Search

The impact of irrigation thresholds based on soil water potentials on total anthocyanin content (TAcy) of cranberry fruit was studied at two sites. Two methods based on spectrophotometric measurements were compared: the acidic solvent extraction (ASE) method and the pH-differential method. TAcy was greater by 21–29 percent when irrigation was managed using a wetter threshold, −5.5 and −6.0 kPa, in comparison with a drier threshold, from −6.5 to −10.0 kPa. The pH-differential method resulted in significantly greater TAcy measurements than the ASE method, although the ASE method was faster and simpler to use.

The optimization of grass filter strip dimensions is important to strike a balance between environmental benefits and loss of agricultural land. The objective of this study was to investigate the temporal evolution of dissolved atrazine, metolachlor, and deethylatrazine (DEA) concentrations as well as the DEA to atrazine ratio (DAR) values in runoff from grass filter strips for the first (four to five) rain events occurring after annual herbicide application over four seasons (1998 to 2001) under natural rain conditions. The 16 plots (65 m long by 3 m wide [213 ft long by 10 ft wide]) were laid in a completely randomized block design with four treatments: 0 m filter strip and grass filter strips of 3, 6, or 9 m (10, 20, or 30 ft). The soil texture was 42% silt, 38% sand, and 20% clay with 3.1% of organic matter; the pH was 6.3; and the slope was 2% to 3%. Runoff-generating plots were left bare in order to maximize runoff. The strips were composed of 45% creeping red fescue (Festuca rubra L.), 45% bent grass (Agrostis alba L.), and 10% perennial ryegrass (Lolium perenne L.). Grass filter strips reduced exported dissolved herbicide concentrations at least by half for the first two rainfalls of each study year. By the end of the sampling period for each season, dissolved concentrations for grass filter strips were most of the time, except for metolachlor in the 6 m grass strip in 1999, less than the criteria for the protection of aquatic life (chronic) (7.8 μg L−1 [7.8 ppb] for metolachlor (provisory) and 1.8 μg L−1 [1.8 ppb] for atrazine), which was not the case with the 0 m strip. Grass filter strips reduced the frequency at which these criteria were exceeded. Generally, exported dissolved herbicide concentrations decreased with time except when a rain event of high intensity occurred. DAR values never exceeded 0.66 and generally showed an increasing trend with time during the sampling periods of 1998 to 2000. Grass filter strips are an efficient mitigation measure against the risk of contamination of surface waters by atrazine and metolachlor.

Herbicides atrazine and metolachlor have been detected in water bodies across the world. The objective of this study was to assess the efficiency of 0-m, 3-m, 6-m and 9-m grass filter strips to reduce masses of dissolved metolachlor, atrazine and deethylatrazine (a degradation product of atrazine) exported in runoff. For that purpose, 16 uncultivated plots (3-m wide × 65-m long) with 0-m-, 3-m-, 6-m- and 9-m-long grass filter strips were setup in a completely randomized block design. During four seasons, masses of dissolved atrazine, metolachlor and deethylatrazine were determined for the first four to five rain events, under natural rain conditions, after atrazine and metolachlor application. Generally, grass filter strips reduced exported herbicide masses by more than 90% and influenced atrazine and metolachlor dissipation kinetics in the field. The 3-m grass filter strip (area ratio source/strip of 22:1) usually provided a reduction in exported herbicide masses similar to the 6- or 9-m grass filter strips. Therefore, under the present experimental soil and climate conditions, a grass filter strip of 3 m would be a good compromise between environmental protection of surface waters against atrazine and metolachlor contamination and conservation of agricultural land use. Such an approach contributes to the acceptability by producers to implement optimized best management practices such as vegetated filter strips for the preservation of the quality of water resources.

The objective was to investigate the impact of vegetated filter strips on exported atrazine and deethylatrazine concentrations [dissolved and sorbed to eroded sediments (>1.5 μm)], the deethylatrazine to atrazine ratio in water and sediments, the ratio of sorbed to dissolved herbicides in runoff and subsurface infiltration as well as field equilibrium state under natural climate during two seasons. We hypothesize that sorption equilibrium was not achieved in 2004 because of the short delay (

Phagocytosis is a highly ordered process orchestrated by signalling through Rho GTPases to locally organise the actin cytoskeleton and drive particle uptake. Specific Rho family members that regulate phagocytosis are not known, as the majority of studies have relied on the use of dominant-negative mutants and/or toxins, which can inactivate multiple Rho GTPases. To identify the relevant GTPases for phagocytosis through the Fcγ receptor (FcγR) and complement receptor 3 (CR3), we depleted 20 Rho proteins individually in an RNA interference (RNAi) screen. We find that distinct GTPase subsets are required for actin polymerisation and uptake by macrophages: FcγR-dependent engulfment requires Cdc42 and Rac2 (but not Rac1), whereas CR3 requires RhoA. Surprisingly, RhoG is required for particle uptake through both FcγR and CR3. RhoG has been previously linked to Rac and Cdc42 signalling in different model systems, but not to RhoA. Interestingly, we find that RhoG is also recruited and activated at phagocytic cups downstream of FcγR and CR3, irrespective of their distinct actin structures and mechanisms of internalisation. Thus, the functional links between RhoG and RhoA downstream of CR3-dependent phagocytosis are new and unexpected. Our data suggest a broad role for RhoG in consolidating signals from multiple receptors during phagocytosis.

