ABCA1 promotes the efflux of bacterial LPS from macrophages and accelerates recovery from LPS-induced tolerance

Macrophages play important roles in both lipid metabolism and innate immunity. We show here that macrophage ATP-binding cassette transporter A1 (ABCA1), a transporter known for its ability to promote apolipoprotein-dependent cholesterol efflux, also participates in the removal of an immunostimulatory bacterial lipid, lipopolysaccharide (LPS). Whereas monocytes require an exogenous lipoprotein acceptor to remove cell-associated LPS, macrophages released LPS in the absence of an exogenous acceptor by a mechanism that was driven, in part, by endogenous apolipoprotein E (apoE). Agents that increased ABCA1 expression increased LPS efflux from wild-type but not ABCA1-deficient macrophages. Preexposure of peritoneal macrophages to LPS for 24 h increased the expression of ABCA1 and increased LPS efflux with a requirement for exogenous apolipoproteins due to suppression of endogenous apoE production. In contrast, LPS preconditioning of ABCA1-deficient macrophages significantly decreased LPS efflux and led to prolonged retention of cell-surface LPS. Although the initial response to LPS was similar in wild-type and ABCA1-deficient macrophages, LPS-induced tolerance was greater and more prolonged in macrophages that lacked ABCA1. Our results define a new role for macrophage ABCA1 in removing cell-associated LPS and restoring normal macrophage responsiveness.     

Phototoxic Action Spectrum on a Retinal Pigment Epithelium Model of Age-Related Macular Degeneration Exposed to Sunlight Normalized Conditions

Among the identified risk factors of age-related macular degeneration, sunlight is known to induce cumulative damage to the retina. A photosensitive derivative of the visual pigment, N-retinylidene-N-retinylethanolamine (A2E), may be involved in this phototoxicity. The high energy visible light between 380 nm and 500 nm (blue light) is incriminated. Our aim was to define the most toxic wavelengths in the blue-green range on an in vitro model of the disease. Primary cultures of porcine retinal pigment epithelium cells were incubated for 6 hours with different A2E concentrations and exposed for 18 hours to 10 nm illumination bands centered from 380 to 520 nm in 10 nm increments. Light irradiances were normalized with respect to the natural sunlight reaching the retina. Six hours after light exposure, cell viability, necrosis and apoptosis were assessed using the Apotox-Glo Triplex™ assay. Retinal pigment epithelium cells incubated with A2E displayed fluorescent bodies within the cytoplasm. Their absorption and emission spectra were similar to those of A2E. Exposure to 10 nm illumination bands induced a loss in cell viability with a dose dependence upon A2E concentrations. Irrespective of A2E concentration, the loss of cell viability was maximal for wavelengths from 415 to 455 nm. Cell viability decrease was correlated to an increase in cell apoptosis indicated by caspase-3/7 activities in the same spectral range. No light-elicited necrosis was measured as compared to control cells maintained in darkness. Our results defined the precise spectrum of light retinal toxicity in physiological irradiance conditions on an in vitro model of age-related macular degeneration. Surprisingly, a narrow bandwidth in blue light generated the greatest phototoxic risk to retinal pigment epithelium cells. This phototoxic spectrum may be advantageously valued in designing selective photoprotection ophthalmic filters, without disrupting essential visual and non-visual functions of the eye.     

Formate increases the F0F1-ATPase activity in Escherichia coli growing on glucose under anaerobic conditions at slightly alkaline pH

Escherichia coli growing on glucose under anaerobic conditions at slightly alkaline pH carries out a mixed-acid fermentation resulting in the production of formate among the other products that can be excreted or further oxidized to H(2) and CO(2). H(2) production is largely dependent on formate dehydrogenase H and hydrogenases 3 and 4 constituting two formate hydrogen lyases, and on the F(0)F(1)-ATPase. In this study, it has been shown that formate markedly increased ATPase activity in membrane vesicles. This activity was significantly (1.8-fold) stimulated by 100mM K(+) and inhibited by N,N(')-dicyclohexylcarbodiimide and sodium azide. The increase in ATPase activity was absent in atp, trkA, and hyf but not in hyc mutants. ATPase activity was also markedly increased by formate when bacteria were fermenting glucose with external formate (30mM) in the growth medium. However this activity was not stimulated by K(+) and absent in atp and hyc but not in hyf mutants. The effects of formate on ATPase activity disappeared when cells were performing anaerobic (nitrate/nitrite) or aerobic respiration. These results suggest that the F(0)F(1)-ATPase activity is dependent on K(+) uptake TrkA system and hydrogenase 4, and on hydrogenase 3 when cells are fermenting glucose in the absence and presence of external formate, respectively.     

