A gel-free proteomic-based method for the characterization of Bordetella pertussis clinical isolates

Bordetella pertussis (Bp) is the etiologic agent of pertussis or whooping cough, a highly contagious respiratory disease occurring primarily in infants and young children. Although vaccine preventable, pertussis cases have increased over the years leading researchers to re-evaluate vaccine control strategies. Since bacterial outer membrane proteins, comprising the surfaceome, often play roles in pathogenesis and antibody-mediated immunity, three recent Bp circulating isolates were examined using proteomics to identify any potential changes in surface protein expression. Fractions enriched for outer membrane proteins were digested with trypsin and the peptides analyzed by nano liquid chromatography-electrospray ionization-mass spectrometry (nLC-ESI-MS), followed by database analysis to elucidate the surfaceomes of our three Bp isolates. Furthermore, a less labor intensive non-gel based antibody affinity capture technology in conjunction with MS was employed to assess each Bp strains' immunogenic outer membrane proteins. This novel technique is generally applicable allowing for the identification of immunogenic surface expressed proteins on pertussis and other pathogenic bacteria.     

The products of mitochondria-bound cytoplasmic polysomes in yeast

Experiments were undertaken to examine the fate and composition of polypeptides synthesized on cytoplasmic polysomes associated with the outer mitochondrial membrane of Saccharomyces cerevisiae. Mitochondria with their associated cytoplasmic polysomes were isolated from growing yeast spheroplasts and placed in a polypeptide chain completion system together with [35S]methionine. Of the total products synthesized in the readout system, 80 to 85% remain associated with the mitochondria after sucrose gradient centrifugation. Most of the labeled products are resistant to papain digestion unless the membranes are disrupted by treatment with detergent or shaking with glass beads. When free cytoplasmic polysomes were translated in the presence of [35S]methionine and incubated with mitochondria, only about 20% of the labeled polypeptides remain associated with the mitochondria; furthermore, most of these products are equally sensitive to papain digestion in the presence or absence of detergent. These results support the view that the cytoplasmic polysomes associated with the outer mitochondrial membrane of yeast facilitate the segregation of newly synthesized proteins into the organelle. The proportion of the alpha, beta, and gamma subunits of the F1-ATPase was determined among the products synthesized by mitochondria-bound and free cytoplasmic polysomes. By double antibody precipitation and immunoreplicate electrophoresis, we find that the proportion of the subunits of F1-ATPase is much greater among the products of the mitochondria-bound polysomes than those synthesized on free polysomes.     

Effects of resistance training on physical function in older disabled women with coronary heart disease.

We studied whether disabled older women with coronary heart disease can perform resistance training at an intensity sufficient to improve measured and self-reported physical function [n = 30, 70.6 +/- 4.5 (SD) yr]. Compared with the controls, the resistance-training group showed significant improvements in overall measured physical function score using the Continuous-Scale Physical Functional Performance Test (+24 vs. +3%). The Continuous-Scale Physical Functional Performance Test measures physical function for 15 practical activities, such as carrying groceries or climbing stairs. Resistance training led to improved measures for domains of upper body strength (+18 vs. +6%), lower body strength (+23 vs. +6%), endurance (+26 vs. +1%), balance and coordination (+29 vs. -2%), and 6-min walk (+15 vs. +7%). Women involved in the flexibility-control group showed essentially no improvement for physical function measures. No changes were observed for body composition, aerobic capacity, or self-reported physical function in either group. In conclusion, disabled older women with coronary heart disease who participate in strength training are able to train at an intensity sufficient to result in improvements in multiple domains of measured physical functional performance, despite no change in lean body mass.     

Regulation of the alternative sigma factor sigma(E) during initiation,adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli

The alternative sigma factor sigma(E) is activated in response to stress in the extracytoplasmic compartment of Escherichia coli. Here we show that sigma(E) activity increases upon initiation of the stress response by a shift to an elevated temperature (43 degrees C) and remains at that level for the duration of the stress. When the stress is removed by a temperature downshift, sigma(E) activity is strongly repressed and then slowly returns to levels seen in unstressed cells. We provide evidence that information about the state of the cell envelope is communicated to sigma(E) primarily through the regulated proteolysis of the inner membrane anti-sigma factor RseA, as the degradation rate of RseA is correlated with the changes in sigma(E) activity throughout the stress response. However, the relationship between sigma(E) activity and the rate of degradation of RseA is complex, indicating that other factors may cooperate with RseA and serve to fine-tune the response.     

Analysis of recombinant acylated pneumococcal surface adhesin A of Streptococcus pneumoniae by mass spectrometry

Streptococcus pneumoniae pneumococcal surface adhesin A (PsaA) is a species-common, immunogenic surface lipoprotein. In this study, the psaA gene was expressed as a nonfusion acylated protein in an Escherichia coli expression system. Yields of pure recombinant PsaA (rPsaA) were 8-10 mg/liter of fermentation culture. Analysis of rPsaA tryptic digests by HPLC-electrospray mass spectrometry (MS) confirmed 98% of the expected protein sequence. GC/MS data demonstrated very similar acylation of native and rPsaA by C12:0-C22:0 fatty acids, with C16 and C18 predominating. Negative ion electrospray MS/MS analysis of the rPsaA lipid anchor released by Pronase-E confirmed that the structure was based on an N-terminal palmitoylcysteine (Pam(3)Cys). Electrospray MS heterogeneity analysis of intact rPsaA indicated that all of the observed heterogeneity could be accounted for by the fatty acid distributions. The availability of well-characterized rPsaA will facilitate the continued research and development of protein-based vaccines for the prevention of pneumococcal disease.