During αMβ2-mediated phagocytosis, the small GTPase Rap1 activates the β2 integrin by binding to a region between residues 732 and 761. Using COS-7 cells transfected with αMβ2, we show that αMβ2 activation by the phorbol ester PMA involves Ser756 of β2. This residue is critical for the local positioning of talin and biochemically interacts with Rap1. Using the CaM (calmodulin) antagonist W7, we found Rap1 recruitment and the inside-out activation of αMβ2 to be affected. We also report a role for CaMKII (calcium/CaM-dependent kinase II) in the activation of Rap1 during integrin activation. These results demonstrate a distinct physiological role for Ser756 of β2 integrin, in conjunction with the actions of talin and Rap1, during αMβ2 activation in macrophages.

Recent column studies suggest that pesticide fate models could be used to estimate the fate of estrogens in soil. Estrogens are detected in livestock manure which is used as a nutrient source on agricultural land. This is the first study to examine estrogen mineralization in a wide range of agricultural soils at the regional-scale. Soil samples were collected from upper and lower landscape positions of 18 agricultural fields in an area spanning 49–60 °N longitude and 110–120 °W latitude and these samples were used to determine 17β-estradiol mineralization parameters in microcosm experiments. Maximum 17β-estradiol mineralization (Max) ranged from 5.8% to 19.2% and was, on average, significantly (P

Addition of an N-substituted indole without any substitution in the 2 and 3 positions to 1,4-naphtoquinone leads to the expected, 2-(3-indolyl)-1,4-naphtoquinone. The latter reacts with some dienophiles via a Diels - Alder reaction and gives new N-substituted naphto-[2,3-C]-carbazole-5-13-quinones.

The small GTPase Rap1 and the cytoskeletal protein talin regulate binding of C3bi-opsonised red blood cells (RBC) to integrin α(M)β(2) in phagocytic cells, although the mechanism has not been investigated. Using COS-7 cells transfected with α(M)β(2), we show that Rap1 acts on the β(2) and not the α(M) chain, and that residues 732-761 of the β(2) subunit are essential for Rap1-induced RBC binding. Activation of α(M)β(2) by Rap1 was dependent on W747 and F754 in the β(2) tails, which are required for talin head binding, suggesting a link between Rap1 and talin in this process. Using talin1 knock-out cells or siRNA-mediated talin1 knockdown in the THP-1 monocytic cell line, we show that Rap1 acts upstream of talin but surprisingly, RIAM knockdown had little effect on integrin-mediated RBC binding or cell spreading. Interestingly, Rap1 and talin influence each other's localisation at phagocytic cups, and co-immunoprecipitation experiments suggest that they interact together. These results show that Rap1-mediated activation of α(M)β(2) in macrophages shares both common and distinct features from Rap1 activation of α(IIb)β(3) expressed in CHO cells.

Estrogenic compounds in livestock manure are also present in soils because manure is land applied on account of its value as an important nutrient source in agricultural production. This is the first study to compare the sorption of 17β-estradiol, estrone, estriol and equol in a wide range of soils. Specifically, for each of these four estrogens, the soil sorption coefficient (Kd) and the sorption coefficient per unit organic carbon (Koc) were determined in 121 surface soils (0–15 cm) obtained from upper-slopes, mid-slopes, and lower-slopes in agricultural fields across seven ecoregions in the Province of Alberta, Canada. Soil organic carbon content (SOC), texture, pH, carbonate content, electrical conductivity and cation exchange capacity were also determined on the air-dried and sieved (2 mm) soil. Both Kd and Koc values significantly increased in the order of Kd-estriol (23 L kg− 1 soil and 1059 L kg− 1, respectively) = Kd-17β-estradiol (23 L kg− 1 soil and 1082 L kg− 1, respectively)

Pathogenic Yersinia species neutralize innate immune mechanisms by injecting type three secretion effectors into immune cells, altering cell signaling. Our study elucidates how one of these effectors, YopO, blocks phagocytosis. We demonstrate using different phagocytic models that YopO specifically blocks Rac-dependent Fcgamma receptor internalization pathway but not complement receptor 3-dependent uptake, which is controlled by Rho activity. We show that YopO prevents Rac activation but does not affect Rac accumulation at the phagocytic cup. In addition, we show that plasma membrane localization and the guanine-nucleotide dissociation inhibitor (GDI)-like domain of YopO cooperate for maximal anti-phagocytosis. Although YopO has the same affinity for Rac1, Rac2, and RhoA in vitro, it selectively interacts with Rac isoforms in cells. This is due to the differential localization of the Rho family G proteins in resting cells; Rac isoforms partially exist as a GDI-free pool at the membrane of resting cells, whereas RhoA is trapped in the cytosol by RhoGDIalpha. We propose that YopO exploits this basic difference in localization and availability to selectively inhibit Rac-dependent phagocytosis.

