The importance of bacterial membrane composition in the structure and function of aurein 2.2 and selected variants

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and 31P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria.     

A propos de l'intervention des aéroions dans les processus de régulation de la lipémie et de la cholestérolémie

Resume Les expériences ont porté sur des lapins repartis en deux groupes parallèles — un groupe étant traité avec des aéroions négatifs en concentrations moderées (n^– = 10 – 40.000; n⁺ = 1.000) et l'autre sans aéroionothérapie (témoins). Par rapport aux animaux témoins (pas exposés aux aéroions),on a enregistré,sous l'influence de l'aéroionothérapie, que: (1)il y a une tendence de normalisation de la motilité spontanée, deprimée par une alimentation contenant un excès de cholestérol(0,3 g/kg/jour); (2) la lipémie et la cholestérolémie sont restées à un niveau plus bas chez les animaux alimentés au cholestérol; (3) la lipémie et la cholestérolémie, de même que l'iode thyroïdien, sont restées aux valeurs voisines de celles normales,chez les animaux nourris avec un excès (20 mg/kg/jour), de substances antithyroïdiennes naturelles (provenant du chou).

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Groups of rabbits were exposed to negative air ions of moderate concentrations (n^– = 10 – 40,000; n⁺ = 1,000) while others served as controls. (1) When rabbits were fed a cholesterol-rich diet (0.3 g/kg/day)causing reduction of motility in the control animals, their motility was normalized under air ion treatment. (2) The blood cholesterol and lipid levels of rabbits on a cholesterol-rich diet were lower with air ion treatment than in controls.(3) When rabbits were fed a diet with thyroid blocking agents from cabbage the blood cholesterol and lipid levels and the thyroid iodine content remained unchanged with air ion treatment.

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Zusammenfassung Gruppen Kaninchen wurden mit negativen Luftionen in mässigen Konzentrationen (n^– = 100 000 – 40 000; n⁺ = 1 000) behandelt, während andere als Kontrollen dienten. (1) Die durch Fütterung von cholesterinreicher Diät (0,3 g/kg/Tag) verminderte Motilität der Tiere wurde unter der Luftionenbehandlung normalisiert. (2) Der durch Fütterung von cholesterinreicher Diät erhöhte Blutcholesterin- und-fettspiegel fiel durch Luftionenbehandlung ab. (3) Der durch Fütterung von strumipriven Substanzen aus Kohl erhöhte Cholesterin- und Fettspiegel im Blut und verminderte Jodgehalt der Schilddrüse wurde durch Luftionenbehandlung nicht beeinflusst.

     

Cervical magnetic stimulation as a method to discriminate between diaphragm and rib cage muscle fatigue

Inspiratory muscle fatigue can probablydetermine hypercapnic respiratory failure. Diaphragm fatigue isdetected by electrical phrenic stimulation (ELS), but there is nosimple tool to assess rib cage muscle (RCM) fatigue. Cervical magneticstimulation (CMS) costimulates the phrenic nerves and RCM. We reasonedthat changes in transdiaphragmatic pressure twitch (Pdi,tw) with CMSand ELS should be different after selective diaphragm vs. RCM fatigue. Five volunteers performed inspiratory resistive tasks while voluntarily uncoupling diaphragm and RCM. BaselinePdi,tw_ELS andPdi,tw_CMS were 28.57 ± 1.68 and 32.83 ± 2.92 cmH₂O. Afterselective diaphragm loading,Pdi,tw_ELS andPdi,tw_CMS were reduced by 39 and26%, with comparable decreases in gastric pressure twitch (Pga,tw).Esophageal pressure twitch (Pes,tw) was better preserved with CMS.Therefore Pes,tw/Pga,tw was lower with ELS than CMS (1.24 ± 0.16 vs. 1.73 ± 0.11, P = 0.05). After selectiveRCM loading, there was no diaphragm fatigue, butPes,tw_CMS was significantlyreduced (30%). These findings support the role of rib cagestiffening by CMS-related RCM contraction in the ELS-CMSdifferences and suggest that CMS can be used to assess RCM fatigue.

     

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.     

