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We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.

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To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.

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A new technique for determining sequence and linkage information of underivatized oligosaccharides is developed using alkaline degradation and matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Alkaline degradation (also known as the "peeling" reaction) is a chemical degradation technique that only cleaves the glycosidic bond at the reducing end by beta-elimination to yield a new reducing end. The reaction products are sampled directly with minimal cleanup and monitored by MALDI-FTMS to elucidate the oligosaccharide sequence. Linkage information is provided by cross-ring cleavage fragmentation of the new reducing ends, created by either MALDI source fragmentation or sustained off-resonance irradiation collision-induced dissociation. This method is illustrated by the successful sequence and linkage determination of neutral, branched, fucosylated, and sialylated oligosaccharides. Experiments on differently linked disaccharides are also performed to determine the specificity of the cross-ring cleavage reactions. The power of this technique is enhanced by the Fourier transform mass analyzer, which provides high-resolution, exact mass, and facile tandem mass spectrometry experiments of MALDI-produced ions.

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A gadolinium-chelated liposomal contrast agent has been prepared, and magnetic resonance imaging (MRI) efficacy has been examined by indirect magnetic resonance lymphography. A lipidic N,N'-dimethylethylenediamine derivative (4) containing a 10,12-diyne-diacyl domain was treated with DTPA anhydride followed by GdCl3 complexation. The complex was confirmed using MALDI spectrometry. An equimolar mixture of the Gd-chelate lipid and a commercially available diyne-PE was formulated as a liposome suspension and irradiated with UV light prior to imaging experiments. Subcutaneous injection of the liposomal gadolinium agent and subsequent MRI of rabbit axillary and popliteal lymph nodes revealed significant contrast enhancement up to 4 h postinjection. To explore the possibility of imaging a DNA transfection event, the gadolinium contrast mixture was formulated with the cationic transfection lipid DOTAP and complexed with the reporter gene encoding luciferase. DNA transfection studies on the NIH3T3 cell line confirmed the transfection activity of the dual-purpose contrast agent and exemplified the potential toward development of an imaging and DNA delivery vehicle.

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Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI/FTMS), operating in the negative ion mode, is used to directly observe sugar alcohol borate complexes in a number of plant fractions. The method involves virtually no sample workup and, in the case of celery phloem sap, requires only 40 nL of sap to observe the borate complex. The isolation and characterization of such soluble borate complexes is important in understanding the distribution of boron in plants. The results show that the complexes are composed of two mannitol or sorbitol ligands (L) complexed to a single borate center (B). In some cases, the boron is complexed to non-alditol monosaccharides. Sustained off-resonance collision irradiation dissociation of the BL2- complex, where L is a mannitol, gives fragments that confirm the proposed structure. Complexes of larger oligosaccharides have also been successfully observed using MALDI/FTMS. Semiempirical molecular orbital calculations (AM1) of the mannitol BL2- complex show that the most favorable configuration is with carbons 3 and 4 of both mannitol residues complexed to the borate. This allows maximum interaction of the remaining hydroxyls with the borate center.

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Boron (B) polyol complexes have been isolated and characterized from the phloem sap of celery (Apium graveolens L.) and the extrafloral nectar of peach (Prunus persica L.). In celery the direct analysis of untreated phloem sap by matrix-assisted laser desorption-Fourier transform mass spectrometry, with verification by high-performance liquid chromagraphy and gas chromatography-mass spectrometry, revealed that B is present in the phloem as the mannitol-B-mannitol complex. Molecular modeling further predicted that this complex is present in the 3,4 3',4' bis-mannitol configuration. In the extrafloral nectar of peach, B was present as a mixture of sorbitol-B-sorbitol, fructose-B-fructose, or sorbitol-B-fructose. To our knowledge, these findings represent the first successful isolation and characterization of soluble B complexes from higher plants and provide a mechanistic explanation for the observed phloem B mobility in these species.

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New compounds bastadin 20 (9), 15,34-O-disulfatobastadin 7 (10), and 10-O-sulfatobastadin 3 (11) were isolated from Ianthella basta collected in Exmouth Gulf, Western Australia. Compounds 10 and 11 exhibited moderate differential activity as SR Ca2+ channel agonists (EC50 13.6 and 100 microM, respectively) of the Ry1R FKBP12 complex, while the potency of 9 was almost half that of 10 (EC50 20.6 microM). The problem of dereplication of bastadins was addressed using 1H-NMR "fingerprinting" of MeO signals in the corresponding permethyl bastadin derivatives.