Background information. The appropriate regulation of cell–cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell–cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell–cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell–cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret. Results. In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell–cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell–cell contacts do not induce filopodia or require Cdc42 function to assemble. Conclusion. We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.

At the onset of mitosis, most adherent cells undergo cell retraction characterised by the disassembly of focal adhesions and actin stress fibres. Mitosis takes place in rounded cells, and the two daughter cells spread again after cytokinesis. Because of the well-documented ability of the small GTPase Rap1 to stimulate integrin-dependent adhesion and spreading, we assessed its role during mitosis. We show that Rap1 activity is regulated during this process. Changes in Rap1 activity play an essential role in regulating cell retraction and spreading, respectively, before and after mitosis of HeLa cells. Indeed, endogenous Rap1 is inhibited at the onset of mitosis; conversely, constitutive activation of Rap1 inhibits the disassembly of premitotic focal adhesions and of the actin cytoskeleton, leading to delayed mitosis and to cytokinesis defects. Rap1 activity slowly increases after mitosis ends; inhibition of Rap1 activation by the ectopic expression of the dominant-negative Rap1[S17A] mutant prevents the rounded cells from spreading after mitosis. For the first time, we provide evidence for the direct regulation of adhesion processes during mitosis via the activity of the Rap1 GTPase.

Rho GTP-binding proteins play a key role as molecular switches in many cellular activities. In response to extracellular stimuli and with the help of regulators (GEF, GAP, Effector, GDI), these proteins serve as switches that interact with their environment in a complex manner. Based on the structure of a published ordinary differential equations (ODE) model, we first present a generic process model for the Rho GTP-binding proteins, and compare it with the ODE model. We then extend the basic model to include the behaviour of the GDI regulators and explore the parameter space for the extended model with respect to biological data from the literature. We discuss the challenges this extension brings and the directions of further research. In particular, we present techniques for modular representation and refinement of process models, where, for example, different Rho proteins with different rates for regulator interactions can be given as instances of the same parametric model.

Actin is the monomeric subunit of actin filaments which form one of the three major cytoskeletal networks in eukaryotic cells. Actin dynamics, be it the polymerisation of actin monomers into filaments or the reverse process, plays a key role in many cellular activities such as cell motility and phagocytosis. There is a growing number of experimental, theoretical and mathematical studies on the components of actin polymerisation and depolymerisation. However, it remains a challenge to develop compositional models of actin dynamics, e.g., by using differential equations. In this paper, we propose compositional process algebra models of actin polymerisation, and present a geometric representation of these models that allows to generate movies reflecting their dynamics.

Phagocytosis is a highly conserved, complex process that has evolved to counter the constant threat posed by pathogens, effete cells and debris. Classically defined as a mechanism for internalising and destroy- ing particles greater than 0.5 mm in size, it is a receptor- mediated, actin-driven process. The best-studied phagocytic receptors are the opsono-receptors, FcgR and CR3. Phagocytic uptake involves actin dynamics including polymerisation, bundling, contraction, se- vering and depolymerisation of actin filaments. Re- cent evidence points to the importance of membrane remodelling during phagocytosis, both in terms of changes in lipid composition and delivery of new membrane to the sites of particle binding. Here we review the molecular mechanisms of phagocytic uptake and some of the strategies developed by microbial pathogens to manipulate this process.

AlphaMbeta2 integrins mediate phagocytosis of opsonized particles in a process controlled by RhoA, Rho kinase, myosin II, Arp2/3, and actin polymerization. AlphaMbeta2, Rho, Arp2/3, and F-actin accumulate underneath bound particles; however, the mechanism regulating Rho function during alphaMbeta2-mediated phagocytosis is poorly understood. We report that the binding of C3bi-opsonized sheep red blood cells (RBCs) to alphaMbeta2 increases Rho-GTP, but not Rac-GTP, levels. Deletion of the cytoplasmic domain of beta2, but not of alphaM, abolished Rho recruitment and activation, as well as phagocytic uptake. Interestingly, a 16-amino acid (aa) region in the membrane-proximal half of the beta2 cytoplasmic domain was necessary for activating Rho. Three COOH-terminal residues (aa 758-760) were essential for beta2-induced accumulation of Rho at complement receptor 3 (CR3) phagosomes. Activation of Rho was necessary, but not sufficient, for its stable recruitment underneath bound particles or for uptake. However, recruitment of active Rho was sufficient for phagocytosis. Our data shed light on the mechanism of outside-in signaling, from ligated integrins to the activation of Rho GTPase signaling.

