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Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections. © 2003 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 71: 141–168, 2003

Chromatographic separations of a series of highly basic peptides on commercially available 300—Å pore size CM 300 weak cation-exchange columns have been compared at various loads, pHs and ionic strengths of the eluent. On analytical columns (250 x 4.1 mm I.D.), mixtures of basic peptides containing 7 – 9 nmole of each component were separated with a 50 mMKH2PO4–KC1 gradient (pH 4.5) and under isocratic conditions (pH 4.5 and 6.5). The isocratic conditions demonstrated the effects of pH and ionic strength on retention time and resolving power on the CM 300 column. The load capacity of a CM 300 preparative column (250 x 10 mm I.D.), studied under gradient conditions (50 mMKH2PO4, 0.2 – 0.4 MKC1, pH 4.5 and 6.5), revealed that its capacity is much greater at pH 6.5. Loads up to 10–20 mg (6.6–13.3 μmol) could be applied before peaks in the crude peptide sample tested were seen to fuse.

The regulatory roles of the components of the troponin-tropomyosin complex in the presence and absence of Mg2+on the acto-S1 ATPase have been examined. The effect of free Mg2+on the inhibition of the acto-S1 ATPase by rabbit skeletal troponin (Tn) was studied at S1 to actin ratios ranging from 0.17:1 to 2.5:1. These studies were performed using two Mg2+concentrations: 2.5 mM Mg2+-2.5 mM ATP, conditions considered to have low free Mg2+; and 5.0 mM Mg2+-2.5 mM ATP, conditions providing a high free Mg2+concentration of ∼2.5 mM. In the presence of high free Mg2+(2.5 mM ATP-5.0 mM MgCl2) the Tn inhibition of acto-S1-TM ATPase increased by approximately 40–50% over a range of S1 to actin ratios of 0.17:1 to 2.5:1. The effect of free Mg2+on increasing quantities of Tn in the absence or presence of tropomyosin was studied independently at two S1 to actin ratios (1:1 and 2:1). In the absence of TM, at 5 mM Mg2+there is an additional 38% (1:1 S1 to actin) or 37% (2:1) decrease in the ATPase activity by Tn compared to 2.5 mM Mg2+. Similarly, in the presence of TM and Tn, Mg2+exerts its effect at both S1 to actin ratios. Significantly, the inhibition by the IT complex in the presence of TM is unaffected by free Mg2+. Furthermore, ultracentrifugation binding studies using14C-iodoacetamide-labeled Tn and TM established that the Tn-TM regulatory complex was firmly bound to F-actin at both Mg2+concentrations, indicating that faciliation of binding to F-actin by Mg2+is not responsible for the increased inhibition. Hence, it is concluded from the data that Mg2+binding and by analogy Ca2+binding to the Ca2+-Mg2+sites of TnC promotes muscle relaxation by inducing inhibition of the actomyosin ATPase, whereas Ca2+binding to the Ca2+-specific sites promotes contraction by potentiating the ATPase. The inhibition of the acto-S1-TM ATPase by TnT has also been further examined. The data indicate that TnT exerts the same level of inhibition upon the ATPase as TnI or Tn. The inhibitory activity requires TM, and occurs to the same extent under conditions where TM alone would have either a potentiating (2:1 S1 to actin) or an inhibitory (1:1 S1 to actin) effect upon the ATPase. In the presence of TM the IT complex is a more effective inhibitor than either TnI, TnT, or Tn. The inhibitory activity of the IT complex is partially released by TnC in the absence of Ca2+. These observations, in conjunction with those by Chong, Asselbergs, and Hodges, which showed that the inhibition by TnT is partially released by TnC plus Ca2+, indicate that the role of TnT involves more than anchoring Tn to the thin filament.