Effect of the cyclobutane cytidine dimer on the properties of Escherichia coli DNA photolyase

Cyclobutane pyrimidine dimer (CPD) photolyases are structure specific DNA-repair enzymes that specialize in the repair of CPDs, the major photoproducts that are formed upon irradiation of DNA with ultraviolet light. The purified enzyme binds a flavin adenine dinucleotide (FAD), which is in the neutral radical semiquinone (FADH(*)) form. The CPDs are repaired by a light-driven, electron transfer from the anionic hydroquinone (FADH(-)) singlet excited state to the CPD, which is followed by reductive cleavage of the cyclobutane ring and subsequent monomerization of the pyrimidine bases. CPDs formed between two adjacent thymidine bases (T< >T) are repaired with greater efficiency than those formed between two adjacent cytidine bases (C< >C). In this paper, we investigate the changes in Escherichia coli photolyase that are induced upon binding to DNA containing C< >C lesions using resonance Raman, UV-vis absorption, and transient absorption spectroscopies, spectroelectrochemistry, and computational chemistry. The binding of photolyase to a C< >C lesion modifies the energy levels of FADH(*), the rate of charge recombination between FADH(-) and Trp(306)(*), and protein-FADH(*) interactions differently than binding to a T< >T lesion. However, the reduction potential of the FADH(-)/FADH(*) couple is modified in the same way with both substrates. Our calculations show that the permanent electric dipole moment of C< >C is stronger (12.1 D) and oriented differently than that of T< >T (8.7 D). The possible role of the electric dipole moment of the CPD in modifying the physicochemical properties of photolyase as well as in affecting CPD repair will be discussed.     

Development of saw palmetto (Serenoa repens) fruit and extract standard reference materials

As part of a collaboration with the National Institutes of Health’s Office of Dietary Supplements and the Food and Drug Administration’s Center for Drug Evaluation and Research, the National Institute of Standards and Technology has developed two standard reference materials (SRMs) representing different forms of saw palmetto (Serenoa repens), SRM 3250 Serenoa repens fruit and SRM 3251 Serenoa repens extract. Both of these SRMs have been characterized for their fatty acid and phytosterol content. The fatty acid concentration values are based on results from gas chromatography with flame ionization detection (GC-FID) and mass spectrometry (GC/MS) analysis while the sterol concentration values are based on results from GC-FID and liquid chromatography with mass spectrometry analysis. In addition, SRM 3250 has been characterized for lead content, and SRM 3251 has been characterized for the content of β-carotene and tocopherols. SRM 3250 (fruit) has certified concentration values for three phytosterols, 14 fatty acids as triglycerides, and lead along with reference concentration values for four fatty acids as triglycerides and 16 free fatty acids. SRM 3251 (extract) has certified concentration values for three phytosterols, 17 fatty acids as triglycerides, β-carotene, and γ-tocopherol along with reference concentration values for three fatty acids as triglycerides, 17 fatty acids as free fatty acids, β-carotene isomers, and δ-tocopherol and information values for two phytosterols. These SRMs will complement other reference materials currently available with concentrations for similar analytes and are part of a series of SRMs being developed for dietary supplements. Contribution of the US Government; not subject to copyright     

Chemical sensing and imaging with metallic nanorods

In this Feature Article, we examine recent advances in chemical analyte detection and optical imaging applications using gold and silver nanoparticles, with a primary focus on our own work. Noble metal nanoparticles have exciting physical and chemical properties that are entirely different from the bulk. For chemical sensing and imaging, the optical properties of metallic nanoparticles provide a wide range of opportunities, all of which ultimately arise from the collective oscillations of conduction band electrons ("plasmons") in response to external electromagnetic radiation. Nanorods have multiple plasmon bands compared to nanospheres. We identify four optical sensing and imaging modalities for metallic nanoparticles: (1) aggregation-dependent shifts in plasmon frequency; (2) local refractive index-dependent shifts in plasmon frequency; (3) inelastic (surface-enhanced Raman) light scattering; and (4) elastic (Rayleigh) light scattering. The surface chemistry of the nanoparticles must be tunable to create chemical specificity, and is a key requirement for successful sensing and imaging platforms.     

