Abstract: Cold acclimation induces an adaptative increase in respiration in brown adipose tissue (BAT). A comparative analysis by two-dimensional differential in-gel electrophoresis of mitochondrial protein patterns found in rat control and cold-acclimated BAT was performed. A total of 58 proteins exhibiting significant differences in their abundance was unambiguously identified. Proteins implicated in the major catabolic pathways were up-regulated as were ATP synthase and mitofilin. Moreover, these results support the fact that adipocytes can balance their ATP synthesis and their heat production linked to UCP1-sustained uncoupling.

Abstract: A promising approach for anticancer strategies is the stabilization of telomeric DNA into a G-quadruplex structure. To explore the intrinsic stabilization of folded G-quadruplexes, we combined electrospray ionization mass spectrometry, ion mobility spectrometry, and molecular modeling studies to study different DNA sequences known to form quadruplexes. Two telomeric DNA sequences of different lengths and two DNA sequences derived from the NHE III1 region of the c-myc oncogene (Pu22 and Pu27) were studied. NH4+ and the ligands PIPER, TMPyP4, and the three quinacridines MMQ1, MMQ3, and BOQ1 were complexed with the DNA sequences to determine their effect on the stability of the G-quadruplexes. Our results demonstrate that G-quadruplex intramolecular folds are stabilized by NH4+ cations and the ligands listed. Furthermore, the ligands can be classified according to their ability to stabilize the quadruplexes and end stacking is shown to be the dominant mode for ligand attachment. In all cases our solvent-free experimental observations and theoretical modeling reveal structures that are highly relevant to the solution-phase structures.

Abstract: The primary structure of 3'-imino[60]fulleryl-3'-deoxythymidine ions is studied using mass spectrometry both in the positive and negative modes. Interaction between the subunits is discussed using collision-induced dissociation (CID) spectra. Collisional activation with argon of the sodiated cations leads to the cleavage of the glycosidic bond and the transfer of a radical hydrogen from the deoxyribose to the thymine. The sodiated thymine is the only fragment observed for low collision energies in the positive mode. In the negative mode, two different ionization mechanisms take place, reduction and deprotonation in the presence of triethylamine. The 2.7 eV electron affinity of C60 and its huge cross section compared to the small cross section and predicted 0.44 eV electron affinity of the thymidine subunit most likely localize the radical electron on the fullerene. On the other hand, deprotonation of the 3'-azido-3'-deoxythymidine (AZT) is known to occur in N-3, the most acidic site of the nucleobase. Consequently, deprotonation causes the negative charge to be initially localized on the thymine. Both types of parent anions give the radical anion C60*- as fragment. The other fragments detected are the dehydrogenated 3'-imino[60]fulleryl-3'-deoxyribose anion, C60NH2-, C60N- and C60H-. Since in negative ion mass spectrometry all fragments include the [60]fullerene unit, this suggests that the fragmentation is driven by the electron affinity of the [60]fullerene, likely responsible for a charge transfer between the deprotonated thymine and the C60.

Abstract: We are confronted daily to unknown microorganisms that have yet to be characterized, detected, and/or analyzed. We propose, in this study, a multidimensional strategy using polyclonal antibodies, consisting of a novel proteomic tool, the ProteomeLab PF2D, coupled to immunological techniques and mass spectrometry (i-PF2D-MS/MS). To evaluate this strategy, we have applied it to Bacillus subtilis, considered here as our unknown bacterial model.

Abstract: Except for fish, no toxicokinetic data on polybrominated diphenyl ethers (PBDEs) is available on relevant animals for the human food chain. In the present work, absorption, elimination through eggs and disposition of PBDEs in laying chickens were studied and compared to dioxin behaviour. Hens were fed with diet containing 3.4 mg/kg feed of PBDEs and 0.95 ng TEQ/kg feed of polychlorodibenzo-p-dioxins (PCDDs) and polychlorodibenzofurans (PCDFs). PBDEs have been demonstrated to show drastically different behaviours from PCDD/Fs and dioxin-like compounds. Excretion of PBDEs increased for two weeks and then decreased to nearly 0%. Sixty-two percent of ingested 2,2',4,4'-tetraBDE (BDE-47) were found in excreta after two weeks, suggesting a reductive debromination of PBDEs in the digestive tract. PBDE level in eggs increased during five weeks and reached 24 microg/g fat. After then, levels decreased to 3 microg/g fat at the end of the trial. PBDE bioconcentration factors estimated for abdominal fat varied from 0.7 for BDE-47 to 2 for BDE-183.

Abstract: We evaluated the impact of two iron and steel plants and two municipal solid waste incinerators (MSWI) in Wallonia (Belgium) on the exposure of residents to dioxins, polychlorinated biphenyls (PCBs), and heavy metals. In total, 142 volunteers living around these facilities were recruited and compared with 63 referents from a rural area with no industrial source of pollution. Information about smoking habits, dietary habits, anthropometric characteristics, residential history, and health status was obtained from a self-administered questionnaire. The volunteers provided blood under fasting conditions in order to evaluate the body burden of dioxins (17 polychlorinated dibenzo-p-dioxins/dibenzofurans [PCDD/Fs] congeners) and PCBs. Samples of blood and urine were also taken for the determination of cadmium, mercury, and lead. After adjustment for covariates, concentrations of cadmium, mercury, and lead in urine or blood were not increased in subjects living in the vicinity of MSWIs or sinter plants by comparison with referents. Residents around the sinter plants and the MSWI located in the industrial area had concentrations of dioxins and PCBs in serum similar to that of referents. By contrast, subjects living in the vicinity of the MSWI in the rural area showed significantly higher serum levels of dioxins (geometric mean, 38 vs. 24 pg TEQ/g fat) and coplanar PCBs (geometric mean, 10.8 vs. 7.0 pg TEQ/g fat). Although age-adjusted dioxin levels in referents did not vary with local animal fat consumption, concentrations of dioxins in subjects living around the incinerators correlated positively with their intake of local animal fat, with almost a doubling in subjects with the highest fat intake. These results indicate that dioxins and coplanar PCBs emitted by MSWIs can indeed accumulate in the body of residents who regularly consume animal products of local origin.

Abstract: Several important cofactors are adenine nucleotides with a vitamin as the catalytic moiety. Here, we report the discovery of the first adenine nucleotide containing vitamin B1: adenosine thiamine triphosphate (AThTP, 1), or thiaminylated ATP. We discovered AThTP in Escherichia coli and found that it accumulates specifically in response to carbon starvation, thereby acting as a signal rather than a cofactor. We detected smaller amounts in yeast and in plant and animal tissues.

Abstract: In this paper, we report the analysis of seven benzopyridoindole and benzopyridoquinoxaline drugs binding to different duplex DNA and triple helical DNA, using an approach combining electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and molecular modeling. The ligands were ranked according to the collision energy (CE(50)) necessary to dissociate 50% of the complex with the duplex or the triplex in tandem MS. To determine the probable ligand binding site and binding mode, molecular modeling was used to calculate relative ligand binding energies in different binding sites and binding modes. For duplex DNA binding, the ligand-DNA interaction energies are roughly correlated with the experimental CE(50), with the two benzopyridoindole ligands more tightly bound than the benzopyridoquinoxaline ligands. There is, however, no marked AT versus GC base preference in binding, as supported both by the ESI-MS and the calculated ligand binding energies. Product ion spectra of the complexes with triplex DNA show only loss of neutral ligand for the benzopyridoquinoxalines, and loss of the third strand for the benzopyridoindoles, the ligand remaining on the duplex part. This indicates a higher binding energy of the benzopyridoindoles, and also shows that the ligands interact with the triplex via the duplex. The ranking of the ligand interaction energies compared with the CE(50) values obtained by MS/MS on the complexes with the triplex clearly indicates that the ligands intercalate via the minor groove of the Watson-Crick duplex. Regarding triplex versus duplex selectivity, our experiments have demonstrated that the most selective drugs for triplex share the same heteroaromatic core.

Abstract: A method to perform absolute quantification of two biomarkers (IGF-1 and IGFBP-3) of growth hormone abuse has been developed. Isotope dilution is used with synthetically labelled peptides as internal standards. Peptide selection and multiple reaction monitoring design are discussed. A simple sample preparation based on the reduction and alkylation of cysteine residues followed by tryptic digestion provides a sufficient digestion of proteins. Serum samples fortified with increasing amounts of target proteins are analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a triple quadrupole mass spectrometer. Specificity is ensured by the selection of sequences with no homology in BLAST, as well as retention time deviation check, and ion ratio monitoring. Linearity is studied in terms of calibration curves. These curves for IGFBP-3 and IGF-1 are generated with mean slopes of 0.055 and 0.065, intercepts of 0.107 and -0.011, and with coefficients of correlation of 0.95 and 0.98, respectively. These curves result from the addition of proteins to the serum. Risks of variations related to potential matrix effects are therefore reduced, as well as probable variations related to the digestion steps. The working concentration ranges are 4-10 ng/microl for IGFBP-3 and 2-8 ng/microl for IGF-1. Preliminary data regarding repeatability show that relative standard deviations (RSDs) range between 13 and 32% for IGFBP-3 and between 7 and 29% for IGF-1.

