Anthony Kennedy

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH(2)) to model membranes. POPC liposomes were used as a simple model for eukaryotic membranes and 4:1 POPC:POPG liposomes were used as a simple model for prokaryotic membranes. CD, ITC and calcein leakage data clearly indicate that compound 1 interacts via very different mechanisms with the two different liposome membranes. Compound 1 exhibits weaker binding and induces less calcein leakage in POPC liposomes than POPC:POPG (4:1 mole ratio) liposomes. The predominant binding mechanism to POPC appears to be limited to surface interactions while the mechanism of binding to 4:1 POPC:POPG most likely involves some type of pore formation.

Electrochemical Anion Recognition By Novel Ferrocenyl Imidazole Systems

A novel class of anion receptors with with C-H•••X- hydrogen bonding is introduced and demonstrated for Cl-, Br-, NO3- and HSO4- recognition. Cyclic voltammetry revealed that novel ferrocenylimidazolium salts, syntheses of which are briefly described, selectively complex and electrochemically recognise guest anions. Futhermore, proton NMR spectroscopy indicated the formation of 1:2 stoichiometric complexes with Cl-, Br- and I- and 1:1 stoichiometric complexes with NO3- and HSO4-.

Physical and thermal properties of blood storage bags: implications for shipping frozen components on dry ice.

BACKGROUND: Frozen blood components are shipped on dry ice. The lower temperature (–70°C in contrast to usual storage at –30°C) and shipping conditions may cause a rent in the storage bag, breaking sterility and rendering the unit useless. The rate of loss can reach 50 to 80 percent. To identify those bags with lower probability of breaking during shipment, the thermal and physical properties of blood storage bags were examined.

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH(2)) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models. These compounds also exhibited very different CD spectra in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG SUVs and LUVs, indicating the formation of different conformations on interaction with the two membrane types. This observation is also supported by ITC and calcein leakage data. ITC data suggested these peptides interact primarily with the surface of zwitterionic LUVs and was further supported by fluorescence experiments where the interactions do not appear to be concentration dependent. In the presence of anionic membranes, the interactions appear more complex and the calorimetric and fluorescence data both imply pore formation is dependent on peptide concentration. Furthermore, evidence suggests that as the length of Spacer# 1 increases the mechanism of pore formation also changes. Based on the observed differences in the mechanisms of interactions with zwitterionic and anionic LUVs these AMPs are potential candidates for further drug development.

CHARACTERIZATION OF THE MAIN PHASE TRANSITION IN 1,2-DIPALMITOYL-PHOSPHATIDYLCHOLINE LUVS BY 1H NMR*

ABSTRACT The main phase transition (Tm) of 100 nm large unilamellar vesicles (LUVs) of 1,2-dipalmitoylphosphatidylcholine (DPPC) was investigated using 1H NMR (proton magnetic resonance) in deuterium oxide, and both DSC (differential scanning calorimetry) and IR (infrared) spectroscopy in water and deuterium oxide. The ability of 1H NMR to determine Tm was demonstrated and the values obtained were in general agreement with those observed with DSC and IR. However, the temperature range of the transition observed by NMR was significantly broader than that observed with either DSC or IR. The effect of deuterium oxide on Tm was studied by comparing results obtained in water and deuterium oxide with DSC and IR. The results showed no significant difference in Tm or temperature range of transition determined in these solvents.

Distinct membrane properties are differentially influenced by cardiolipin content and acyl chain composition in biomimetic membranes

