Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.     

Electrical measurements of bilayer membranes formed by Langmuir-Blodgett deposition on single-crystal silicon

Bilayer membranes on solid supports are used for fundamental studies of biophysical properties and for the development of biosensors and other devices. Here we report on electrically addressable bilayer membranes formed by Langmuir-Blodgett (LB)-based deposition on single-crystal silicon. The incorporation of a polymer cushion ensures high lipid mobility in both the lower and upper leaflet, allowing the potential for combined investigations of electrical, structural, and dynamic characteristics of membrane-associated proteins. Impedance spectroscopy is used to demonstrate that the lipid bilayers are robust and reproducible with an impedance of about 10(4) Omega cm2 and a capacitance of about 0.8 microF cm(-2). The ability to characterize ion channels is demonstrated using the model system gramicidin. These results demonstrate that artificial bilayers formed by LB deposition have many unique advantages for electrical measurements of membranes and their components.     

Typical aromatic noncovalent interactions in proteins: A theoretical study using phenylalanine

A systematic study of CH ··· π, OH ··· π, NH ··· π, and cation ··· π interactions has been done using complexes of phenylalanine in its cationic, anionic, neutral, and zwitterionic forms with CH₄, H₂O, NH₃, and NH at B3LYP, MP2, MPWB1K, and M06‐2X levels of theory. All noncovalent interactions are identified by the presence of bond critical points (bcps) of electron density (ρ( r )) and the values of ρ( r ) showed linear relationship to the binding energies (E_total). The estimated E_total from supermolecule, fragmentation, and ρ( r ) approaches suggest that cation ··· π interactions are in the range of 36 to 46 kcal/mol, whereas OH ··· π, and NH ··· π interactions have comparable strengths of 6 to 27 kcal/mol and CH ··· π interactions are the weakest (0.62–2.55 kcal/mol). Among different forms of phenylalanine, cationic form generally showed the highest noncovalent interactions at all levels of theory. Cooperativity of multiple interactions is analyzed on the basis of ρ( r ) at bcps which suggests that OH ··· π and NH ··· π interactions show positive, whereas CH ··· π and cation ··· π interactions exhibit negative cooperativity with respect to the side chain hydrogen bond interactions. In general, side chain interactions are strengthened as a result of aromatic interaction. Solvation has no significant effect on the overall geometry of the complex though slight weakening of noncovalent interactions by 1–2 kcal/mol is observed. An assessment of the four levels of theory studied herein suggests that both MPWB1K and M06‐2X give better performance for noncovalent interactions. The results also support the fact that B3LYP is inadequate for the study of weak interactions. © 2008 Wiley Periodicals, Inc. J Comput Chem 2009     

Optical coherence and theoretical study of the excitation dynamics of a highly symmetric cyclophane-linked oligophenylenevinylene dimer

Optoelectronic properties of a polyphenylenevinylene-based oligomer and its paracylophane-linked dimer are studied using a variety of experimental and theoretical techniques. Despite the symmetrical structure and redshifted absorption of the dimer versus the monomer, an exciton picture is not the most appropriate. Electronic structure calculations establish changes in charge density upon optical excitation and show localized excitations that cannot be accounted for by a simple Frenkel exciton model. Visible frequency pump-probe anisotropy measurements suggest that the dimer should be considered as a three-level system with a fast, approximately 130 fs, internal conversion from the higher to lower energy excited electronic state. Signatures of nuclear relaxation processes are compared for electric field-resolved transient grating and two-dimensional photon echo spectra. These measurements reveal that nuclear relaxation occurs on similar time scales for the monomer and dimer. The connection between the spectral phase of four-wave mixing signals and the time dependent width of a nuclear wave packet is discussed. Semiempirical electronic structure and metropolis Monte Carlo calculations show that the dominant line broadening mechanisms for the monomer and dimer are associated with inter-ring torsional coordinates. Together, the theoretical calculations and electric field-resolved four-wave mixing experiments suggest that while the structure of dimer is more rigid than that of monomer, the difference in their rigidities is not sufficient to slow down excited state relaxation of dimer with respect to the monomer.     

Improved curve fitting procedures to determine equilibrium binding constants

For ligand-biomacromolecule titration experiments it has been traditional practice to extract parameters such as the equilibrium binding constant K and the number of bases per ligand binding site n with relatively labour intensive methods, usually based on single wavelength data, such as the difference method by Rodger and Nordén coupled together with a Scatchard plot. Presented in this paper are both the theory and a least squares fitting method to derive parameters such as K and n more directly from all spectral non-linear experimental data. Both the case of non competitive binding of a metal complex ligand to DNA and the case of displacement by a metal complex ligand of an ethidium marker attached to the DNA are considered. This work may be applied directly to reduce experimental data produced by a spectropolarimeter (for circular or linear dichroism) or a spectrophotometer (for fluorescence or UV-Vis spectroscopy).     