Abstract: We report on the development and validation under ISO 17025 criteria of a multi-residue confirmatory method to identify and quantify 17 widely chemically different pesticides (insecticides: Carbofuran, Methiocarb, Pirimicarb, Dimethoate, Fipronil, Imidacloprid; herbicides: Amidosulfuron, Rimsulfuron, Atrazine, Simazine, Chloroturon, Linuron, Isoxaflutole, Metosulam; fungicides: Diethofencarb) and 2 metabolites (Methiocarb sulfoxide and 2-Hydroxytertbutylazine) in honey. This method is based on an on-column liquid-liquid extraction (OCLLE) using diatomaceous earth as inert solid support and liquid chromatography (LC) coupled to mass spectrometry (MS) operating in tandem mode (MS/MS). Method specificity is ensured by checking retention time and theoretical ratio between two transitions from a single precursor ion. Linearity is demonstrated all along the range of concentration that was investigated, from 0.1 to 20 ng g(-1) raw honey, with correlation coefficients ranging from 0.921 to 0.999, depending on chemicals. Recovery rates obtained on home-made quality control samples are between 71 and 90%, well above the range defined by the EC/657/2002 document, but in the range we had fixed to ensure proper quantification, as levels found in real samples could not be corrected for recovery rates. Reproducibility is found to be between 8 and 27%. Calculated CCalpha and CCbeta (0.0002-0.943 ng g(-1) for CCalpha, and 0.0002-1.232 ng g(-1) for CCbeta) show the good sensitivity attained by this multi-residue analytical method. The robustness of the method has been tested in analyzing more than 100 raw honey samples collected from different areas in Belgium, as well as some wax and bee samples, with a slightly adapted procedure.

Abstract: Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite state-of-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.

Abstract: DNA multiply charged anions stored in a quadrupole ion trap undergo one-photon electron ejection (oxidation) when subjected to laser irradiation at 260 nm (4.77 eV). Electron photodetachment is likely a fast process, given that photodetachment is able to compete with internal conversion or radiative relaxation to the ground state. The DNA [6-mer]3- ions studied here show a marked sequence dependence of electron photodetachment yield. Remarkably, the photodetachment yield (dG6 > dA6 > dC6 > dT6) is inversely correlated with the base ionization potentials (G < A < C < T). Sequences with guanine runs show increased photodetachment yield as the number of guanine increases, in line with the fact that positive holes are the most stable in guanine runs. This correlation between photodetachment yield and the stability of the base radical may be explained by tunneling of the electron through the repulsive Coulomb barrier. Theoretical calculations on dinucleotide monophosphates show that the HOMO and HOMO-1 orbitals are localized on the bases. The wavelength dependence of electron detachment yield was studied for dG63-. Maximum electron photodetachment is observed in the wavelength range corresponding to base absorption (260-270 nm). This demonstrates the feasibility of gas-phase UV spectroscopy on large DNA anions. The calculations and the wavelength dependence suggest that the electron photodetachment is initiated at the bases and not at the phosphates. This also indicates that, although direct photodetachment could also occur, autodetachment from excited states, presumably corresponding to base excitation, is the dominant process at 260 nm. Excited-state dynamics of large DNA strands still remains largely unexplored, and photo-oxidation studies on trapped DNA multiply charged anions can help in bridging the gap between gas-phase studies on isolated bases or base pairs and solution-phase studies on full DNA strands.

Abstract: Uncoupling protein 1 (UCP1) is a mitochondrial inner membrane protein that dissipates the proton electrochemical gradient built up by the respiratory chain. Its activity is stimulated by free fatty acids and inhibited by purine nucleotides. Here we investigated how active and regulated recombinant UCP1 expressed in yeast at approximately 1 and approximately 10 microg/mg of total mitochondrial proteins induced changes in the mitochondrial proteome and in oxygen free radical production. Using two-dimensional differential in-gel electrophoresis (2D-DIGE), we found that most of the proteins involved in the response to ectopically expressed UCP1 are related to energy metabolism. We also quantified the cellular H(2)O(2) release in the absence or in the presence of UCP1. Our results suggest that UCP1 has a dual influence on free radical generation. On one side, FFA-activated UCP1 was able to decrease the superoxide anion production, demonstrating that a decrease in the generation of reactive oxygen species is an obligatory outcome of UCP1 activity even in a heterologous context. On the other side, an increase in UCP1 content was concomitant with an increase in the basal release of superoxide anion by mitochondria as a side consequence of the overall increase in oxidative metabolism.

Abstract: Since foodstuffs from animal origin and particularly poultry products have been pointed out several times as reservoir of dioxins and related compounds, notably in Belgium few years ago, food chain safety issues appeared. Although food chain contamination incidents occurred many times through contaminated feedstuffs consumption in commercial chicken farms, very few studies have been carried out on transfer of dioxins and related compounds from commercial feed to hens. The present work continues a preliminary study on dioxin transfer in laying chickens carried out in our lab and available on-line on November 2004 in Environment International. In this work, absorption of dioxins were not preferential for 2,3,7,8-substituted congeners, increase with increasing number of chlorines, and was not linearly dependent of the octanol/water partition. Only 2,3,7,8-congeners were found in all organs studied, and these latter showed the same congener profile and similar lipid-normalized concentration, except for the liver. Abdominal fat and liver seemed to be the major storage sites and the liver preferentially retained highly chlorinated congeners. Unfortunately in this previous trial, laying process stopped very early for unknown reason leading to a considerable lost of information. In the present toxicokinetic study, more complete gastrointestinal absorption, excretion in eggs and bioaccumulation of dioxins in different tissues were investigated in chickens fed for 14 weeks with a 9 ng TEQ/kg contaminated feed. Stable levels were reached after 7 weeks in excreta and 9 weeks in eggs. During the whole trial, gastrointestinal absorption ranged between 41% and 91% depending on the congener. At steady state conditions, excretion of 1,2,3,4,6,7,8-HpCDD, OCDD, 1,2,3,4,6,7,8-HpCDF and OCDF exceeded 100% demonstrating excretion from tissues of these congeners which were also the most abundant in feed. 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 2,3,4,7,8-PeCDF, 1,2,3,6,7,8-HxCDF, 1,2,3,7,8,9-HxCDF and 2,3,4,6,7,8-HxCDF seemed to be metabolized more efficiently. Lipid adjusted concentrations and pattern were unexpectedly similar in the abdominal fat and the liver. On the contrary, eggs and breast muscles showed different pattern with higher level for high chlorinated congeners. When extrapolating our results, we found that a feed containing 0.750 ng TEQ/kg of dioxins (European norm for feedstuff) would cause a level lower than the maximum threshold level of 3 pg TEQ/g fat for chicken eggs fixed by European Communities. Nevertheless, a concentration lower than 0.6 ng TEQ/kg in feed would be needed to produce breast muscles less contaminated than 2 pg TEQ/g fat authorized in European.

Abstract: The energy-dissipating alternative oxidase (AOX) from Hansenula anomala was expressed in Saccharomyces cerevisiae. The recombinant AOX was functional. A comparative analysis by two-dimensional differential in-gel electrophoresis (2D-DIGE) of mitochondrial protein patterns found in wild-type and recombinant AOX strains was performed. 60 proteins exhibiting a significant difference in their abundance were identified. Interestingly, proteins implicated in major metabolic pathways such as Krebs cycle and amino acid biosynthesis were up-regulated. Surprisingly, an up-regulation of the respiratory-chain complex III was associated with a down-regulation of the ATP synthase complex.

Abstract: Chemical ecology is the study of how particular chemicals are involved in interactions of organisms with each other and with their surroundings. In order to reduce insect attack, plants have evolved a variety of defence mechanisms, both constitutive and inducible, while insects have evolved strategies to overcome these plant defences (such as detoxification enzymes). A major determinant of the influence of evolutionary arms races is the strategy of the insect: generalist insect herbivores, such as Myzus persicae aphid, need more complex adaptive mechanisms since they need to respond to a large array of different plant defensive chemicals. Here we studied the chemical ecology of M. persicae associated with different plant species, from Brassicaceae and Solanaceae families. To identify the involved adaptation systems to cope with the plant secondary substances and to assess the differential expression of these systems, a proteomic approach was developed. A non-restrictive approach was developed to identify all the potential adaptation systems toward the secondary metabolites from host plants. The complex protein mixtures were separated by two-dimensional electrophoresis methods and the related spots of proteins significantly varying were selected and identified by mass spectrometry (ESI MS/MS) coupled with data bank investigations. Fourteen aphid proteins were found to vary according to host plant switch; ten of them were down regulated (proteins involved in glycolysis, TCA cycle, protein and lipid synthesis) while four others were overexpressed (mainly related to the cytoskeleton). These techniques are very reliable to describe the proteome from organisms such as insects in response to particular environmental change such as host plant species of herbivores.

Abstract: We hereby explore the effects of irradiating DNA polyanions stored in a quadrupole ion trap mass spectrometer with an optical parametric oscillator laser between 250 and 285 nm. We studied DNA 6-20-mer single strands and 12-base pair double strands. In all cases, laser irradiation causes electron detachment from the multiply charged DNA anions. Electron photodetachment efficiency directly depends on the number of guanines in the strand, and maximum efficiency is observed between 260 and 275 nm. Subsequent collision-induced dissociation (CID) of the radical anions produced by electron photodetachment results in extensive fragmentation. In addition to neutral losses, a large number of fragments from the w, d, a*, and z* ion series are obtained, contrasting with the w and (a-base) ion series observed in regular CID. The major advantage of this technique, coined electron photodetachment dissociation (EPD) is the absence of internal fragments, combined with good sequence coverage. EPD is therefore a highly promising approach for de novo sequencing of oligonucleotides. EPD of nucleic acids is also expected to give specific radical-induced strand cleavages, with conservation of other fragile bonds, including noncovalent bonds. In effect, preliminary results on a DNA hairpin and on double strands suggest that EPD could also be used to probe intra- and intermolecular interactions in nucleic acids.

