Characterization of a p53-related activation domain in Adr1p that is sufficient for ADR1-dependent gene expression.

The yeast transcriptional activator Adr1p controls expression of the glucose-repressible alcohol dehydrogenase gene (ADH2), genes involved in glycerol metabolism, and genes required for peroxisome biogenesis and function. Previous data suggested that promoter-specific activation domains might contribute to expression of the different types of ADR1-dependent genes. By using gene fusions encoding the Gal4p DNA binding domain and portions of Adr1p, we identified a single, strong acidic activation domain spanning amino acids 420-462 of Adr1p. Both acidic and hydrophobic amino acids within this activation domain were important for its function. The critical hydrophobic residues are in a motif previously identified in p53 and related acidic activators. A mini-Adr1 protein consisting of the DNA binding domain of Adr1p fused to this 42-residue activation domain carried out all of the known functions of wild-type ADR1. It conferred stringent glucose repression on the ADH2 locus and on UAS1-containing reporter genes. The putative inhibitory region of Adr1p encompassing the protein kinase A phosphorylation site at Ser-230 is thus not essential for glucose repression mediated by ADR1. Mini-ADR1 allowed efficient derepression of gene expression. In addition it complemented an ADR1-null allele for growth on glycerol and oleate media, indicating efficient activation of genes required for glycerol metabolism and peroxisome biogenesis. Thus, a single activation domain can activate all ADR1-dependent promoters.

Awareness with recall during general anesthesia: incidence and risk factors.

UNLABELLED: We studied the associated factors and incidence of awareness during general anesthesia and the nature of subsequent psychiatric disorders. Patients older than 12 yr undergoing surgery under general anesthesia in a secondary care hospital during 1 yr were included in the study. The doses of anesthetics were calculated for the patients with and without awareness. There were 4818 operations under general anesthesia; 2612 (54%) patients were interviewed. Ten (0.4% of those interviewed) patients were found to have undisputed awareness, and there were nine (0.3%) patients with possible awareness. The doses of isoflurane (P < 0.01) and propofol (P < 0.05) were smaller in patients with awareness. Five patients with awareness underwent a psychiatric evaluation. One patient experienced sleep disturbances afterward, but the other four patients did not have any after effects. In conclusion, awareness is a rare complication of general anesthesia associated with small doses of anesthetics. IMPLICATIONS: In an interview of 2612 patients after general anesthesia, 10 (0.4%) patients with awareness and 9 (0.3%) patients with possible awareness were found. A predisposing factor was small doses of the principal anesthetic. In a psychiatric interview, a large proportion of the patients with awareness were found to have suffered from depression in the past.

On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase.

Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.