l-selectin gene polymorphisms and complications of sickle cell disease.

We had found high expression of L-selectin and alphaMbeta2 integrin on leukocytes in patients with complications of sickle cell disease (SCD). In non-SCD patients, L-selectin polymorphisms are associated with vasculopathy and nephropathy. Our objective was to determine if L-selectin gene polymorphisms affect leukocyte expression of the protein, or the development of complications in SCD. By polymerase chain reaction with sequence-specific primers incorporating mismatches at the 3'-end, we analysed DNA from 142 HbSS patients and 102 healthy, racially matched, HbAA controls; to detect the F206L, T49S, and P213S L-selectin gene polymorphisms. All patients were assessed for complications of SCD. Steady-state expression of L-selectin on leukocytes was measured by flow cytometry in 44 patients. We excluded HbSS patients on hydroxyurea, with any other disease, pregnancy, or HbF > or = 10%. There were no significant differences in distribution of F206L, T49S or P213S l-selectin gene polymorphisms between patients and controls (chi(2) = 0.1, P > 0.05). There was no association between any of these gene polymorphisms and high expression of L-selectin by leukocytes, or the development of complications in SCD (chi(2) = 2.37, P > 0.05). The findings suggest that these three gene polymorphisms do not predispose to high leukocyte expression of L-selectin, or development of complications in SCD.

Export of cytochrome P450 105D1 to the periplasmic space of Escherichia coli.

CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell extract and whole-cell activity studies showed that the periplasmically located CYP105D1 competently catalyzed NADH-dependent oxidation of the xenobiotic compounds benzo[a]pyrene and erythromycin, further revealing the presence in the E. coli periplasm of endogenous functional redox partners. This system offers substantial advantages for the application of P450 enzymes to whole-cell biotransformation strategies, where the ability of cells to take up substrates or discard products may be limited.

Targeting of active human cytochrome P4501A1 (CYP1A1) to the periplasmic space of Escherichia coli.

Native human cytochrome P4501A1 (CYP1A1) was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase. The chimeric P450 construct was placed under the transcriptional control of the native phoA promoter in a prokaryotic expression vector. Induction of the hemoprotein by heterologous expression in E. coli following growth in a phosphate-limited medium resulted in abundant synthesis of recombinant CYP1A1 as detected by reduced CO-difference spectra. Furthermore, the signal-appended CYP1A1 was translocated across the bacterial inner membrane by the sec-dependent pathway and processed to yield authentic, heme-incorporated P450 within the periplasmic space. In vitro and whole-cell metabolic activity studies showed that the periplasmically-located CYP1A1 competently catalysed NADPH-dependent benzo[a]pyrene 3-hydroxylation and 7-ethoxyresorufin O-deethylation. The means to localise cytochromes P450 in the periplasm offers an ability to produce high levels of protein, attributable to the less hostile nature of the compartment, and therein the enzymes for posttranslational assembly of heme with the translocated protein.