5 mmol/l NaH2PO4, 0.5 mmol/l MgSO2, 2 mmol/l NaHCO3, 1.5 mmol/l CaCl2, 10 mmol/l HEPES, 5.6 mmol/l glucose, and 0.5% BSA. The isolated cells were washed twice in cold KRBH. This procedure yielded ~0.5–0.7  106 islet cells per pancreas. Cell suspensions were used either for immunocytochemistry or for -cell sorting. Culture of pancreatic islet cells and immunocytochemistry. Culture of islet cells was performed on glass coverslips coated with 10 µg/ml polyornithine and placed in a Petri dish. Islet cells were resuspended in culture medium RPMI-1640 supplemented with 5.6 mmol/l glucose, 10% heat-inactivated fetal calf serum, 100 µg/ml gentamycin, 2 mmol/l L-glutamine, and 10 mmol/l HEPES. Then, 50  103 cells were plated onto the glass coverslips and were allowed to adhere for 24 h at 37°C in 95% O2/5% CO2 and saturated humidity. At the end of the culture period and 6 h before the experiment, the glucose concentration in the medium was lowered to 2.8 mmol/l. Then, immunocytochemistry studies were performed. Cell preparations were rinsed and incubated for 30 min with 16.7 mmol/l glucose plus 10 µmol/l forskolin (stimulating condition). Four coverslips were randomly chosen, and double immunolabeling experiments for PSA-NCAM and an islet hormone were performed on attached cells. Cells were fixed in 4% paraformaldehyde and incubated overnight at 4°C with the mouse anti–PSANCAM antibody (mouse IgM monoclonal antibody that recognizes specifically -2,8–linked PSA chains with >12 residues; ascites fluid was used at a dilution of 1:4). Cells were rinsed in PBS–1% BSA and were fixed again in 4% paraformaldehyde for 30 min at 4°C. Cells were washed in PBS–1% BSA and then incubated for 1 h with the second antibody, an anti-mouse IgM coupled to FITC (1:100; Caltag, San Francisco, CA). Cells were washed twice in PBS–1% BSA and reincubated for 1 h with primary antibodies in the presence of Triton X-100 (guinea pig anti-insulin, 1:1,000; rabbit anti-glucagon, 1:1,000; rabbit anti-somatostatin, 1:700; or rabbit antipancreatic polypeptide, 1:1500; ICN Biochemicals). Texas red–conjugated second antibodies were then applied for 45 min (goat anti–guinea pig and anti-rabbit IgG, 1:150; Vector Laboratories, Burlingame, CA). Fluorescence-activated cell sorting of -cells. -Cells were sorted either with or without PSA-NCAM labeling. PSA-NCAM labeling was performed as follows: islet cell suspension was incubated with the mouse anti–PSA-NCAM antibody (1:4) for 30 min. Cells were rinsed twice with KRBH–1% BSA buffer and reincubated for 15 min with the second antibody (anti-mouse IgM coupled to phycoerythrin, 1:150; Caltag). After two washes with KRBH–1% BSA, cells were resuspended at a density of 1  106 cells/ml in KRBH–1% BSA. Control cells were incubated only with the second antibody. Cells were kept on ice in S126

