Eddie L. Chang

Cobalt Complexes as Antiviral and Antibacterial Agents

Metal ion complexes are playing an increasing role in the development of antimicrobials. We review here the antimicrobial properties of cobalt coordination complexes in oxidation state 3+. In addition to reviewing the cobalt complexes containing polydentate donor ligands, we also focus on the antimicrobial activity of the homoleptic [Co(NH3)6]3+ ion.

Purification and characterization of a liposomal-forming tetraether lipid fraction

Polar lipid E, PLE, a native tetraether lipid mixture from Sulfolobus acidocaldarius is shown to spontaneously form multilamellar liposomes in aqueous media. PLE lipids were isolated as a single fraction from the crude lipid extract of S. acidocaldarius on a reverse-phase column followed by TLC and methanol precipitation. The methanol-precipitated mixture was able to form liposomes, whereas the non-precipitated material did not. It has thus been demonstrated, for the first time, that tetraether lipids from S. acidocaldarius can form liposomes, per se, in aqueous media.

Encapsulation of hemoglobin in phospholipid vesicles

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 Å as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4°C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4°C.

Destabilization of a lipid non-bilayer phase by high pressure

Abstract Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H11) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.

Metal–chelator polymers as reactive adsorbents for organophosphate hydrolysis

Abstract We describe the synthesis and hydrolytic properties of three monomeric Cu(II)–1,4,7-triazacyclononane chelators, containing one to three polymerizable N -vinylbenzyl substituents, and their insoluble polymerized forms. The rates of hydrolysis of methyl parathion (MeP) and bis-(4-nitrophenyl)phosphate (BNPP), expressed as their apparent Michaelis–Menten kinetic constants, were determined. The reported phosphodiester and phosphotriester catalytic rates are among the highest reported for insoluble polymers, with apparent catalytic constants of 1.0×10 −2 and 5.5×10 −4 s −1 for MeP and BNPP, respectively. Unusual substrate inhibition was also observed at high substrate concentrations for the three polymers that were cross-linked using trimethylolpropane trimethacrylate (TRIM), whereas polymers formed from the pure monomers or from a mixture of monomeric and TRIM polymerizations did not exhibit substrate inhibition at the same concentrations.

Monitoring viral RNA in infected cells with LNA flow-FISH

We previously showed the feasibility of using locked nucleic acid (LNA) for flow cytometric-fluorescence in situ hybridization (LNA flow-FISH) detection of a target cellular mRNA. Here we demonstrate how the method can be used to monitor viral RNA in infected cells. We compared the results of the LNA flow-FISH with other methods of quantifying virus replication, including the use of an enhanced green fluorescent protein (EGFP) viral construct and quantitative reverse-transcription polymerase chain reaction. We found that an LNA probe complementary to Sindbis virus RNA is able to track the increase in viral RNA over time in early infection. In addition, this method is comparable to the EGFP construct in sensitivity, with both peaking around 3 h and at the same level of infected cells. Finally, we observed that the LNA flow-FISH method responds to the decrease in levels of viral RNA caused by antiviral medication. This technique represents a straightforward way to monitor viral infection in cells and is easily applicable to any virus.

Towards achieving selectivity in metal ion binding by fixing ligand–chelator complex geometry in polymers

Abstract The synthesis of polymers with pre-organized metal ion binding sites for optimal binding of selected metal ions is reported. An acyclic N -vinylbenzyl substituted chelator, triethylenetetramine, is complexed with copper(II) ions and crosslinked with matrix monomer TRIM [2-ethyl-2-(hydroxymethyl) propane-1, 3-diol trimethacrylate] to form the polymer. UV–visible spectra indicate that the coordination geometry of the monomeric metal–ligand complex is highly conserved in the polymer. Unlike macrocyclic ligands, the acyclic ligating molecules demonstrate flexibility in forming chelation rings that are specific to metal-ion size and geometry. The complexation step is used as a means to optimize the chelator conformation for copper ions. The resulting polymers, after removal of metal ions, show selectivity towards the templated metal ions. The small size of the metal ions facilitated their access to the templated sites embedded in the porous polymers. This scheme has the potential to become a generalized procedure for making metal ion selective polymers.

Unusual pressure dependence of the lateral motion of pyrene-labeled phosphattdylcholine in bipolar lipid vesicles

The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 degrees C. At temperatures > 48 degrees C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes.

Photon correlation spectroscopy study on the stability of small unilamellar DPPC vesicles.

The growth in size of dipalmitoylphosphatidylcholine small unilamellar vesicles (SUV) below Tm has been studied by photon correlation spectroscopy and differential scanning calorimetry. We see an initial fast rise of the hydrodynamic diameter of the vesicles followed by a slower increase. We assign the slow component of the size change to fusion of SUV. The order of the kinetics appears to be higher than first order. The estimated half lifetime of the fusion is approximately 67 h. The diameters for the fast and slow processes at t = O are 756 and 256 A, respectively, while as t leads to infinity the diameters increase to 1,570 and 733 A, respectively.

Temporal Transcriptional Response during Infection of Type II Alveolar Epithelial Cells with Francisella tularensis Live Vaccine Strain (LVS) Supports a General Host Suppression and Bacterial Uptake by Macropinocytosis

Background: The mechanism of Francisella LVS entry into A549 cells is unknown. Results: Microarrays were performed at early infection time points, supported by phenotypic observations and inhibition experiments. Conclusion: Francisella LVS may enter A549 cells by macropinocytosis and quiets the host infection response. Significance: Francisella LVS induces significant host cell signaling at very early time points after the bacterias interaction with the cell. Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2–6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacterias interaction with the cell.