Despite their homology, the regulation of WASP and WAVE, activators of Arp2/3-dependent actin polymerization, has always been thought to be different. Several recent studies have revealed new aspects of their regulation, highlighting its complexity and the crucial role of post-translational modifications. New data also suggest additional functions for WASP family proteins, pushing us to reconsider existing models.

Rap1 belongs to the Ras subgroup of small GTP-binding proteins. Whereas its early history has focused on its biochemical homology to Ras and the alleged functional antagonism between these two small GTPases, recent cellular evidence suggests that endogenous Rap1 plays a unique, Ras-independent role in eukaryotic cells. Activated by virtually all receptor types and second messengers, Rap1 controls adhesion-related functions such as phagocytosis,cell-cell contacts and functional activation of integrins through inside-out signalling. Whereas the precise mechanism by which its downstream effectors exert these diverse functions is unknown, Rap1 seems to fulfil the evolutionarily conserved function of patterning the eukaryotic cell, thus enabling it to respond to its environment, in particular through cytoskeletal remodelling.

Phagocytosis through Fcγ receptor (FcγR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway [1]. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcγR phagocytosis, while Rho controls actin assembly at CR phagosomes [2, 3]. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho → ROK → myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcγR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcγR phagocytosis [4–6], to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.

Opa adhesins of pathogenic Neisseria species target four members of the human carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. CEACAM receptors mediate opsonization-independent phagocytosis of Neisseria gonorrhoeae by human granulocytes and each receptor individually can mediate gonococcal invasion of epithelial cells. We show here that gonococcal internalization occurs by distinct mechanisms depending on the CEACAM receptor expressed. For the invasion of epithelial cell lines via CEACAM1 and CEACAM6, a pathogen-directed reorganization of the actin cytoskeleton is not required. In marked contrast, ligation of CEACAM3 triggers a dramatic but localized reorganization of the host cell surface leading to highly efficient engulfment of bacteria in a process regulated by the small GTPases Rac1 and Cdc42, but not Rho. Two tyrosine residues of a cytoplasmic immune receptor tyrosine-based activating motif of CEACAM3 are essential for the induction of phagocytic actin structures and subsequent gonococcal internalization. The granulocyte-specific CEACAM3 receptor has properties of a single chain phagocytic receptor and may thus contribute to innate immunity by the elimination of Neisseria and other CEACAM-binding pathogens that colonize human mucosal surfaces.

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 CrkII, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induced Rho GTPase-dependent formation of actin filament bundles. We propose that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism.

R-Ras has a high degree of sequence homology to Ras and to other members of the Ras subfamily including Rap, TC21 and M-Ras. Activated versions of Ras and TC21 are highly transforming in a variety of cell lines and mutated forms of both proteins have been found in human tumours. R-Ras interacts with many of the same proteins as Ras and TC21, including c-Raf1, and can induce transformed foci, although this activity is weak compared to Ras and appears to be cell-type specific. Here, we have investigated R-Ras signalling pathways in a variety of cell types. We find that microinjection of activated R-Ras into quiescent fibroblasts stimulates cell cycle progression through G(1) phase and subsequent DNA synthesis. However, unlike Ras, R-Ras does not activate the ERK MAP kinase pathway nor does it activate the JNK or p38/Mpk2 MAP kinase pathways. Microinjection of R-Ras into PC12 cells does not induce terminal differentiation, but instead causes extensive cell spreading, consistent with R-Ras having a role in integrin activation. Finally, in a macrophage cell line, R-Ras activates the (α)(M)(β)(2)integrin via the small GTPase Rap1, leading to phagocytosis of opsonized red blood cells, whereas Ras does not. These results indicate that R-Ras has an important role in the regulation of cell growth and adhesion, but that this is mediated through downstream signals distinct from those used by Ras.

FGD1, the gene responsible for the inherited disease faciogenital dysplasia, encodes a guanine nucleotide exchange factor (GEF) that specifically activates the p21 GTPase Cdc42. In order, FGD1 is composed of a proline-rich N-terminal region, adjacent GEF and pleckstrin homology (PH) domains, a FYVE-finger domain and a second C-terminal PH domain (PH2), structural motifs involved in signaling and subcellular localization. Fgd1, the mouse FGD1 ortholog, is expressed in regions of active bone formation within osteoblasts and in the osteoblast-like cell line MC3T3-E1, a finding consistent with its role in skeletal formation. Here, we use subcellular fractionation studies to show that endogenous Fgd1 protein is localized in the cytosolic and Golgi and plasma membrane fractions of mouse calvarial cells. Immunocytochemical studies performed with osteoblast-like MC3T3-E1 cells and other mammalian cell lines confirm the localization of Fgd1 and show that the proline-rich N-terminal region is necessary and sufficient for Fgd1 subcellular localization to the plasma membrane and Golgi complex. In contrast, the FYVE-finger and PH2 domains do not appear to direct the localization of Fgd1 or the activation of Cdc42. In addition, microinjection studies indicate that the N-terminal Fgd1 domain inhibits filopodia formation, suggesting that this region down-regulates GEF function. These results characterize the function of the Fgd1 domains for both protein localization and Cdc42 activation and indicate that the Fgd1 Cdc42GEF protein is involved in the regulation of Cdc42 activity at the subcortical actin cytoskeleton and Golgi complex.

