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  • Articles  (23)
  • American Geophysical Union  (9)
  • Cambridge University Press  (9)
  • Blackwell Science Ltd  (5)
  • 2005-2009  (14)
  • 2000-2004  (9)
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  • Articles  (23)
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  • 1
    Electronic Resource
    Electronic Resource
    The type III protein translocation system of enteropathogenic Escherichia coli involves EspA–EspB protein interactions (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 35 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, use a type III secretion system to deliver effector proteins across the bacterial cell wall. In EPEC, four proteins, EspA, EspB, EspD and Tir are known to be exported by a type III secretion system and to be essential for ‘attaching and effacing’ (A/E) lesion formation, the hallmark of EPEC pathogenicity. EspA was recently shown to be a structural protein and a major component of a large, transiently expressed, filamentous surface organelle which forms a direct link between the bacterium and the host cell. In contrast, EspB is translocated into the host cell where it is localized to both membrane and cytosolic cell fractions. EspA and EspB are required for translocation of Tir to the host cell membrane suggesting that they may both be components of the translocation apparatus. In this study, we show that EspB co-immunoprecipitates with the EspA filaments and that, during EPEC infection of HEp-2 cells, EspB localizes closely with EspA. Using a number of binding assays, we also show that EspB can bind and be copurified with EspA. Nevertheless, binding of EspA filaments to the host cell membranes occurred even in the absence of EspB. These results suggest that following initial attachment of the EspA filaments to the target cells, EspB is delivered into the host cell membrane and that the interaction between EspA and EspB may be important for protein translocation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01814.x
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  • 2
    Electronic Resource
    Electronic Resource
    Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ (2005)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 55 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 – amino acid residues 1–55 in the mature protein, and D2 – amino acid residues 90–170), each comprising a three-stranded β-sheet flanked by two α-helices. A flexible region (residues 60–85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJD1 and EscJD2 in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJD1 and EscJD2 in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04508.x
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  • 3
    Electronic Resource
    Electronic Resource
    SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD (2004)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 52 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04101.x
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  • 4
    Electronic Resource
    Electronic Resource
    3D structure of EspA filaments from enteropathogenic Escherichia coli (2003)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 49 (2003), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The type III secretion system (TTSS) is a modular apparatus assembled by many pathogenic Gram-negative bacteria and is designed to translocate proteins through the bacterial cell wall into the eukaryotic host cell. The conserved components of the TTSS comprise stacks of rings spanning the inner and outer bacterial membrane and a narrow, needle-like structure projecting outwards. The TTSS of enteropathogenic E. coli is unique in that one of the translocator proteins, EspA, polymerizes to form an extension to the needle complex which interacts with the host cell. In this study we present the 3D structure of EspA filaments to c. 26 Å resolution determined from electron micrographs of negatively stained preparations by image processing. The structure comprises a helical tube with a diameter of 120 Å enclosing a central channel of 25 Å diameter through which effector proteins may be transported. The subunit arrangement corresponds to a one-start helix with 28 subunits present in five turns of the helix and an axial rise of 4.6 Å per subunit. This is the first report of a 3D structure of a filamentous extension to the TTSS.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03555.x
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  • 5
    Electronic Resource
    Electronic Resource
    CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map (2003)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 47 (2003), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Map is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir-specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone–effector pairings, binding is apparent at the amino-terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp-2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2003.03290.x
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  • 6
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    Direct numerical simulation of stenotic flows. Part 2. Pulsatile flow (2007)
    Cambridge University Press
    Details
    Publication Date: 2007-06-14
    Description: Direct numerical simulations (DNS) of stenotic flows under conditions of steady inlet flow were discussed in Part 1 of this study. DNS of pulsatile flow through the 75% stenosed tube (by area) employed for the computations in Part 1 is examined here. Analogous to the steady flow results, DNS predicts a laminar post-stenotic flow field in the case of pulsatile flow through the axisymmetric stenosis model, in contrast to previous experiments, in which intermittent disturbed flow regions and turbulent breakdown were observed in the downstream region. The introduction of a stenosis eccentricity, that was 5% of the main vessel diameter at the throat, resulted in periodic, localized transition to turbulence. Analysis in this study indicates that the early and mid-acceleration phases of the time period cycle were relatively stable, with no turbulent activity in the post-stenotic region. However, towards the end of acceleration, the starting vortex, formed earlier as the fluid accelerated through the stenosis at the beginning of acceleration, started to break up into elongated streamwise structures. These streamwise vortices broke down at peak flow, forming a turbulent spot in the post-stenotic region. In the early part of deceleration there was intense turbulent activity within this spot. Past the mid-deceleration phase, through to minimum flow, the inlet flow lost its momentum and the flow field began to relaminarize. The start of acceleration in the following cycle saw a recurrence of the entire process of a starting structure undergoing turbulent breakdown and subsequent relaminarization of the post-stenotic flow field. Peak wall shear stress (WSS) levels occurred at the stenosis throat, with the rest of the vessel experiencing much lower levels. Turbulent breakdown at peak flow resulted in a sharp amplification of instantaneous WSS magnitudes across the region corresponding to the turbulent spot, accompanied by large axial and circumferential fluctuations, even while ensemble-averaged axial shear stresses remained mostly low and negative. WSS levels dropped rapidly after the mid-deceleration phase, when the relaminarization process took over, and were almost identical to laminar, axisymmetric shear levels through most of the acceleration phase.
