Ability of Escherichia coli O157:H7 isolates to colonize the intestinal tract of conventional adult CD1 mice is transient

In an attempt to improve upon a current mouse model of intestinal colonization by Escherichia coli O157:H7 used in this laboratory for vaccine development, nine clinical isolates of the pathogen were screened for their ability to persist in the intestinal tract of conventional adult CD-1 mice. None of the test isolates of E. coli O157:H7 were capable of colonizing these mice for a period of more than two weeks. Most of the isolates appeared to be benign for the experimental host, but one isolate was lethal. This virulence correlated with the ability of the latter isolate to produce large quantities of Shiga-like toxin 2 in vitro.     

The effect of estrogen on muscle damage biomarkers following prolonged aerobic exercise in eumenorrheic women

This study assessed the influence of estrogen (E₂) on muscle damage biomarkers [skeletal muscle - creatine kinase (CK); cardiac muscle - CK-MB] responses to prolonged aerobic exercise. Eumenorrheic women (n=10) who were physically active completed two 60-minute treadmill running sessions at ∼60-65% maximal intensity during low E₂ (midfollicular menstrual phase) and high E₂ (midluteal menstrual phase) hormonal conditions. Blood samples were collected prior to exercise (following supine rest), immediately post-, 30 min post-, and 24 hours post-exercise to determine changes in muscle biomarkers. Resting blood samples confirmed appropriate E₂ hormonal levels Total CK concentrations increased following exercise and at 24 hours post-exercise were higher in the midfollicular low E₂ phase (p<0.001). However, CK-MB concentrations were unaffected by E₂ level or exercise (p=0.442) resulting in the ratio of CK-MB to total CK being consistently low in subject responses (i.e., indicative of skeletal muscle damage). Elevated E₂ levels reduce the CK responses of skeletal muscle, but had no effect on CK-MB responses following prolonged aerobic exercise. These findings support earlier work showing elevated E₂ is protective of skeletal muscle from exercise-induced damage associated with prolonged aerobic exercise.     

A phylogenetic Gibbs sampler that yields centroid solutions for cis-regulatory site prediction

MOTIVATION: Identification of functionally conserved regulatory elements in sequence data from closely related organisms is becoming feasible, due to the rapid growth of public sequence databases. Closely related organisms are most likely to have common regulatory motifs; however, the recent speciation of such organisms results in the high degree of correlation in their genome sequences, confounding the detection of functional elements. Additionally, alignment algorithms that use optimization techniques are limited to the detection of a single alignment that may not be representative. Comparative-genomics studies must be able to address the phylogenetic correlation in the data and efficiently explore the alignment space, in order to make specific and biologically relevant predictions. RESULTS: We describe here a Gibbs sampler that employs a full phylogenetic model and reports an ensemble centroid solution. We describe regulatory motif detection using both simulated and real data, and demonstrate that this approach achieves improved specificity, sensitivity, and positive predictive value over non-phylogenetic algorithms, and over phylogenetic algorithms that report a maximum likelihood solution. AVAILABILITY: The software is freely available at http://bayesweb.wadsworth.org/gibbs/gibbs.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.     

Parenteral immunization with a glycoconjugate vaccine containing the O157 antigen of Escherichia coli O157:H7 elicits a systemic humoral immune response in mice, but fails to prevent colonization by the pathogen.

The results of the present study show that whereas both BALB/c and C57BL/6 mice parenterally inoculated with a horse serum albumin-Escherichia coli O157 antigen conjugate vaccine develop systemic, specific antibodies to the carrier protein, only the former mice routinely develop antibodies to the carbohydrate O157 moiety. However, little convincing evidence was found to show that these antibodies transuded into the intestinal tract either naturally or in response to an oral inoculum of the pathogen. Moreover, this vaccination procedure failed to protect mice against intestinal colonization following oral challenge with the pathogen. Thus, the results of this study suggest that parenteral vaccination might be an unsuitable strategy for combatting E. coli O157:H7 organisms located in the gut.     

Chlamydia trachomatis major outer membrane protein epitopes expressed as fusions with LamB in an attenuated aro A strain of Salmonella typhimurium; their application as potential immunogens.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is the focus of attention for chlamydial vaccine design, particularly those serovar- and subspecies-specific epitopes which provoke neutralizing immune responses. Selected surface-exposed B-cell epitopes of MOMP, incorporating B-subspecies specificities, were expressed as fusions with LamB, an inducible outer-membrane transport protein of Escherichia coli. These recombinant chlamydial-LamB proteins were correctly transported to the outer membrane of both E. coli and an aro A mutant of Salmonella typhimurium. The immunogenicity of the constructs was investigated in a mouse model of chlamydial salpingitis. After oral immunization, recombinant S. typhimurium were recovered from the livers of mice for up to two weeks, and a serum IgG response was induced both to the Salmonella and to the inserted chlamydial epitopes. By contrast, intravenous inoculation was ineffective. Although these LamB fusions proved only weakly immunogenic, this approach should be useful for investigating the ability of attenuated S. typhimurium vaccines incorporating chlamydial epitopes to stimulate protective mucosal immunity in the mouse model of chlamydial salpingitis.