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The study was done between April 1989 and December 1990. There were 122 patients with cfu counts values at any time available, but the numbers of patients with counts at each sampling time decreased by 33 27% ; of the 122 at 2 days and finally by 54 44% ; at the 28-day period Table 1 ; . In some instances, sputum gave a nega.
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To a 3-fold stimulation of the enzyme with ECra values concentration causing a half-maximal response ; of 50 p~ and 40 p ~respectively. Unfortunately, we have been unable to con, firm the presence of a dopamine-sensitive cyclase in Helix homogenates orwashed particulate fractionsassayed under a variety of experimental conditions, including those described byOsborne 5 ; notshown ; . Also, thestimulation of the enzyme by 5HT has never, in our hands, exceeded a doubling of the basal enzyme activity. The reasons for these discrepancies are unclear; neurotransmitter-sensitive adenylate cyclase preparations are, however, notoriously fickle. The stimulationof Helix adenylate cyclase activity by 5HT in awashed particulate preparation was enhanced by the presence of GTP Table III ; , which as previously reported 9.
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1a-AR mutations, the affinities of a panel of agonists Fig. 2 ; was determined in additional competition binding studies. Ki values for five 1-AR agonists are shown in Table I. Unlike the results obtained with the antagonists, mutagenesis of either phenylalanine residue in the 1a-AR had considerable effects on the binding affinities of agonists at this receptor. In COS-1 cells, overexpressed 1-ARs display single-site competition curves with Hill coefficients near unity. The addition of GTP analogues does not change the slope significantly and were not used in these competition studies. The affinity of the endogenous agonist epinephrine was reduced 12.5-fold by the F163Q mutation p 0.01 ; and by a factor of 8-fold by the F187A mutation p 0.01 ; , suggesting that each phenylalanine residue is directly involved in stabilizing the catecholamine in the agonist binding pocket of the 1a-AR. Furthermore, we argue that the impaired binding of epinephrine at the mutated 1a-AR is not the result of changes in the global conformation of the receptor since the affinities of the antagonists were not effected by either mutation. Therefore, both the Phe-163 and Phe-187 residues of the 1a-AR constitute novel and specific agonist-receptor point contacts in the binding pocket of the 1-ARs. Like epinephrine, the affinities of the synthetic phenethylamines, phenylephrine and methoxamine Fig. 2 ; , were also reduced by each phenylalanine mutation. A 6-fold decrease in affinity for phenylephrine p 0.05 ; and a 12-fold decrease in the affinity of methoxamine p 0.01 ; were observed at 1aARs displaying the F163Q mutation Table I ; . The F187A mutation had greater effects on the binding of the endogenous agonist epinephrine than on either of the phenethylamines. Phenylephrine and methoxamine binding was reduced by factors of just 3- and 4-fold, respectively, by the F187A mutation p 0.05 ; , as opposed to the greater than 8-fold decrease in affinity for epinephrine induced by the same mutation. Single mutagenesis of either the Phe-163 or Phe-187 residue of the 1a-AR illustrates that both amino acids provide significant contributions to the binding and stabilization of the agonist in the binding pocket of the receptor. To prove that these interactions between each phenylalanine residue and the catechol ring of the agonist are independent of one another, we constructed a mutant receptor expressing both mutations F163Q F187A 1a-AR ; . As observed with the single mutations, the binding affinity of the 1a-AR-selective antagonists was not altered by the combination of these mutations, indicating that, for instance, epivir tablets.
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