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Cabomba is a small water lily genus that is native to the New World. Studies of pollen development and associated changes in the anther yield valuable characters for considering the evolution of reproductive biology in seed plants. Here we characterized the complete ontogenetic sequence for pollen in Cabomba caroliniana. Anthers at the microspore mother cell, tetrad, free microspore, and mature pollen grain stages were studied using scanning electron, transmission electron, and light microscopy. Tetragonal and decussate tetrads both occur in C. caroliniana, indicating successive microsporogenesis. The exine is tectate-columellate, and the infratectal columellae are the first exine elements to form, followed by a continuous tectum and a thin foot layer. A lamellate endexine initiates in the early free microspore stage, but becomes compressed in mature grains. Tectal microchannels and sculptural rods also initiate during the early free microspore stage, and significant pollenkitt deposition follows, supporting the hypothesis that these elements function in entomophily. The tapetum is morphologically amoeboid, with migratory tapetal cells directly contacting developing free microspores within the anther locule. Results from this study illustrate the importance of including ontogenetic data in analyzing pollen characters and in developing evolutionary and ecological hypotheses. The new palynological data also emphasize the character plasticity that occurs in basal angiosperms.

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The physiologic and pathologic functions of sphingosine kinase (SK) require translocation to specific membrane compartments. We tested the hypothesis that interactions with actin filaments regulate the localization of SK1 to membrane surfaces, including the plasma membrane and phagosome. Macrophage activation is accompanied by a marked increase in association of SK1 with actin filaments. Catalytically-inactive (CI)- and phosphorylation-defective (PD)-SK1 mutants exhibited reductions in plasma membrane translocation, colocalization with cortical actin filaments, membrane ruffling, and lamellipodia formation, compared with wild-type (WT)-SK1. However, translocation of CI- and PD-SK1 to phagosomes were equivalent to WT-SK1. SK1 exhibited constitutive- and stimulus-enhanced association with actin filaments and F-actin-enriched membrane fractions in both intact macrophages and a novel in vitro assay. In contrast, SK1 bound G-actin only under stimulated conditions. Actin inhibitors disrupted SK1 localization and modulated its activity. Conversely, reduction of SK1 levels or activity via RNA interference or specific chemical inhibition resulted in dysregulation of actin filaments. Thus, the localization and activity of SK1 are coordinately regulated with actin dynamics during macrophage activation.

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To gain insight into the role of luxSHi in disease pathogenesis, we inactivated that gene in several non-typeable Haemophilus influenzae isolates with an antibiotic resistance cassette. Gene inactivation was confirmed by PCR and by Southern blot analysis in each strain. Culture filtrates from luxSHi mutants contained a decreased amount of autoinducer-2 (AI-2) activity in comparison to the wild-type isolates using the Vibrio harveyi BB170 bioassay. Culture filtrates from Escherichia coli strain DH5alpha expressing a cloned luxSHi contained 350-fold more AI-2 activity per cell than E. coli DH5alpha containing the vector alone. The growth rate in several liquid media, and the cell density after overnight growth were not significantly different between the parents and the luxSHi mutants. Two clinical H. influenzae and their luxSHi mutants produced an identical biofilm in a flow system. Invasion of human cells by the luxSHi mutants, in comparison to the wild-type parents was strain-dependent, and cell type-dependent, but the luxSHi mutants tended to be more invasive. The luxSHi mutant of an otitis media isolate, strain R3157 appeared more virulent in the chinchilla model of otitis media: there were more bacteria in the middle ear, a greater inflammatory response and more goblet cell hyperplasia 10 days after the inoculation. We conclude that the H. influenzae homologue of luxS modulates certain virulence traits.

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Mycobacterium tuberculosis (M.tb) is a leading cause of global infectious mortality. The pathogenesis of tuberculosis involves inhibition of phagosome maturation, leading to survival of M.tb within human macrophages. A key determinant is M.tb-induced inhibition of macrophage sphingosine kinase (SK) activity, which normally induces Ca2+ signaling and phagosome maturation. Our objective was to determine the spatial localization of SK during phagocytosis and its inhibition by M.tb. Stimulation of SK activity by killed M.tb, live Staphylococcus aureus, or latex beads was associated with translocation of cytosolic SK1 to the phagosome membrane. In contrast, SK1 did not associate with phagosomes containing live M.tb. To characterize the mechanism of phagosomal translocation, live cell confocal microscopy was used to compare the localization of wild-type SK1, catalytically inactive SK1G82D, and a phosphorylation-defective mutant that does not undergo plasma membrane translocation (SK1S225A). The magnitude and kinetics of translocation of SK1G82D and SK1S225A to latex bead phagosomes were indistinguishable from those of wild-type SK1, indicating that novel determinants regulate the association of SK1 with nascent phagosomes. These data are consistent with a model in which M.tb inhibits both the activation and phagosomal translocation of SK1 to block the localized Ca2+ transients required for phagosome maturation.

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