the dark until fluorescence-activated cell sorting. Islet cells were sorted using a FACStarplus (Becton Dickinson, San Jose, CA). An argon laser (163; Spectra Physics, Mountain View, CA) illuminated the cells at 488 nm. An interference filter detected the emission light at 510–540 nm that was taken as the flavin adenine dinucleotide (FAD) cell content. A second filter at 580 nm detected the emission light of phycoerythrin. The cells were fed to the FACS at the rate of 1,000 cells per second. For cell sorting of -cells unlabeled with PSANCAM, autofluorescence for FAD (fluorescence channel 1 [Fl-1]) and cell size (forward side scatter [FSC]) were used for the discrimination between -cells and non–-cells. In the basal condition (5.6 mmol/l glucose), -cells presented a 50% higher FAD content than the non–-cells (16). -Cells were also 50% larger (17). Window 1 was set to enclose the population of -cells. -Cells labeled for PSA-NCAM were sorted using the autofluorescence for FAD (Fl1) and the fluorescence for phycoerythrin (Fl-2). Two windows were set: one to enclose the high-labeled -cell subpopulation and the other to enclose the low-labeled -cell subpopulation. Quality of sorting was verified by subsequent FACS analysis of samples of the purified cells. The data were arranged in dot plots and histograms with the attached FACStarplus statistical analysis software (Becton Dickinson). Levels of light scatter (cell size) and immunofluorescence light were expressed in log scale. In vitro insulin release studies. After purification, -cells were resuspended in RPMI-1640 culture medium supplemented as above and plated in a mini bacteriological culture dish. For aggregate formation, -cells were incubated for 1.5 h in a rotary incubator at 30 cycles/min. Cells were then cultured for 18 h and washed in KRBH containing 0.7% FFA-BSA. The cell viability rate was assayed by neutral red (0.01%) and was always between 90 and 95% (18). Flow columns for perifusion were filled with 1 ml Bio-Gel P2 fine (BioRad, Richmond, CA). After a 30-min equilibration period with KRBH–0.7% FFABSA with 2.8 mmol/l glucose, 2  105 cells were laid on top of the matrix, covered with 200 µl Bio-Gel P2, and perifused at 200 µl/min at 37°C. -Cells were first equilibrated for 20 min in KRBH–0.7% FFA-BSA containing basal glucose (2.8 mmol/l) and then exposed for 15 min to 2.8 mmol/l glucose, 30 min to 8.3 or 16.7 mmol/l glucose, and 20 min to 2.8 mmol/l glucose. Insulin radioimmunoassay. Insulin was measured by a radioimmunoassay kit (Cis Bio International, Gif-sur-Yvette, France) using 125I-porcine insulin tracer and tube coated with anti-porcine guinea pig antiserum. Rat insulin standard was obtained from Linco Research (St. Charles, MO). The lower limit of the assay was 75 pmol/l with a coefficient of variation within the assay of 6% and between the assay of 8%. Data presentation and statistical methods. Data are presented as means ± SE. Statistical significance was determined by analysis of variance. P < 0.05 was considered significant.

RESULTS

Expression of PSA-NCAM in islet cells. Using a double immunocytochemistry approach, we found that only -cells expressed PSA-NCAM surface immunoreactivity under stimulating conditions for insulin secretion (Fig. 1A). We never observed double labeling for PSA-NCAM and glucagon, somatostatin, or pancreatic polypeptide under this stimulating condition (Fig. 1B–D). Sorting total -cells from non–-cells. Figure 2A illustrates the dot plot of FAD fluorescence (Fl-1) and cell size (FSC) distribution in the islet cell population. In addition to the small signal originating from cell debris and cell clusters, two distinct populations of cells were clearly identified, as described by Van de Winkel et al. (16). The separation between these two populations is also detectable in Fig. 2B, in which the only discriminant parameter is cellular FAD. We verified by immunocytochemical staining that the population of large cells with a high FAD content was 90% pure for -cells, whereas the population of small cells with a low FAD content was 90% pure for non–-cells (-cells, -cells, and PP cells) (data not shown). Distribution of PSA-NCAM among -cells. Figure 3A represents the distribution of fluorescence for FAD (Fl-1) and phycoerythrin (Fl-2) in the islet cell population. In the -cell population, PSA-NCAM surface staining (FL-2) showed a shift toward higher fluorescence values when compared DIABETES, VOL. 50, SUPPLEMENT 1, FEBRUARY 2001

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FIG. 1. PSA-NCAM in islet cells revealed by fluorescence microscopy. Superimposed digitized images show PSA-NCAM labeling in green and islet hormone labeling in red. The yellow pseudo-color indicates colocalization of PSA-NCAM and insulin. A: Cell immunostaining for insulin. B: Cell immunostaining for glucagon. C: Cell immunostaining for somatostatin. D: Cell immunostaining for pancreatic polypeptide.

with the non–-cell population. When the dot plot of FAD and phycoerythrin fluorescence distributions was expressed in a three-dimensional figure (Fig. 3B), the PSA-NCAM labeling in the -cell population appeared to be heterogeneous, with some -cells exhibiting an intense staining for PSA-NCAM and others displaying weak labeling. Differences in fluorescence intensity for PSA-NCAM between total unlabeled -cells and total labeled -cells are depicted in Fig. 3C. The fluorescence intensity of the unlabeled -cell population was 7 ± 1 (n = 5) (arbitrary units), whereas the median of the fluorescence intensity for the PSA-NCAM–labeled -cell population was 25 ± 5 (n = 5). This value was used to sort the labeled -cell population into two groups. The first represents the -cell subpopulation with high PSA-NCAM staining (Fig. 3A, window 2); the second represents the population with low PSANCAM staining (Fig. 3A, window 3). Study of insulin release. The window R1 was used to sort the total -cells (Fig. 2A). In response to both 8.3 and 16.7 mmol/l glucose, there was a clear increase in insulin release by reaggregated total -cells (Fig. 4). Insulin secretion was dose-dependent, with the release at 8.3 mmol/l glucose increased twofold and the release at 16.7 mmol/l glucose DIABETES, VOL. 50, SUPPLEMENT 1, FEBRUARY 2001

increased threefold compared with 2.8 mmol/l glucose (Fig. 4). In both subpopulations sorted according to PSANCAM abundance, insulin response to glucose was dosedependent (Fig. 4). In high PSA-NCAM–labeled -cells, insulin release was significantly higher than that in total -cells and much higher than that in low PSA-NCAM–labeled -cells (Fig. 4). DISCUSSION

