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The sugar-phosphate backbone of DNA near single-stranded (ss)–double-stranded (ds) junctions likely fluctuates within a broad distribution of conformations to permit the proper binding of genome regulatory proteins that function at these sites. In this work we use absorbance, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS) to study the local conformations and conformational disorder within chromophore-labeled DNA constructs. These constructs employ dimers of the fluorescent chromophore Cy3 that are site-specifically incorporated into the sugar-phosphate backbones of DNA strands at ss–ds DNA fork junctions. We show that these data can be analyzed to determine the local conformations of the (Cy3)2 dimer, and the degree of conformational disorder. Our analysis employs an essential-state Holstein–Frenkel Hamiltonian model, which takes into account the internal electronic-vibrational motions within each Cy3 chromophore, and the resonant electronic interaction that couples the two chromophores together. Our results suggest that this approach may be applied generally to understand local backbone conformation and conformational disorder at ss–ds DNA fork junctions. [ABSTRACT FROM AUTHOR]

Gas-to-liquid mass transfer of hydrogen (H2) was investigated in a gas–liquid reactor with a continuous gas phase, a batch liquid phase, and liquid mixing regimes relevant to assessing kinetics of microbial H2 consumption. H2 transfer was quantified in real-time with a H2 microsensor for no mixing, moderate mixing [100 rotations per minute (rpm)], and rapid mixing (200 rpm). The experimental results were simulated by mathematical models to find best-fit values of volumetric mass transfer coefficients—kLa—for H2, which were 1.6/day for no mixing, 7/day for 100 rpm, and 30/day for 200 rpm. Microbiological H2-consumption experiments were conducted with Methanobacterium bryantii M.o.H. to assess effects of H2 mass transfer on microbiological H2-threshold studies. The results illustrate that slow mixing reduced the gas-to-liquid H2 transfer rate, which fell behind the rate of microbiological H2 consumption in the liquid phase. As a result, the liquid-phase H2 concentration remained much lower than the liquid-phase H2 concentration that would be in equilibrium with the gas-phase H2 concentration. Direct measurements of the liquid-phase H2 concentration by an in situ probe demonstrated the problems associated with slow H2 transfer in past H2 threshold studies. The findings indicate that some of the previously reported H2-thresholds most likely were over-estimates due to slow gas-to-liquid H2 transfer. Essential requirements to conduct microbiological H2 threshold experiments are to have vigorous mixing, large gas-to-liquid volume, large interfacial area, and low initial biomass concentration. [ABSTRACT FROM AUTHOR]

The morphology of nanodomain structures in binary polymer blends of a random copolymer and a homopolymer is determined using electronic excitation transport (EET) studies. The experimental system employed is a copolymer, 6.5% atactic poly(methyl methacrylate-co-2-vinyl naphthalene) [P(MMA-2VN)], in atactic poly(vinyl acetate) (PVAc). The naphthalene groups serve as chromophores in the EET experiments. The mixtures are prepared such that initially the P(MMA-2VN) chains are randomly distributed in the PVAc matrix. The nanodomains are formed while low-concentration mixtures of the P(MMA-2VN) in PVAc are held at constant temperature in the melt state (T>=Tg), above the temperature at which phase separation occurs. In the melt the chains diffuse, and P(MMA-2VN) chains aggregate until the temperature is quenched below Tg. The structures of the resulting domains are examined with time-resolved fluorescence depolarization measurements, and the data are analyzed using an analytical theory to model EET among interacting polymer chains. The agreement between theory and data is very good. The results of the analysis indicate that the nanodomains correspond to aggregates with a characteristic size equal to the radius of gyration of the copolymer, Rg. The number of P(MMA-2VN) chains in aggregates prepared under different conditions is determined. © 1995 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Monte Carlo (MC) simulations and an analytical theory are presented to describe electronic excitation transport (EET) among static chromophores constrained to lie on the surfaces of spherical micelles. Both donor–trap (DT) and donor–donor (DD) EET are examined for two types of systems: probe molecules on the surfaces of isolated (low concentration) micelles, and probes on the surfaces of interacting (concentrated) micelles. The EET dynamics are described by the function, 〈G s(t)〉, the probability of finding the excitation on the originally excited chromophore. For the isolated micelle calculations, the excitation dynamics depend on the distribution of probes on a single hard sphere surface. For the interacting micelle calculations, the hard sphere structure is accounted for by using the radial pair distribution function, [ATOTHER]@sg[/ATOTHER](r). Both single micelle and many micelle DT calculations do not involve approximations. Consequently, the DT expressions agree exactly with the MC calculations. For the DD calculations, a first order cumulant approximation is used to obtain analytically tractable solutions to 〈G s(t)〉. Padé approximants of the cumulant solution, accurate over a broad range of chromophore number and Förster interaction strengths, are used to describe DD EET on isolated micelles. For DD EET in many micelle systems, the first order cumulant approach is shown to be a suitable method for intermicelle structural studies. Both the cumulant and MC calculations are simultaneously compared to time resolved flourescence depolarization measurements performed on octadecylrhodamine B(ODRB)/triton X-100/water systems made in previous investigations. [ABSTRACT FROM AUTHOR]

Abstract: In situ bioreduction of soluble hexavalent uranium U(VI) to insoluble U(IV) (as UO2) has been proposed as a means of preventing U migration in the groundwater. This work focuses on the bioreduction of U(VI) and precipitation of U(IV). It uses anaerobic batch reactors with Desulfovibrio vulgaris, a well‐known sulfate, iron, and U(VI) reducer, growing on lactate as the electron donor, in the absence of sulfate, and with a 30‐mM bicarbonate buffering. In the absence of sulfate, D. vulgaris reduced  >90% of the total soluble U(VI) (1 mM) to form U(IV) solids that were characterized by X‐ray diffraction and confirmed to be nano‐crystalline uraninite with crystallite size 2.8 ± 0.2 nm. pH values between 6 and 10 had minimal impact on bacterial growth and end‐product distribution, supporting that the mono‐nuclear, and poly‐nuclear forms of U(VI) were equally bioavailable as electron acceptors. Electron balances support that H2 transiently accumulated, but was ultimately oxidized via U(VI) respiration. Thus, D. vulgaris utilized H2 as the electron carrier to drive respiration of U(VI). Rapid lactate utilization and biomass growth occurred only when U(VI) respiration began to draw down the sink of H2 and relieve thermodynamic inhibition of fermentation. [ABSTRACT FROM AUTHOR]

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (~1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions. [ABSTRACT FROM AUTHOR]

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR. [ABSTRACT FROM AUTHOR]