Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, ␣/␤-hydrolase, AMPdependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likely ole genes. Selected microorganisms from the phyla Verucomicrobia, Planctomyces, Chloroflexi, Proteobacteria, and Actinobacteria were tested experimentally and shown to produce longchain olefinic hydrocarbons. However, different species from the same genera sometimes lack the ole genes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containing ole genes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds. Xanthomonas campestris produced the greatest number of distinct olefin products, 15 compounds ranging in length from C 28 to C 31 and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on the oleA gene in experiments with Shewanella oneidensis MR-1 ole gene deletion mutants containing native or heterologous oleA genes expressed in trans. A strain deleted in oleABCD and containing oleA in trans produced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a ␤-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.

Lack of septation of the cardiac outflow tract (OFT) results in persistent truncus arteriosus (PTA), a form of congenital heart disease. The outflow myocardium expands through addition of cells originating from the pharyngeal mesoderm referred to as secondary/anterior heart field, whereas cardiac neural crest (CNC) cellderived mesenchyme condenses to form an aortopulmonary septum. We show for the first time that a mutation in Wnt5a in mice leads to PTA. We provide evidence that Wnt5a is expressed in the pharyngeal mesoderm adjacent to CNC cells in both mouse and chicken embryos and in the myocardial cell layer of the conotruncus at the time when CNC cells begin to form the aortopulmonary septum in mice. Although expression domains of secondary heart field markers are not altered in Wnt5a mutant embryos, the expression of CNC cell marker PlexinA2 is significantly reduced. Stimulation of CNC cells with Wnt5a protein elicits Ca 2ϩ transients, suggesting that CNC cells are capable of responding to Wnt5a. We propose a novel model in which Wnt5a produced in the OFT by cells originating from the pharyngeal mesoderm signals to adjacent CNC cells during formation of the aortopulmonary septum through a noncanonical pathway via localized intracellular increases in Ca

A polyolefinic hydrocarbon was found in nonpolar extracts of Shewanella oneidensis MR-1 and identified as 3,6,9,12,15,19,22,25,28-hentriacontanonaene (compound I) by mass spectrometry, chemical modification, and nuclear magnetic resonance spectroscopy. Compound I was shown to be the product of a head-to-head fatty acid condensation biosynthetic pathway dependent on genes denoted as ole (for olefin biosynthesis). Four ole genes were present in S. oneidensis MR-1. Deletion of the entire oleABCD gene cluster led to the complete absence of nonpolar extractable products. Deletion of the oleC gene alone generated a strain that lacked compound I but produced a structurally analogous ketone. Complementation of the oleC gene eliminated formation of the ketone and restored the biosynthesis of compound I. A recombinant S. oneidensis strain containing oleA from Stenotrophomonas maltophilia strain R551-3 produced at least 17 related long-chain compounds in addition to compound I, 13 of which were identified as ketones. A potential role for OleA in head-to-head condensation was proposed. It was further proposed that long-chain polyunsaturated compounds aid in adapting cells to a rapid drop in temperature, based on three observations. In S. oneidensis wild-type cells, the cellular concentration of polyunsaturated compounds increased significantly with decreasing growth temperature. Second, the oleABCD deletion strain showed a significantly longer lag phase than the wild-type strain when shifted to a lower temperature. Lastly, compound I has been identified in a significant number of bacteria isolated from cold environments.

Cyclosporin A (CSA) is commonly used to prevent graft-versus-host disease. The influence of CSA on T-cell function has been extensively investigated; however, the effect of CSA on natural killer (NK) cells is less understood. NK cells were cultured with IL-2 and IL-15 with and without CSA for 1 week. Compared with controls, CSA-treated cultures showed fewer CD56+CD16+KIR+ NK cells and a reciprocal increase in CD56+CD16−KIR− cells. These changes were due mainly to a reduced proliferation of the CD56dim NK-cell subpopulation and a relative resistance of CD56bright NK cells to CSA. Following coculture with K562 targets, CSA-exposed NK cells differed from controls and lacked Ca2+ oscillations, nuclear factor of activated T cells (NFAT) dephosphorylation, and NFAT nuclear translocation. NK cells cultured in CSA retained cytotoxicity against K562, Raji, and KIR ligand-expressing lymphoblastoid cells. NK cells cultured in CSA showed increases in NKp30 and reductions in NKp44 and NKG2D. Following IL-12 and IL-18 stimulation, CSA-treated NK cells showed more IFN-γ–producing cells. Using in vitro NK-cell differentiation, progenitor cells gave rise to more CD56+KIR− NK cells in the presence of CSA than controls. Collectively, these studies show that CSA influences NK-cell function and phenotype, which may have important implications for graft-versus-leukemia effects.