The main conclusions from this study are the specificity of PSA-NCAM expression in pancreatic -cells and the correlation between abundance of PSA-NCAM and insulin response to glucose. Kiss et al. (14) demonstrated that the insulin-secreting cell line INS-1 and primary islet cells express PSA-NCAM. Here, we show that PSA-NCAM expression is strictly restricted to the -cells under conditions known to induce maximal insulin secretion. This is in agreement with previous studies showing that PSA-NCAM is present on the membrane of the insulin secretory granules and can be mobilized at the cell surface when insulin secretion is stimulated by high glucose, whereas it is largely hampered by the addition of calciumS127

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FIG. 2. Distribution of FAD content and cell size in islet cells evaluated by a FACS. A: Dot plot analysis of dissociated islet cells examined for their FAD content (Fl-1) and cell size (FSC); large high-fluorescent total -cells were sorted using window 1. B: Dot plot analysis of islet cells examined for their FAD content (Fl-1); no fluorescence was detected for Fl-2 in -cells not labeled for PSA-NCAM. Fluorescence intensities are expressed in arbitrary units on a logarithmic scale.

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FIG. 3. PSA-NCAM distribution in islet cells evaluated by a FACS. A: Dot plot analysis of dissociated islet cells examined for their FAD content (Fl-1) and phycoerythrin fluorescence (Fl-2), which represents PSA-NCAM labeling. -Cells presenting a high fluorescence for FAD and a low fluorescence for PSA-NCAM were sorted using window 2. -Cells presenting a high fluorescence for both FAD and PSA-NCAM parameters were sorted using window 3. B: Three-dimensional representation of the dot plot in A. Note that PSA-NCAM staining is heterogeneous among the -cell population. C: Histogram for phycoerythrin fluorescence (Fl-2) of high and low PSA-NCAM–labeled -cells. Fluorescence intensities are in arbitrary units on a logarithmic scale. The arrow indicates the median. S128

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FIG. 4. Insulin release in response to 2.8, 8.3, and 16.7 mmol/l glucose from total -cells sorted for their FAD content and from -cell subpopulations sorted according to their PSA-NCAM content. Insulin release from total -cells (), high-labeled -cells (), and low-labeled -cells () is shown. Aggregated -cells (2  105 cells/column) were perifused for 15 min at 2.8 mmol/l glucose, 30 min at 8.3 or 16.7 mmol/l glucose, and 20 min at 2.8 mmol/l glucose. Data are means ± SE of four independent experiments for total -cells and three independent experiments for PSA-NCAM–labeled -cells.

channel blockers to the culture medium (14). From these data, it was inferred that the expression of PSA-NCAM at the cell membrane correlates with -cell activity and, more precisely, regulated exocytosis pathway. The role of PSA-NCAM in adult -cells is not known. However, because of the intrinsic propriety of the molecule, one could speculate that increased expression of PSA-NCAM at the surface of -cells could influence contacts between -cells and other islet cells. This result could facilitate secretion of insulin into the circulation from -cells able to establish connections with capillaries (19). In insulin granules, PSA-NCAM may contribute to cation storage (in particular Mg2+) because of the electronegativity of the molecule necessary for convertase action (20). More recently, the role of PSA-NCAM in islet function has been highlighted in NCAM knockout mice, in which NCAM deficiency produced almost total loss of PSA content (13). In these mice, a large fraction of -cells was degranulated, suggesting impaired insulin secretion. These data argue for the involvement of PSA-NCAM in the normal turnover of insulincontaining secretory granules (21). Based on the above, we studied the relationship between PSA-NCAM abundance and -cell activity and used this molecule as a marker to sort -cell subpopulations. -Cells can be purified from non–-cells according to their higher FAD content and larger size when compared with non–-cells (16,17). When cells were sorted for their abundance for PSANCAM, a shift toward higher fluorescence values was observed in the -cell population compared with non– -cells. Moreover, the PSA-NCAM labeling appeared to be heterogeneous in the whole -cell population, with some exhibiting an intense staining for PSA-NCAM, whereas others displayed weak labeling. To verify whether the abundance for PSA-NCAM on -cell subpopulations was correlated with different functional activity, we performed perifusions of sorted -cell subpopulations after reaggregation, because it is known that glucose-induced insulin secretion in single cells is severely impaired (22). There was good correlation between PSA-NCAM content of sorted -cells and their abilDIABETES, VOL. 50, SUPPLEMENT 1, FEBRUARY 2001