    Print ISSN: 0022-1120
    Electronic ISSN: 1469-7645
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics , Physics
    Published by Cambridge University Press
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  • 7
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    Direct numerical simulation of stenotic flows. Part 1. Steady flow (2007)
    Cambridge University Press
    Details
    Publication Date: 2007-06-14
    Description: Direct numerical simulations (DNS) of steady and pulsatile flow through 75% (by area reduction) stenosed tubes have been performed, with the motivation of understanding the biofluid dynamics of actual stenosed arteries. The spectral-element method, providing geometric flexibility and high-order spectral accuracy, was employed for the simulations. The steady flow results are examined here while the pulsatile flow analysis is dealt with in Part 2 of this study. At inlet Reynolds numbers of 500 and 1000, DNS predict a laminar flow field downstream of an axisymmetric stenosis and comparison to previous experiments show good agreement in the immediate post-stenotic region. The introduction of a geometric perturbation within the current model, in the form of a stenosis eccentricity that was 5% of the main vessel diameter at the throat, resulted in breaking of the symmetry of the post-stenotic flow field by causing the jet to deflect towards the side of the eccentricity and, at a high enough Reynolds number of 1000, jet breakdown occurred in the downstream region. The flow transitioned to turbulence about five diameters away from the stenosis, with velocity spectra taking on a broadband nature, acquiring a -5/3 slope that is typical of turbulent flows. Transition was accomplished by the breaking up of streamwise, hairpin vortices into a localized turbulent spot, reminiscent of the turbulent puff observed in pipe flow transition, within which r.m.s. velocity and turbulent energy levels were highest. Turbulent fluctuations and energy levels rapidly decayed beyond this region and flow relaminarized. The acceleration of the fluid through the stenosis resulted in wall shear stress (WSS) magnitudes that exceeded upstream levels by more than a factor of 30 but low WSS levels accompanied the flow separation zones that formed immediately downstream of the stenosis. Transition to turbulence in the case of the eccentric stenosis was found to be manifested as large temporal and spatial gradients of shear stress, with significant axial and circumferential variations in instantaneous WSS.
    Print ISSN: 0022-1120
    Electronic ISSN: 1469-7645
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics , Physics
    Published by Cambridge University Press
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  • 8
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    Reassessing the New Madrid Seismic Zone (2000)
    American Geophysical Union
    Details
    Publication Date: 2000-01-01
    Print ISSN: 0096-3941
    Electronic ISSN: 2324-9250
    Topics: Geosciences
    Published by American Geophysical Union
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  • 9
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    [Comment on “Should Memphis build for California's earthquakes?”] from A.D. Frankel (2003)
    American Geophysical Union
    Details
    Publication Date: 2003-07-22
    Print ISSN: 0096-3941
    Electronic ISSN: 2324-9250
    Topics: Geosciences
    Published by American Geophysical Union
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  • 10
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    On the Rayleigh–Bénard problem: dominant compressibility effects (2006)
    Cambridge University Press
    Details
    Publication Date: 2006-09-28
    Description: We study the linear temporal hydrodynamic stability in the Rayleigh-Bénard problem for a compressible fluid (a perfect gas) under marginally super-adiabatic conditions, i.e. when the ambient temperature gradient only slightly exceeds the adiabatic gradient and then only within the fluid adjacent to the upper (cold) wall. The onset of convection in this limit demonstrates some unique features which differ qualitatively from those of the familiar Boussinesq approximation. Thus, the ensuing convection is effectively confined to a narrow domain of the fluid close to the upper wall and is characterized by large wavenumbers. Furthermore, these distinct attributes persist with diminishing temperature difference, implying that the prevailing generalized Boussinesq approximation (based on the use of the potential temperature gradient) is non-uniform in the present limit. This non-uniformity is resolved in terms of the small yet significant variations of fluid properties (which are commonly neglected). We comment on the analogy between the present problem and the Taylor-Couette problem for a viscous incompressible fluid within a narrow gap between counter-rotating cylinders. We briefly discuss the potential relevance of the present limit to some recent observations of the onset of convection within near-critical fluids. © 2006 Cambridge University Press.
    Print ISSN: 0022-1120
    Electronic ISSN: 1469-7645
    Topics: Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics , Physics
    Published by Cambridge University Press
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