Molecular Association of the Neural Adhesion Molecules L1 and N-CAM in the Surface Membrane of Neuroblastoma Cells is Shown by Chemical Cross-linking.

The two neural cell adhesion molecules L1 and N-CAM could be shown to be associated in the surface membrane of cultured neuroblastoma cells by chemical cross-linking with 3,3'-dithiobis(sulphosuccinimidyl-propionate) and subsequent immunopurification and precipitation using antibodies to L1 and N-CAM. Glycoproteins recognized in neuroblastoma cells by antibodies to mouse liver membranes were not chemically cross-linked to L1 or N-CAM. These observations suggest that a molecular association between the two molecules may be the basis for their functional cooperativity (Kadmon et al., 1990a,b, J. Cell Biol., 110, 193-208; 209-218).

Two Monoclonal Antibodies Recognizing Carbohydrate Epitopes on Neural Adhesion Molecules Interfere with Cell Interactions.

We have studied two monoclonal antibodies raised against crude fractions of membrane glycoproteins from adult mouse brain and found them to react with two carbohydrate epitopes expressed on several neural cell adhesion molecules. Other identified and unidentified glycoproteins from different cell types, organs and species were also recognized by these antibodies. Both epitopes are N-glycosidically linked mannosidic or hybrid type oligosaccharides and co-expressed on all the glycoproteins so far tested. In spite of their remarkable similarities, the glycan epitopes are different as shown by ELISA competition assays. In microexplant outgrowth and cell adhesion assays, both antibodies interfere with neural cell adhesion, migration, and neurite outgrowth. These observations, together with previous studies on the L2/HNK-1 glycan (Künemund et al., 1988), indicate that adhesion molecules carry various carbohydrate epitopes mediating different cell interactions in in vitro assays.

Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell line.

We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (BAY 73-6691), the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC50 = 55 nM) and murine (IC50 = 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl)[2-[(4-phenethyl-benzyl)oxy]phenethyl]amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity.