Genetic system in Pseudomonas alcaligenes NCIB 9867

Abstract Genetic transfer of both auxotrophic and catabolic markers was detected in filter matings of mutant strains of Pseudomonas alcaligenes NCIB 9867. Bidirectional transfer of auxotrophic markers was demonstrated in most of the crosses. Strains could either act as donors or recipients. Polarized transfer of auxotrophic markers was observed in some crosses. There was low co-inheritance of both 2,5X⁺ catabolic marker and auxotrophic markers. No evidence could be presented indicating the involvement of the indigenous 33-kb plasmid in the genetic transfer process. Partial sensitivity to DNase was observed in some of the crosses. Maximum frequency of recombinant formation obtained with mating cultures from stationary growth phase suggested an influence of physiological states on genetic transfer. As transfer did not appear to be due to classical transformation or to be plasmid-mediated, the likely mechanism could involve the release of DNA upon intimate cell-to-cell contact. The gene transfer system may be useful for linkage analysis of closely linked genes.     

Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.     

Experimental mapping of DNA duplex shape enabled by global lineshape analyses of a nucleotide-independent nitroxide probe

Sequence-dependent variation in structure and dynamics of a DNA duplex, collectively referred to as ‘DNA shape’, critically impacts interactions between DNA and proteins. Here, a method based on the technique of site-directed spin labeling was developed to experimentally map shapes of two DNA duplexes that contain response elements of the p53 tumor suppressor. An R5a nitroxide spin label, which was covalently attached at a specific phosphate group, was scanned consecutively through the DNA duplex. X-band continuous-wave electron paramagnetic resonance spectroscopy was used to monitor rotational motions of R5a, which report on DNA structure and dynamics at the labeling site. An approach based on Pearson''s coefficient analysis was developed to collectively examine the degree of similarity among the ensemble of R5a spectra. The resulting Pearson''s coefficients were used to generate maps representing variation of R5a mobility along the DNA duplex. The R5a mobility maps were found to correlate with maps of certain DNA helical parameters, and were capable of revealing similarity and deviation in the shape of the two closely related DNA duplexes. Collectively, the R5a probe and the Pearson''s coefficient-based lineshape analysis scheme yielded a generalizable method for examining sequence-dependent DNA shapes.     

Cupping: from a biomechanical perspective

The effectiveness of the cupping technique, a treatment modality in Traditional Chinese Medicine, in stimulating acupuncture points for pain relief was examined in this paper from a biomechanical perspective. Parametric studies including the effects of vacuum pressure, loading rate, friction coefficient at the cup-skin interface, and size and shape of the cup were carried out using a model based on the finite-element method. The anatomical structures of skin, fat, and muscle were modelled. All the soft-tissue layers were assumed to be nonlinearly elastic and viscoelastic. The rim of the cup was also modelled to study the interaction between cup and skin; the cup rim was assumed to be rigid. The simulation results showed that the stresses in the soft tissue were increased for increasing applied vacuum pressures and that the effects of cupping were mostly limited to the region enclosed by the cup. The simulations also indicated that the magnitude of the applied vacuum may have had direct implications for the severity of bruising of the skin following cupping treatment. Most significantly, the simulation results contradicted the established practice of cup size selection according to the depth of the disorder. Experimental verification of the proposed multi-layered finite-element model is presented. The nature of the bruising inherent to the cupping treatment is also explained by the proposed model.     

A Novel Family of Apicomplexan Glideosome-associated Proteins with an Inner Membrane-anchoring Role