Abstract: We report on the use of a state-of-the-art method for the measurement of selected polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls in human serum specimens. The sample preparation procedure is based on manual small size solid-phase extraction (SPE) followed by automated clean-up and fractionation using multi-sorbent liquid chromatography columns. SPE cartridges and all clean-up columns are disposable. Samples are processed in batches of 20 units, including one blank control (BC) sample and one quality control (QC) sample. The analytical measurement is performed using gas chromatography coupled to isotope dilution high-resolution mass spectrometry. The sample throughput corresponds to one series of 20 samples per day, from sample reception to data quality cross-check and reporting, once the procedure has been started and series of samples keep being produced. Four analysts are required to ensure proper performances of the procedure. The entire procedure has been validated under International Organization for Standardization (ISO) 17025 criteria and further tested over more than 1500 unknown samples during various epidemiological studies. The method is further discussed in terms of reproducibility, efficiency and long-term stability regarding the 35 target analytes. Data related to quality control and limit of quantification (LOQ) calculations are also presented and discussed.

Abstract: We propose a multi-dimensional strategy, associating immunodetection to a protein fractionating two-dimensional liquid chromatography tool, for serological characterization of microbial antigens. The originality of such immunoproteomic approaches resides in their application in large-scale studies for rapid serotyping of micro-organisms, evaluation of immunomes and could be proposed in the development and monitoring of vaccines.

Abstract: The first part of the present study focused on the development of an alternative automated sample preparation method for the measurement of selected polybrominated diphenyl ethers (PBDEs) in fish and shellfish. A previously developed automated method has been further optimized and simplified to decrease blank levels and cost. Sorbent and solvent quantities have been significantly reduced without altering extract quality. The second part of the study consisted of comparing tandem-in-time quadrupole ion storage mass spectrometry (QISTMS/MS) to high resolution mass spectrometry (HRMS) for the isotope dilution (ID) measurement step after gas chromatography (GC) separation. Both mass spectrometric methods performed similarly in terms of accuracy but better precision was observed for HRMS. Although better sensitivity can be attained with the high resolution sector instrument, method limits of quantification (mLOQs) were very similar for both approaches as they were dependent on the procedural blanks levels. The mLOQ values ranged between 0.04 and 3.56 ng/g fat, depending on the congener. They allowed the unambiguous identification and quantification of all target analytes, except for BDE-183, in most considered fish extracts. An analytical procedure based on rapid automated sample preparation and QISTMS/MS appeared to be suitable for the measurement of PBDEs in fish and shellfish speciment under quality assurance/quality control (QA/QC) criteria.

Abstract: BACKGROUND: Consistent average length differences between species and chromosome arm differences within species indicate that telomere length is genetically determined. This seems to contradict an observed large variation in lengths of the same human telomere between metaphases of the same individual. We examined the extent to which the variation in the telomeres of the human X and Y chromosomes is heritable, induced, or technical in origin. METHODS: Metaphase chromosomes were stained by fluorescence in situ hybridization with a telomere repeat-specific probe, and fluorescence intensities of the X and Y chromosomes were measured. If telomere length variation is predominantly genetically determined and a 50% probability of meiotic recombination between the pseudo-autosomal regions of Yp and Xp in the father is taken into account, one expects an equal chance that the Yp telomere of a son is derived from his father's Xp or Yp telomere. This implies that the Yp/Yq telomere ratios in fathers and sons will be identical in the absence of paternal meiotic recombination and different when recombination occurs. RESULTS: Among five father-son pairs, four showed similar Yp/Yq ratios (P > 0.05), whereas one pair exhibited a large difference in the Yp/Yq ratio that was attributable to a significantly longer Xp than Yp telomere in the father and a presumptive meiotic exchange between X and Y during paternal meiosis. Further, the Xq telomere exhibited a generally shorter telomere length than the others. CONCLUSIONS: The high variation in telomere length appeared to be intracellular (between sister chromatids) and, hence, technical in nature. We found no measurable induced variation in the cells studied, implying that, if induced variation exists, it is small compared with the technical variation.

Abstract: Twelve chickens raised according to commercial standards were fed a diet containing about 30 ng total toxic equivalent (TEQ)/kg for 10 weeks. Persistent pollutants were introduced into the poultry feed via recycled oil to mimic contamination conditions closely resembling those occurring during the Belgian crisis five years ago. Absorption of congeners with the same chlorination degree did not seem to depend on the substitution, demonstrating that unlike for cows, no preferential absorption for 2,3,7,8-substituted compounds could be remarked for chickens. As already observed, absorption decreased with increasing number of chlorines and was not linearly dependent on the octanol/water partition coefficient. On the other hand, no real differences were observed in the absorption of coplanar polychlorinated biphenyls (c-PCBs) with regard to degree of chlorination. When monitored during the course of experiment, concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and c-PCBs in excreta reached an apparent steady state after 5 weeks. Only 2,3,7,8-substituted dioxins or furans were found in tissues and eggs. All organs showed the same congener profile and similar lipid-normalized concentration, except for the liver. Bioconcentration factors were evaluated, highlighting that the liver preferentially retained highly chlorinated congeners. No depletion of dioxin and PCB concentration was observed after 8 and 14 weeks of control diet, but high inter-individual variation occurs.

Abstract: The majority of DNA-binding small molecules known thus far stabilize duplex DNA against heat denaturation. A high, drug-induced increase in the melting temperature (Tm) of DNA is generally viewed as a good criterion to select DNA ligands and is a common feature of several anticancer drugs such as intercalators (e.g., anthracyclines) and alkylators (e.g., ecteinascidin 743). The reverse situation (destabilization of DNA to facilitate its denaturation) may be an attractive option for the identification of therapeutic agents acting on the DNA structure. We have identified the tumor-active benzoacronycine derivative S23906-1 [(+/-)-cis-1,2-diacetoxy-6-methoxy-3,3,14-trimethyl-1,2,3,14-tetrahydro-7H-benzo[b]pyrano[3,2]acridin-7-one] as a potent DNA alkylating agent endowed with a helicase-like activity. Using complementary molecular approaches, we show that covalent binding to DNA of the diacetate compound S23906-1 and its monoacetate analogue S28687-1 induces a marked destabilization of the double helix with the formation of alkylated ssDNA. The DNA-bonding properties and effects on DNA structure of a series of benzoacronycine derivatives, including the dicarbamate analogue S29385-1, were studied using complementary biochemical (electromobility shift assay, nuclease S1 mapping) and spectroscopic (fluorescence and Tm measurements) approaches. Alkylation of guanines in DNA by S28687-1 leads to a local denaturation of DNA, which becomes susceptible to cleavage by nuclease S1 and significantly decreases the Tm of DNA. The drug also directly alkylates single-strand DNA, but mass spectrometry experiments indicate that guanines in duplexes are largely preferred over single-stranded structures. This molecular study expands the repertoire of DNA-binding mechanisms and provides a new dimension for DNA recognition by small molecules.

Abstract: AIMS: To evaluate the contribution of tobacco smoking to dioxin accumulation. METHODS: Dioxin (17 PCDD/F) concentrations in fasting blood from 251 subjects (161 never smokers, 54 past smokers, and 36 current smokers) were quantified. RESULTS: Whereas serum dioxin concentrations of male smokers were on average 40% higher than those of non-smokers, in women, smoking was associated with significantly lower serum dioxin levels. A synergistic potentiation of dioxin metabolism by tobacco smoke in women is postulated to explain these paradoxical findings. CONCLUSIONS: Current smoking is associated with gender dependent effects on dioxin body burden and is a potential source of confounding in human studies using blood dioxins as indicators of exposure.

Abstract: Among the different analytical tools proposed as an alternative to the very expensive gas chromatography high-resolution mass spectrometry (GC-HRMS) analyses of polychlorodibenzo-p-dioxin and polychlorodibenzofurans, Chemically Activated LUciferase gene eXpression (CALUX) in vitro cell bioassay is very promising. It allows the analyses of a high number of samples since it is relatively fast, inexpensive, and sensitive. However, this technique is not yet widely applied for screening or environmental monitoring. The main reasons are probably the lack of validation and the difficulty in interpreting the global biological response of the bioassay. In this paper, the strict quality control criteria set up for the validation of CALUX are described. The validation has shown good repeatability (relative standard deviation (RSD) = 9%) and good within-lab reproducibility (RSD = 15%) of the results. The quantification limit, in the conditions applied in this paper, is 1.25 pg CALUX-TEQ/g fat. Comparison of CALUX and GC-HRMS analysis was made forvarious marine matrixes (fishes, mussels, starfishes, sea birds, and marine mammals). Good correlations are usually observed, but there are systematic differences between the results. Attempts are made to identify the origin of the discrepancy between the two methods.

