Michelle V. Buchanan

The hydrogen economy

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Frog Embryo Teratogenesis Assay: Xenopus (FETAX) — A Short-Term Assay Applicable to Complex Environmental Mixtures

There is no question that a rapid and inexpensive screening tool is needed to assess potential teratogenicity. The now classical Ames test (Ames, 1975) and other tests screen for potential mutagenicity and carcinogenicity. Although a number of relatively rapid bioassays, ranging from the invertebrates through lower vertebrates to mammals, as well as cell, organ, and embryo culture systems, have been used to examine teratogenicity, none has been adequately validated or widely accepted for routine use. The tier-testing approach, which uses rapid screens to detect potential hazard and indicate need for further testing, has been useful and expedient. Comparable tests, however, are not yet applicable for teratogenesis testing despite the fact that there are a number of methods available. Among these is one which we submit may be a valid model for preliminary assessment of potential teratogens. This model, referred to as FETAX (Frog Embryo Teratogenesis Assay: Xenopus), has been applied to examine the relative teratogenic risk of a variety of chemicals and complex mixtures. The mixtures we have tested come from coal-conversion and shale-oil technologies and their effects have been compared to those of similar materials derived from natural petroleums.

Chemical composition of fingerprints for gender determination.

This work investigates the chemical nature of fingerprints to ascertain whether differences in chemical composition or the existence of chemical markers can be used to determine personal traits, such as age, gender, and personal habits. This type of information could be useful for reducing the pool of potential suspects in criminal investigations when latent fingerprints are unsuitable for comparison by traditional methods. Fingertip residue that has been deposited onto a bead was extracted with a solvent such as chloroform. Samples were analyzed by gas chromatography/mass spectrometry (GC/MS). The chemical components identified include fatty acids, long chain fatty acid esters, cholesterol and squalene. The area ratios of ten selected components relative to squalene were calculated for a small preliminary experiment that showed a slight gender difference for three of these components. However, when the experiment was repeated with a larger, statistically designed experiment no significant differences between genders were detected for any of the component ratios. The multivariate Hotellings T2 test that tested all ten-component ratios simultaneously also showed no gender differences at the 5% significance level.

Structural Characterization of Normal and Modified Oligonucleotides by Matrix-assisted Laser Desorption Fourier Transform Mass Spectrometry.

Matrix-assisted UV laser desorption Fourier transform mass spectrometry (266 nm, nicotinic acid matrix) can be used for the detailed structural characterization of normal and modified oligonucleotides. The negative ion spectra for these compounds revealed abundant (M − H) - ions as well as fragment ions that provided the information necessary to determine oligomer sequence and to differentiate isomers. The nicotinic acid matrix was required for the production of (M − H) - ions for the oligonucleotide dimers, trimers, tetramers, and hexamers examined in this study. Elimination of the nicotinic acid matrix resulted in complete loss of the (M −H) - ions as well as most of the larger fragment ions for the oligomers. The primary fragmentation pathway was observed to be phosphate ester bond cleavage with the resulting charge retained on the 3’ end of the oligomer and enabled isomeric differentiation of compounds such as d(S’-CGCG-3’) and d(S’-CCGG-3’). Collisioninduced dissociation experiments of the (M − H) - ions for these compounds confirmed the preferential loss of nucleotides from the 5’ end of the oligomers. The presence and location of modifications such as methyl and ethyl alkyl groups to the oligonucleotides could also be identified.

Applications of mass spectrometry to DNA sequencing

The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.

Investigation of UV matrix-assisted laser desorption fourier transform mass spectrometry for peptides

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Ultraviolet matrix-assisted laser desorption can be used to enhance formation of [M + H]+, [M + Na]+, and [M + K)+ ions from small peptides for Fourier transform mass spectrometry (FTMS). In accord with laser desorption (LD) time-of-flight experiments, matrices such as nicotinic acid and 2-pyrazinecarboxylic acid exhibit strong enhancement effects (i.e., formation of abundant protonated and cationized molecules for the analyte with virtually no fragment ions) for 266 nm LD/FTMS, whereas pyrazinedicarboxylic acid provides no matrix enhancement at this wavelength. Both sinapinic acid and coumarin-120 provide strong matrix enhancement effects for the 355-nm LD of peptides. For the small peptides examined in this study, no significant differences in the abundance of fragment ions were observed between the 266- and 355-nm wavelengths. Matrix-assisted LD/FTMS is useful for the generation and characterization of ions corresponding to protonated and cationized molecules from virtually all biological compounds with molecular weights up to 2000. The lack of observation of biological ions with m/ z > 2500 may be related to inefficient trapping of these laser-desorbed ions or instrumental detection limitations of FTMS and is under further investigation.