Centriolar SAS-5 is required for centrosome duplication in C. elegans

Centrosomes, the major microtubule-organizing centres (MTOCs) of animal cells, are comprised of a pair of centrioles surrounded by pericentriolar material (PCM). Early in the cell cycle, there is a single centrosome, which duplicates during S-phase to direct bipolar spindle assembly during mitosis. Although crucial for proper cell division, the mechanisms that govern centrosome duplication are not fully understood. Here, we identify the Caenorhabditis elegans gene sas-5 as essential for daughter-centriole formation. SAS-5 is a coiled-coil protein that localizes primarily to centrioles. Fluorescence recovery after photobleaching (FRAP) experiments with green fluorescent protein (GFP) fused to SAS-5 (GFP-SAS-5) demonstrated that the protein shuttles between centrioles and the cytoplasm throughout the cell cycle. Analysis of mutant alleles revealed that the presence of SAS-5 at centrioles is crucial for daughter-centriole formation and that ZYG-1, a kinase that is also essential for this process, controls the distribution of SAS-5 to centrioles. Furthermore, partial RNA-interference (RNAi)-mediated inactivation experiments suggest that both sas-5 and zyg-1 are dose-dependent regulators of centrosome duplication.     

Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli

Proton translocation, coupled to formate oxidation and hydrogen evolution, was studied in anaerobically grown fermenting Escherichia coli JW136 carrying hydrogenase 1 (hya) and hydrogenase 2 (hyb) double deletions. Rapid acidification of the medium by EDTA-treated anaerobic suspension of the whole cells or its alkalization by inverted membranes was observed in response to application of formate. The formate-dependent proton translocation and 2H(+)-K(+) exchange coupled to H(2) evolution were sensitive to the uncoupler, carbonylcyanide-m-chlorophenylhydrazone, and to copper ions, inhibitors of hydrogenases. No pH changes were observed in a suspension of formate-pulsed aerobically grown ("respiring") cells. The apparent H(+)/formate ratio of 1.3 was obtained in cells oxidizing formate. The 2H(+)-K(+) exchange of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes does take place in JW136 cell suspension. Hydrogen formation from formate by cell suspensions of E. coli JW136 resulted in the formation of a membrane potential (Deltapsi) across the cytoplasmic membrane of -130 mV (inside negative). This was abolished in the presence of copper ions, although they had little effect on the value of Deltapsi generated by E. coli under respiration. We conclude that the hydrogen production by hydrogenase 3 is coupled to formate-dependent proton pumping that regulates 2H(+)-K(+) exchange in fermenting bacteria.     

Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events

Positive-sense single-stranded RNA viruses have developed strategies to exploit cellular resources at the expense of host mRNAs. The genomes of these viruses display a variety of structures at their 5' and 3' ends that differentiate them from cellular mRNAs. Despite this structural diversity, viral RNAs are still circularized by juxtaposition of their 5' and 3' ends, similar to the process used by cellular mRNAs. Also reminiscent of the mechanisms used by host mRNAs, translation of viral RNAs involves the recruitment of translation initiation factors. However, the roles played by these factors likely differ from those played by cellular mRNAs. In keeping with the general parsimony typical of RNA viruses, these host factors also participate in viral RNA replication. However, the dual use of host factors requires that viral RNA template utilization be regulated to avoid conflict between replication and translation. The molecular composition of the large ribonucleoprotein complexes that form the viral RNA replication and translation machineries likely evolves over the course of infection to allow for switching template use from translation to replication.