Self-assembly of fibronectin into fibrillar networks underneath dipalmitoyl phosphatidylcholine monolayers: role of lipid matrix and tensile forces.
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The cell-mediated assembly of fibronectin (Fn) into fibrillar matrices is a complex multistep process that is incompletely understood because of the chemical complexity of the extracellular matrix and a lack of experimental control over molecular interactions and dynamic events. We have identified conditions under which Fn assembles into extended fibrillar networks after adsorption to a dipalmitoyl phosphatidylcholine (DPPC) monolayer in contact with physiological buffer. We propose a sequential model for the Fn assembly pathway, which involves the orientation of Fn underneath the lipid monolayer by insertion into the liquid expanded (LE) phase of DPPC. Attractive interactions between these surface-anchored proteins and the liquid condensed (LC) domains leads to Fn enrichment at domain edges. Spontaneous self-assembly into fibrillar networks, however, occurs only after expansion of the DPPC monolayer from the LC phase though the LC/LE phase coexistence. Upon monolayer expansion, the domain boundaries move apart while attractive interactions among Fn molecules and between Fn and domain edges produce a tensile force on the proteins that initiates fibril assembly. The resulting fibrils have been characterized in situ by using fluorescence and light-scattering microscopy. We have found striking similarities between fibrils produced under DPPC monolayers and those found on cellular surfaces, including their assembly pathways. (+info)
Stable packaging of phage PRD1 DNA requires adsorption protein P2, which binds to the IncP plasmid-encoded conjugative transfer complex.
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The double-stranded DNA bacteriophage PRD1 uses an IncP plasmid-encoded conjugal transfer complex as a receptor. Plasmid functions in the PRD1 life cycle are restricted to phage adsorption and DNA entry. A single phage structural protein, P2, located at the fivefold capsid vertices, is responsible for PRD1 attachment to its host. The purified recombinant adsorption protein was judged to be monomeric by gel filtration, rate zonal centrifugation, analytical ultracentrifugation, and chemical cross-linking. It binds to its receptor with an apparent K(d) of 0.20 nM, and this binding prevents phage adsorption. P2-deficient particles are unstable and spontaneously release the DNA with concomitant formation of the tail-like structure originating from the phage membrane. We envisage the DNA to be packaged through one vertex, but the presence of P2 on the other vertices suggests a mechanism whereby the injection vertex is determined by P2 binding to the receptor. (+info)
Bacterial adhesion at synthetic surfaces.
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A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ. m(-2). Protein adsorption experiments were performed with (3)H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface. (+info)
Display of polyhistidine peptides on the Escherichia coli cell surface by using outer membrane protein C as an anchoring motif.
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A novel cell surface display system was developed by employing Escherichia coli outer membrane protein C (OmpC) as an anchoring motif. Polyhistidine peptides consisting of up to 162 amino acids could be successfully displayed on the seventh exposed loop of OmpC. Recombinant cells displaying polyhistidine could adsorb up to 32.0 micromol of Cd(2+) per g (dry weight) of cells. (+info)
Entry of rotaviruses is a multistep process.
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The infection of epithelial cells by some animal rotavirus strains requires the presence of sialic acid (SA) on the cell surface. Recently, we isolated rhesus rotavirus variants, named nar, whose infectivity, like that of human rotaviruses, is not dependent on SA. In this work, we have determined the binding properties of these SA-dependent and -independent rotavirus strains to MA104 cells. The half-time of attachment of the SA-dependent porcine rotavirus YM and reassortant virus DS1xRRV was found to be about 10 times longer in neuraminidase-treated cells than in untreated cells. On the other hand, human rotaviruses Wa and DS1, and the variant nar3, bound to cells two to three times more rapidly in the absence of SA. To investigate whether the SA-independent cellular structure recognized by the variant and human rotaviruses was the same, we used an infection assay designed to detect competition for cell surface molecules at both attachment and post-attachment steps. In this assay, human rotavirus Wa efficiently competed the infectivity of YM in untreated cells and that of the variant nar3 in untreated, as well as neuraminidase-treated, cells. This competition was nonreciprocal, since YM and nar3 did not compete, but rather increased three- to fivefold the infectivity of Wa. In contrast, a two-direction competition between the variant nar3 and DS1xRRV was found. Similar results were obtained when psoralen-inactivated viruses were used as competitors, indicating that the competition observed was during the early stages of infection. Altogether, these results suggest the existence of multiple interactions between rotaviruses and the cell surface and revealed the existence of common steps during the entry of human and animal rotavirus strains. (+info)
A novel virus-host cell membrane interaction. Membrane voltage-dependent endocytic-like entry of bacteriophage straight phi6 nucleocapsid.