ity to release insulin in response to glucose. Indeed, high PSA-NCAM–labeled -cells secreted much more insulin in response to glucose than low PSA-NCAM–labeled -cells. This secretion was obvious in response to 8.3 mmol/l glucose, which represented relatively low glucose stimulation of insulin release, and even more so in response to 16.7 mmol/l glucose. Thus, PSA-NCAM appears to be a good tool to sort different -cell subpopulations and to identify -cell subpopulations according to their functional activity. These data support studies suggesting that PSA-NCAM could be involved in islet function (14,21). The concept of -cell heterogeneity proposes that pancreatic -cells differ in their individual sensitivity to glucose (22) and are recruited when glucose concentration increases (23). Because the most responsive -cells are those that are the most metabolically active, it is possible to sort the -cell subpopulations according to their NAD(P)H content (22). However, this concept has been questioned by Mercan and Malaisse (24), who denied the existence of subpopulation heterogeneity on the basis of measurement of cellular calcium variations in response to glucose in isolated -cells (24). Using PSA-NCAM as a marker of -cell activity, we provide here further and direct evidence of at least two different -cell subpopulations that diverge in their ability to secrete insulin in response to glucose. PSA-NCAM belongs to a family of molecules involved in the development of the endocrine pancreas (21,25). The expression of epithelial NCAM is high in pancreatic epithelial cells in the human fetus compared with the adult (25). This molecule allows adhesion between epithelial cells, permitting the transmission of signals that induce morphogenesis of the endocrine pancreas (25). In NCAM-deficient mice, the normal segregation of islet endocrine cells is altered during development (21), thus underlining the important role of NCAM in islet cell organization. PSA is a large negatively charged residue that contributes to the anti-adhesion effect of NCAM and promotes cell migration (26). These data strongly support that, combined with its role in the secretory process, PSAS129

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NCAM could be involved in the differentiation of new islets with functional -cells. Therefore, this molecule could be an important tool for discriminating between fully functional and less functional -cells in the regenerating pancreas. ACKNOWLEDGMENTS

C.B.-K. was supported by a fellowship from the Institut de Recherches Internationales Servier. The authors gratefully acknowledge Dr. Cécile Tourel for expert technical advice for cell perifusions. REFERENCES 1. Parsons JA, Brelje TC, Sorenson RL: Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion. Endocrinology 130: 1459–1466, 1992 2. Klöppel G, Löhr M, Habich K, Oberholzer M, Heitz P: Islet pathology and pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Path Res 4:110–4125, 1985 3. Unger RH: Diabetic hyperglycemia: link to impaired glucose transport in pancreatic -cells. Science 251:1200–1205, 1991 4. Portha B, Giroix MH, Serradas P, Morin L, Saulnier C, Bailbe D: Cellular basis for glucose refractoriness of pancreatic -cells in rat models of non-insulin dependent diabetes. Diabetes Metab 20:108–115, 1994 5. Pipeleers D, Kiekens R, Ling Z, Wilikens A, Schuit F: Physiologic relevance of heterogeneity in the pancreatic -cell population. Diabetologia 37 (Suppl. 2):S57–S64, 1994 6. Edelman G: CAMs and immunoglobulins: cell adhesion and the evolutionary origins of immunity. Immunol Rev 100:11–45, 1987 7. Finne J, Finne U, Deagostini-Bazin H, Goridis C: Occurrence of alpha-2-8 linked polysialosyl units in a neural cell adhesion molecule. Biochem Biophys Res Commun 112:482–487, 1983 8. Rutishauser U, Acheson A, Hall AK, Mann DM, Sunshine J: The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science 240:53–57, 1988 9. Rutishauser U, Landmesser L: Polysialic acid in the vertebrate nervous system: a promoter of plasticity in cell-cell interactions. Trends Neurosci 19:422– 427, 1996 10. Walsh F, Meiri K, Doherty P: Cell signalling and CAM-mediated neurite outgrowth. Soc Gen Physiol 52:221–226, 1997

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