Abstract: The gas-phase hydrogen/deuterium (H/D) exchange kinetics of DNA G-quadruplexes has been investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The quadruplex [(TGGGGT)4 . 3NH4+] undergoes very fast H/D exchange, in both the positive and in the negative ion modes, compared to DNA duplexes and other quadruplexes tested, and compared to the corresponding single-stranded TGGGGT. Substitution of NH4+ for K+ did not alter this fast H/D exchange, indicating that the hydrogens of the ammonium ions are not those exchanged. However, stripping of the interior cations of the quadruplex by source collision-induced dissociation (CID) in the positive ion mode showed that the presence of the inner cations is essential for the fast exchange to be possible. Molecular dynamics simulations show that the G-quadruplex is very rigid in the gas phase with NH4+ ions inside the tetrads. We suggest that the fast H/D exchange is favored by this rigid quadruplex conformation. This example illustrates that the concept that compact DNA structures exchange H for D slower than unfolded ones is a misconception.

Abstract: Ascididemin and Meridine are two marine compounds with pyridoacridine skeletons known to exhibit interesting antitumour activities. These molecules have been reported to behave like DNA intercalators. In this study, dialysis competition assay and mass spectrometry experiments were used to determine the affinity of ascididemin and meridine for DNA structures among duplexes, triplexes, quadruplexes and single-strands. Our data confirm that ascididemin and meridine interact with DNA but also recognize triplex and quadruplex structures. These molecules exhibit a significant preference for quadruplexes over duplexes or single-strands. Meridine is a stronger quadruplex ligand and therefore a stronger telomerase inhibitor than ascididemin (IC50=11 and >80 muM, respectively in a standard TRAP assay).

Abstract: The complete understanding of the morphological differentiation of streptomycetes is an ambitious challenge as diverse sensors and pathways sensitive to various environmental stimuli control the process. Germination occupies a particular position in the life cycle as the good achievement of the process depends on events occurring both during the preceding sporulation and during germination per se. The cyclic AMP receptor protein (crp) null mutant of Streptomyces coelicolor, affected in both sporulation and germination, was therefore presented as a privileged candidate to highlight new proteins involved in the shift from dormant to germinating spores. Our multidisciplinary approach-combining in vivo data, the analysis of spores morphological properties, and a proteome study-has shown that Crp is a central regulatory protein of the life cycle in S. coelicolor; and has identified spores proteins with statistically significant increased or decreased expression that should be listed as priority targets for further investigations on proteins that trigger both ends of the life cycle.

Abstract: Steatosis encompasses the accumulation of droplets of fats into hepatocytes. In this work, we performed a comparative analysis of mitochondrial protein patterns found in wild-type and steatosis-affected liver using the novel technique two-dimensional differential in-gel electrophoresis (2D-DIGE). A total of 56 proteins exhibiting significant difference in their abundances were unambiguously identified. Interestingly, major proteins that regulate generation and consumption of the acetyl-CoA pool were dramatically changed during steatosis. Many proteins involved in the response to oxidative stress were also affected.

Abstract: A comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC x GC-TOF-MS) experimental setup was tested for the measurement of seven 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs), ten 2,3,7,8-substituted polychlorinated dibenzofurans (PCDFs), four non-ortho-polychlorinated biphenyls (PCBs), eight mono-ortho-PCBs, and six indicator PCBs (Aroclor 1260) in foodstuff samples. A 40m RTX-500 (0.18mm I.D., 0.10 microm df) was used as the first dimension (1D) and a 1.5 m BPX-50 (0.10mm I.D., 0.10 microm df) as the second dimension (2D). The GC x GC chromatographic separation was completed in 45 min. Quantification was performed using 13C-label isotope dilution (ID). Isotope ratios of the selected quantification ions were checked against theoretical values prior to peak assignment and quantification. The dynamic working range spanned three orders of magnitude. The lowest detectable amount of 2,3,7,8-TCDD was 0.2 pg. Fish, pork, and milk samples were considered. On a congener basis, the GC x GC-ID-TOF-MS method was compared to the reference GC-ID high resolution mass spectrometry (HRMS) method and to the alternative GC-ID tandem-in-time quadrupole ion storage mass spectrometry (QIST-MS/MS). PCB levels ranged from low picogram (pg) to low nanogram (ng) per gram of sample and data compared very well between the different methods. For all matrices, PCDD/Fs were at a low pg level (0.05-3 pg) on a fresh weight basis. Although congener profiles were accurately described, RSDs of GC x GC-ID-TOF-MS and GC-QIST-MS/MS were much higher than for GC-ID-HRMS, especially for low level pork and milk. On a toxic equivalent (TEQ) basis, all methods, including the dioxin-responsive chemically activated luciferase gene expression (DR-CALUX) assay, produced similar responses. A cost comparison is also presented.

Abstract: The impact of a recently closed old municipal solid waste incinerator (MSWI) on polychlorodibenzo-p-dioxin (PCDD), polychlorodibenzofuran (PCDF), and polychlorinated biphenyl (PCB) levels in the surrounding environment and resident serum has been studied in a small rural area of France. Studied soils and eggs from chickens foraging on these soils were sampled in the vicinity of the MSWI underthe prevailing wind stream, while comparison samples were collected outside the assumed impact zone. PCB levels observed in soils and eggs did not differ statistically from comparison sites. This confirmed the low impact of MSWI PCB emission on environmental media, compared to other well-known sources. PCDD/PCDF levels in soils and eggs were significantly higher than in comparison samples, pointing out the impact of MSWI emission on the surrounding environment. The high dioxin concentrations in eggs set aside for private consumption would increase the dioxin intake for the studied population. Blood specimens of 10 nonoccupationally exposed volunteers who had lived within a 2 km radius of the incinerator for at least 25 years have been analyzed. When adjusted for age, PCB and PCDD/F blood levels were higher than general European populations and comparable to a similarly exposed Belgian population.

Abstract: The sintering process is among the major sources of the very toxic polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in the environment. At the industrial scale, it has been shown that dust collected on the grid, which supports the feed, contains PCDD/Fs amounts between the values found in the bottom of the cake and the values found on dust collected during gas sampling in the wind boxes. This fact suggests that the grid, containing 25wt.% of chromium, could have a catalytic activity in PCDD/Fs formation during the sintering process. This research tries to study this potential role. The de novo synthesis of PCDD/Fs is simulated at laboratory scale by thermal treatments of samples mixed with grid filings or Cr2O3. The thermal experiments performed with E.S.P. dust (dust collected in the electrostatic precipitator of a sintering plant) or graphite mixed with grid filings do not allow to confirm a role of the grid in PCDD/Fs formation during the industrial process. On the other hand, it has been shown that Cr2O3 can be considered as a catalyst in the de novo synthesis of PCDD/Fs. This compound takes place in the two steps of the de novo synthesis: the degradation of the carbon matrix as well as the chlorination reactions.

Abstract: This review addresses the determination of the internal energy of ions produced by electrospray ionization (ESI) sources, and the influence of the internal energy on analyte fragmentation. A control of the analyte internal energy is crucial for several applications of electrospray mass spectrometry, like structural studies, construction of reproducible and exportable spectral libraries, analysis of non-covalent complexes. Sections II and III summarize the Electrospray mechanisms and source design considerations which are relevant to the problem of internal energy, and Section IV gives an overview of the inter-relationships between ion internal energy, reaction time scale, and analyte fragmentation. In these three sections we tried to make the most important theoretical elements understandable by all ESI users, and their understanding requires a minimal background in physical chemistry. We then present the different approaches used to experimentally determine the ion internal energy, as well as various attempts in modeling the internal energy uptake in electrospray sources. Finally, a tentative comparison between electrospray and other ionization sources is made. As the reader will see, although many reports appeared on the subject, the knowledge in the field of internal energy of ions produced by soft ionization sources is still scarce, because of the complexity of the system, and this is what makes this area of research so interesting. The last section presents some perspectives for future research.

Abstract: Past years, many efforts have been dedicated to the development of alternative analytical methods for the measurement of dioxins in various types of matrices. Polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) are compounds that are present in samples at part-per-billion (ppb) or part-per-trillion (ppt) level. Their measurement requires the use of very sensitive analytical methods. Gas chromatography (GC) coupled to quadrupole ion storage mass spectrometry (QISTMS), fast GC (FGC) coupled to time-of-flight mass spectrometry (TOFMS) and comprehensive two-dimensional gas chromatography (GC x GC) coupled to TOFMS are the more promising tools challenging the reference GC high resolution mass spectrometry (HRMS) based on sector instruments. We report herein some of the advances we achieved in the past years in our laboratory on the development of alternative measurement methods for those compounds.

Abstract: Congener-specific analyses of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs) were performed on twenty-eight non-pooled fast food samples collected in Belgium, Switzerland, Czech Republic, United States of America and Australia. PCDD/F and PCB concentrations for the four investigated types of meals were very low. PCDD/F values ranged from non-detected to 1.40 pg WHO-TEQ/g fat and from 0.79 to 2.08 pg WHO-TEQ/g fat for lower and upper bound, respectively. Major contributors to the PCDD/F TEQ were 1,2,3,4,7,8-HxCDD, 1,2,3,6,7,8-HxCDD, 2,3,7,8-TCDF and 2,3,4,7,8-PeCDF. The relative contribution of PCBs to the total TEQ was 68%. For adults, an average estimated intake was 6.7 pg WHO-TEQ/kg bw/month, including consumption of all types of analyzed meals, representing 9.5% of the PTMI. For child, a value of 14.5 pg WHO-TEQ/kg bw/month was obtained, representing 20.6% of the PTMI.

