Puroindoline a, a wheat endosperm-specific protein containing a tryptophan-rich domain, was reported to have antimicrobial activities. We found that a 13-residue fragment of puroindoline a (FPVTWRWWKWWK G-NH 2 ) (puroA) exhibits activity against both gram-positive and gram-negative bacteria. This suggests that puroA may be a bactericidal domain of puroindoline a. PuroA interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the microbicidal effect of puroA may be due to interactions with bacterial membranes. A variety of biophysical and biochemical methods, including fluorescence spectroscopy and microcalorimetry, were used to examine the mode of action of puroA. These studies showed that puroA is located at the membrane interface, probably due to its high content of Trp residues that have a high propensity to partition into the membrane interface. The penetration of these Trp residues in negatively charged phospholipid vesicles resembling bacterial membranes was more extensive than the penetration in neutral vesicles mimicking eukaryotic membranes. Peptide binding had a significant influence on the phase behavior of the former vesicles. The three-dimensional structure of micellebound puroA determined by two-dimensional nuclear magnetic resonance spectroscopy indicated that all the positively charged residues are oriented close to the face of Trp indole rings, forming energetically favorable cation-interactions. This characteristic, along with its well-defined amphipathic structure upon binding to membrane mimetic systems, allows puroA to insert more deeply into bacterial membranes and disrupt the regular membrane bilayer structure.

Lactoferricins are highly basic bioactive peptides that are released in the stomach through proteolytic cleavage of various lactoferrin proteins. Here we have determined the solution structure of human lactoferricin (LfcinH) by conventional two-dimensional nuclear magnetic resonance methods in both aqueous solution and a membrane mimetic solvent. Unlike the 25-residue bovine lactoferricin (LfcinB), which adopts a somewhat distorted antiparallel ␤ sheet, the longer LfcinH peptide shows a helical content from Gln14 to Lys29 in the membrane mimetic solvent but a nonexistent ␤-sheet character in either the N-or C-terminal regions of the peptide. The helical characteristic of the LfcinH peptide resembles the conformation that this region adopts in the crystal structure of the intact protein. The LfcinH structure determined in aqueous solution displays a nascent helix in the form of a coiled conformation in the region from Gln14 to Lys29. Numerous hydrophobic interactions create the basis for the better-defined overall structure observed in the membrane mimetic solvent. The 49-residue LfcinH peptide isolated for these studies was found to be slightly longer than previously reported peptide preparations and was found to have an intact peptide bond between residues Ala11 and Val12. The distinct solution structures of LfcinH and LfcinB represent a novel difference in the physical properties of these two peptides, which contributes to their unique physiological activities.The bactericidal effects of lactoferrin and lactoferricin (Lfcin) have been the subject of intensive study for at least two decades. Originally, the antimicrobial properties of intact lactoferrins were believed to be related to the iron-scavenging ability of this iron-binding protein (6,43). Subsequent studies have shown, however, that potent antimicrobial properties reside in the highly basic N-terminal regions of these proteins, which are not involved in iron binding (8, 16). As a result, further study was given to the isolation of the lactoferricin peptide produced in vivo by digestion of bovine lactoferrin (27,40). More recently, additional work has focused upon the identification of key antimicrobial segments and enhancement of the activity of the various shorter synthetic human lactoferricin (LfcinH) peptide analogs by using amino acid substitutions and various activity assays (7,10,11,28,30,37,42,47). These studies have established not only that LfcinH has antimicrobial, antiviral, and antifungal activities but also that it is capable of stimulating the immune system and neutralizing endotoxin.Other research has focused on determining how lactoferricin functions with specific target systems. For example, lactoferricin can effectively interact with porins (34) and DNA (39), bind to lipopolysaccharide and teichoic acid (5), reduce the charge potential at the bacterial membrane as well as the pH gradient (1), and display antiviral properties (2-4, 23, 24). Lactoferricin B has also been found to translocate into the bacterial cytoplasm (19), where it ...

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11-154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103-239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1-96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676-10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.

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Background: A novel symmetric posterior condylar design and option of a femoral component with an outer zirconium oxide layer were introduced to a successful asymmetric condylar total knee arthroplasty system in 2005. Given the paucity of information on this modified design, we performed a study to determine its early to midterm survivorship and performance. Methods: Patients who received the Genesis II Symmetric Posterior Condyle or Legion Primary total knee (cobalt-chromium [CoCr] or oxidized zirconium-niobium [OxZr]) (Smith & Nephew) implanted at the study centre between March 2007 and December 2013 were enrolled into a prospective database. We retrospectively reviewed the database and performed survival analysis using Kaplan-Meier techniques. Results: There were 2178 patients (1359 women [62.4%]; mean age 64.6 yr; mean body mass index 35.0) with 2815 knee replacements available for analysis. Survival rates were 98.2% (95% confidence interval [CI] 97.6%-98.7%) for failure for any reason at 2 years and 96.8% (95% CI 96.0%-97.7%) at 5 years. Age (hazard ratio [HR] 0.97, 95% CI 0.94-0.997) and female sex (HR 0.45, 95% CI 0.27-0.75) were protective, whereas body mass index (HR 1.02, 95% CI 0.99-1.05) and OxZr implant (HR 1.11, 95% CI 0.57-2.18) did not influence survivorship. Oxford Knee Score values improved from a mean of 40.2 to 21.8 by 2 years (p < 0.001), with no difference between the CoCr and OxZr groups. Conclusion: The symmetric posterior condylar posterior-stabilized knee offers excellent midterm survivorship. Implant bearing surface did not have an influence on survivorship to 5 years, and, thus, use of OxZr implants may not be justified. Contexte : Un nouveau modèle à condyles postérieurs symétriques et l'option d'un élément fémoral pourvu d'une couche d'oxyde de zirconium ont été introduits avec succès dans un système de prothèse totale du genou à condyles asymétriques en 2005. Compte tenu du manque de renseignements concernant ce modèle modifié, nous avons procédé à une étude pour en déterminer la survie et le rendement à court et à moyen terme. Méthodes : Les patients ayant reçu la prothèse à condyles postérieurs symétriques Genesis II ou une prothèse totale primaire Legion (au cobalt-chrome [CoCr] ou au zirconium-niobium oxydé [OxZr]) (Smith & Nephew) au centre de recherche entre mars 2007 et décembre 2013 ont été inscrits dans une base de données prospective. Nous avons procédé à une revue rétrospective de la base de données et effectué une analyse de survie à l'aide de techniques de Kaplan-Meier. Résultats : On a dénombré 2178 patients (dont 1359 femmes [62,4 %]; âge moyen 64,6 ans; indice de masse corporelle moyen 35,0) totalisant 2815 arthroplasties du genou pour analyse. Les taux de survie ont été de 98,2 % (intervalle de confiance [IC] à 95 % 97,6 %-98,7 %) pour le paramètre défaillance de toutes causes à 2 ans et de 96,8 % (IC à 95 % 96,0 %-97,7 %) à 5 ans. L'âge (risque relatif [RR] 0,97, IC à 95 % 0,94-0,997) et le fait d'être de sexe féminin (RR 0,45, IC à 95 % 0,27-0,75) ont conféré un e...