Requirements for Vav Guanine Nucleotide Exchange Factors and Rho GTPases in FcγR- and Complement-Mediated Phagocytosis

SummaryVav guanine nucleotide exchange factors (GEFs) have been implicated in cell adhesion by integrin and immune response receptors through the regulation of Rho GTPases. Here, we examine the role of Vav and Rho GTPases in phagocytosis by using primary murine macrophages. The genetic deletion of Rac1 and Rac2 prevents phagocytosis mediated by integrin and Fcγ receptors (FcγR), whereas the genetic deletion of Vav1 and Vav3 only prevents integrin-mediated phagocytosis through the complement receptor αMβ2. In addition, a Rac1/2 or Vav1/3 deficiency blocks Arp2/3 recruitment and actin polymerization at the complement-induced phagosome, indicating that these proteins regulate early steps in phagocytosis. Moreover, constitutively active Rac is able to rescue actin polymerization and complement-mediated phagocytosis in Vav-deficient macrophages. These studies indicate that Rac is critical for complement- and FcγR-mediated phagocytosis. In contrast, Vav is specifically required for complement-mediated phagocytosis, suggesting that Rac is regulated by GEFs other than Vav downstream of the FcγR

Power laws in microrheology experiments on living cells: comparative analysis and modelling

We compare and synthesize the results of two microrheological experiments on the cytoskeleton of single cells. In the first one, the creep function J(t) of a cell stretched between two glass plates is measured after applying a constant force step. In the second one, a micrometric bead specifically bound to transmembrane receptors is driven by an oscillating optical trap, and the viscoelastic coefficient $G_e(\omega)$ is retrieved. Both $J(t)$ and $G_e(\omega)$ exhibit power law behavior: $J(t)= A(t/t_0)^\alpha$ and $\bar G_e(\omega)\bar = G_0 (\omega/\omega_0)^\alpha$, with the same exponent $\alpha\approx 0.2$. This power law behavior is very robust ; $\alpha$ is distributed over a narrow range, and shows almost no dependance on the cell type, on the nature of the protein complex which transmits the mechanical stress, nor on the typical length scale of the experiment. On the contrary, the prefactors $A_0$ and $G_0$appear very sensitive to these parameters. Whereas the exponents $\alpha$ are normally distributed over the cell population, the prefactors $A_0$ and $G_0$ follow a log-normal repartition. These results are compared with other data published in the litterature. We propose a global interpretation, based on a semi-phenomenological model, which involves a broad distribution of relaxation times in the system. The model predicts the power law behavior and the statistical repartition of the mechanical parameters, as experimentally observed for the cells. Moreover, it leads to an estimate of the largest response time in the cytoskeletal network: $\tau_m \approx 1000$ s.Comment: 47 pages, 14 figures // v2: PDF file is now Acrobat Reader 4 (and up) compatible // v3: Minor typos corrected - The presentation of the model have been substantially rewritten (p. 17-18), in order to give more details - Enhanced description of protocols // v4: Minor corrections in the text : the immersion angles are estimated and not measured // v5: Minor typos corrected. Two references were clarifie

Identification and characterization of a new true lipase isolated through metagenomic approach