Abstract: The sintering process is among the major sources of PCDD/Fs in the environment. This research studies the influence of the raw materials in this type of industrial plant on the amounts of PCDD/Fs generated. Particular interest is given to coke, which constitutes the principal source of carbon for the de novo synthesis of PCDD/Fs, and to the dust collected in the electrostatic precipitator (E.S.P. dust), usually recycled in the raw materials. The de novo synthesis of PCDD/Fs is simulated at the laboratory scale by thermal treatments of the samples. The use of a particular coke as a fuel does not drastically reduce the formation of PCDD/Fs. Actually, the global amounts of PCDD/Fs generated from the graphite and the two cokes tested are very similar. Only modifications in the fingerprint are observed. On the other hand, the addition of 10 wt % dust collected in the electrostatic precipitator leads to the formation of amounts of PCDD/Fs multiplied by a factor larger than 10(3). These results imply caution against the recycling of this E.S.P. dust in the raw materials.

Abstract: Congener-specific analyses of 7 polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 4 non-ortho (coplanar) polychlorinated biphenyls (cPCBs) were performed on 35 samples of commercial long-life pasteurised cows' milk issued from eight different brands available in Walloon supermarkets (Belgium). The observed congener profile was characteristic of milk samples issued from industrialised countries with good inter and intra-brand reproducibility's. The PCDDs to PCDFs ratio was equal to 1.8 in concentration. The toxic equivalent (TEQ based on WHO-TEF) value for PCDD/Fs in all analysed milks was 1.09+/-0.30 pg TEQ/g fat (range 0.86-1.59), which is below the recommended EU non-commercialisation threshold value of 3 pg TEQ PCDD/Fs/g of milk fat. The mean TEQ value including cPCBs was 2.23+/-0.55 pg TEQ/g fat. These PCBs actually contributed for 49+/-8.6% of the total TEQ. Among PCDD/Fs and cPCBs, tetrachloro dibenzo-p-dioxin (TCDD), pentachloro dibenzo-p-dioxin (PeCDD), pentachloro dibenzofurans (PeCDFs) and 3,3',4,4',5-pentachloro biphenyl (PCB-126) were the most important contributors to the TEQ. Estimated daily intake (EDI) due to consumption of such milks was 0.34 pg TEQ/kg of body weight/day for PCDD/Fs and 0.69 pg TEQ/kg of body weight/day when cPCBs were included.

Abstract: Cryptolepine, the main alkaloid present in the roots of Cryptolepis sanguinolenta, presents a large spectrum of biological properties. It has been reported to behave like a DNA intercalator with a preference for GC-rich sequences. In this study, dialysis competition assay and mass spectrometry experiments were used to determine the affinity of cryptolepine and neocryptolepine for DNA structures among duplexes, triplexes, quadruplexes and single strands. Our data confirm that cryptolepine and neocryptolepine prefer GC over AT-rich duplex sequences, but also recognize triplex and quadruplex structures. These compounds are weak telomerase inhibitors and exhibit a significant preference for triplexes over quadruplexes or duplexes.

Abstract: Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell-depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.

Abstract: Dioxins and polychlorinated biphenyls (PCBs) are persistent organic pollutants widely distributed in the food chain, which is the main source of human exposure. Their effects on human health at background exposure levels are still poorly understood. Recent epidemiological evidence suggests a possible association between these pollutants and diabetes. We report here the results of a population-based study in Belgium on 257 (142 women and 115 men) environmentally exposed subjects, including 10 cases of endometriosis and nine cases of diabetes. Seventeen 2,3,7,8-polychlorinated dibenzodioxins/dibenzofurans (PCDD/Fs or dioxins), four coplanar PCBs (International Union of Pure and Applied Chemistry [IUPAC] nos 77, 81, 126 and 169) and 12 PCB markers (IUPAC nos 3, 8, 28, 52, 101, 118, 138, 153, 180, 194, 206 and 209) were quantified in serum fat from fasting blood samples in order to estimate the body burden of these pollutants. Whilst no difference was found between women with endometriosis and their controls, diabetic patients had significantly increased serum levels of dioxins, coplanar PCBs and the 12 PCB markers. After adjustment for age and other covariates, serum total toxic equivalent activity (sum of PCDD/Fs and coplanar PCBs) and 12 PCB marker concentrations in diabetics were 62% (p = 0.0005) and 39% (p = 0.0067) higher, respectively, than in controls. The risk of diabetes was significantly increased in subjects in the top decile for adjusted concentrations of dioxins (odds ratio 5.1, 95% confidence interval [CI] 1.18-21.7), coplanar PCBs (odds ratio 13.3, 95% CI 3.31-53.2) or 12 PCB markers (odds ratio 7.6, 95% CI 1.58-36.3). These findings warrant further studies to assess the significance of the associations between diabetes and environmental exposure to polychlorinated pollutants.

Abstract: To evaluate the human exposure impact of municipal waste incinerators, dioxin and coplanar polychlorinated biphenyl (PCB) concentrations were determined in blood of 84 subjects who resided approximately 18 yr in the vicinity of two old incinerators, one located in a rural area (n=51) and the other in an industrial area (n=33). These subjects were compared with 63 controls from an unpolluted area. While no change was found in contaminant levels in residents living around the incinerator in the industrial area, subjects residing around the incinerator in the rural area possessed significantly higher serum levels of dioxins (38 vs. 24 pg TEQ/g fat) and coplanar PCBs (10 vs. 7 pg TEQ/g fat) than controls. These results were confirmed by multiple-regression analysis, showing that residence around the incinerator in the rural area (partial r2=.18) was the major contributor to dioxin accumulation followed by age (partial r2=.07). A two-way analysis of variance (ANOVA) on age-adjusted dioxin levels revealed a significant interaction between residence around incinerators and the consumption of fat from local origin, especially bovine and poultry products. Although age-adjusted dioxin levels in controls did not vary with local animal fat consumption, concentrations of dioxins in subjects living around the incinerators increased proportionally to their intake of local animal fat, with almost a doubling in subjects with a fat intake higher than 150 g fat/wk. Extrapolation from these data suggests that a significant increase of dioxin body burden is likely to occur only when dioxin emissions exceed 5 ng TEQ/Nm3, a threshold considerably above most emissions standards currently in force.

Abstract: A method for determining the equilibrium association constant of a complexation reaction A + B left harpoon over right harpoon AB by electrospray ionization mass spectrometry is described. The method consists in measuring the relative intensities of the peaks corresponding to A and to AB in equimolar A-B solutions at different concentrations C(0). The results are fitted by a non-linear least-squares procedure, with the two variable parameters being the equilibrium association constant K(a) and a factor R, defined by I(AB)/I(A) = R x [AB]/[A]. The factor R is the ratio between the response factors of AB and A, and corrects for the relative electrospray responses of the complex and the free substrate A, mass discrimination of instrumental origin and/or moderate in-source dissociation. The method is illustrated with the following two systems: complexes between a double-stranded 12-base pair oligonucleotide and minor groove binders, and cyclodextrin complexes with alpha,omega-dicarboxylic acids. For the oligonucleotide complexes, it is found that the response of the complex is not dramatically different to the response of the free oligonucleotide duplex, as the double helix conformation is disturbed by the drug only to a minor extent. In the case of cyclodextrin complexes, these complexes were found to have a much higher response than free cyclodextrin. This may be due to the fact that cyclodextrin is neutral in solution, whereas the complex is charged, but it can also stem from the fact that a significant proportion of the complex is in a non-inclusion geometry. The present method requires the exact determination of the concentrations of the reactants and is applicable to 1 : 1 complexes.

Abstract: The present paper describes a calibration of the ion effective temperatures as a function of the resonant activation amplitude in a quadrupole ion trap mass spectrometer. MS/MS experiments are performed on leucine enkephalin (M + H)+, bradykinin (M + H)+, (M + 2H)2+, and (M + 3H)3+, and ubiquitin (M + 11H)11+. For each amplitude, the effective temperature is calculated as the temperature that would give the same dissociation rate constant as the one observed and is calculated using published Arrhenius parameters. The effective temperature is found to be linearly dependent on the activation amplitude on the range investigated. The dependence of the slope and of the intercept of the T(eff) = f (amplitude) functions on the parent ion m/z is examined and an equation is derived to calibrate the ion effective temperature between 365 and 600 K. Below 365 K, a deviation from linearity is expected. Above 600 K, the validity of the equation will depend on whether the rapid energy exchange limit is still reached. Calculating backward, the Arrhenius parameters from the measured dissociation rates using this calibration show excellent agreement with the published values. The calibration can therefore be used to determine Arrhenius activation parameters from dissociation kinetics under resonant activation in quadrupole ion trap mass spectrometers.

Abstract: Electrospray mass spectrometry (ESI-MS) was used to monitor the kinetics of duplex formation between the human telomeric DNA quadruplex and its complementary strand; the complexation of telomestatin to the G-quadruplex delays the unwinding of the quadruplex structure and formation of the duplex.