The Bio-SANS instrument at the High Flux Isotope Reactor of Oak Ridge National Laboratory

Small-angle neutron scattering (SANS) is a powerful tool for characterizing complex disordered materials, including biological materials. The Bio-SANS instrument of the High Flux Isotope Reactor of Oak Ridge National Laboratory (ORNL) is a high-flux low-background SANS instrument that is, uniquely among SANS instruments, dedicated to serving the needs of the structural biology and biomaterials communities as an open-access user facility. Here, the technical specifications and performance of the Bio-SANS are presented. Sample environments developed to address the needs of the user program of the instrument are also presented. Further, the isotopic labeling and sample preparation capabilities available in the Bio-Deuteration Laboratory for users of the Bio-SANS and other neutron scattering instruments at ORNL are described. Finally, a brief survey of research performed using the Bio-SANS is presented, which demonstrates the breadth of the research that the instruments user community engages in.

Methyl guanine isomer distinction by hydrogen / deuterium exchange using a fourier transform mass spectrometer.

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Differentiation of the seven isomers of methyl guanine has been accomplished by monitoring gas-phase hydrogen/deuterium (H/D) exchange reactions of the protonated molecular ions with deuterium oxide (D2O) in a Fourier transform mass spectrometer. In each case a distinctive reaction rate for the first H/D exchange was observed, and exchanges of up to three deuterium atoms occurred with characteristic ion abundances that could be used to differentiate the isomers. O6-Methyl guanine, for example, showed only one slow H/D exchange with D2O, whereas l-methyl guanine exchanged two hydrogen atoms at a significantly faster rate. On comparison of the possible resonance structures of each protonated isomer with the experimental information about the number and rate of H/D exchanges observed, a reaction mechanism involving a concerted proton abstraction-deuterium cation donation was proposed.

Embryotoxic and teratogenic effects of aqueous extracts of tar from a coal gasification electrostatic precipitator

Aqueous extracts of tar from a coal gasification electrostatic precipitator were tested for its toxic and teratogenic potential in vitro on embryos of the amphibian Xenopus laevis. The 96-h LC50 and EC50 were determined to be 0.83% and 0.48%, respectively. The developmental stage of normal-appearing exposed embryos is not affected by increasing concentrations of the extract. Embryo growth, however, is significantly reduced at concentrations as low as 0.25%. Motility and pigmentation were effectively reduced relative to controls by extract concentrations of 0.5% and greater. Exposed embryos are shorter and stockier than controls. Malformations of head, eyes, viscera, and spine are common, and cartilage formation is abnormal. The epidermis is often hyperplastic, and large blisters occur over the somatic surface. The severity of abnormal development is directly related to the concentration of the toxicant to which the embryos are exposed. Chemical analysis shows that the aqueous extracts contain phenols, furans, monoaromatic and diaromatic hydrocarbons, and mono- and diazaarenes and/or monoaromatic amines.

Chemical characterization of fingerprints from adults and children

The observation that the fingerprints of children disappear from surfaces more quickly than those of adults initiated a study to characterize the chemical components in fingerprints. Samples were obtained from about 50 individuals ranging in age from three to 64 by extracting chemicals from the fintertips using rubbing alcohol. Using combined gas chromatography/mass spectrometry, a wide range of compounds were identified. It was found that the chemical compositions of fingerprints were quite different in children and adults. In general, the samples obtained from children contained higher levels of relatively volatile free fatty acids. Samples from adults were found to have higher concentrations of less volatile long chain esters of fatty acids. These esters are thought to originate from sebaceous glands located on the face and the levels of these compounds increase substantially after puberty. In addition to these compounds, a variety of other compounds were observed that could be used to develop improved methods for fingerprint detection at a crime scene. Further, the observation of specific compounds raises the possibility of being able to identify personal traits (gender, habits, diseases, etc.) via the analysis of components in fingerprints and/or skin.