A major class of nicotinic receptors in the nervous system is one that binds ␣-bungarotoxin and contains the ␣7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the ␣7 gene and have binding sites for the toxin, but previous attempts to elicit currents from the putative receptors have failed. Using whole-cell patch-clamp recording techniques and rapid application of agonist, we find a rapidly desensitizing acetylcholine-induced current in the cells that can be blocked by ␣-bungarotoxin. The current amplitude varies dramatically among three populations of PC12 cells but correlates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with ␣7 transcript; cells with high levels of ␣7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reverse transcription-PCR reveal no defects in ␣7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length ␣7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain ␣7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from ␣7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind ␣-bungarotoxin and contain ␣7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.

Of the 15 nicotinic ACh receptor genes identified in vertebrates, only four (alpha 1, beta 1, gamma, and delta) have been shown to be expressed in embryonic skeletal muscle at early times. In mammalian muscle a fifth gene (epsilon) replaces the gamma gene in expression at later times. The remaining 10 nicotinic receptor genes identified to date (alpha 2-alpha 8, beta 2-beta 4) are expressed in the nervous system and are considered neuronal genes. Using RNase protection assays, we show here that four of the neuronal-type genes (alpha 4, alpha 5, alpha 7, and beta 4) are expressed in developing chick skeletal muscle. Two of them (alpha 4 and alpha 7) decline substantially in transcript abundance between embryonic days 11 and 17, as does alpha 1, while the other two (alpha 5 and beta 4) show only moderate decreases over the same time period. At embryonic day 8, alpha 7 transcripts are nearly 20% as abundant as alpha 1 transcripts. In situ hybridizations confirm the presence of alpha 7 transcripts in muscle cells both in cell culture and in embryonic tissue. No evidence was found for expression of the alpha 2, alpha 3, alpha 8, or beta 3 genes in muscle. Immunoprecipitations and immunoblot analysis using subunit-specific monoclonal antibodies reveal alpha 7 protein in muscle, and the amount of protein rises and declines with the amount of alpha 7 mRNA during development. Sucrose gradient analysis demonstrates that the alpha 7 protein is present in muscle as a species of 10S, the size expected for a nicotinic receptor. The alpha 7 species in muscle binds alpha-bungarotoxin but does not contain alpha 1 subunits, indicating that the two kinds of alpha-type gene products segregate during assembly. The results suggest that neuronal AChRs may play a role in early muscle development.

A novel series of non-carbohydrate imidazole-based selectin inhibitors has been discovered via high-throughput screening using a P-selectin ELISA-based assay system. The initial lead 1 had an IC(50) of 17 microM in the P-selectin ELISA; this potency was significantly improved via an extensive SAR exploration. One of the current lead compounds (29) has an IC(50) of 300 nM in a P-selectin ELISA; it also has good activity in P- and E-selectin cell adhesion assays and shows efficacy in vivo. These compounds represent a novel series of sLe(X) mimetics with antiinflammatory activity. Their unique profile supports our interest in their further evaluation as drug candidates for the treatment of inflammation. Herein we describe the syntheses, optimization, and SAR of this series of novel potent selectin antagonists.

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp 187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R-and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52؊72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg 2؉, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11; FBPase) 1 catalyzes a tightly regulated step of gluconeogenesis, the hydrolysis of fructose 1,6-bisphosphate (F16P 2 ) to fructose 6-phosphate (F6P) and P i (1, 2). AMP and F26P 2 (binding to allosteric and active sites, respectively) inhibit FBPase, while simultaneously activating its counterpart in glycolysis, fructose-6-phosphate 1-kinase (3, 4). Biosynthesis and degradation of F26P 2 is subject to hormonal control principally by glucagon and insulin (4, 5 FBPase is a homotetramer (subunit M r of 37,000 (11)) and exists in at least two distinct quaternary conformations called R and T (12Ϫ14). AMP induces the transition from the active R-state to the inactive (or less active) T-state. Substrates or products in combination with metal cations stabilize the Rstate conformation. A proposed mechanism for allosteric regulation of catalysis involves three conformational states of loop 52Ϫ72 called engaged, disengaged, and disordered (15). AMP alone or with F26P 2 stabilizes a disengaged loop (16, 17), whereas metals with products stabilize an engaged loop (10, 17Ϫ19). In active forms of the enzyme, loop 52Ϫ72 probably cycles between its engaged and disordered conformations (15,18). Fluorescence from a tryptophan reporter group at position 57 is consistent with the conformational states for loop 52Ϫ72, observed in crystal structures (20,21). Presumably, the engaged, disengaged, and disordered conformations of loop 52Ϫ72 are possible in both the R-and T-states of FBPase, but only the engaged and disordered conformers of the R-st...

Second messenger regulation of neuronal acetylcholine receptors (AChRs) was investigated in a mouse fibroblast cell line, M10, stably transfected with chicken α4 and β2 cDNAs. Both forskolin and 8‐bromo‐cyclic adenosine 3′,5′‐monophosphate (cAMP) induced large increases in the numbers of AChRs. The increases were due in part to increased transcription and translation of the α4 and β2 genes. Blockade of protein synthesis with cycloheximide, however, revealed that forskolin also exerts a post‐translational effect, increasing the number of surface receptors by twofold. Immunoblot analysis of sucrose gradient fractions confirmed that the cells had a large fraction of unassembled subunits potentially available for receptor assembly. The post‐translational effect of forskolin was blocked by H‐89, an inhibitor of cAMP‐dependent protein kinase, and by okadaic acid, an inhibitor of phosphatases 1 and 2A. Nicotine also acted post‐translationally to induce a twofold increase in the number of surface receptors, but the mechanism differed from that utilized by forskolin, since the effects of the two agents were additive and were differentially affected by okadaic acid. The results suggest that protein phosphorylation‐dephosphorylation mechanisms act post‐translationally to increase the number of neuronal AChRs maintained on the cell surface. This could be achieved by increasing the efficiency of receptor assembly, transport, or stabilization on the cell surface. © 1996 John Wiley & Sons, Inc.