In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering

Raman micro-spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast-like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non-tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non-tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non-tumour bone cells. A supervised classification model (LDA) was built and showed high cross-validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non-tumour cells, with 96% of the cells being correctly classified either as tumour or non-tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells.     

Triplex mediated delivery of a platinum complex to a specific DNA target site

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a site for complexation with K(2)PtCl(4). Due to the low reactivity of dT toward a platinum source, we chose dT(8) and dT(15) as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT(8) sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT(15) sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.     

The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity

Mutagens in urban air pollution come from anthropogenic sources (especially combustion sources) and are products of airborne chemical reactions. Bacterial mutation tests have been used for large, multi-site, and/or time series studies, for bioassay-directed fractionation studies, for identifying the presence of specific classes of mutagens, and for doing site- or source-comparisons for relative levels of airborne mutagens. Early research recognized that although carcinogenic PAHs were present in air samples they could not account for the majority of the mutagenic activity detected. The mutagenicity of airborne particulate organics is due to at least 500 identified compounds from varying chemical classes. Bioassay-directed fractionation studies for identifying toxicants are difficult to compare because they do not identify all of the mutagens present, and both the analytical and bioassay protocols vary from study to study. However, these studies show that the majority of mutagenicity is usually associated with moderately polar/highly polar classes of compounds that tend to contain nitroaromatic compounds, aromatic amines, and aromatic ketones. Smog chamber studies have shown that mutagenic aliphatic and aromatic nitrogen-containing compounds are produced in the atmosphere when organic compounds (even non-mutagenic compounds) are exposed to nitrogen oxides and sunlight. Reactions that occur in the atmosphere, therefore, can have a profound effect on the genotoxic burden of ambient air. This review illustrates that the mutagenesis protocol and tester strains should be selected based on the design and purpose of the study and that the correlation with animal cancer bioassay results depends upon chemical class. Future emphasis needs to be placed on volatile and semi-volatile genotoxicants, and on multi-national studies that identify, quantify, and apportion mutagenicity. Initial efforts at replacing the Salmonella assay for ambient air studies with some emerging technology should be initiated.     

Preparation and characterization of a 5'-deazaFAD T491V NADPH-cytochrome P450 reductase

NADPH-cytochrome P450 reductase is a flavoprotein which contains both an FAD and FMN cofactor. Since the distribution of electrons is governed solely by the redox potentials of the cofactors, there are nine different ways the electrons can be distributed and hence nine possible unique forms of the protein. More than one species of reductase will exist at a given level of oxidation except when the protein is either totally reduced or oxidized. In an attempt to unambiguously characterize the redox properties of the physiologically relevant FMNH(2) form of the reductase, the T491V mutant of NADPH-cytochrome P450 reductase has been reconstituted with 5'-deazaFAD which binds to the FAD-binding site of the reductase with a K(d) of 94 nM. The 5'-deazaFAD cofactor does not undergo oxidation or reduction under our experimental conditions. The molar ratio of FMN to 5'-deazaFAD in the reconstituted reductase was 1.1. Residual FAD accounted for less than 5% of the total flavins. Addition of 2 electron equivalents to the 5'-deazaFAD T491V reductase from dithionite generated a stoichiometric amount of the FMN hydroquinone form of the protein. The 5'-deazaFAD moiety remained oxidized under these conditions due to its low redox potential (-650 mV). The 2-electron-reduced 5'-deazaFAD reductase was capable of transferring only a single electron from its FMN domain to its redox partners, ferric cytochrome c and cytochrome b(5). Reduction of the cytochromes and oxidation of the reductase occurred simultaneously. The FMNH(2) in the 5'-deazaFAD reductase autoxidizes with a first-order rate constant of 0.007 s(-)(1). Availability of a stable NADPH-cytochrome P450 reductase capable of donating only a single electron to its redox partners provides a unique tool for investigating the electron-transfer properties of an intact reductase molecule.     

MHC class I gene conversion mutations alter the CD8 T cell repertoire

MHC class I molecules are highly polymorphic within populations. This diversity is thought to be the result of selective maintenance of new class I alleles formed by gene conversion. It has been proposed that rare alleles are maintained by their ability to confer resistance to common pathogens. Investigation has focused on differences in the presentation of foreign Ags by class I alleles, but the majority of peptides presented by class I molecules are self peptides used in shaping the naive T cell repertoire. We propose that the key substrate for the natural selection of class I gene conversion variants is the diversity in immune potential formed by new alleles. We show that T cells compete with each other for niches in the thymus and spleen during development, and that competition between different clones is dramatically affected by class I mutations. We also show that peripheral naive T cells proliferate preferentially in the presence of the class I variant that directed T cell development. The data argue that class I gene conversion mutations dramatically affect both the development and the maintenance of the naive CD8 T cell repertoire.     

Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry

Phthalates are widely used as industrial solvents and plasticizers, with global use exceeding four million tons per year. We improved our previously developed high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometric (HPLC-APCI-MS/MS) method to measure urinary phthalate metabolites by increasing the selectivity and the sensitivity by better resolving them from the solvent front, adding three more phthalate metabolites, monomethyl phthalate (mMP), mono-(2-ethyl-5-oxohexyl)phthalate (mEOHP) and mono-(2-ethyl-5-hydroxyhexyl)phthalate (mEHHP); increasing the sample throughput; and reducing the solvent usage. Furthermore, this improved method enabled us to analyze free un-conjugated mono-2-ethylhexyl phthalate (mEHP) by eliminating interferences derived from coelution of the glucuronide-bound, or conjugated form, of the mEHP on measurements of the free mEHP. This method for measuring phthalate metabolites in urine involves solid-phase extraction followed by reversed-phase HPLC-APCI-MS/MS using isotope dilution with (13)C(4) internal standards. We further evaluated the ruggedness and the reliability of the method by comparing measurements made by multiple analysts at different extraction settings on multiple instruments. We observed mMP, monoethyl phthalate (mEP), mono-n-butyl phthalate (mBP), monobenzyl phthalate (mBzP), mEHP, mEHHP and mEOHP in the majority of urine specimens analyzed with DEHP-metabolites mEHHP and mEOHP present in significantly higher amounts than mEHP.