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is a potent activator of macrophages. Besides inducing many transcriptional pathways, LPS also elicits rapid morphological changes such as cell spreading. Here we have investigated the signaling pathway that controls macrophage beta2-integrin-dependent spreading in response to LPS. We show that inhibition of the adapter protein MyD88, the interleukin-1 receptor-associated kinase Irak, the p38 mitogen-activated protein kinase, or the Ras-like GTPase Rap1 blocks LPS-induced spreading. In addition, Irak activates p38 and stimulates p38-dependent spreading. The activation of p38 by Irak requires Irak's kinase activity. We find that p38 controls spreading independently of its role in transcription but rather through activation of Rap1. Together, our results suggest that beta2-integrin-dependent spreading of macrophages in response to LPS is controlled by a linear signaling pathway via MyD88, Irak, p38, and Rap1.

BACKGROUND: beta2 integrins mediate many aspects of the inflammatory and immune responses, including adhesion of leukocytes to the endothelium, complement-mediated phagocytosis in macrophages and neutrophils, and antigen-specific conjugate formation between cytotoxic T cells and their targets. A variety of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha), platelet-activating factor (PAF), and lipopolysaccharide (LPS) and other bacterial products induce the functional activation of beta2 integrins, but the signaling events that link membrane receptors to integrin activation are poorly understood. RESULTS: We report here that expression of the constitutively active small GTPases Rap1 or R-ras, but not Ras or RalA, is sufficient for functional activation of alphaMbeta2, the complement receptor 3 (CR3), in macrophages, allowing phagocytosis of C3bi- opsonized targets. Inhibition of Rap1, but not other Ras-like or Rho- like small GTPases, abolishes activation of alphaMbeta2 induced by phorbol esters, LPS, TNF-alpha or PAF. Finally, Rap1 activation specifically controls the binding properties of alphaMbeta2 towards its physiological ligand, namely the complement-opsonized phagocytic targets. CONCLUSIONS: In macrophages, the Rap1 GTPase regulates activation of the alphaMbeta2 integrin in response to a wide variety of inflammatory mediators

Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.

The complement and immunoglobulin receptors are the major phagocytic receptors involved during infection. However, only immunoglobulin-dependent uptake results in a respiratory burst and an inflammatory response in macrophages. Rho guanosine triphosphatases (molecular switches that control the organization of the actin cytoskeleton) were found to be essential for both types of phagocytosis. Two distinct mechanisms of phagocytosis were identified: Type I, used by the immunoglobulin receptor, is mediated by Cdc42 and Rac, and type II, used by the complement receptor, is mediated by Rho. These results suggest a molecular basis for the different biological consequences that are associated with phagocytosis.

We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-γ)-treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis . The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies ( ops-Brucella ) or with a control nonimmune serum ( c-Brucella ). The involvement of the produced NO in the killing of intracellular B. suis was evaluated. B. suis survived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-γ enhanced the antibrucella activity of phagocytes, with this effect being greater in ops-Brucella infection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-α) mRNAs was induced in both c-Brucella - and ops-Brucella -infected cells and was strongly potentiated by IFN-γ. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-γ-treated cells infected with c-Brucella or ops-Brucella ; however, expression of iNOS protein and production of NO were detected only in IFN-γ-treated cells infected with ops-Brucella . These discrepencies between iNOS mRNA and protein levels were not due to differences in TNF-α production. The iNOS inhibitor N ω-nitro- l -arginine methyl ester increased B. suis multiplication specifically in IFN-γ-treated cells infected with ops-Brucella , demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing that B. suis was sensitive to NO killing. Together our data indicate that in B. suis -infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fcγ receptors. They also support the possibility that in mice, NO favors the elimination of Brucella , providing that IFN-γ and antibrucella antibodies are present, i.e., following expression of acquired immunity.

Few studies on natural estrogens have been conducted in agricultural ecosystems. High (up to 58 ng/L) estrone concentrations were measured shortly after applications of manure in a small agricultural watershed. No other estrogens (17s-estradiol, estrone or estriol) were detected afterward (first three rainfalls after applications). Results suggest that aquatic organisms in agricultural watersheds could be exposed to estrone shortly after manure applications, probably from leaching of land-applied animal wastes, but that this exposure would be short-lived.

GTP-binding proteins of the Rho family regulate each other's activities by largely elusive mechanisms. Now, an unexpected signalling pathway has been identified in fibroblasts that links Rac activation to the inhibition of Rho activity, through the release of oxygen radicals.

