Development of a systems analysis approach for resolving the structure of biodegrading soil systems

An experimental and mathematical method is developed for the microbial systems analysis of polyaromatic hydrocarbon (PAH)-degrading mixed cultures in PAH-contaminated “town gas” soil systems. Frequency response is the primary experimental and data analysis tool used to probe the structure of these complicated systems. The objective is to provide a fundamental protocol for evaluating the performance of specific mixed microbial cultures on specific soil systems by elucidating the salient system variables and their interactions. Two well-described reactor systems, a constant volume stirred tank reactor (CSTR) and a plug flow differential volume reactor, are used in order to remove performance effects that are related to reactor type as opposed to system structure. These two reactor systems are well-defined systems that can be described mathematically and represent the two extremes of one potentially important system variable, macroscopic mass transfer. The experimental and mathematical structure of the protocol is described, experimental data is presented, and data analysis is demonstrated for the stripping, sorption, and biodegradation of napththalene.     

POTENTIATION OF MEROCYANINE 540-MEDIATED PHOTODYNAMIC THERAPY BY SALICYLATE and RELATED DRUGS

Abstract— Simultaneous exposure to merocyanine 540 (MC540) and light of a suitable wavelength kills leukemia, lymphoma and neuroblastoma cells but is relatively well tolerated by normal pluripotent hematopoietic stem cells. This differential phototoxic effect has been exploited in preclinical models and a phase I clinical trial for the extracorporeal purging of autologous bone marrow grafts. Salicylate is known to potentiate the MC540-mediated photokilling of tumor cells. Assuming that salicylate induces a change in the plasma membrane of tumor cells (but not normal hematopoietic stem cells) that enhances the binding of dye molecules it has been suggested that salicylate may provide a simple and effective means of improving the therapeutic index of MC540-mediated photodynamic therapy. We report here on a direct test of this hypothesis in a murine model of bone marrow transplantation as well as in clonal cultures of normal murine hematopoietic progenitor cells. In both systems, salicylate enhanced the MC540-sensitized photoinactivation of leukemia cells and normal bone marrow cells to a similar extent and thus failed to improve the therapeutic index of MC540 significantly. On the basis of a series of dye-binding studies, we offer an alternative explanation for the potentiating effect of salicylate. Rather than invoking a salicylate-induced change in the plasma membrane of tumor cells, we propose that salicylate displaces dye molecules from serum albumin, thereby enhancing the concentration of free (active) dye available for binding to tumor as well as normal hematopoietic stem cells.     

Oil-in-water emulsions stabilized by highly charged polyelectrolyte-grafted silica nanoparticles

Fully sulfonated poly(styrenesulfonate) brushes were grown from the surface of colloidal silica particles and used to prepare stable trichloroethylene-in-water and heptane-in-water Pickering emulsions. These particles were highly charged and colloidally stable in water but could not be dispersed in trichloroethylene or heptane. Both two-phase (emulsion plus neat water) and three-phase (emulsion separating neat oil and water phases) systems were observed, with water-continuous emulsion phases in all cases. Emulsion phases containing as much as 83% (v/v) oil were stable for over six months. Poly(styrenesulfonate)-grafted particles were very efficient emulsifiers; stable emulsion phases were prepared when using as little as 0.04 wt% particles. The emulsifying effectiveness of the poly(styrenesulfonate)-grafted silica particles can be attributed to the hydrophobicity of the vinylic polymer backbone that makes this highly charged polyelectrolyte unusually surface active at the oil/water interface.     

Conformational dependence of the intrinsic acidity of the aspartic acid residue sidechain in N-acetyl- -aspartic acid-N′-methylamide

The sidechain conformational potential energy hypersurfaces (PEHS) for the γ_L, β_L, α_L, and α_D backbone conformations of N-acetyl- -aspartate-N′-methylamide were generated. Of the 81 possible conformers initially expected for the aspartate residue, only seven were found after geometric optimizations at the B3LYP/6-31G(d) level of theory. No stable conformers could be located in the δ_L, _L, γ_D, δ_D, and _D backbone conformations. The ‘adiabatic’ deprotonation energies for the endo and exo forms of N-acetyl- -aspartic acid-N′-methylamide were calculated by comparing their optimized relative energies against those found for the seven stable conformers of N-acetyl- -aspartate-N′-methylamide. Sidechain conformational PEHSs were also generated for the estimation of ‘vertical’ deprotonation energies for both endo and exo forms of N-acetyl- -aspartic acid-N′-methylamide. All backbone–sidechain (N–H⁻O–C) and backbone–backbone (N–HO=C) hydrogen bond interactions were analyzed. A total of two backbone–backbone and four backbone–sidechain interactions were found for N-acetyl- -aspartate-N′-methylamide. The deprotonated sidechain of N-acetyl- -aspartate-N′-methylamide may allow the aspartyl residue to form strong hydrogen bond interactions (since it is negatively charged) which may be significant in such processes as protein–ligand recognition and ligand binding. As a primary example, the molecular geometry of the aspartyl residue may be important in peptide folding, such as that in the RGD tripeptide.     

A critical comparison of approximation methods and models for equilibrium properties of low-barrier hydrogen bonds

Recent experimental evidence has led to the conclusion that short, strong hydrogen bonds can stabilize transition states of enzyme catalyzed biochemical reactions. Evidence for such hydrogen bonds is the low value of the isotopic fractionation factor, phi, which is defined as the equilibrium constant for the generic reaction, R-H + DOH <--> R-D + HOH, where H is the hydrogen atom participating in the low-barrier hydrogen bond in a molecule R-H. In this work we assess two approximation methods for computing the isotopic fractionation factors for single and multidimensional systems containing a low-barrier hydrogen bond. These methods are WKB and an approach that corrects the classical partition function via a quantum correction factor. We find that the latter approach is universally accurate and applicable in both single and multidimensional systems containing a low-barrier hydrogen bond. We also assess two different models for the coupling of a molecule's low-barrier hydrogen bond to other degrees of freedom, both internal and external to the molecule, and show that each leads to a lowering of the fractionation factor.     

Unrestricted perfect pairing: the simplest wave-function-based model chemistry beyond mean field

The perfect pairing (PP) approximation from generalized valence bond theory is formulated in an unrestricted fashion for both closed- and open-shell systems using a coupled cluster ansatz. In the model chemistry proposed here, active electron pairs are correlated, but the unpaired or radical electrons remain uncorrelated, leading to a linear number of decoupled cluster amplitudes which can be solved for analytically. The alpha and beta spatial orbitals are variationally optimized independently. This minimal treatment of electron-electron correlation noticeably improves upon symmetry-breaking problems and other pathologies in Hartree-Fock (HF) theory and may be computed using the resolution of the identity approximation at only a factor of several times more effort than HF itself. PP also generally predicts improved molecular structures over HF. This compact, correlated wave function potentially provides a useful starting point for dynamical correlation corrections.     

Mediation of ultrafast light-harvesting by a central dimer in phycoerythrin 545 studied by transient absorption and global analysis

We report ultrafast femtosecond transient absorption measurements of energy-transfer dynamics for the antenna protein phycoerythrin 545, PE545, isolated from a unicellular cryptophyte Rhodomonas CS24. The phycoerythrobilins are excited at both 485 and 530 nm, and the spectral response is probed between 400 and 700 nm. Room-temperature measurements are contrasted with measurements at 77 K. An evolution-associated difference spectra (EADS) analysis is combined with estimations of bilin spectral positions and energy-transfer rates to obtain a detailed kinetic model for PE545. It is found that sub pulse-width dynamics include relaxation between the exciton states of a chromophore dimer (beta 50/60) located in the core of the protein. Energy transfer from the lowest exciton state of the phycoerythrobilin (PEB) dimer to one of the periphery 15,16-dihydrobiliverdin (DBV) bilins is found to occur on a time scale of 250 fs at room temperature and 960 fs at 77 K. A host of energy-transfer dynamics involving the beta 158, beta 82, and alpha 19 bilins occur on a time scale of 2 ps at room temperature and 3 ps at 77 K. A final energy transfer occurs between the red-most DBV bilins with a time scale estimated to be approximately 30 ps. The role of the centrally located phycoerythrobilin dimer is seen as crucial-spectrally as it expands the cross-section of absorption of the protein; structurally as it sits in the middle of the protein acting as an intermediary trap; and kinetically, as the internal conversion and subsequent red-shift of the excitation is extremely fast.     

