Ceramide 1-phosphate, a mediator of phagocytosis

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.     

Identification of Site-Specific Adaptations Conferring Increased Neural Cell Tropism during Human Enterovirus 71 Infection

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP1₉₇ Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.     

Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.     

Restricted movement by mottled sculpin (pisces: cottidae) in a southern Appalachian stream

1. We used direct observation and mark‐recapture techniques to quantify movements by mottled sculpins (Cottus bairdi) in a 1 km segment of Shope Fork in western North Carolina. Our objectives were to: (i) quantify the overall rate of sculpin movement, (ii) assess variation in movement among years, individuals, and sculpin size classes, (iii) relate movement to variation in stream flow and population size structure, and (iv) quantify relationships between movement and individual growth rates. 2. Movements were very restricted: median and mean movement distances for all sculpin size classes over a 45 day period were 1.3 and 4.4 m respectively. Nevertheless, there was a high degree of intrapopulation and temporal variation in sculpin movement. Movement of juveniles increased with discharge and with the density of large adults. Movement by small and large adults was not influenced by stream flow, but large adults where more mobile when their own density was high. Finally, there were differences in the growth rates of mobile and sedentary sculpins. Mobile juveniles grew faster than sedentary individuals under conditions of low flow and high density of large adults, whereas adults exhibited the opposite pattern. 3. Our results support the hypothesis that juvenile movement and growth is influenced by both intraspecific interactions with adults and stream flow. In contrast, adult movement appears to be influenced by competitive interactions among residents for suitable space. The relationship between movement and growth may provide a negative feedback mechanism regulating mottled sculpin populations in this system.     

Winter Conditions and Land Cover Structure the Subnivium,A Seasonal Refuge beneath the Snow

In seasonally snow-covered environments, many organisms endure winter by using the subnivium, a below-snow thermally stable seasonal refugium. Because the insulation of snow is dependent on snow depth and density, the stability of temperatures within the subnivium varies across land cover types. Additionally, across much of the Northern Hemisphere snow extent, depth and duration are generally decreasing while snow density is increasing due to climate change. These changes are likely to destabilize the thermal profile of the subnivium, although they have not yet been quantified. To explore the effects of land cover and climate change on the subnivium, we measured snow pack characteristics (depth and density), and ambient and subnivium temperatures from three different land cover types (prairie, deciduous forest, and coniferous forest) and within a micro-greenhouse (2.5 x 2.5 x 2 m) that maintained a temperature of 5°C warmer than outdoor ambient temperatures, and automatically opened during snow events throughout the winter of 2013/14. We found that the mean daily subnivium temperature was significantly colder in the deciduous cover type than the prairie cover type, and that prairie had higher maximum subnivium temperatures than both of the other cover types. Our climate change simulation revealed that, although ambient temperatures within the micro-greenhouse were 5°C warmer than outside the greenhouse, the daily minimum subnivium temperature was significantly lower inside the greenhouse. Our findings suggest that climate change could have considerable effects on the refuge quality of the subnivium, and that some cover types appear to be more susceptible to these effects than others.     

Neutrophil C3bi receptors: formation of membrane clusters during cell triggering requires intracellular granules

Video-intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescein- or rhodamine-labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double-labeling studies were conducted by first labeling with anti-CR3 followed by attachment to LPS/serum-treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum-treated slides. To determine the contribution of granule-associated CR3, we have studied neutrophils defective in receptor up-regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3-modified surfaces. The patient's neutrophils were defective in CR3 up-regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum-treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3-bearing granules at local sites of adhesion.