<p>Abstract</p> <p>Background</p> <p>Metagenomics, the application of molecular genomics to consortia of non-cultivated microbes, has the potential to have a substantial impact on the search for novel industrial enzymes such as esterases (carboxyl ester hydrolases, EC 3.1.1.1) and lipases (triacylglycerol lipases, EC 3.1.1.3). In the current work, a novel lipase gene was identified from a fosmid metagenomic library constructed with the "prokaryotic-enriched" DNA from a fat-contaminated soil collected from a wastewater treatment plant.</p> <p>Results</p> <p>In preliminary screening on agar containing 1% tributyrin, 2661 of the approximately 500,000 clones in the metagenomic library showed activity. Of these, 127 showed activity on agar containing 1% tricaprylin, while 32 were shown to be true lipase producers through screening on agar containing 1% triolein. The clone with the largest halo was further characterized. Its lipase gene showed 72% identity to a putative lipase of <it>Yersinia enterocolitica </it>subsp. <it>palearctica </it>Y11. The lipase, named LipC12, belongs to family I.1 of bacterial lipases, has a chaperone-independent folding, does not possess disulfide bridges and is calcium ion dependent. It is stable from pH 6 to 11 and has activity from pH 4.5 to 10, with higher activities at alkaline pH values. LipC12 is stable up to 3.7 M NaCl and from 20 to 50°C, with maximum activity at 30°C over a 1 h incubation. The pure enzyme has specific activities of 1722 U/mg and 1767 U/mg against olive oil and pig fat, respectively. Moreover, it is highly stable in organic solvents at 15% and 30% (v/v).</p> <p>Conclusions</p> <p>The combination of the use of a fat-contaminated soil, enrichment of prokaryotic DNA and a three-step screening strategy led to a high number of lipase-producing clones in the metagenomic library. The most notable properties of the new lipase that was isolated and characterized were a high specific activity against long chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability and stability in water-miscible organic solvents and at high salt concentrations. These characteristics suggest that this lipase has potential to perform well in biocatalytic processes, such as for hydrolysis and synthesis reactions involving long-chain triacylglycerols and fatty acid esters.</p

Comparison and relative utility of inequality measurements: as applied to Scotland’s child dental health

This study compared and assessed the utility of tests of inequality on a series of very large population caries datasets. National cross-sectional caries datasets for Scotland’s 5-year-olds in 1993/94 (n = 5,078); 1995/96 (n = 6,240); 1997/98 (n = 6,584); 1999/00 (n = 6,781); 2002/03 (n = 9,747); 2003/04 (n = 10,956); 2005/06 (n = 10,945) and 2007/08 (n = 12,067) were obtained. Outcomes were based on the d3mft metric (i.e. the number of decayed, missing and filled teeth). An area-based deprivation category (DepCat) measured the subjects’ socioeconomic status (SES). Simple absolute and relative inequality, Odds Ratios and the Significant Caries Index (SIC) as advocated by the World Health Organization were calculated. The measures of complex inequality applied to data were: the Slope Index of Inequality (absolute) and a variety of relative inequality tests i.e. Gini coefficient; Relative Index of Inequality; concentration curve; Koolman and Doorslaer’s transformed Concentration Index; Receiver Operator Curve and Population Attributable Risk (PAR). Additional tests used were plots of SIC deciles (SIC10) and a Scottish Caries Inequality Metric (SCIM10). Over the period, mean d3mft improved from 3.1(95%CI 3.0–3.2) to 1.9(95%CI 1.8–1.9) and d3mft = 0% from 41.1(95%CI 39.8–42.3) to 58.3(95%CI 57.8–59.7). Absolute simple and complex inequality decreased. Relative simple and complex inequality remained comparatively stable. Our results support the use of the SII and RII to measure complex absolute and relative SES inequalities alongside additional tests of complex relative inequality such as PAR and Koolman and Doorslaer’s transformed CI. The latter two have clear interpretations which may influence policy makers. Specialised dental metrics (i.e. SIC, SIC10 and SCIM10) permit the exploration of other important inequalities not determined by SES, and could be applied to many other types of disease where ranking of morbidity is possible e.g. obesity. More generally, the approaches described may be applied to study patterns of health inequality affecting worldwide populations

Filamin-A Regulates Neutrophil Uropod Retraction through RhoA during Chemotaxis

Filamin-A (FLNa) has been shown to be a key cross-linker of actin filaments in the leading edge of a motile melanoma cell line, however its role in neutrophils undergoing chemotaxis is unknown. Using a murine transgenic model in which FLNa is selectively deleted in granulocytes, we report that, while neutrophils lacking FLNa show normal polarization and pseudopod extension, they exhibit obvious defects in uropod retraction. This uropod retraction defect was found to be a direct result of reduced FLNa mediated activation of the small GTPase RhoA and myosin mediated actin contraction in the FLNa null cells. This results in a neutrophil recruitment defect in FLNa null mice. The compensatory increase in FLNb levels that was observed in the FLNa null neutrophils may be sufficient to compensate for the lack of FLNa at the leading edge allowing for normal polarization, however this compensation is unable to regulate RhoA activated tail retraction at the rear of the cell