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Collision-induced dissociation (CID) is used in an external source Fourier transform mass spectrometer (FTMS) equipped with matrix-assisted laser desorption/ionization (MALDI) to study a number of complex, branched oligosaccharides. The relative dissociation thresholds for various oligosaccharide fragmentation pathways have been calculated in terms of kinetic and center-of-mass frame energy. For two isomers of difucosyllacto-N-hexaose, the loss of the fucose sugar is always the lowest energy fragment observed and occurs at the same energy for both isomers when the oligosaccharide is coordinated to a sodium ion. When the oligosaccharide is complexed to cesium, the threshold for the removal of the fucose moiety increases, indicating that the cesium is involved in a coordination complex that stabilizes the sugar. MS/ MS/MS is performed on a sugar, mannose core, which does not readily fragment during MALDI. In all the sugars examined, CID produces additional structural information relative to MALDI/FTMS.

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A design is presented involving two separate vacuum chambers to provide nearly simultaneous capabilities of liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) in an external source Fourier transform mass spectrometer. The instrument consists of two vacuum chambers, one with five stages of differential pumping for a combined LSIMS/MALDI source. The chamber dedicated to ESI was formerly a three-stage chamber with LSIMS and electron ionization. Two additional stages were added with the ESI source. LSIMS and MALDI have similar vacuum requirements and were moved to a newly built chamber with two stages of pumping. We present our first results obtained on the new vacuum chamber. Data presented for the MALDI source show that, with only two stages of pumping, and with shorter radio frequency-only quadrupole rods for ion injection, spectra comparable to those obtained on the formerly three-stage instrument can be obtained. Characterization of the MALDI source and data on linear, cyclic, and branched oligosaccharides are given. Finally, the design of the secon chamber is proposed as a low-cost prototype for an external source FTMS instrument.

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Gas-phase noncovalently bound complexes are probed by hydrogen/deuterium exchange. The complexes, composed of a protonated amino acid and a monosaccharide, are investigated to observe the effects of complexation on the rates of exchange. Rate constants are determined and compared for complexed and uncomplexed amino acids. The overall rate constant, which corresponds to exchange of a specific number of hydrogens, is deconvoluted to yield site-specific rate constants. Complexation of amino acids with saccharides significantly decreases the rate constants of the exchange. Results of molecular orbital calculations are provided to explain the decrease in the rates.

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A systematic approach is outlined for optimization of enantiomeric separations in free solution capillary electrophoresis using chiral mobile-phase additives. Maximum electrophoretic mobility difference between the enantiomers occurs when the concentration of free selector is equal to the reciprocal of the average binding constant. General equations and data analysis methods are presented to relate mobilities to equilibrium constants in simple and competitive binding equilibria and used to determine thermodynamic parameters for host-guest complexation of tioconazole enantiomers with a range of cyclodextrin selectors. Selectivities are found to be in the reverse order of binding constants in the series dimethyl-beta-cyclodextrin (K1 = 6.9 x 10(3) M-1, alpha = 1.10) to hydroxypropyl-beta-cyclodextrin (K1 = 0.72 x 10(3) M-1, alpha = 1.29). For beta-cyclodextrin (K1 = 1.32 x 10(3)M-1, alpha = 1.20), delta H zero provides the dominant contribution to binding but delta delta H zero and T delta delta S zero terms give comparable contributions to the selectivity. Addition of alcohol does not affect the selectivity, but allows displacement of the optimum separation conditions to higher cyclodextrin concentration through either competitive binding (with cyclohexanol) or preferential solvation of reactants (with methanol).

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Capillary electrophoresis has been used to determine binding constants of tioconazole enantiomers with hydroxypropyl-beta-cyclodextrin (HP-beta-CD), after correcting for changes in mobility with increasing viscosity. The predicted and observed values of enantiomeric mobility difference were found to be maximum at a concentration of HP-beta-CD equal to the reciprocal of the average binding constant.

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