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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive ‘attaching and effacing’ lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (α, β, γ, δ and ε) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin-binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site-directed mutagenesis. We used the yeast two-hybrid system and gel overlays to study intimin–Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an I237/897A substitution (positions numbered according to Int280α/whole intimin α) in intimin α did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing lesions (AE) on epithelial cells. The genes involved in the formation of the AE lesions are contained within a pathogenicity island named the locus of enterocyte effacement (LEE). The LEE comprises 41 open reading frames organized in five major operons: LEE1, LEE2, LEE3, LEE4 and tir. The first gene of the LEE1 operon encodes a transcription activator of the other LEE operons that is called the LEE-encoded regulator (Ler). The LEE2 and LEE3 operons are divergently transcribed with overlapping −10 promoter regions, and gene fusion studies have shown that they are both activated by Ler. Deletion analysis, using lacZ reporter fusions, of the LEE2 and LEE3 promoters demonstrated that deletions extending closer to the LEE2 transcription start site than −247 bp lead to loss of activation by Ler, whereas only 70 bp upstream of the LEE3 transcription start site is required for Ler-mediated activation. We have purified Ler as a His-tagged protein and used it to perform DNA-binding assays with LEE2 and LEE3. We observed that Ler bound to a DNA fragment containing the −300 to +1 region of LEE2; however, it failed to bind to a DNA fragment containing the −300 to +1 region of LEE3, suggesting that Ler activates both operons by only binding to the regulatory region upstream of LEE2. The Ler-activatable LEE3::lacZ fusions extended to what would be −246 bp of the LEE2 operon. A lacZ fusion from the −300 to +1 region of LEE3 failed to be activated by Ler, consistent with our hypothesis that Ler activates the expression of LEE2 and LEE3 by binding to a region located downstream of the LEE3 transcription start site. DNase I footprinting revealed that Ler protected a region of 121 bp upstream of LEE2. Purified Ler mutated in the coiled-coil domain was unable to activate transcription and to bind to the LEE2 regulatory region. These data indicate that Ler may bind as a multimer to LEE2 and activate both divergent operons by a novel mechanism potentially involving changes in the DNA structure.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science, Ltd
    Molecular microbiology 45 (2002), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Summary The pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature. Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiled-coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.
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  • 4
    Publication Date: 2002-08-01
    Description: We used recordings of the M 6.8 Nisqually earthquake and its M (sub L) 3.4 aftershock to study site response and basin effects for 35 locations in Seattle, Washington. We determined site amplification from Fourier spectral ratios of the recorded horizontal ground motions, referenced to a soft-rock site. Soft-soil sites (generally National Earthquake Hazard Reduction Program [NEHRP] class E) on artificial fill and young alluvium have the largest 1-Hz amplifications (factors of 3-7) for both the mainshock and aftershock. These amplifications are correlated with areas of higher damage from the mainshock to major buildings and liquefaction. There are several indications of nonlinear response at the soft-soil sites for the mainshock ground motions, despite relatively modest peak accelerations in the S waves of 15%-22%g. First, the mainshock spectral ratios do not show amplification at 2-8 Hz as do the aftershock spectral ratios. Spectral peaks at frequencies below 2 Hz generally occur at lower frequencies for the mainshock spectral ratios than for the aftershock ratios. At one soft-soil site, there is a clear shift of the resonant frequency to a lower frequency for the mainshock compared with the aftershock. The frequency of this resonance increases in the coda of the mainshock record, indicating that the site response during the weaker motions of the coda is more linear than that of the initial S wave. Three of the soft-soil sites display cusped, one-sided mainshock accelerograms after the S wave. These soft-soil sites also show amplification at 10-20 Hz in the S wave, relative to the rock site, that is not observed for the aftershock. The cusped waveforms and 10-20-Hz amplification are symptomatic of nonlinear response at the soft-soil sites. These sites had nearby liquefaction. The largest amplifications for 0.5 Hz occur at soft-soil sites on the southern portion of the Seattle Basin. Stiff-soil sites (NEHRP classes D and C) on Pleistocence-age glacial deposits display similar spectral amplification for the mainshock and aftershock, indicating approximately linear response. The stiff-soil sites generally have moderate amplification (factors of 1.1-2.4) at 0.5 and 1 Hz. Amplifications at 1 and 5 Hz for all sites generally increase with decreasing shear-wave velocity measured in the top 30 m (V (sub s) 30). However, larger amplifications at 0.5 and 1 Hz for sites with similar V (sub s) 30 values are observed for sites in the Seattle Basin, illustrating the amplification from the deeper (〉30 m) sediments and the contribution from basin surface waves. Record sections for the mainshock and aftershock show that basin surface waves produce the peak velocities for many of the sites in the Seattle Basin and often dominate the amplitude at 1 Hz and lower frequencies.
