Conserved features of the sequences of dopamine receptors and of homologous G-protein-coupled receptors point to regions, and amino acid residues within these regions, that contribute to their ligand binding sites. Differences in binding specificities among the catecholamine receptors, however, must stem from their nonconserved residues. Using the substituted-cysteine accessibility method, we have identified the residues that form the surface of the water-accessible binding-site crevice in the dopamine D2 receptor. Of approximately 80 membrane-spanning residues that differ between the D2 and D4 receptors, only 20 were found to be accessible, and 6 of these 20 are conservative aliphatic substitutions. In a D2 receptor background, we mutated the 14 accessible, nonconserved residues, individually or in combinations, to the aligned residues in the D4 receptor. We also made the reciprocal mutations in a D4 receptor background. The combined substitution of four to six of these residues was sufficient to switch the affinity of the receptors for several chemically distinct D4-selective antagonists by three orders of magnitude in both directions (D2- to D4-like and D4- to D2-like). The mutated residues are in the second, third, and seventh membrane-spanning segments (M2, M3, M7) and form a cluster in the binding-site crevice. Mutation of a single residue in this cluster in M2 was sufficient to increase the affinity for clozapine to D4-like levels. We can rationalize the data in terms of a set of chemical moieties in the ligands interacting with a divergent aromatic microdomain in M2-M3-M7 of the D2 and D4 receptors.

Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

In the current era, continuous electroencephalographic monitoring demonstrates early postoperative seizures in 11.2% of a heterogeneous cohort of neonates and infants with complex congenital heart defects. Increasing duration of DHCA was identified as a predictor of seizures. However, the incidence of seizures in children with limited duration of DHCA was similar to that in infants undergoing continuous cardiopulmonary bypass alone.

Abstract. Torpedo californica acetylcholine receptor (AChR) tx-, ~-, 3'-, and tS-subunit cDNAs were each stably introduced into muscle and/or fibroblast cell lines using recombinant retroviral vectors and viral infection, or using SV-40 vectors and DNA-mediated cotransfection. The expressed proteins were characterized in terms of their molecular mass, antigenicity, posttranslational processing, cell surface expression, stability in fibroblasts, stability in differentiated and undifferentiated muscle cells, and ability (of a) to bind o~-bungarotoxin (BuTx). We demonstrated that the ct, ~, .,/, and t5 polypeptides acquired one, one, two, and three units of oligosaccharide, respectively. If all four subunits were expressed in the same cell, fully functional cell surface AChRs were produced which had a K~ for BuTx of 7.8 x 10-" M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual o~ polypeptides was only '~4 x 10 -7 M. The half-lives of the or, 3', and t5 subunits at 37°C were all found to be quite short (,x,43 min), while the half-life of the ~ subunit was found to be even shorter ('~12 min). The unique half-life of the/3 subunit suggests that it might perform a key regulatory role in the process of AChR subunit assembly. One stable fibroblast cell line was established by transfection that expressed ~, -y, and t5 subunits simultaneously. When this cell line was infected with a retroviral o~ recombinant, fully functional cell surface AChRs were produced. The successful expression of this pentameric protein complex combining transfection and infection techniques demonstrates one strategy for stably introducing the genes of a heterologous multisubunit protein complex into cells.

. INVITED COMMENTARYI read with interest the article by Chen and colleagues [1]. A 10% incidence of perioperative stroke is a possible partial explanation for less than ideal neurodevelopmental outcomes in congenital heart disease (CHD) [2,3]. Additional explanations are genetic causes [4], fetal development with CHD, and injury that is not measured by imaging. If we are to improve neurologic outcomes, there are four areas in which practice improvement needs to become uniform or further explored: (1) prenatal diagnosis and maternal care, (2) postnatal preoperative care, (3) operative interventional strategies, and (4) postoperative care. Specifics of these are briefly discussed. 1.Can fetal diagnosis and maternal care for all having CHD impact birth weight and thereby mitigate this as a risk? 2.Can fetal triage to a congenital cardiac liaised high-risk obstetrics and neonatal team preclude cardiorespiratory instability in more babies and thus lessen preoperative stroke? 3.Risk was shown to be modifiable in the operating room. A higher hematocrit (28% vs 26%) on cardiopulmonary bypass (CPB) was associated with a lower incidence of stroke. This corroborates previous findings that hematocrit on CPB is important [5]. Not all factors believed to be a risk in the operating room were actually a risk. Circulatory arrest, as performed and compared, did not impact neurodevelopment. Fruitful areas for further study will include hybrid stage I versus conventional stage I palliation for single ventricle and arch obstruction, further human studies on pCO2 management strategy for CPB, rate of CPB flow, and others. 4.Postoperative measurements, such as systemic oxygen delivery [6], length of stay in the intensive care unit, and other factors are also important and potentially modifiable.Survival with CHD has become an expectation. It is our duty to ensure that those unfortunate enough to have CHD not only survive, but live. Continued critical data analysis in the areas outlined, trials, and modification of practice will allow us to help our patients live-in the fullest sense. AbstractBackground-The prevalence of perioperative stroke in infants undergoing operations for congenital heart disease has not been well described. The objectives of this study were to determine the prevalence of stroke as assessed by postoperative brain magnetic resonance imaging (MRI), characterize the neuroanatomic features of focal ischemic injury, and identify risk factors for its development.