Livestock manure applied to agricultural land is one of the ways natural steroid estrogens enter soils. To examine the impact of long-term solid beef cattle (Bos Taurus) manure on soil properties and 17β-estradiol sorption and mineralization, this study utilized a soil that had received beef cattle manure over 35 years. The 17β-estradiol was strongly sorbed and sorption significantly increased (P < 0.05) with increasing soil organic carbon content (SOC) and with an increasing annual rate of beef cattle manure. The 17β-estradiol mineralization half-life was significantly negatively correlated, and the total amount of 17β-estradiol mineralized at 90 days (MAX) was significantly positively correlated with 17β-estradiol sorption. The long-term rate of manure application had no significant effect on MAX, but the addition of fresh beef cattle manure in the laboratory resulted in significantly (P < 0.05) smaller MAX values. None of the treatments showed MAX values exceeding one-third of the 17β-estradiol applied.

The adaptor protein Nck has been shown to link receptor ligation to actin-based signalling in a diverse range of cellular events, such as changes in cell morphology and motility. It has also been implicated in phagocytosis. However, its molecular role in controlling actin remodelling associated with phagocytic uptake remains to be clarified. Here, we show that Nck, which is recruited to phagocytic cups, is required for Fcγ receptor (FcγR)- but not complement receptor 3 (CR3)-induced phagocytosis. Nck recruitment in response to FcγR ligation is mediated by the phosphorylation of tyrosine 282 and 298 in the ITAM motif in the cytoplasmic tail of the receptor. In the absence of FcγR phosphorylation, there is also no recruitment of N-WASP or Cdc42 to phagocytic cups. Nck promotes FcγR-mediated phagocytosis by recruiting N-WASP to phagocytic cups. Efficient phagocytosis, however, only occurs, if the CRIB domain of N-WASP can also interact with Cdc42. Our observations demonstrate that Nck and Cdc42 collaborate to stimulate N-WASP-dependent FcγR-mediated phagocytosis.

Metolachlor and atrazine are herbicides used in corn agriculture and detected in surface- and groundwater. Vegetated filters reduce masses of herbicides in runoff, but less attention has been given to their impact on degradation products and subsurface infiltrated water. The objective was to study the temporal evolution of dissolved metolachlor, atrazine and deethylatrazine concentrations in runoff and subsurface infiltrated water with two types of vegetated filters over 2 years for the first three rains following herbicide application. Runoff and subsurface infiltrated at 90-cm water samples from 12 plots of 30 × 5 m in a completely randomized block design of four replicates of three treatments-control without filter, 5-m-long grass filter, 5-m-long grass and tree filter were analysed using gas chromatography/mass spectrometry. Controls in runoff generally had the highest average atrazine, as high as 739 μg L−1, and metolachlor average concentrations, as high as 1,725 μg L−1. The first rain after application was mainly responsible for atrazine and metolachlor exports. Vegetated filters reduced concentrations of atrazine and metolachlor in runoff below their respective Canadian criterion of 1.8 μg L−1 for atrazine and 7.8 μg L−1 for metolachlor (provisory) (CCME 2002) for the protection of aquatic life when rain did not occur shortly after application. With the need to increase food production and agricultural yields to sustain the increasing world population came the need to develop efficient mitigation tools such as vegetated filter strips to reduce the ecotoxicologial impacts of pesticides. The present study is among the few that examined herbicide degradation products and subsurface infiltrated water under filter strips in order to provide new knowledge on the relationship between herbicide loss pathways and the environmental benefits of these strips. Such knowledge will provide much needed information to modellers, decision makers, ecotoxicologists and agronomists involved in the regulation, design and implementation of vegetated filter strips for the protection of water quality.

International audience; Salmonella can invade non-phagocytic cells through its type III secretion system (T3SS-1), which induces a Trigger entry process. This study showed that Salmonella enterica, subspecies enterica serovar Enteritidis can also invade cells via the Rck outer membrane protein. Rck was necessary and sufficient to enable non-invasive E. coli and Rck-coated beads to adhere to and invade different cells. Internalization analysis of latex beads coated with different Rck peptides showed that the peptide containing amino acids 140-150 promoted adhesion, whereas amino acids between 150 and 159 modulated invasion. Expression of dominant-negative derivatives and use of specific inhibitors demonstrated the crucial role of small GTPases Rac1 and Cdc42 in activating the Arp2/3 complex to trigger formation of actin-rich accumulation, leading to Rck-dependent internalization. Finally, scanning and transmission electron microscopy with Rck-coated beads and E. coli expressing Rck revealed microvillus-like extensions that formed a Zipper-like structure, engulfing the adherent beads and bacteria. Overall, our results provide new insights into the Salmonella T3SS-independent invasion mechanisms and strongly suggest that Rck induces a Zipper-like entry mechanism. Consequently, Salmonella seems to be the first bacterium found to be able to induce both Zipper and Trigger mechanisms to invade host cells.

