Leukocyte adhesion to vascular endothelium contributes to vaso-occlusion and widespread organ damage in sickle cell disease (SCD). Previously, we found high expression of the adhesion molecules alphaMbeta2 integrin and L-selectin in HbSS individuals with severe disease. Since membrane n-6 and n-3 polyunsaturated fatty acids modulate cell adhesion, inflammation, aggregation and vascular tone, we investigated the fatty acid composition of mononuclear cells (MNC) and platelets of HbSS patients in steady state (n=28) and racially matched, healthy HbAA controls with similar age and sex distribution living in the same environment (n=13). MNC phospholipids of the patients had lower levels of docosahexaenoic acid (DHA, p<0.01) and increased arachidonic acid (AA, p<0.005) relative to HbAA controls. Similarly, platelets from HbSS patients had less eicosapentaenoic acid (EPA, p<0.05) and more AA (p<0.05) in choline phosphoglycerides (CPG), with reduced DHA (p<0.05) in ethanolamine phosphoglycerides. Platelet CPG had lower DHA levels in SCD patients with complications compared to those without (p<0.05). Reduced cell content of EPA and DHA relative to AA favours the production of aggregatory and proinflammatory eicosanoids that activate leukocytes and platelets. This facilitates inflammation, leukocyte adhesion, platelet aggregation and vaso-occlusion in SCD.

No abstract

No abstract

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.Cytochromes P450 (CYPs) are a superfamily of enzymes capable of an unprecedented array of catalytic activities (4, 12). Distinct members are engaged in biosynthetic reactions within many organisms, while others have a role in the detoxification of foreign compounds. The latter substrates include medicines, pollutants, pesticides, carcinogens, perfumes, and herbicides representing considerable applied importance for pharmacology and toxicology. CYPs show a high degree of stereo-and regiospecificity for their reactions, which have wider industrial applications. For example, fungal CYPs are used in the production of corticosteroids (19), and a CYP enzyme from a Streptomyces sp. is exploited in statin production (17). Many of the CYP enzymes have very broad substrate ranges, and among the widest range is that of CYP105D1 from Streptomyces griseus (ATCC 13273), encompassing pharmaceuticals, agrochemicals, and environmental pollutants (16,21). This enzyme has been employed in Streptomyces whole-cell biotransformations for the preparation of a number of valuable drug metabolites (3).CYP105D1 has previously been expressed as an active recombinant cytosolic form in Escherichia coli using the IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible tac promoter (20). Selective permeability of E. coli to many substrates and products can cause problems when using whole-cell systems. For such reasons, cell wall mutants of Salmonella enterica serovar Typhimurium were developed for use in mutagenesis tests (10). One approach to overcome these problems could be to engineer CYPs that can be exported to the periplasm or the cell exterior. Previous studies with cytochrome b 5 have shown that cytosolic hemoproteins can be efficiently exported for their enhanced accumulation in the less hostile environments of the periplasm (see, for example, references 5 and 9). Based on these observations, we genetically manipulated an S. griseus CYP (CYP105D1) to attempt export to the periplasm. MATERIALS AND METHODSBacteria and plasmids. The E. coli DH5␣ strain was used for ...