The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton.Apicomplexan parasites cause a multitude of illnesses through infection of both human and livestock hosts. Members of this phylum include the opportunistic human parasites Toxoplasma gondii and Cryptosporidium parvum, pathogens of livestock, including Theileria annulata and Eimeria tenalla, and most notably the Plasmodium species, the causative agents of malaria in humans. Infection with P. falciparum results in ∼1–3 million deaths and a further 500 million infections annually (1).During various stages of the Apicomplexan lifecycle the parasites require motility to migrate through their insect and vertebrate hosts and to invade and internalize themselves within targeted host cells (2–4). The parasite''s unique mechanism of gliding motility is powered by an Apicomplexan-specific motor complex termed the actin-myosin motor (5), which resides between the outer plasma membrane and inner membrane complex (IMC)⁴ (6). The IMC is a continuous patchwork of flattened vesicular cisternae located directly beneath the plasma membrane and overlying the cytoskeletal network (7, 8). The IMC appears to arise from Golgi-associated vesicles flattened during parasite maturation to form large membranous sheets, which envelope the parasite and leave only a small gap at the extreme parasite apex (9).The myosin component of the actin-myosin motor has previously been defined as a tetrameric complex consisting of a class XIV myosin termed Myo-A (10), a myosin tail interacting protein (also called myosin light chain) (7) and the two glideosome-associated proteins GAP45 and GAP50 (11). These motor components are linked to the outer IMC membrane via the membrane proteins GAP45/50 (11). Between the plasma membrane and the IMC are actin filaments held in place through aldolase-mediated contact with the C-terminal tails of plasma membrane-spanning adhesive proteins whose ectodomains bind substrate and host cells (2). To power the forward movement of apicomplexan zoite stages, myosin pulls the actin filaments and their attached adhesins rearward. For this to succeed the GAP-myosin complex must presumably be fixed to the IMC, possibly via interactions with unidentified proteins linking the motor to the underlying cytoskeleton. Studies of fluorescently tagged GAP50 confirm it is relatively immobile within the IMC, however attempts to identify potential anchoring proteins have not been successful and have instead indicated that GAP50 may be immobilized by the lipid-raft like properties of the IMC membranes (12).The actin-myosin complex is confined to the outer IMC membrane while the opposing innermost IMC membrane is studded with 9 nm intramembranous particles, revealed by electron microscopy of freeze fractured Toxoplasma tachyzoites and Plasmodium ookinetes (13, 14). The size of these particles suggests that the proteins involved are likely to form high molecular weight complexes that overlay the parasite''s cytoskeletal network and possibly anchor the IMC to the cytoskeleton (12–15). Due to the close apposition of the inner and outer IMC membranes (14, 16), it is possible that the intramembranous particles could bridge the IMC lumen and interact with the GAP-myosin complex contributing to its stabilization within the IMC.To identify putative proteins that might be components of the intramembranous particles, we examined data from the detergent-resistant membrane (DRM) proteome of schizont-stage P. falciparum parasites containing developing merozoites (17, 18). DRMs, or lipid-rafts, were of considerable interest, because they appeared to harbor proteins involved in host cell invasion such as glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins. Our data also indicated that P. falciparum schizont-stage DRMs contained the IMC proteins PfGAP45/50 (17), and recent studies in T. gondii have also suggested that the IMC is enriched in DRMs (12). Another study indicated that when P. falciparum DRM protein complexes were separated by blue native gel electrophoresis, a band was produced containing PfGAP45/50 and PfMyo-A as well as a novel six-pass transmembrane protein (PlasmoDB: PFD1110w, GenBank^TM: {"type":"entrez-protein","attrs":{"text":"CAD49269","term_id":"23498297","term_text":"CAD49269"}}CAD49269) (18). This protein was related to another six-pass transmembrane DRM protein (PlasmoDB: MAL13P1.130, GenBank^TM: {"type":"entrez-protein","attrs":{"text":"CAD52385","term_id":"23615394","term_text":"CAD52385"}}CAD52385) we had previously identified in P. falciparum schizont-stage DRMs (17).We show here that MAL13P1.130 and PFD1110w, termed PfGAPM1 and PfGAPM2 (glideosome-associated protein with multiple-membrane spans), respectively, belong to a family of proteins specific to the Apicomplexa and demonstrate that P. falciparum GAPM proteins, and their orthologues in T. gondii, localize to the parasite IMC. The GAPMs form high molecular weight complexes that are resistant to dissociation and solubilization by a variety of common detergents and could therefore be components of the intramembranous particles seen in electron microscopy. When isolated by immunoprecipitation, the GAPM complexes co-purify with components of the actin-myosin motor and particularly the parasite cytoskeletal network suggesting GAPMs could anchor the IMC to the cytoskeleton and perhaps even play a role in tethering the motor to cytoskeleton.     

Interdependence of Laminin-mediated Clustering of Lipid Rafts and the Dystrophin Complex in Astrocytes

