Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+-dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.     

Homologous recombination: from model organisms to human disease

Recent experiments show that properly controlled recombination between homologous DNA molecules is essential for the maintenance of genome stability and for the prevention of tumorigenesis.     

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.     

Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.     

Extracellular calcium affects the membrane currents of cultured human keratinocytes.

Electrophysiologic properties of cultured human keratinocytes were studied using the patch voltage-clamp technique. Undifferentiated, proliferative keratinocytes grown in low Ca2+ medium had an average resting membrane potential of -24 mV. Voltage-clamp experiments showed that these cells had two membrane ionic currents: a large voltage-independent leak conductance, and a smaller voltage-dependent Cl- current that activated with depolarization. Increasing the extracellular Ca2+ concentration from 0.15 to 2 mM resulted in a doubling of the magnitude of the voltage-gated current and a shift in current activation to more negative potentials. Since levels of extracellular Ca2+ can alter the morphology and differentiation state of keratinocytes, the finding of a Ca2(+)-activated Cl- current in these cells suggests a role for this conductance in the initiation of differentiation.     

Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes

Cytosolic sialidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Ultrogel AcA-34 column chromatography and had an apparent molecular weight of 4 x 10(4). On incubation with micellar ganglioside GT1b, (molecular weight of the micelle, 3.5 x 10(5)) under the conditions used for the enzyme assay, brain cytosolic sialidase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 X 10(5), and a ganglioside/protein ratio (w/w) of 4:1. This is consistent with a stoichiometric combination of one ganglioside micelle and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides GD1a and GD1b, which, like GT1b, are potential substrates for the enzyme and GM1, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after interacting with ganglioside micelles. GT1b-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with GD1a and GD1b.     

Megafauna decline have reduced pathogen dispersal which may have increased emergent infectious diseases

The Late Quaternary extinctions of megafauna (defined as animal species > 44.5 kg) reduced the dispersal of seeds and nutrients, and likely also microbes and parasites. Here we use body-mass based scaling and range maps for extinct and extant mammal species to show that these extinctions led to an almost seven-fold reduction in the movement of gut-transported microbes, such as Escherichia coli (3.3–0.5 km² d⁻¹). Similarly, the extinctions led to a seven-fold reduction in the mean home ranges of vector-borne pathogens (7.8–1.1 km²). To understand the impact of this, we created an individual-based model where an order of magnitude decrease in home range increased maximum aggregated microbial mutations 4-fold after 20 000 yr. We hypothesize that pathogen speciation and hence endemism increased with isolation, as global dispersal distances decreased through a mechanism similar to the theory of island biogeography. To investigate if such an effect could be found, we analysed where 145 zoonotic diseases have emerged in human populations and found quantitative estimates of reduced dispersal of ectoparasites and fecal pathogens significantly improved our ability to predict the locations of outbreaks (increasing variance explained by 8%). There are limitations to this analysis which we discuss in detail, but if further studies support these results, they broadly suggest that reduced pathogen dispersal following megafauna extinctions may have increased the emergence of zoonotic pathogens moving into human populations.     

Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor: ASSOCIATION WITH CELL PROTECTION FROM APOPTOSIS*

Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A₄) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes β-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation.