Two ZnF-UBP domains in isopeptidase T (USP5).

Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme's hydrolytic activity toward ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored monoubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163-291), which binds the substrate's unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1-156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.

Tariquidar, a selective P-glycoprotein inhibitor, does not potentiate loperamide's opioid brain effects in humans despite full inhibition of lymphocyte P-glycoprotein.

BACKGROUND: Loperamide, a potent opioid, has been used as an in vivo probe to assess P-glycoprotein activity at the blood-brain barrier, because P-glycoprotein inhibition allows loperamide to cross the blood-brain barrier and exert its central opioid effects. In humans, studies with nonselective and moderately potent inhibitors resulted in mild opioid effects but were confounded by the concurrent inhibition of loperamide's metabolism. The authors studied the effect of the highly selective, potent P-glycoprotein inhibitor tariquidar on loperamide's central opioid effects. METHODS: In a randomized, double-blind, crossover study, nine healthy subjects received on 2 study days oral loperamide (32 mg) followed by an intravenous infusion of either tariquidar (150 mg) or placebo. Central opioid effects (pupil diameter, sedation) were measured for 12 h, and blood samples were drawn up to 48 h after drug administration to determine plasma loperamide concentrations and ex vivo P-glycoprotein activity in T lymphocytes. Values for pupil diameter and loperamide concentrations were plotted over time, and the areas under the curves on the tariquidar and placebo study day were compared within each subject. RESULTS: Tariquidar did not significantly affect loperamide's central effects (median reduction in pupil diameter area under the curve, 6.9% [interquartile range, -1.4 to 12.1%]; P = 0.11) or plasma loperamide concentrations (P = 0.12) but profoundly inhibited P-glycoprotein in lymphocytes by 93.7% (95% confidence interval, 92.0-95.3%). CONCLUSION: These results suggest that despite full inhibition of lymphocyte P-glycoprotein, the selective P-glycoprotein inhibitor tariquidar does not potentiate loperamide's opioid brain effects in humans.

Crystal structures of monomeric actin bound to cytochalasin D.

The fungal toxin cytochalasin D (CD) interferes with the normal dynamics of the actin cytoskeleton by binding to the barbed end of actin filaments. Despite its widespread use as a tool for studying actin-mediated processes, the exact location and nature of its binding to actin have not been previously determined. Here we describe two crystal structures of an expressed monomeric actin in complex with CD: one obtained by soaking preformed actin crystals with CD, and the other obtained by cocrystallization. The binding site for CD, in the hydrophobic cleft between actin subdomains 1 and 3, is the same in the two structures. Polar and hydrophobic contacts play equally important roles in CD binding, and six hydrogen bonds stabilize the actin-CD complex. Many unrelated actin-binding proteins and marine toxins target this cleft and the hydrophobic pocket at the front end of the cleft (viewing actin with subdomain 2 in the upper right corner). CD differs in that it binds to the back half of the cleft. The ability of CD to induce actin dimer formation and actin-catalyzed ATP hydrolysis may be related to its unique binding site and the necessity to fit its bulky macrocycle into this cleft. Contacts with residues lining this cleft appear to be crucial to capping and/or severing. The cocrystallized actin-CD structure also revealed changes in actin conformation. An approximately 6 degrees rotation of the smaller actin domain (subdomains 1 and 2) with respect to the larger domain (subdomains 3 and 4) results in small changes in crystal packing that allow the D-loop to adopt an extended loop structure instead of being disordered, as it is in most crystal structures of actin. We speculate that these changes represent a potential conformation that the actin monomer can adopt on the pathway to polymerization or in the filament.

Beta-1-adrenoceptor genetic variants and ethnicity independently affect response to beta-blockade.