    Print ISSN: 0037-1106
    Electronic ISSN: 1943-3573
    Topics: Geosciences , Physics
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  • 5
    Publication Date: 2001-01-01
    Print ISSN: 0895-0695
    Electronic ISSN: 1938-2057
    Topics: Geosciences
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  • 6
    Publication Date: 2000-03-01
    Print ISSN: 0895-0695
    Electronic ISSN: 1938-2057
    Topics: Geosciences
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  • 7
    Publication Date: 2004-09-01
    Print ISSN: 0895-0695
    Electronic ISSN: 1938-2057
    Topics: Geosciences
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  • 8
    Publication Date: 2001-04-01
    Print ISSN: 0037-1106
    Electronic ISSN: 1943-3573
    Topics: Geosciences , Physics
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  • 9
    Publication Date: 2001-02-01
    Description: We installed a dense array of 41 digital seismographs in San Jose, California, to evaluate in detail the effects of a deep sedimentary basin and shallow sedimentary deposits on earthquake ground motions. This urban array is located near the eastern edge of the Santa Clara Valley and spans the Evergreen sedimentary basin identified by gravity data. Average station spacing is 1 km, with three stations initially spaced 110 m apart. Despite the high-noise urban environment, the stations of the array successfully triggered on and recorded small local earthquakes (M 2.5-2.8 at 10-25 km distance) and larger regional events such as the M 5.0 Bolinas earthquake (90 km distance), M 4.6-5.6 earthquakes near Mammoth Lakes (270 km distance), M 4.9-5.6 events in western Nevada (420 km distance) and the M 7.1 Hector Mine earthquake (590 km distance). Maps of spectral ratios across the array show that the highest amplitudes in all frequency bands studied (0.125-8 Hz) are generally observed at stations farther from the eastern edge of the Santa Clara Valley. Larger spectral amplitudes are often observed above the western edge of the Evergreen Basin. Snapshots of the recorded wavefield crossing the array for regional events to the east reveal that large, low-frequency (0.125-0.5 Hz) arrivals after the S-wave travel from south to north across the array. A moving-window, cross-correlation analysis finds that these later arrivals are surface waves traveling from the south. The timing and propagation direction of these arrivals indicates that they were likely produced by scattering of incident S waves at the border of the Santa Clara Valley to the south of the array. It is remarkable that the largest low-frequency phases at many of the valley sites for regional events to the east are basin surface waves coming from a direction about 70 degrees different from that of the epicenters. Basin surface waves emanating from the eastern edge of the valley are also identified by the cross-correlation analysis.
    Print ISSN: 0037-1106
    Electronic ISSN: 1943-3573
    Topics: Geosciences , Physics
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  • 10
    Publication Date: 2000-10-01
    Description: We used the 3D finite-difference method to model observed seismograms of two earthquakes (M (sub L) 4.9 and 3.5) in the Seattle region and to simulate ground motions for hypothetical M 6.5 and M 5.0 earthquakes on the Seattle fault, for periods greater than 2 sec. A 3D velocity model of the Seattle Basin was constructed from studies that analyzed seismic-reflection surveys, borehole logs, and gravity and aeromagnetic data. The observations and the simulations highlight the importance of the Seattle Basin on long-period ground motions. For earthquakes occurring just south of the basin, the edge of the basin and the variation of the thickness of the Quaternary deposits in the basin produce much larger surface waves than expected from flat-layered models. The data consist of seismograms recorded by instruments deployed in Seattle by the USGS and the University of Washington (UW). The 3D simulation reproduces the peak amplitude and duration of most of the seismograms of the June 1997 Bremerton event (M (sub L) 4.9) recorded in Seattle. We found the focal mechanism for this event that best fits the observed seismograms in Seattle by combining Green's functions determined from the 3D simulations for the six fundamental moment couples. The February 1997 event (M (sub L) 3.5) to the south of the Seattle Basin exhibits a large surface-wave arrival at UW whose amplitude is matched by the synthetics in our 3D velocity model, for a source depth of 9 km. The M 6.5 simulations incorporated a fractal slip distribution on the fault plane. These simulations produced the largest ground motions in an area that includes downtown Seattle. This is mainly caused by rupture directed up dip toward downtown, radiation pattern of the source, and the turning of S waves by the velocity gradient in the Seattle basin. Another area of high ground motion is located about 13 km north of the fault and is caused by an increase in the amplitude of higher-mode Rayleigh waves caused by the thinning of the Quaternary deposits.
    Print ISSN: 0037-1106
    Electronic ISSN: 1943-3573
    Topics: Geosciences , Physics
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