Multifunctional mixed SAMs that promote both cell adhesion and noncovalent DNA immobilization

The ability of DNA strands to influence cellular gene expression directly and to bind with high affinity and specificity to other biological molecules (e.g., proteins and target DNA strands) makes them a potentially attractive component of cell culture substrates. On the basis of the potential importance of immobilized DNA in cell culture and the well-defined characteristics of alkanethiol self-assembled monolayers (SAMs), the current study was designed to create multifunctional SAMs upon which cell adhesion and DNA immobilization can be independently modulated. The approach immobilizes the fibronectin-derived cell adhesion ligand Arg-Gly-Asp-Ser-Pro (RGDSP) using carbodiimide activation chemistry and immobilizes DNA strands on the same surface via cDNA-DNA interactions. The surface density of hexanethiol-terminated DNA strands on alkanethiol monolayers (30.2-69.2 pmol/cm2) was controlled using a backfill method, and specific target DNA binding on cDNA-containing SAMs was regulated by varying the soluble target DNA concentration and buffer characteristics. The fibronectin-derived cell adhesion ligand GGRGDSP was covalently linked to carboxylate groups on DNA-containing SAM substrates, and peptide density was proportional to the amount of carboxylate present during SAM preparation. C166-GFP endothelial cells attached and spread on mixed SAM substrates and cell adhesion and spreading were specifically mediated by the immobilized GGRGDSP peptide. The ability to control the characteristics of noncovalent DNA immobilization and cell adhesion on a cell culture substrate suggests that these mixed SAMs could be a useful platform for studying the interaction between cells and DNA.     

A GUM-compliant uncertainty budget for the analysis of total carbon dioxide (TCO<Subscript>2</Subscript>) in equine plasma

Samples of plasma from chosen horses are collected prior to racing for the analysis of total carbon dioxide (TCO₂). Five or six (dependent on jurisdiction) tubes of blood are taken as required for a screen, followed (if positive) by confirmation (two tubes in quadruplicate) and, further confirmation (two tubes in quadruplicate) at a second referee laboratory. The threshold limit prescribed by the rules of racing is an amount concentration of 36.0 mM, with further action ensuing if the reported concentration is greater than 37.2 mM. Using QC data and an analysis of the measurement procedure, the combined standard measurement uncertainty of a measurement result was estimated to be 0.22 mM. This leads to a conclusion that the odds of incorrectly deciding that a sample at the threshold limit exceeded the action concentration, are 13 000 000:1 against. The combined standard measurement uncertainty could be reduced to 0.20 mM by combining results from the first and referee laboratories. An allowance for uncertainty of measurement of 1.0 mM is proposed, which leads to odds of 185 000:1 against concluding a sample is over the threshold limit when, in fact, it is not.     

Development of glycyl radical parameters for the OPLS-AA/L force field

On the basis of quantum chemical calculations C(alpha)-glycyl radical parameters have been developed for the OPLS-AA/L force field. The molecular mechanics hypersurface was fitted to the calculated quantum chemical surface by minimizing their molecular mechanics parameter dependent sum-of-squares deviations. To do this, a computer program in which the molecular mechanics energy derivatives with respect to the parameters were calculated analytically was developed, implementing the general method of Lifson and Warshel (J Chem Phys 1968, 49, 5116) for force field parameter optimization. This program, in principle, can determine the optimal parameter set in one calculation if enough representative value points on the quantum chemical potential energy surface are available and there is no linear dependency between the parameters. Some of the parameters in quantum calculations, including several new torsion types around a bond as well as angle parameters at a new central atom type, are not completely separable. Consequently, some restrictions and/or presumptions were necessary during parameter optimization. The relative OPLS-AA energies reproduced those calculated quantum chemically almost perfectly.     

X‐ray reflectivity from curved liquid interfaces

X‐ray reflectivity studies of the structure of liquid–vapour and liquid–liquid interfaces at modern sources, such as free‐electron lasers, are currently impeded by the lack of dedicated liquid surface diffractometers. It is shown that this obstacle can be overcome by an alternative experimental approach that uses the natural curvature of a liquid drop for variation of the angle of incidence. Two modes of operation are shown: (i) sequential reflectivity measurements by a nanometre beam and (ii) parallel acquisition of large ranges of a reflectivity curve by micrometre beams. The feasibility of the two methods is demonstrated by studies of the Hg/vapour, H₂O/vapour and Hg/0.1 M NaF interface. The obtained reflectivity curves match the data obtained by conventional techniques up to 5α_c in micro‐beam mode and up to 35α_c in nano‐beam mode, allowing observation of the Hg layering peak.     

On the Fiber Stretch in Shearing Deformations of Fibrous Soft Materials

The behavior of the fiber stretch in simple shear of soft materials fiber-reinforced with a single family of oriented parallel fibers is examined. The analysis is purely kinematical and the results are valid for both compressible and incompressible materials. It is shown that for a given amount of shear, for all fiber orientation angles in the range \(0 < \theta < \pi /4\), the fiber stretch increases with increasing \(\theta\) whereas in the range \(\pi /4 < \theta < \pi /2\), this is no longer the case and there is a particular fiber orientation for which the fiber stretch is a maximum. For a particular amount of shear corresponding to a special angle of shear (a “magic” angle of \(35.26^{\circ}\)), the fiber-orientation angle at which the fiber stretch is a maximum is its geometric complement namely a magic angle of \(54.74^{\circ}\). The results are also valid for torsion of a circular cylinder reinforced with a single family of helically wound fibers.