Role of glucocorticoid receptor-mediated mechanisms in cocaine memory enhancement.

The basolateral amygdala (BLA) is a critical site for the reconsolidation of labile contextual cocaine memories following retrieval-induced reactivation/destabilization. Here, we examined whether glucocorticoid receptors (GR), which are abundant in the BLA, mediate this phenomenon. Rats were trained to lever press for cocaine reinforcement in a distinct environmental context, followed by extinction training in a different context. Rats were then briefly exposed to the cocaine-paired context (to elicit memory reactivation and reconsolidation) or their home cages (no reactivation control). Exposure to the cocaine-paired context elicited greater serum corticosterone concentrations than home cage stay. Interestingly, the GR antagonist, mifepristone (3-10 ng/hemisphere), administered into the BLA after memory reactivation produced a further, dose-dependent increase in serum corticosterone concentrations during the putative time of cocaine-memory reconsolidation but produced an inverted U-shaped dose-effect curve on subsequent cocaine-seeking behavior 72 h later. This effect was anatomically selective, dependent on memory reactivation (i.e., not observed after home cage exposure), and did not reflect protracted hyperactivity. However, the effect was also observed when mifepristone was administered after novelty stress that mimics drug context-induced hypothalamic-pituitary-adrenal (HPA) axis activation without explicit memory reactivation. Together, these findings suggest that, similar to explicit memory retrieval, a stressful event is sufficient to destabilize cocaine memories and permit their manipulation. Furthermore, BLA GR stimulation exerts inhibitory feedback upon HPA axis activation and thus suppresses cocaine-memory reconsolidation.

Differences between cystic fibrosis transmembrane conductance regulator and HisP in the interaction with the adenine ring of ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is a member of the ATP-binding cassette transporter family. The most conserved features of this family are the nucleotide-binding domains. As in other members of this family, these domains bind and hydrolyze ATP; in CFTR this opens and closes the channel pore. The recent crystal structures of related bacterial transporters show that an aromatic residue interacts with the adenine ring of ATP to stabilize nucleotide binding. CFTR contains six aromatic residues that are candidates to coordinate the nucleotide base. We mutated each to cysteine and examined the functional consequences. None of the mutations disrupted channel function or the ability to discriminate between ATP, GTP, and CTP. We also applied [2-(triethylammonium)ethyl] methanethiosulfonate to covalently modify the introduced cysteines. The mutant channels CFTR-F429C, F430C, F433C, and F1232C showed no difference from wild-type CFTR, indicating that either the residues were not accessible to modification, or cysteine modification did not affect function. Although modification inactivated CFTR-Y1219C more rapidly than wild-type CFTR, and inactivation of CFTR-F446C was nucleotide-dependent; failure of these mutations to alter gating suggested that Tyr(1219) and Phe(446) were not important for nucleotide binding. The results suggest that ATP binding may not involve the coordination of the adenine ring by an aromatic residue analogous to that in some bacterial transporters. Taken together with earlier work, this study points to a model in which most of the binding energy for ATP is contributed by the phosphate groups.

Delineation of two functionally distinct gammaPDE binding sites on the bovine retinal cGMP phosphodiesterase by a mutant gammaPDE subunit.

The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In this work, we describe a full length, doubly point-mutated gamma subunit, C68S, Y84C gammaPDE, which binds to PDE with increased affinity but has a decreased ability to inhibit the enzyme. Fluorescence studies monitoring the competition between wild-type gammaPDE and the C68S, Y84C gammaPDE mutant suggest that the mutant gammaPDE binds with high affinity to only half of the total sites occupied by wild-type gammaPDE. Competition studies between wild-type gammaPDE and the mutant further suggest that the wild-type protein is able to fully inhibit PDE activity even when the mutant gammaPDE occupies its high-affinity binding site on PDE. Taken together, our findings are consistent with a model in which there are two distinguishable binding sites for gammaPDE on the PDE enzyme but that only one of the two sites mediates PDE inhibition.

Real time conformational changes in the retinal phosphodiesterase gamma subunit monitored by resonance energy transfer.

The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT). In order to characterize conformational changes in the 87-amino acid gammaPDE subunit that may accompany the activation of the holoenzyme, gammaPDE was labeled with the fluorescent probes 5-iodoacetamidofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements. 5-Iodoacetamidofluorescein specifically labeled a cysteine residue at position 68 and served as a resonance energy transfer donor. The site of modification of eosin-5-isothiocyanate, which served as the resonance energy transfer acceptor, was determined to be within the first seven residues of the amino terminus of gammaPDE. Energy transfer between the labeled sites on free, unbound gammaPDE indicated that they were separated by a distance of 63 A, consistent with a random conformation. Upon binding the catalytic alphabeta subunits of the PDE, the distance between the two probes on gammaPDE increased to 77 A. Binding of the labeled gammaPDE by alphaT.guanosine 5'-3-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated. These data are consistent with the view that the binding of activated alphaT to gammaPDE, which is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the gammaPDE molecule. They also support a model for PDE activation that places active alphaT in a complex with the holoenzyme.

A liveborn case of 49,XXXY, + 18.

The first case of a liveborn male infant trisomic for both the X and the No 18 chromosome is presented. The patient had multiple congenital anomalies many of which were similar in appearance to patients with trisomy 18. The proband died after 2 days. Both maternal and paternal karyotypes were normal.

Incidence of chromosomal rearrangements in couples with reproductive loss.

We report on 50 couples with reproductive loss did not have any detectable chromosome abnormality. A history of a previous child with multiple congenital abnormalities may be significant in identifying couples with a structural rearrangement. Only by studying more families can this hypothesis be tested. Studies of abortus tissue reveal a high percentage of chromosome abnormalities but a very low incidence of unbalanced translocations. Cytogenetic studies are indicated in a couple which has a past history of spontaneous abortions and a previous child with multiple congenital anomalies.