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Studies on the virus-cell interactions have proven valuable in elucidating vital cellular processes. Interestingly, certain virus-host membrane interactions found in eukaryotic systems seem also to operate in prokaryotes (Bamford, D.H., M. Romantschuk, and P. J. Somerharju, 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1467-1473; Romantschuk, M., V.M. Olkkonen, and D.H. Bamford. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1821-1829). straight phi6 is an enveloped double-stranded RNA virus infecting a gram-negative bacterium. The viral entry is initiated by fusion between the virus membrane and host outer membrane, followed by delivery of the viral nucleocapsid (RNA polymerase complex covered with a protein shell) into the host cytosol via an endocytic-like route. In this study, we analyze the interaction of the nucleocapsid with the host plasma membrane and demonstrate a novel approach for dissecting the early events of the nucleocapsid entry process. The initial binding of the nucleocapsid to the plasma membrane is independent of membrane voltage (DeltaPsi) and the K(+) and H(+) gradients. However, the following internalization is dependent on plasma membrane voltage (DeltaPsi), but does not require a high ATP level or K(+) and H(+) gradients. Moreover, the nucleocapsid shell protein, P8, is the viral component mediating the membrane-nucleocapsid interaction. (+info)
Modification of liposomes with N-substituted polyacrylamides: identification of proteins adsorbed from plasma.
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Liposomes prepared from DMPC (80%) and cholesterol (20%) were modified with a series of hydrophobically modified N-substituted polyacrylamides, namely, poly[N-isopropylacrylamide] (PNIPAM), poly[N,N-bis(2-methoxyethyl) acrylamide] (PMEAM), and poly[(3-methoxypropyl)acrylamide] (PMPAM). The hydrophobic group, N-[4-(1-pyrenylbutyl)-N-n-octadecylamine was attached to one end of the polymer chains to serve as an anchor for incorporation into the liposome bilayer. Liposome-polymer interactions were confirmed using fluorescence spectroscopy and chemical analysis. Microscopy revealed differences in aggregation tendency between unmodified and polymer-modified liposomes. Proteins adsorbed to liposome surfaces during exposure to human plasma were identified by immunoblot analysis. It was found that both unmodified and polymer-modified liposomes adsorb a wide variety of plasma proteins. Contact phase coagulation proteins, complement proteins, cell-adhesive proteins, serine protease inhibitors, plasminogen, antithrombin III, prothrombin, transferrin, alpha(2)-microglobulin, hemoglobin, haptoglobin and beta-lipoprotein as well as the major plasma proteins were all detected. Some differences were found between the unmodified and polymer-modified liposomes. The unmodified liposomes adsorbed plasminogen mainly as the intact protein, whereas on the modified liposomes plasminogen was present in degraded form. Also, the liposomes modified with PNIPAM in its extended conformation (below the lower critical solution temperature) appeared to adsorb less protein than those containing the 'collapsed' form of PNIPAM (above the LCST). (+info)
Reversible molecular adsorption based on multiple-point interaction by shrinkable gels.
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A general approach is presented for creating polymer gels that can recognize and capture a target molecule by multiple-point interaction and that can reversibly change their affinity to the target by more than one order of magnitude. The polymers consist of majority monomers that make the gel reversibly swell and shrink and minority monomers that constitute multiple-point adsorption centers for the target molecule. Multiple-point interaction is experimentally proven by power laws found between the affinity and the concentration of the adsorbing monomers within the gels. (+info)