Estrogens are a family of feminizing hormones that are excreted by vertebrates. It has been documented that their presence in surface waters, even in the ng/L range, can have detrimental impacts on fish reproduction. Two competitive enzyme-linked immunosorbent assays using rabbit polyclonal antibodies were developed: one for 17beta-estradiol and a second one for 17beta-estradiol (E2)+estrone (E1)+estriol (E3). Two different conjugates were synthesized using the Mixed-anhydride (for the 17beta-estradiol ELISA) and the Mannich (for the E1 + E2 + E3 ELISA) reactions. The 17beta-estradiol ELISA was highly specific with an IC(50) of 243 ng/mL for 17beta-estradiol. The E1 + E2 + E3 ELISA exhibited cross-reactivity with estrone (85%) and estriol (62%) with an IC(50) of 18 ng/mL for 17beta-estradiol. Cross-reactivity was tested against 13 chemically related compounds and both immunoassays showed significant cross-reactivity with two estradiol conjugates: beta estradiol-17-valerate and beta estradiol-3-benzoate (from 57 to 84 %) for which, to our knowledge, there are currently no commercially available ELISA. Characteristics (sensitivity, inter and intra assay variation, and cross-reactivity) of the E1 + E2 + E3 ELISA were further compared to those from a commercial Estriol ELISA. The commercial ELISA was more specific, sensitive and its inter-assay variation was less (9.5% compared to 10% for the E1 + E2 + E3 ELISA) but the E1 + E2 + E3 ELISA had less intra-assay variation (4% compared to 5% for the commercial ELISA). Finally, a solid-phase extraction method compatible with the E1 + E2 + E3 immunoassay demonstrated that this combined approach of extraction and immunoassay had good potential for determining estrogen concentrations in environmental samples such as surface water in urban and agricultural ecosystems.

In many areas of intensive corn production, atrazine and metolachlor are among the most commonly found herbicides in surface and ground water. This 2-yr study compared the impact of grass and grass+tree buffer strips on the exported masses of atrazine, metolachlor, and a degradation product of atrazine, desethylatrazine (DEA). The experimental system consisted of four replicate plots in a three-way completely randomized design (no buffer zone, grass buffer zone, and grass+tree buffer strips). The field plots were 5 m wide and 30 m long and grown in corn. The grass and grass+tree buffer strips were 5 m and had the same grass vegetation except for eight young hybrid poplars. Over the 2-yr study, surface runoff and subsurface infiltration water (under the buffer strip) were collected after the initial three rainfall events after herbicide application. Dissolved atrazine, metolachlor, and DEA were analyzed by gas chromatography/mass spectrometry. The presence of buffer strips decreased the exported masses of atrazine and metolachlor in surface runoff. A three-way ANOVA with treatment (type of buffer strip), water (surface runoff or subsurface infiltration), and time between herbicide application and rainfall event as factors showed a significant reduction (40-60% in 2004 and 75-95% in 2005) in the total (surface runoff+infiltrated water) exported masses of atrazine and metolachlor in the presence of buffer strips. Rainfall events after herbicide application were different between the 2 yr and greatly affected the flow distribution (e.g., subsurface infiltration) and the leached herbicide concentrations. No significant difference in the capacity to reduce herbicide exports was observed between grass and grass+tree buffer strip treatments; the poorly developed young poplar biomass at the time of the study may partly explain this observation.

A genetic pathway controlling the removal of apoptotic corpses has now been identified in the nematode. Mammalian homologs of the components of this pathway are already known to have key roles in cell motility and integrin-mediated signaling, suggesting that the three processes are mechanistically related.

We introduce a natural language interface for building stochastic p calculus models of biological systems. In this language, complex constructs describing biochemical events are built from basic primitives of association, dissociation and transformation. This language thus allows us to model biochemical systems modularly by describing their dynamics in a narrative-style language, while making amendments, refinements and extensions on the models easy. We give a formal semantics for this language and a translation algorithm into stochastic p calculus that delivers this semantics. We demonstrate the language on a model of Fcg receptor phosphorylation during phagocytosis. We provide a tool implementation of the translation into a stochastic p calculus language, Microsoft Research’s SPiM, which can be used for simulation and analysis. 1 2