Astrocyte endfeet surrounding blood vessels are active domains involved in water and potassium ion transport crucial to the maintenance of water and potassium ion homeostasis in brain. A growing body of evidence points to a role for dystroglycan and its interaction with perivascular laminin in the targeting of the dystrophin complex and the water-permeable channel, aquaporin 4 (AQP4), at astrocyte endfeet. However, the mechanisms underlying such compartmentalization remain poorly understood. In the present study we found that AQP4 resided in Triton X-100-insoluble fraction, whereas dystroglycan was recovered in the soluble fraction in astrocytes. Cholesterol depletion resulted in the translocation of a pool of AQP4 to the soluble fraction indicating that its distribution is indeed associated with cholesterol-rich membrane domains. Upon laminin treatment AQP4 and the dystrophin complex, including dystroglycan, reorganized into laminin-associated clusters enriched for the lipid raft markers GM1 and flotillin-1 but not caveolin-1. Reduced diffusion rates of GM1 in the laminin-induced clusters were indicative of the reorganization of raft components in these domains. In addition, both cholesterol depletion and dystroglycan silencing reduced the number and area of laminin-induced clusters of GM1, AQP4, and dystroglycan. These findings demonstrate the interdependence between laminin binding to dystroglycan and GM1-containing lipid raft reorganization and provide novel insight into the dystrophin complex regulation of AQP4 polarization in astrocytes.The basement membrane is a specialized extracellular matrix (ECM)² composed of collagen, fibronectin, perlecan, agrin, and laminin. Several studies have focused on the involvement of these ECM molecules in the formation and maturation of neuromuscular junctions (1–4) and interneuronal synapses (5). More recently, much effort has been made by our group and others to understand the role of these molecules at the interface of astroglia and blood vessels (6–8). Laminin is highly expressed at the perivascular ECM, and the laminin receptor, dystroglycan (α-DG), together with many other components of the dystrophin-associated protein (DAP) complex, is particularly enriched at astrocyte endfeet abutting the blood vessels (9–11). The binding of laminin to α-DG at these specialized astrocyte domains in brain plays a key role in the polarized distribution of components of the DAP complex (6, 12).Multiple lines of evidence indicate that the DAP complex is crucial for the functional distribution both of the water-permeable channel, AQP4, and the inwardly rectifying potassium channel, Kir4.1, at astrocyte endfeet. Indeed, mutations in the dystrophin gene, deletion of α-syntrophin, or loss of laminin binding to α-DG caused by a mutation in the Large1 glycosyltransferase result in a dramatic reduction of the expression of AQP4 and Kir4.1 at perivascular astrocyte endfeet (6, 7, 12–15). The mislocalization of AQP4 in the dystrophin mutant and α-syntrophin null mice results in delayed onset of brain edema and K⁺ clearance (16–18). Collectively, these studies highlight a cooperative role of the ECM and both the extracellular and cytoplasmic components of the DAP complex in the proper targeting of proteins to functional domains of astrocytes leading to the regulation of electrolyte balance and fluid movement.Although the role of DG in targeting other members of the DAP complex (6) as well as AQP4 and Kir4.1 to astrocyte endfeet has been well established (12), the mechanisms underlying this highly organized distribution remain poorly understood. In C2C12 myotubes, agrin triggers AChR clustering, a DG-dependent process, through the coalescence of lipid rafts, which is necessary for proper AChR gating functions (19–21). In oligodendrocytes, laminin induces the relocalization of α6β1 integrin to lipid rafts containing PDGFαR, thereby providing a potential mechanism for the incorporation of cell survival signals (22). Lipid rafts are defined as small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. These small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions (23). Indeed, the immunological synapse is a good example where rafts are brought together to form large functional membrane domains (24). At the immunological synapse, agrin induces the clustering of lipid rafts and their colocalization with CD3 and CD28 complex surface antigens as well as with Lck tyrosine kinase leading to T cell activation (24). Together, these studies provide evidence for a functional role of ligand-induced clustering of lipid rafts.We have previously shown that laminin induces the coclustering of the DAP complex with Kir4.1 and AQP4 in glial cell cultures (8, 25). Moreover, in vivo studies have shown that the perivascular localization of these channels and several components of the DAP complex at astrocyte endfeet require the interaction of laminin with α-DG (6, 12). In light of these data we asked whether lipid rafts contribute to the laminin-DG-dependent compartmentalization of the DAP complex and AQP4 to key active domains of astrocytes. We show here using fluorescently labeled cholera toxin subunit B (CtxB), a common marker for GM1-containing lipid rafts, that laminin induces a dramatic reorganization of GM1 into large clusters or macrodomains that colocalize extensively with components of the DAP complex in cortical astrocyte cultures. Laminin-mediated clustering of AQP4 is dependent both on cholesterol-sensitive lipid rafts and the DAP complex bringing novel insight into ECM-dependent membrane domain organization and the mechanisms underlying the polarized distribution of these proteins in astrocytes.     

Entrapment of intracytosolic bacteria by septin cage-like structures

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.     

Different Types of Door-Opening Motions as Contributing Factors to Containment Failures in Hospital Isolation Rooms

Hospital isolation rooms are vital for the containment (when under negative pressure) of patients with, or the protection (when under positive pressure) of patients, from airborne infectious agents. Such facilities were essential for the management of highly contagious patients during the 2003 severe acute respiratory syndrome (SARS) outbreaks and the more recent 2009 A/H1N1 influenza pandemic. Many different types of door designs are used in the construction of such isolation rooms, which may be related to the space available and affordability. Using colored food dye as a tracer, the qualitative effects of door-opening motions on the dissemination of potentially contaminated air into and out of a single isolation room were visualized and filmed using Reynolds-number-equivalent, small-scale, water-tank models fitted with programmable door-opening and moving human figure motions. Careful scaling considerations involved in the design and construction of these water-tank models enabled these results to be accurately extrapolated to the full-scale situation. Four simple types of door design were tested: variable speed single and double, sliding and hinged doors, in combination with the moving human figure. The resulting video footage was edited, synchronized and presented in a series of split-screen formats. From these experiments, it is clear that double-hinged doors pose the greatest risk of leakage into or out of the room, followed by (in order of decreasing risk) single-hinged, double-sliding and single-sliding doors. The relative effect of the moving human figure on spreading any potential contamination was greatest with the sliding doors, as the bulk airflows induced were large relative to those resulting from these door-opening motions. However, with the hinged doors, the airflows induced by these door-opening motions were significantly greater. Further experiments involving a simulated ventilated environment are required, but from these findings alone, it appears that sliding-doors are far more effective for hospital isolation room containment.