Abstract: A strategy for determination of polybrominated diphenyl ethers, polychlorinated dibenzodioxins, polychlorinated dibenzofurans and polychlorinated biphenyls on fatty matrices has been established. After extraction, the proposed method allows the purification and the fractionation of all target groups of compounds in a simple multi-step automated clean-up. Furthermore, their subsequent analysis is carried out using a single benchtop mass spectrometer, in four separate injections. Required sensitivity considering levels found in the environment is attained using electron impact ionisation followed by tandem in time mass spectrometry. The whole method has been evaluated on standard solution and quality control samples consisting of fortified beef fat. Sensitivity, selectivity, accuracy and repeatability were tested with satisfactory results.

Abstract: The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.

Abstract: This work describes a method to use relative fragmentation yields to characterize the internal energy distribution of ions produced by matrix-enhanced laser desorption/ionization mass spectrometry (MELDI-MS, see: Wright LG, Cooks RG, Wood KL. Biomed. Mass Spectrom. 1985; 12: 153-162). Assuming that the fragmentation proceeds statistically and that the collisions in the source lead to a Boltzmann-like distribution of the internal energy, a characteristic parameter, the effective temperature, is introduced to describe the internal energy distribution of the ions observed. The hypotheses, advantages and drawbacks of the implementation of the method that uses substituted benzylpyridinium salts as thermometer ions are discussed. Use is made of two matrices that produce no matrix cations in MELDI and are suitable for small cationic salts. The actual value of this effective temperature significantly depends on an accurate determination of the threshold dissociation energies and on the time spent in the source, in addition to the statistical hypothesis itself. The method could be applied to normalize spectra in order to compare results issued from different instruments.

Abstract: The polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are highly toxic compounds produced by some natural processes and different human activities. Waste incineration as well as steel and iron industries, in particular, the sintering process, are among the principal sources of these pollutants. In this paper, two inhibitors, triethanolamine (TEA) and monoethanolamine (MEA) are tested relating to their abilityto prevent the de novo formation of PCDD/Fs on sinter plant fly ash. The amounts of both PCDDs and PCDFs, formed by thermal treatment of the fly ash, decrease when inhibitors are added. The best results, up to 90% reduction of the PCDD/ Fs formation, are obtained when MEA is mixed with the fly ash at the highest concentration tested (2 wt %). The addition of inhibitors modifies the PCDFs/PCDDs ratios and, under some experimental conditions, the PCDD/Fs homologue distributions, suggesting that more than one pathway for the de novo formation of PCDD/Fs exist. On the other hand, no modification in the PCDD/Fs isomer distributions is observed as a result of the addition of inhibitors, in accordance with the possible thermodynamic control of these distributions. The temperature tested, 325 and 400 degrees C, does not affect the inhibition activity; however, longer reaction times (4 h instead of 2 h) give better percentages of PCDD/Fs reduction. The results suggest that the two inhibitors and especially MEA can reduce the PCDD/Fs formation on sinter plant fly ash under various conditions of temperature and reaction time, making them suitable for use in the real process. Tests performed in parallel at a real sinter plant are in good agreement with the laboratory experiments and confirm that the use of inhibitors is an appropriate technique for the prevention of PCDD/Fs emissions from sintering processes.

Abstract: It is generally believed that homeostatic responses regulate T-cell recovery after peripheral stem cell transplantation (PSCT). We studied in detail immune recovery in relation to T-cell depletion and clinical events in a group of adult patients who underwent PSCT because of hematologic malignancies. Initially, significantly increased proportions of dividing naive, memory, and effector CD4(+) and CD8(+) T cells were found that readily declined, despite still very low numbers of CD4(+) and CD8(+) T cells. After PSCT, increased T-cell division rates reflected immune activation because they were associated with episodes of infectious disease and graft-versus-host disease (GVHD). T-cell receptor excision circles (TRECs) were measured to monitor thymic output of naive T cells. Mean TREC content normalized rapidly after PSCT, long before naive T-cell numbers had significantly recovered. This is compatible with the continuous thymic production of TREC(+) naive T cells and does not reflect homeostatic increases of thymic output. TREC content was decreased in patients with GVHD and infectious complications, which may be explained by the dilution of TRECs resulting from increased proliferation. Combining TREC and Ki67 analysis with repopulation kinetics led to the novel insight that recovery of TREC content and increased T-cell division during immune reconstitution after transplantation are related to clinical events rather than to homeostatic adaptation to T-cell depletion. (Blood. 2002;99:3449-3453)

Abstract: Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues, and recent data suggest that it may act as a phosphate donor for the phosphorylation of some proteins. In the mammalian brain, ThTP synthesis is rapid, but its steady-state concentration remains low, presumably because of rapid hydrolysis. In this report we purified a soluble thiamine triphosphatase (ThTPase; EC ) from calf brain. The bovine ThTPase is a 24-kDa monomer, hydrolyzing ThTP with virtually absolute specificity. Partial sequence data obtained from the purified bovine enzyme by tandem mass spectrometry were used to search the GenBank data base. A significant identity was found with only one human sequence, the hypothetical 230-amino acid protein MGC2652. The coding regions from human and bovine brain mRNA were amplified by reverse transcription-PCR, cloned in Escherichia coli, and sequenced. The human open reading frame was expressed in E. coli as a GST fusion protein. Transformed bacteria had a high isopropyl-beta-d-thiogalactopyranoside-inducible ThTPase activity. The recombinant ThTPase had properties similar to those of human brain ThTPase, and it was specific for ThTP. The mRNA was expressed in most human tissues but at relatively low levels. This is the first report of a molecular characterization of a specific ThTPase.

Abstract: Congener-specific analyses of 7 polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 4 non-ortho (coplanar) polychlorinated biphenyls (cPCBs) were performed on 197 foodstuffs samples of animal origin from Belgium during years 2000 and 2001. All investigated matrices (except horse) present background levels lower than the Belgian non-commercialization value of 5 pg TEQ/g fat. Pork was the meat containing the lowest concentration of both PCDD/Fs and cPCBs. The mean background concentration of 2,3,7,8-TCDD toxicity equivalent in milk was 1.1 pg/g of fat, with a congener distribution typical of non-contaminated milk. The relative contribution of 2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF to the PCDD/Fs TEQ was 85+/-7.9% for all investigated matrices. The cPCBs contribution to the total TEQ was 47+/-19.0% for products of terrestrial species and 69+/-20.0% for aquatic species. Once the contribution of cPCBs was added to the TEQ, few foodstuffs such as horse, sheep, beef, eggs and cheese presented levels above the future European guidelines that currently only include PCDD/Fs but will be re-evaluated later in order to include 'dioxin-like' PCBs. Based on levels measured in the samples, the estimation of the dietary intake was 65.3 pg WHO-TEQ/day for PCDD/Fs only (1.00 pg WHO-TEQ/kg bw/day, for a 65 kg person) and 132.9 pg WHO-TEQ/day if cPCBs were included (2.04 pg WHO-TEQ/kg bw/day, for a 65 kg person). Meat (mainly beef), dairy products, and fish each account for roughly one third of the intake.

Abstract: Accelerated telomere shortening has been proposed as a possible long-term risk of allogeneic bone marrow transplantation (allo-BMT). In this study we monitored telomere length in white blood cells (WBC), granulocytes, and naïve and memory CD4+ T lymphocytes in recipients of allo-BMT at long-term follow-up. Peripheral blood was collected from 10 allo-BMT recipients and donors at a median interval of 18 years after allo-BMT. Telomere length was determined using Southern blot analysis. Similar to results previously reported at short-term follow-up, a small difference in telomere length (0.1-0.3 kb) between recipients and donors was detected in WBC, granulocytes and naïve CD4+ T cells. Our data therefore provide no evidence for sustained telomere shortening in leucocytes, and render the possibility of long-term haematopoietic graft failure unlikely. In addition, we observed two phenomena that may be related to involution of the thymus. First, the number of naïve CD4+ T cells in the blood was significantly lower in recipients (0.4 x 10(9)/l) than in donors (0.7 x 10(9)/l) (P < 0.05). Second, telomeres in memory CD4+ T cells from recipients were on average 0.6 kb shorter than those from donors (P = 0.01). The latter may be related to the reported rapid peripheral expansion of memory T cells immediately after transplantation.

Abstract: New antibiotics were recently developed, among which are the (fluoro)quinolones. This paper presents an analytical method which allows the determination of 11 (fluoro)quinolones in swine kidneys: norfloxacin, ofloxacin, cinoxacin, oxolinic acid, nalidixic acid, flumequine, enrofloxacin, enoxacin, ciprofloxacin, danofloxacin and marbofloxacin. The procedure involves a rapid and efficient pre-treatment by solid-phase extraction (recoveries 83-98%), followed by the sensitive and selective determination of all compounds in a single run using LC-ESI-MS-MS. Multiple reaction monitoring (MRM) was used for selective detection of each (fluoro)quinolone. Quinine was selected as internal standard. The accuracy of the method, expressed as recovery, was between 89 and 109%; the repeatability had a maximum RSD lower than 15%. The limits of detection (LOD) were much lower than the respective Maximum Residue Limits (MRL)/4.

