Publications

Cumene hydroperoxide-mediated (CuOOH-mediated) inactivation of cytochromes P450 (CYPs) results in destruction of their prosthetic heme to reactive fragments that irreversibly bind to the protein. We have attempted to characterize this process structurally, using purified, 14C-heme labeled, recombinant human liver P450 3A4 as the target of CuOOH-mediated inactivation, and a battery of protein characterization approaches [chemical (CNBr) and proteolytic (lysylendopeptidase-C) digestion, HPLC-peptide mapping, microEdman sequencing, and mass spectrometric analyses]. The heme-peptide adducts isolated after CNBr/lysylendopeptidase-C digestion of the CuOOH-inactivated P450 3A4 pertain to two distinct P450 3A4 active site domains. One of the peptides isolated corresponds to the proximal helix L/Cys-region peptide 429-450 domain and the others to the K-region (peptide 359-386 domain). Although the precise residue(s) targeted remain to be identified, we have narrowed down the region of attack to within a 17 amino acid peptide (429-445) stretch of the 55-amino acid proximal helix L/Cys domain. Furthermore, although the exact structures of the heme-modifying fragments and the nature of the adduction remain to be established conclusively, the incremental masses of approximately 302 and 314 Da detected by electrospray mass spectrometric analyses of the heme-modified peptides are consistent with a dipyrrolic heme fragment comprised of either pyrrole ring A-D or B-C, a known soluble product of peroxidative heme degradation, as a modifying species.

CONTEXT

The association between environmental tobacco smoke (ETS) exposure and respiratory symptoms has not been well established in adults.

OBJECTIVE

To study the respiratory health of bartenders before and after legislative prohibition of smoking in all bars and taverns by the state of California.

DESIGN

Cohort of bartenders interviewed before and after smoking prohibition.

SETTING AND PARTICIPANTS

Bartenders at a random sample of bars and taverns in San Francisco.

MAIN OUTCOME MEASURES

Interviews assessed respiratory symptoms, sensory irritation symptoms, ETS exposure, personal smoking, and recent upper respiratory tract infections. Spirometric assessment included forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) measurements.

RESULTS

Fifty-three of 67 eligible bartenders were interviewed. At baseline, all 53 bartenders reported workplace ETS exposure. After the smoking ban, self-reported ETS exposure at work declined from a median of 28 to 2 hours per week (P<.001). Thirty-nine bartenders (74%) initially reported respiratory symptoms. Of those symptomatic at baseline, 23 (59%) no longer had symptoms at follow-up (P<.001). Forty-one bartenders (77%) initially reported sensory irritation symptoms. At follow-up, 32 (78%) of these subjects had resolution of symptoms (P<.001). After prohibition of workplace smoking, we observed improvement in mean FVC (0.189 L; 95% confidence interval [CI], 0.082-0.296 L; 4.2% change) and, to a lesser extent, mean FEV1 (0.039 L; 95% CI, -0.030 to 0.107 L; 1.2% change). Complete cessation of workplace ETS exposure (compared with continued exposure) was associated with improved mean FVC (0.287 L; 95% CI, 0.088-0.486; 6.8% change) and mean FEV1 (0.142 L; 95% CI, 0.020-0.264 L; 4.5% change), after controlling for personal smoking and recent upper respiratory tract infections.

CONCLUSION

Establishment of smoke-free bars and taverns was associated with a rapid improvement of respiratory health.

To assess the roles of the active site residues Glu160 and Asp181 of human FEN-1 nuclease in binding and catalysis of the flap DNA substrate and in vivo biological processes of DNA damage and repair, five different amino acids were replaced at each site through site-directed mutagenesis of the FEN-1 gene. The mutants were then expressed in Escherichia coli and purified using a His-tag. Even though the mutants bind to the flap DNA to different degrees, most of the mutants lost flap nuclease activity with the exception of an E160D mutant. This mutant retained wild type-like binding ability, specificity, and partial catalytic activity. Detailed steady state and pre-steady state kinetic analysis revealed that the functional deficiency of this mutant was due to retardation of the endonucleolytic cleavage. When the mutant enzyme E160D was expressed in yeast, it partially complements the biological functions of the homologous yeast gene, RAD27, and reverses the hyper-temperature lethality and hypersensitivity to methyl methanesulfonate, in a manner corresponding to the in vitro activity.