Individual optimization: Mechanisms shaping the optimal reaction norm

In general, optimal reaction norms in heterogeneous populations can be obtained only by iterative numerical procedures (McNamara, 1991; Kawecki and Stearns, 1993). We consider two particular, but biologically plausible and analytically tractable cases of individual optimization to gain insight into the mechanisms which shape the optimal reaction norm of fecundity in relation to an environmental variable or an individual trait. In the first case, we assume that the quality of the environment (e.g. food abundance) or the quality of the individual (e.g. body size) is fixed during its entire life; it may also be a heritable individual trait. In the second case, individual quality is assumed to change randomly such that the probability distribution of quality in the next year is the same for the parent and for her offspring. For these two cases, we obtain analytical expressions for the shape of the optimal reaction norm, which are heuristically interpretable in terms of underlying selective mechanisms. It is shown that better quality may reduce the optimal fecundity. This outcome is particularly likely if better quality increases a fecundity-independent factor of parental survival in a long-lived species with fixed quality. This revised version was published online in July 2006 with corrections to the Cover Date.     

NMR structural studies of the antibiotic lipopeptide daptomycin in DHPC micelles

Daptomycin is a cyclic anionic lipopeptide that exerts its rapid bactericidal effect by perturbing the bacterial cell membrane, a mode of action different from most other currently commercially available antibiotics (except e.g. polymyxin and gramicidin). Recent work has shown that daptomycin requires calcium in the form of Ca²⁺ to form a micellar structure in solution and to bind to bacterial model membranes. This evidence sheds light on the initial steps in the mechanism of action of this novel antibiotic. To understand how daptomycin goes on to perturb bacterial membranes, its three-dimensional structure has been determined in the presence of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micelles. NMR spectra of daptomycin in DHPC were obtained under two conditions, namely in the presence of Ca²⁺ as used by Jung et al. [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57] to solve the calcium-conjugated structure of daptomycin in solution and in a phosphate buffer as used by Rotondi and Gierasch [K.S. Rotondi, L.M. Gierasch, A well-defined amphipathic conformation for the calcium-free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution, Biopolymers 80 (2005) 374-85] to solve the structure of apo-daptomycin. The structures were calculated using molecular dynamics time-averaged refinement. The different sample conditions used to obtain the NMR spectra are discussed in light of fluorescence data, lipid flip-flop and calcein release assays in PC liposomes, in the presence and absence of Ca²⁺ [D. Jung, A. Rozek, M. Okon, R.E.W. Hancock, Structural transitions as determinants of the action of the calcium-dependent antibiotic daptomycin, Chem. Biol. 11 (2004) 949-57]. The implications of these results for the membrane perturbation mechanism of daptomycin are discussed.     

Bactericidal activity of testicular macrophages

The purpose of these studies was to determine if testicular macrophages are capable of bactericidal activity. Testicular macrophages were isolated from adult Wistar rats and studied in vitro. Studies were designed to determine if these cells could kill pathogenic gram-negative organisms and if these cells secreted lysozyme, an enzyme involved with the lysis of the cell wall of gram-positive bacteria. The regulation of lysozyme secretion by hormones and lipopolysaccharide was also studied. The secretion of this enzyme by testicular macrophages was also compared to enzyme secretion by macrophages isolated from other tissues. We also studied the secretion of superoxide anion, which is known to be involved in cytotoxic reactions. It was found that testicular macrophages were capable of killing up to approximately 38% of a virulent encapsulated strain of Klebsiella pneumoniae within 1 h. This process was in part dependent upon the presence of immune serum generated against these organisms but could not be mimicked by control serum or immune serum tested in the absence of macrophages. Testicular macrophages secreted lysozyme in culture for at least 8 days; however, macrophages from the peritoneal cavity and lung secreted significantly more lysozyme under the same conditions. Lipopolysaccharide suppressed lysozyme secretion in a dose-dependent manner, whereas neither follicle-stimulating hormone, testosterone, nor leuteininzing hormone had an effect on lysozyme secretion. Finally, testicular macrophages secreted superoxide anion in a manner similar to peritoneal macrophages. These studies indicate that testicular macrophages have the capability to mount an appropriate defense against pathogenic bacteria by opsonization-dependent phagocytosis, the secretion of lysozyme, and the production of super oxide anion.