The appropriate regulation of cell-cell adhesion is an important event in the homoeostasis of different cell types. In epithelial cells, tight adhesion mediated by E-cadherin receptors is essential for the differentiation and functionality of epithelial sheets. Upon assembly of cadherin-mediated cell-cell contacts, it is well established that the small GTPases Rho and Rac are activated and are necessary for junction stability. However, the role of the small GTPase Cdc42 in cadherin adhesion is less clear. Cdc42 can be activated by E-cadherin in a breast tumour cell line, but the requirement for Cdc42 function for new junction assembly or maintenance has been contradictory. Cdc42 participation in cell-cell contacts has been inferred from the presence of filopodia, the typical F-actin structure induced by Cdc42 activation, as cells approach each other to establish cell-cell contacts. Yet, under these conditions, the contribution of migration to filopodia protrusion cannot be excluded and the results are difficult to interpret.In the present study, we set out to address (a) whether Cdc42 is activated by new E-cadherin cell-cell contacts when junction assembly occurs without prior migration and (b) whether Cdc42 function is necessary for cadherin stability. We found that junction formation in confluent keratinocytes or upon E-cadherin clustering decreased Cdc42-GTP levels. In the absence of serum- and migration-induced Cdc42 activation, we demonstrated that cell-cell contacts do not induce filopodia or require Cdc42 function to assemble.We conclude that Cdc42 does not participate in the early events that initiate stable cadherin adhesion in keratinocytes. Yet, it is feasible that Cdc42 may be activated at later time points or by other receptors. Cdc42 can then participate in additional functions during polarization, such as Golgi re-positioning or basolateral trafficking.

Phagocytosis is the process by which cells internalize particulate material, and is of central importance to immunity, homeostasis and development. Here, we study the internalization of immunoglobulin G-coated particles in cells transfected with Fcgamma receptors (FcgammaRs) through the formation of an enveloping phagocytic cup. Using confocal microscopy, we precisely track the location of fluorescently tagged FcgammaRs during cup growth. Surprisingly, we found that phagocytic cups growing around identical spherical particles showed great variability even within a single cell and exhibited two eventual fates: a cup either stalled before forming a half-cup or it proceeded until the particle was fully enveloped. We explain these observations in terms of a mechanical bottleneck using a simple mathematical model of the overall process of cup growth. The model predicts that reducing F-actin concentration levels, and hence the deforming force, does not necessarily lead to stalled cups, a prediction we verify experimentally. Our analysis gives a coherent explanation for the importance of geometry in phagocytic uptake and provides a unifying framework for integrating the key processes, both biochemical and mechanical, occurring during cup growth.

We deleted exon 2 in human skeletal beta-tropomyosin (h beta-SK tropomyosin) using an improved adaptation of polymerase chain reaction (PCR) technology. The first PCR product was used to prime the full-length cDNA, leading to an exon 2-deleted h beta-SK tropomyosin. This new protein, des-(39-80)-tropomyosin, could then be expressed in Escherichia coli and purified to homogeneity. At the nucleotide level, the junction between exons 1 and 3 has been precisely made in the PCR product. The mutated protein was purified using high-performance liquid chromatography. Des-(39-80)-tropomyosin revealed new immunological properties but was still recognized by certain antitropomyosin antibodies. Furthermore, the structural characteristics of the mutated tropomyosin fit those of the full-length tropomyosin. This new adaptation of PCR technology appears to be suitable for every kind of mutation inside a cloned DNA molecule, and one mutation primer per mutation is sufficient.

A key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) remain extracellular while colonizing the gut mucosa by attaching and effacing mechanism. EPEC use the type three secretion system effector protein EspF to prevent their own uptake into macrophages. EPEC can also block in trans the internalization of IgG-opsonized particles. In this study, we show that EspJ is the type three secretion system effector protein responsible for trans-inhibition of macrophage opsono-phagocytosis by both EPEC and EHEC. While EspF plays no role in trans-inhibition of opsono-phagocytosis, espJ mutants of EPEC or EHEC are unable to block uptake of opsonized sheep red blood cells (RBC), a phenotype that is rescued upon complementation with the espJ gene. Importantly, ectopic expression of EspJ(EHEC) in phagocytes is sufficient to inhibit internalization of both IgG- and C3bi-opsonized RBC. These results suggest that EspJ targets a basic mechanism common to these two unrelated phagocytic receptors. Moreover, EspF and EspJ target independent aspects of the phagocytic function of mammalian macrophages in vitro.

The cytoskeletal, actin-binding protein talin has been previously implicated in phagocytosis in Dictyostelium discoideum and mammalian phagocytes. However, its mechanism of action during internalization is not understood. Our data confirm that endogenous talin can occasionally be found at phagosomes forming around IgG- and C3bi-opsonized red blood cells in macrophages. Remarkably, talin knockdown specifically abrogates uptake through complement receptor 3 (CR3, CD11b/CD18, alpha(M)beta(2) integrin) and not through the Fc gamma receptor. We show that talin physically interacts with CR3/alpha(M)beta(2) and that this interaction involves the talin head domain and residues W747 and F754 in the beta(2) integrin cytoplasmic domain. The CR3/alpha(M)beta(2)-talin head interaction controls not only talin recruitment to forming phagosomes but also CR3/alpha(M)beta(2) binding activity, both in macrophages and transfected fibroblasts. However, the talin head domain alone cannot support phagocytosis. Our results establish for the first time at least two distinct roles for talin during CR3/alpha(M)beta(2)-mediated phagocytosis, most noticeably activation of the CR3/alpha(M)beta(2) receptor and phagocytic uptake.