OBJECTIVES: Black patients may be less responsive to beta-blockers than whites. Genetic variants in the beta1-adrenergic receptor (beta1-AR) associated with lesser response to beta-blockers are more common in blacks than in whites. The purpose of this study was to determine whether ethnic differences in response to beta-blockade can be explained by differing distributions of functional genetic variants in the beta1-AR. METHODS: We measured sensitivity to beta-blockade by the attenuation of exercise-induced tachycardia in 165 patients (92 whites), who performed a graded bicycle exercise test before and 2.5 h after oral atenolol (25 mg). We determined heart rate at rest and at three exercise levels from continuous ECG recordings and calculated the area under the curve. We also measured plasma atenolol concentrations and determined genotypes for variants of the beta1-AR (Ser49Gly, Arg389Gly) and alpha2C-AR (del322-325). The effects of ethnicity, genotype, and other covariates on the heart rate reduction after atenolol were estimated in multiple regression analyses. RESULTS: Atenolol resulted in a greater reduction in exercise heart rate in whites than in blacks (P=0.006). beta1-AR Arg389 (P=0.003), but not the alpha2C-AR 322-325 insertion allele (P=0.31), was independently associated with a greater reduction in heart rate area under the curve. Ethnic differences in sensitivity to atenolol remained significant (P=0.006) after adjustment for beta1-AR and alpha2C-AR genotypes. CONCLUSION: Ethnic differences in sensitivity to the beta1-blocker atenolol persist even after accounting for different distributions of functional genetic beta1-AR variants, suggesting that additional, as yet unidentified factors contribute to such ethnic differences.

Clinical sedation scores as indicators of sedative and analgesic drug exposure in intensive care unit patients.

BACKGROUND: It is unclear how best to measure sedative/analgesic drug exposure in the clinical care of critically ill patients. Large doses and prolonged use of sedatives and analgesics in the intensive care unit (ICU) may lead to oversedation and adverse effects, including delirium and long-term cognitive impairment. These issues are of particular concern in elderly patients (aged > or =65 years), who account for at least half of all ICU admissions and nearly two thirds of ICU days. OBJECTIVE: This pilot study explored the relationships between clinical sedation scores, sedative/analgesic drug doses, and plasma drug concentrations in critically ill patients, the majority of whom were elderly. METHODS: This was a prospective, observational study conducted in a 500-bed, tertiary care community hospital. Study patients included a cohort of mechanically ventilated, medical ICU patients who were admitted to the hospital between April and June 2004 who required use of fentanyl, lorazepam, or propofol. Sedative/analgesic medications were administered according to clinical guidelines. Patients' sedation levels were measured twice daily using the Richmond Agitation-Sedation Scale (RASS). Dosing of fentanyl, lorazepam, and propofol was recorded. Blood was sampled twice daily for up to 5 days to analyze plasma drug concentrations. To specifically evaluate the effects of acute, extended (rather than chronic) sedative and analgesic exposure, the study focused on an ICU population receiving these agents for at least 48 hours but <2 weeks. RESULTS: Eighteen medical ICU patients (11 females, 7 males; mean [SD] age, 66.1 [18.1] years) on mechanical ventilation comprised the study cohort. Fifteen patients were aged >62 years, and 11 of those were aged > or =71 years. Plasma drug concentrations (median and interquartile range) were as follows: fentanyl--2.1 ng/mL, 0.9-3.4 ng/mL; lorazepam--266 ng/mL, 112-412 ng/mL; and propofol--845 ng/mL, 334-1342 ng/mL. Maximum concentrations were 3- to 12-fold higher than medians (fentanyl, 7.3 ng/mL; lorazepam, 3108 ng/mL; propofol, 10,000 ng/mL). Medication doses were only moderately correlated with RASS scores (Spearman rho): fentanyl--rho = -0.39, P = 0.002; lorazepam--rho = -0.28, P = 0.001; and propofol--rho = -0.46, P < 0.001. Plasma drug concentrations of fentanyl and lorazepam demonstrated moderate correlations with sedation scores (fentanyl--rho = -0.46, P = 0.002; lorazepam: rho = -0.49, P < 0.001), while propofol concentrations correlated poorly with sedation scores (rho = -0.18, P = 0.07). Associations between interval drug doses and plasma concentrations were as follows: fentanyl, rho = 0.84; lorazepam, rho = 0.76; and propofol, rho = 0.61 (all, P < 0.001). Instructive examples of discrepant dose versus plasma concentration profiles and drug interactions are provided, including 3 cases with patients aged > or =64 years. CONCLUSIONS: Elderly patients are commonly encountered in the ICU setting. Only moderate correlations existed between clinical sedation levels and dose or plasma concentration of sedative/analgesic medications in this study population. Further work is needed to understand appropriate and feasible measures of exposure to sedatives/analgesics relating to clinical outcomes.