Abstract: A fast automated extraction and clean-up procedure for low-level analysis of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (cPCBs) in biological fluids is presented. Online extraction of prepared fluids is carried out using disposable octadecyl bonded (C(18)) solid-phase extraction columns. Extracts are then cleaned up through disposable multi-layer silica (acidic, basic and neutral) and dispersed PX-21 carbon columns. This new methodology is compared with classical Soxhlet extraction and manual solid-phase extraction in terms of repeatability, reproducibility, accuracy and recovery rates for reference and certified materials. Robustness is evaluated on different matrices, such as cow's milk, breast milk and human serum. As a consequence of the reduced number of reusable glassware used, as well as lowering of solvent consumption, recorded blank levels are decreased in favor of limits of detection (LODs). Total analysis time and cost are further reduced using simultaneous sample preparation units and the sample throughput is increased compared to classical methods. As a result, this new approach appears to be suitable for the fast sample preparation often required for such fluids in case of emergency foodstuffs analysis or during large epidemiological studies.

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Abstract: Congener-specific analyses of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and non-ortho (coplanar) polychlorinated biphenyls (cPCBs) were performed on 20 non-pooled breast milk samples collected in or close to an industrial area of Wallonia (Belgium). PCDD/F concentrations ranged between 16.0 and 52.1 pg TEQ/g fat, with a mean value of 29.4 pg TEQ/g fat. If coplanar PCBs (77, 126, 169) are included in TEQ calculations, levels ranged between 22.2 and 100.2 pg TEQ/g fat, with a mean value of 40.8 pg TEQ/g fat. It appears that 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,4,7,8-PeCDF and PCB-126 account for more than 90% of the TEQ. Estimated PCDD/F dietary intake is 76 pg TEQ/kg body weight (bw)/day. This value is almost 20 times higher than the World Health Organization tolerable daily intake. A value of 103 pg TEQ/kg bw/day represents the intake of PCDDs, PCDFs and cPCBs (no mono-ortho PCBs included).

Abstract: The structural selectivity of the DNA-binding antitumor drug ditercalinium was investigated by competition dialysis with a series of nineteen different DNA substrates. The 7H-pyridocarbazole dimer was found to bind to double-stranded DNA with a preference for GC-rich species but can in addition form stable complexes with triplex and quadruplex structures. The preferential interaction of the drug with four-stranded DNA structures was independently confirmed by electrospray mass spectrometry and a detailed analysis of the binding reaction was performed by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR study showed that the kinetic parameters for the interaction of ditercalinium with the human telomeric quadruplex sequence are comparable to those measured with a duplex sequence. Slow association and dissociation were observed with both the quadruplex and duplex structures. The newly discovered preferential binding of ditercalinium to the antiparallel quadruplex sequence d(AG(3)[T(2)AG(3)](3)) provides new perspectives for the design of drugs that can bind to human telomeres.

Abstract: Alpha-cyclodextrin complexes with linear alpha,omega-dicarboxylic acids were investigated by electrospray mass spectrometry. These hydrophobic complexes are known to have an equilibrium binding constant that increases with the diacid chain length. However, the electrospray mass spectrometry (ES-MS) spectra showed that the relative intensity of the complex did not vary significantly with chain length. This contradiction is caused by a contribution of nonspecific adducts to the signal of the complex in ES-MS. In order to estimate the contribution of nonspecific adducts to the total intensity of the complexes with alpha-cyclodextrin, the comparison was made between alpha-cyclodextrin and maltohexaose, the latter being incapable of making inclusion complexes in solution. The signal observed for complexes between diacids and maltohexaose can only result from nonspecific electrostatic aggregation, and is found to be more favorable with the shorter diacids. This is also supported by MS/MS experiments. A procedure is described which allows estimation of the contribution of the nonspecific complex in the spectra of the complexes with alpha-cyclodextrin by using the relative intensity of the complex with maltohexaose. The contribution of the specific complex to the total signal intensity is found to increase with the diacid chain length, which is in agreement with solution behavior.

Abstract: Electrospray mass spectrometry was evaluated regarding the reliability of the determination of the stoichiometries and equilibrium association constants from single spectra. Complexes between minor groove binders (Hoechst 33258, Hoechst 33342, DAPI, netropsin and berenil) and 12mer oligonucleotide duplexes with a central sequence (A/T)4 flanked by G/C base pairs were chosen as model systems. To validate the electrospray ionization mass spectrometry (ESI-MS) method, comparisons were made with circular dichroism and fluorescence spectroscopy measurements. ESI-MS allowed the detection of minor (2 drug + DNA) species for Hoechst 33258, Hoechst 33342, DAPI and berenil with duplex d(GGGG(A/T)4GGGG). d(CCCC(A/T)4CCCC), which were undetectable with the other techniques. Assuming that the duplexes and the complexes have the same electrospray response factors, the equilbrium association constants of the 1:1 and 2:1 complexes were determined by ESI-MS, and the values show a good quantitative agreement with fluorescence determined constants for Hoechst 33258 and Hoechst 33342. It is also shown that ESI-MS can quickly give reliable information on the A/T sequence selectivity of a drug: the signal of a complex is directly related to the affinity of the drug for that particular duplex. The potential of ESI-MS as a qualitative and quantitative affinity screening method is emphasized.

Abstract: The host-guest interactions play a very important role in chemical and biological processes. It is therefore important to be able to characterize these complexes. Electrospray mass spectrometry can be used to characterize the complex formation. It provides information on the mass and the charge of these ionic complexes. In this article, we show that the use of ab initio and semiempirical calculations, in addition to the results obtained by electrospray mass spectrometry, reveal to be a promising tool for the study of these noncovalent complexes. In this article, host-guest complexes formed by macropolycyclic polyammonium host molecules and dicarboxylic acids are studied.

Abstract: Fly ash, collected in the electrostatic precipitator of a sinter plant in Belgium, has been examined and characterized in terms of its behavior with respect to thermal polychlorodibenzo-p-dioxins (PCDD) and polychlorodibenzofurans (PCDF) formation. Thermal experiments of the fly ash were conducted in a flow of air. The temperature was varied from 250 to 450 degrees C, and the reaction time varied from 30 min to 6 h. For comparison, the oxidative degradation of carbon in the fly ash was studied by differential scanning calorimetry (DSC) in the temperature range from 50 to 500 degrees C. Besides the known maximum of formation of PCDD/Fs around 325 degrees C generally found on experiments with incinerator fly ash, a second maximum of formation around 400 degrees C is observed on the sinter fly ash used in this study. DSC measurements on the fly ash show that the oxidative degradation of carbon appears at these two different temperatures confirming that the de novo synthesis on this kind of fly ash take place at two different optimum temperatures. About the reaction time, already after 30 min, an important quantity of PCDD/Fs is formed; the fast increase in PCDD/Fs amount is followed by a slower formation rate between 2 and 4 h. At longer reaction time, the formation slows down, and decomposition reactions become important. Analysis of homologue distribution indicates that the profile of PCDD/Fs is independent of the reaction time but that an increase of the temperature leads to a rise of lower chlorinated species. In all experiments, PCDF are formed preferentially (total PCDF/PCDD ratios larger than 5). The PCDF/PCDD ratio is clearly independent of the reaction time. Concerning the temperature, the apparently better stability of PCDF at high temperature (PCDF/PCDD ratio higher at high temperature) results in the fact of different PCDF/PCDD ratios for the different family and modifications of homologue distribution with the temperature. The isomer distribution shows little reaction time or temperature dependency, which is an argument in favor of a thermodynamic control of the isomer distribution during de novo formation of PCDD/Fs. Differences within the isomer distribution patterns of PCDD/Fs obtained from the laboratory de novo synthesis experiments and the original fly ash, reflecting the formation under the industrial process, suggest a different mechanism of formation in the sinter plant for the PCDD and PCDF. The de novo synthesis is sufficient to explain the PCDF formation in the real process, but synthesis from precursors must play a role for the PCDD formation.

Abstract: A fast clean-up procedure for the low level analysis of polychlorinated dibenzo-p-dioxins. polychlorinated dibenzofurans and coplanar polychlorinated biphenyls in highly fatty biological matrices using high capacity disposable multi-layer silica columns is presented. Results were compared with gel permeation chromatography for removal of lipids. Analytical criteria such as recovery rates, repeatability, reproducibility and robustness are evaluated through a broad range of biological matrices and reference materials analysis. The final proposed procedure for the complete analysis, including pressurized liquid extraction, Power-Prep system clean-up and GC-high-resolution MS analysis requires only 48 h, and allows the simultaneous preparation of up to 10 samples.

Abstract: Rapid screening of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans using quadrupole ion storage tandem-in-time mass spectrometry (QISTMS) conjointly with polyclonal antibody immunoassay has been considered. The optimisation of the fragmentation of the parent ion in the trap has been completed. The analysis of fly ashes from a municipal waste incinerator contaminated at different levels has then been realised. Results obtained using QISTMS, HRMS and immunoassay are compared.

Abstract: Electrospray ionization with in-source collisionally induced dissociation has been used to probe the gas phase stability of an oligonucleotide duplex and its complexes with some minor groove binding drugs. On the basis of the arguments developed in detail by Drahos et al. (J. Mass Spectrom. 1999; 34:1373), this type of experiment can also be described as 'thermal denaturation in the gas phase'. We found that the gas phase denaturation curves were very similar to the solution phase denaturation curves determined by the traditional UV spectrophotometric method and, by analogy with the melting temperature T(m) which characterizes the stability in solution, we define a melting voltage V(m) to characterize the stability in the gas phase. A comparison of the T(m) and V(m) relative values suggests that the structure of the complexes is conserved during the electrospray process which transfers the ions from the solution to the gas phase.

