Cytochrome P450BM-3, a bacterial fatty acid monoxygenase, resembles the eukaryotic microsomal P450's and their flavoprotein reductase in primary structure and function. The three-dimensional structure of the hemoprotein domain of P450BM-3 was determined by x-ray diffraction and refined to an R factor of 16.9 percent at 2.0 angstrom resolution. The structure consists of an alph and a beta domain. The active site heme is accessible through a long hydrophobic channel formed primarily by the beta domain and the B' and F helices of the alpha domain. The two molecules in the asymmetric unit differ in conformation around the substrate binding pocket. Substantial differences between P450BM-3 and P450cam, the only other P450 structure available, are observed around the substrate binding pocket and the regions important for redox partner binding. A general mechanism for proton transfer in P450's is also proposed.

Based on this comparison we believe that all P450s will be found to possess the same tertiary structure. The ability to precisely predict other P450 substrate-contact residues is limited by the extreme structural heterogeneity in the substrate-recognition regions. The central I-helix structures of P450terp and P450BM-3 suggest a role for helix-associated solvent molecules as a source of catalytic protons, distinct from the mechanism for P450cam. We suggest that the P450 molecular dipole might aid in both redox-partner docking and proton recruitment for catalysis.

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Clarifying the signals that lead to dendritic cell (DC) development and identifying cellular intermediates on their way to DC differentiation are essential steps to understand the dynamic regulation of number, localization, and functionality of these cells. In the past decade, much knowledge on cytokines, transcription factors, and successive progenitors involved in steady-state and demand-adapted DC development was gained. From the stage of multipotent progenitors, DCs are generated from Flt3(+) intermediates, irrespective of lymphoid or myeloid commitment, making fms-related tyrosine kinase 3 ligand one of the major regulators for DC development. Additional key cytokines involved are granulocyte-macrophage colony-stimulating factor (GM-CSF) and M-CSF, with each being essential for particular DC subsets and leading to specific activation of downstream transcription factors. In this review, we seek to draw an integrative view on how instructive cytokine signals acting on intermediate progenitors might lead to the generation of specific DC subsets in steady-state and during inflammation. We hypothesize that the lineage potential of a progenitor might be determined by the set of cytokine receptors expressed that make it responsive to further receive lineage instructive signals. Commitment to a certain lineage might consequently occur when lineage-relevant cytokine receptors are further upregulated and others for alternative lineages are lost. Along this line, we emphasize the role that diverse microenvironments have in influencing the generation of DC subsets with specific functions throughout the body.

SummaryLFAH12, an oleate 12-hydroxylase gene from Lesquerella fendleri (L.) was isolated on the basis of nucleotide sequence similarity to an oleate hydroxylase gene from Ricinus communis (L.). Transgenic Arabidopsis plants containing the Lesquerella gene under transcriptional control of the cauliflower mosaic virus 35S promoter accumulated ricinoleic, lesquerolic and densipolic acids in seeds, but not in leaves or roots. However, hydroxylase activity was detectable in crude extracts of vegetative tissues. The discrepancy between the presence of activity and the lack of hydroxy fatty acids suggests selective removal and breakdown of hydroxy fatty acids in vegetative organs. High levels of LFAH12 mRNA accumulation did not lead to correspondingly high levels of protein accumulation, suggesting that accumulation of the hydroxylase may be controlled post-transcriptionally. Expression of the L. fendleri gene in transgenic plants of a fad2 mutant of Arabidopsis, which is deficient in cytoplasmic oleate ∆12 desaturase activity, resulted in partial suppression of the mutant phenotype in roots. Thus, unlike the hydroxylase from R. communis, the L. fendleri enzyme has both hydroxylase and desaturase activities. Fusion of the 5Ј flanking region of the LFAH12 gene to the β-glucuronidase coding sequence resulted in a high level of early seedspecific expression of β-glucuronidase activity in transgenic Arabidopsis plants.