Does cytotoxicity of metallointercalators correlate with cellular uptake or DNA affinity?

The cytotoxicity of the metallointercalators, [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1R,2R-diaminocyclohexane)](2+) ([56MERR]) and [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1S,2S-diaminocyclohexane)](2+) ([56MESS]), towards A549 human lung cancer cells was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value obtained following exposure of A549 cells to [56MESS] for 4 h was approximately three times smaller than that obtained when [56MERR] was administered under the same conditions, indicating that the former complex displayed greater cytotoxicity. Both IC(50) values were greater than that obtained after exposure of A549 cells to cisplatin, demonstrating that the latter compound was the most cytotoxic of the three examined. Microprobe synchrotron radiation X-ray fluorescence (SR-XRF) analyses of metallointercalator-treated A549 cells showed that platinum became localised in DNA-rich regions of the nucleus. In contrast, when the same cells were treated with cisplatin the metal became distributed throughout the cell. Determination of the maximum concentration of platinum present inside the cells using graphite furnace atomic absorption spectrophotometry (GFAAS) of platinum-treated cells suggested that there was greater uptake of [56MERR] compared to [56MESS] by the A549 cells, and that platinum uptake did not account for the greater toxicity of [56MESS], as assessed by the MTT assay. Electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic studies of solutions containing either [56MERR] or [56MESS], and a duplex hexadecamer molecule, showed the two metallointercalators displayed very similar affinity towards the nucleic acid. Overall these results indicate that the difference in cytotoxicity of the two platinum metallointercalators is probably the result of variations in their interactions with other cellular components.     

A new reagent for direct difluoromethylation

Molecular scaffolds containing alkylfluorine substituents are desired in many areas of chemical research from materials to pharmaceuticals. Herein, we report the invention of a new reagent (Zn(SO(2)CF(2)H)(2), DFMS) for the innate difluoromethylation of organic substrates via a radical process. This mild, operationally simple, chemoselective, and scalable difluoromethylation method is compatible with a range of nitrogen-containing heteroarene substrates of varying complexity as well as select classes of conjugated π-systems and thiols. Regiochemical comparisons suggest that the CF(2)H radical generated from the new reagent possesses nucleophilic character.     

Detection of colorectal metastases in patients being treated with chemotherapy utilising SPIO-MRI: a radiological–pathological study

Objective

Chemotherapy commonly causes liver injury through sinusoidal obstructive syndrome and steatosis. Chemotherapy-induced liver injury may make it more difficult to detect metastases secondary to reduced contrast between the injured liver and metastases. The aim of this study was to determine the sensitivity of superparamagnetic iron oxide (SPIO) contrast-enhanced imaging in patients who have undergone chemotherapy prior to liver surgery.

Methods

Local ethics committee approval was obtained. Thirty-one patients with hepatic metastases completing preoperative chemotherapy were prospectively recruited. Images were reviewed independently by two blinded observers who identified and localized lesions with a four-point confidence scale. The alternative free-response receiver operator characteristic method was used to analyze the results.

Results

The sensitivity in detecting colorectal metastases following chemotherapy was 78% and 76%, respectively, for observers 1 and 2 (95% confidence interval: 71%–85% and 68%–82%). The areas under the alternative free-response receiver operator curves were 0.73 and 0.80 for observers 1 and 2, respectively.

Conclusion

Compared to previously published work on chemotherapy-naïve patients, it is clear that the sensitivity of SPIO-enhanced magnetic resonance imaging (MRI) in detecting colorectal metastases following chemotherapy is reduced. It is therefore critical that all imaging — pre-, during and postchemotherapy — is reviewed when reporting liver MRI prior to surgery.     

Site-directed coordination chemistry with P22 virus-like particles

Protein cage nanoparticles (PCNs) are attractive platforms for developing functional nanomaterials using biomimetic approaches for functionalization and cargo encapsulation. Many strategies have been employed to direct the loading of molecular cargos inside a wide range of PCN architectures. Here we demonstrate the exploitation of a metal-ligand coordination bond with respect to the direct packing of guest molecules on the interior interface of a virus-like PCN derived from Salmonella typhimurium bacteriophage P22. The incorporation of these guest species was assessed using mass spectrometry, multiangle laser light scattering, and analytical ultracentrifugation. In addition to small-molecule encapsulation, this approach was also effective for the directed synthesis of a large macromolecular coordination polymer packed inside of the P22 capsid and initiated on the interior surface. A wide range of metals and ligands with different thermodynamic affinities and kinetic stabilities are potentially available for this approach, highlighting the potential for metal-ligand coordination chemistry to direct the site-specific incorporation of cargo molecules for a variety of applications.