David J. Sukovich

Wnt5a Is Required for Cardiac Outflow Tract Septation in Mice

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) cell–derived 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 Ca2+ 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 Ca2+.

Widespread Head-to-Head Hydrocarbon Biosynthesis in Bacteria and Role of OleA

ABSTRACT 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, α/β-hydrolase, AMP-dependent 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 long-chain 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 C28 to C31 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 β-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.

Structure, Function, and Insights into the Biosynthesis of a Head-to-Head Hydrocarbon in Shewanella oneidensis Strain MR-1

ABSTRACT 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.

Genomic and Biochemical Studies Demonstrating the Absence of an Alkane-Producing Phenotype in Vibrio furnissii M1

ABSTRACT Vibrio furnissii M1 was recently reported to biosynthesize n-alkanes when grown on biopolymers, sugars, or organic acids (M. O. Park, J. Bacteriol. 187:1426-1429, 2005). In the present study, V. furnissii M1 was subjected to genomic analysis and studied biochemically. The sequence of the 16S rRNA gene and repetitive PCR showed that V. furnissii M1 was not identical to other V. furnissii strains tested, but the level of relatedness was consistent with its assignment as a V. furnissii strain. Pulsed-field gel electrophoresis showed chromosomal bands at approximately 3.2 and 1.8 Mb, similar to other Vibrio strains. Complete genomic DNA from V. furnissii M1 was sequenced with 21-fold coverage. Alkane biosynthetic and degradation genes could not be identified. Moreover, V. furnissii M1 did not produce demonstrable levels of n-alkanes in vivo or in vitro. In vivo experiments were conducted by growing V. furnissii M1 under different conditions, extracting with solvent, and analyzing extracts by gas chromatography-mass spectrometry. A highly sensitive assay was used for in vitro experiments with cell extracts and [14C]hexadecanol. The data are consistent with the present strain being a V. furnissii with properties similar to those previously described but lacking the alkane-producing phenotype. V. furnissii ATCC 35016, also reported to biosynthesize alkanes, was found in the present study not to produce alkanes.

Rho kinase modulates postnatal adaptation of the pulmonary circulation through separate effects on pulmonary artery endothelial and smooth muscle cells

At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial P(O)₂ (Pa(O₂)) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease.

Acute normoxia increases fetal pulmonary artery endothelial cell cytosolic Ca2+ via Ca2+-induced Ca2+ release

To test the hypothesis that an acute increase in O2 tension increases cytosolic calcium ([Ca2+]i) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (<10 passages) were isolated from the intralobar pulmonary artery (PA) of fetal sheep and maintained under hypoxic conditions (Po2, 25 Torr). Using the calcium-sensitive dye fura-2, we demonstrated that acute normoxia (Po2 = 120 Torr) increased PAECs [Ca2+]i by increasing the rate of entry of extracellular calcium. In the presence of either ryanodine or 2-aminoethoxy-diphenylborate (2APB), normoxia did not lead to a sustained increase in PAECs [Ca2+]i Whole-cell patch clamp studies demonstrated that acute normoxia causes PAEC membrane depolarization. When loaded with the nitric oxide (NO)–sensitive dye, DAF - FM, acute normoxia increased PAEC fluorescence. In PAECs derived from fetal lambs with pulmonary hypertension, an acute increase in O2 tension had no effect on either [Ca2+]i or NO production. Hypoxia increases loading of acetylcholine-sensitive calcium stores, as hypoxia potentiated the response to acetylcholine We conclude that acute normoxia increases [Ca2+]i and NO production in normotensive but not hypertensive fetal PAECs via extracellular calcium entry and calcium release from calcium-sensitive intracellular stores.