Cardiolipin (CL) has a critical role in maintaining mitochondrial inner membrane structure. In several conditions such as heart failure and aging, there is loss of CL content and remodeling of CL acyl chains, which are hypothesized to impair mitochondrial inner membrane biophysical organization. Therefore, this study discriminated how CL content and acyl chain composition influenced select properties of simple and complex mitochondrial mimicking model membranes. We focused on monolayer excess area/molecule (a measure of lipid miscibility), bilayer phase transitions, and microdomain organization. In monolayer compression studies, loss of tetralinoleoyl [(18:2)4] CL content decreased the excess area/molecule. Replacement of (18:2)4CL acyl chains with tetraoleoyl [(18:1)4] CL or tetradocosahexaenoyl [(22:6)4] CL generally had little influence on monolayer excess area/molecule; in contrast, replacement of (18:2)4CL acyl chains with tetramyristoyl [(14:0)4] CL increased monolayer excess area/molecule. In bilayers, calorimetric studies showed that substitution of (18:2)4CL with (18:1)4CL or (22:6)4CL lowered the phase transition temperature of phosphatidylcholine vesicles whereas (14:0)4CL had no effect. Finally, quantitative imaging of giant unilamellar vesicles revealed differential effects of CL content and acyl chain composition on microdomain organization, visualized with the fluorescent probe Texas Red DHPE. Notably, microdomain areas were decreased by differing magnitudes upon lowering of (18:2)4CL content and substitution of (18:2)4CL with (14:0)4CL or (22:6)4CL. Conversely, exchanging (18:2)4CL with (18:1)4CL increased microdomain area. Altogether, these data demonstrate that CL content and fatty acyl composition differentially target membrane physical properties, which has implications for understanding how CL regulates mitochondrial activity and the design of CL-specific therapeutics.

Conservation of chemically degraded waterlogged wood with sugars

Abstract The effectiveness of two non-reducing sugars, both analogues of sucrose, to conserve degraded waterlogged wood was examined. The two sugars examined are trehalose and sucralose, both stable and relatively unreactive. The ability of these sugars to conserve a series of degraded tongue depressors was measured by determining the anti-shrink efficiency of each at various concentrations and comparing them to sucrose. The findings of this study indicate that both sucralose and trehalose may be effective conservation treatments for waterlogged archaeological wood and that at moderate concentrations the performance of both is comparable to sucrose. However, sucralose has a lower solubility, and concentrations higher than 60% w/v were not examined, whereas concentrations of up to 100% w/v of trehalose were studied. At these higher concentrations trehalose performed as well, if not better than sucrose, although there were crystalline deposits on the wood surface at these higher concentrations. With modifications and careful control, both of these sugars may be suitable conservation alternatives to sucrose due to their long-term stability and resistance to hydrolysis.

White particulate matter found in blood collection bags consist of platelets and leukocytes

BACKGROUND:  In late January 2003, some blood centers and hospitals throughout the US voluntarily sus‐pended the use of some RBC and plasma units for trans‐fusion due to the presence of unknown white particulate matter (WPM) in these units. To better understand the WPM phenomena, a number of technologies were used to establish the nature of the particulates observed in Terumo Collection sets.

The Interaction of DMSO with Model Membranes. II. Direct Evidence of DMSO Binding to Membranes: An NMR Study

Abstract Modern techniques in nuclear magnetic resonance (NMR) allow investigators to probe molecular interactions with greater sensitivity and speed than ever before. Exploiting the nuclear Overhauser effect (NOE), the intermolecular interactions between dimethylsulfoxide (DMSO) and lipid vesicles were investigated. The DMSO methyl proton signal varies with experimental mixing time suggesting the system behaves in a manner similar to that of a ligand weakly binding to a macromolecule.

The Interaction of DMSO with Model Membranes. I. Comparison of DMSO and d6-DMSO: A DSC and IR Investigation

Abstract The effect of deuterated solvents on the main phase transition (Tm) temperature of large unilamellar vesicles (LUVs) of 1,2-dipalmitoyl phosphatidylcholine (DPPC) was examined by infrared spectroscopy and differential scanning calorimetry. There was no significant difference in the Tm, ΔH of the main transition, or cooperativity when water was replaced by deuterium oxide (D2O) in the preparation of the vesicles or when dimethylsulfoxide (DMSO) was replaced by hexadeutero-dimethylsulfoxide (d6-DMSO) in either D2O or water. These results provide the necessary advance for further structural, kinetic, and thermodynamic investigations of DMSO interactions with membranes and lipid vesicles.