Abstract: This study investigates the replacement of Soxhlet extraction by supercritical fluid extraction (SFE) or accelerated solvent extraction (ASE) for the removal of dioxins from municipal waste incinerator fly ash. SFE is very matrix dependent; higher percent recoveries versus Soxhlet extraction can be obtained for low-carbon-level fly ash, but only a few percent of dioxins can be extracted from high-carbon-level fly ash. The addition of large quantities of toluene in the extraction cell prior to extraction of high-carbon fly ash improves the recovery of the lowest chlorinated dioxins (approximately 90%), but a maximum of 20% of the octachlorodibenzo-p-dioxins can be extracted. Since large quantities of toluene are needed to improve the recoveries, ASE with toluene was tested. Recoveries similar to Soxhlet extraction can be obtained in 2 h at 80 degrees C. Increasing the temperature to 150 degrees C increases the extraction rate and yields recoveries of approximately 110-160% compared to 48-h Soxhlet extraction for all congeners for both low- and high-carbon fly ashes. These results question the choice of Soxhlet extraction as a reference method for dioxin determination.

Abstract: Electrospray ionization mass spectrometry was used to investigate the complex formation between a double-stranded oligonucleotide and various antitumor drugs belonging to two categories: intercalators (ethidium bromide, amsacrine and ascididemin) and minor groove binders (Hoechst 33258, netropsin, distamycin A, berenil and DAPI). The goal of this study was to determine whether the relative intensities in the mass spectra reflect the relative abundances of the species in the solution phase. The full-scan mass spectra suggest non-specific binding for the intercalators and specific binding for the minor groove binders. The preferential stoichiometries adopted by each minor groove binder were determined by studying the influence of the drug concentration on the spectra. We obtained 2:1 > 1:1 for distamycin, 1:1 > 2:1 for Hoechst 33258 and DAPI and only the 1 : 1 complex for netropsin and berenil. These features reflect their known behavior in solution. The compared tandem mass spectra of the 1 : 1 complexes with Hoechst 33258 and netropsin, when correlated with published crystallographic data, suggest the possibility of inferring some structural information. The relative binding affinities of the drug for the considered duplex were deduced with two by two competition experiments, assuming that the relative intensities reflect the composition of the solution phase. The obtained affinity scale is netropsin > distamycin A > DAPI > Hoechst 33258 > berenil. These examples show some of the potential uses of mass spectrometry as a useful tool for the characterization of specific drug binding to DNA, and possibly a rapid drug screening method requiring small amounts of materials.

Abstract: The blaIMP gene coding for the IMP-1 metallo-beta-lactamase produced by a Pseudomonas aeruginosa clinical isolate (isolate 101/1477) was overexpressed via a T7 expression system in Escherichia coli BL21 (DE3), and its product was purified to homogeneity with a final yield of 35 mg/liter of culture. The structural and functional properties of the enzyme purified from E. coli were identical to those of the enzyme produced by P. aeruginosa. The IMP-1 metallo-beta-lactamase exhibits a broad-spectrum activity profile that includes activity against penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems. Only monobactams escape its action. The enzyme activity was inhibited by metal chelators, of which 1,10-o-phenanthroline and dipicolinic acid were the most efficient. Two zinc-binding sites were found. The zinc content of the P. aeruginosa 101/1477 metallo-beta-lactamase was not pH dependent.

Abstract: A new method was developed which fits a thermal internal energy distribution to ions formed by electrospray ionization. The molecular ion survival yield was measured and determined by RRKM calculations as a function of temperature. The ('characteristic') temperature was determined when the calculated and measured molecular ion survival yields were equal. The 'characteristic' temperatures were very similar (average RSD errors were 8%) for a set of analogous compounds (benzylpyridine salts), and the resulting thermal internal energy distributions were close to those determined by De Pauw's method. The validity of the method was also checked performing blackbody infrared radiation and on-resonance excitation experiments on a Fourier transform ion cyclotron resonance instrument with benzylpyridine salts and leucine enkephalin. The results strongly suggest that internal energy distributions in electrospray ionization are very close to thermal distributions. It was found that the characteristic temperature increases linearly with the cone voltage. It is suggested that the characteristic temperature can be used as a quantitative measure to control and standardize conditions in electrospray ionization. Copyright 1999 John Wiley & Sons, Ltd.

Abstract: The abundance distribution of multiply charged ions produced during the electrospray process is thought to depend on the initial conformational state of the analyte in solution and on solution chemistry, but with some proteins, including apomyoglobin, the correlation between solution and mass spectrometry data is unclear. In this study, we compare our results obtained by mass spectrometry with available data describing conformations and average number of charges of apomyoglobin in solution. A good correlation between average number of charges in solution and in the mass spectrum is only found for the pH 4.2 solution. We propose that the discrepancies between solution and electrospray ionization mass spectrometry observed with apomyoglobin under other solution conditions can be mainly explained by (1) a preferential fragmentation of the highly charged ions in the source and (2) a variation of pH in the droplets during the electrospray process.

Abstract: Telomeres are G/C-rich repetitive DNA sequences at the end of all eukaryotic chromosomes. The loss of telomeric repeat sequences during cell divisions has been proposed as a possible mechanism for cell senescence. The standard procedure for measurement of telomere length is Southern blot (SB) hybridization with a telomere-specific probe. However, in using this technique no information can be obtained on variation in telomeric fragments due to interchromosomal, intrachromosomal, and intercellular differences. Lansdorp et al. (Hum Mol Genet 5:685-691, 1996) developed a method to measure individual telomeres, using in situ hybridization on metaphase chromosomes, employing peptide nucleic acid (PNA) probes and digital fluorescence microscopy. In this paper we describe a method that can be used to assess telomeric length in interphase cells. An algorithm was developed to measure the total intranuclear fluorescence in situ hybridization (FISH) signal, which features accurate correction for the local autofluorescence. Application of this methodology to samples of fetal liver, umbilical cord blood, and adult bone marrow cells showed a gradual decrease of average telomeric length. Southern blot analysis and PNA FISH measurements on chromosomes in the same samples showed similar results. Advantages of interphase measurements include the possibility of studying nonproliferating cells, thus avoiding selection and cell culturing.

Abstract: Before being transported to the cytoplasm, intron-containing pre-mRNAs have to be spliced somewhere in the cell nucleus. Efficient splicing requires an ordered assembly of splicing factors onto the pre-mRNAs. To accomplish this, intron containing genes may be preferentially localized at nuclear sites enriched for splicing factors or alternatively, splicing factors may circulate throughout the nucleus and have the ability to associate with randomly positioned nascent transcripts. Combined detection of HCMV-IE mRNA/DNA and splicing factors in rat 9G cells that can be induced for IE gene expression shows that IE genes are not associated with speckled regions enriched for splicing factors when transcriptionally inactive, but 'attract' splicing factors when transcriptionally activated. This process proved reversible after transcription inhibition. IE transcripts appeared to be retained near the transcription site in track-like domains by splicing factors associated with them until splicing has been completed. Double-hybridization experiments revealed that a substantial part of the accumulated transcripts contain a poly(A) tail suggesting that most, if not all, IE transcripts are polyadenylated at the site of transcription. These results indicate that RNA processing may occur independent of the position of the gene in the cell nucleus relative to speckle domains.

Abstract: A method was developed for the analysis of 7-(2-hydroxyethyl)guanine (7HEG), the major DNA adduct formed after exposure to ethylene oxide (EO). The method is based on DNA neutral thermal hydrolysis, adduct micro-concentration, and final characterization and quantification by HPLC coupled to single-ion monitoring electrospray mass spectrometry (HPLC/SIR-ESMS). The method was found to be selective, sensitive, and easy to handle with no need for enzymatic digestion or previous sample derivatization. Detection limit was found to be close to 1 fmol of adduct injected (10(-10) M), thus allowing the detection of approximately three modified bases on 10(8) intact nucleotides in blood sample analysis. Quantification results are shown for 7HEG after calf thymus DNA and blood exposure to various doses of EO, in both cases obtaining clear dose-response relationships.

Abstract: The circadian pacemaker of the suprachiasmatic nuclei (SCN) is entrained to the environmental light-dark cycle via the retinohypothalamic tract (RHT). It is unknown whether light activates or suppresses firing of the retinal ganglion cells which mediate photic entrainment. We therefore electrically stimulated the optic nerves and the anterior optic chiasm of hamsters with free-running activity rhythms in continuous darkness. These electrical stimulations are thought to induce a release of neurotransmitter at the RHT terminals. Electrical stimulation mimicked the phase dependent shifts induced by light pulses. The phase shifts were significantly larger than the shifts induced by sham stimulation in the same animals or by electrical stimulation in animals with an electrode outside the optic nerves and chiasm. Our results indicate that the retinal ganglion cells which project to the SCN are activated by light. Intraperitoneal administration of MK-801, a non-competitive antagonist of the NMDA-receptor, attenuated the phase delays induced by electrical stimulation in the early subjective night. This suggests that an excitatory amino acid mediates the effects of light upon the circadian pacemaker.

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Abstract: Mass spectrometry is becoming an important method in structure determination of biopolymers. Determination of the nature and the number of the various monomers is an important information for the elucidation of fragmentation patterns. We present here a matrix designed to allow both identification and semi-quantitative measurement of amino acids residues in peptides.

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