A multiresidue pesticide monitoring procedure using gas chromatography/mass spectrometry and selected ion monitoring for the determination of pesticides containing nitrogen, sulfur, and/or oxygen in fruits and vegetables

A procedure for the analysis of over 100 pesticides that only contain combinations of carbon, hydrogen, nitrogen, sulfur, and oxygen (i.e., no phosphorous or halogen atoms) has been developed. The procedure employs gas chromatography with a mass selective detector (GC/MSD), electron impact ionization, and selected ion monitoring. This GC/MSD procedure provided lower limits of quantitation and increased confirmational data compared to the traditional element-selective GC procedures that are commonly used for the detection of this "class" of pesticides. These analytical improvements were demonstrated by 26 pesticides that were detected at ng/g levels in a variety of fruit and vegetable matrixes using this procedure; these pesticides were missed by the traditional element-selective GC procedures. Validation of the procedure was performed using acetone extraction with solid-phase extraction cleanup. Twenty representative target pesticides were used to demonstrate repeatability and linearity of the chromatographic response and recovery from fruit and vegetable matrixes.     

Photochemical attachment of organic monolayers onto H-terminated Si(111): radical chain propagation observed via STM studies

Photochemical reactions of terminal alkenes with hydrogen-terminated silicon surfaces are being used by many groups to produce covalently attached organic monolayers with a wide range of terminal functionalities. Despite the considerable activity in this area, the mechanism for these reactions has not been definitively established. Here we present STM and HREELS data on a sequence of partially reacted samples, showing the progress of the reaction. The attachment reaction is found to proceed via formation of irregularly shaped islands that appear to grow by a pseudorandom walk process. These data support a radical chain propagation mechanism previously suggested for this reaction. However, since the photons employed here (447 nm) lack sufficient energy for Si-H bond cleavage, an alternate mechanism for initiating the chain reaction appears to be required.     

Suicide inactivation of dioldehydratase by glycolaldehyde and chloroacetaldehyde: an examination of the reaction mechanism

High-level ab initio calculations have been used to study the mechanism for the inactivation of diol dehydratase (DDH) by glycolaldehyde or 2-chloroacetaldehyde. As in the case of the catalytic substrates of DDH, e.g., ethane-1,2-diol, the 5'-deoxyadenosyl radical (Ado*) is able to abstract a hydrogen atom from both substrate analogues in the initial step on the reaction pathway, as evidenced by comparable energy barriers. However, in subsequent step(s), each substrate analogue produces the highly stable glycolaldehyde radical. The barrier for hydrogen atom reabstraction by the glycolaldehyde radical is calculated to be too high ( approximately 110 kJ mol-1) to allow Ado* to be regenerated and recombine with the cob(II)alamin radical, the latter therefore remaining tightly bound to DDH. As a consequence, the catalytic pathway is disrupted, and DDH becomes an impotent enzyme. Interconversion of equivalent structures of the glycolaldehyde radical via the symmetrical cis-ethanesemidione radical is calculated to require 38 kJ mol-1. EPR indications of a symmetrical cis-ethanesemidione structure are likely to be the result of formation of an equilibrium mixture of glycolaldehyde radical structures, this equilibration being facilitated by partial deprotonation of the glycolaldehyde radical by the carboxylate of an amino acid residue within the active site of DDH.