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PubMed2006

[Research advances in ecotoxicology and risk assessment of chemical mixtures at low doses].

Chemicals in the environment often exist as mixtures and at low doses, whose ecotoxicological diagnosis and risk assessment are hard to be done with the help of the research results on the ecotoxicity of high-level exposure of single chemicals. This paper reviewed the research advances in ecotoxicology and risk assessment of low-dose chemical mixture contamination, with the focus on the molecular toxicological methods and the risk assessment approaches and schemes of simple and complex mixtures. Comments were given on the development directions of low-dose chemical mixture research, including the necessity of searching for sensitive endpoints, involving the approaches of other disciplines, increasing data accumulation, and setting up a comprehensive and identical risk assessment system.

Dose-Response Relationship, Drug↗
PubMed2001

Characterization of isolated fractions of dissolved organic matter from natural waters and a wastewater effluent.

Dissolved organic matter (DOM) was concentrated from natural waters and the effluent of a wastewater treatment plant using a portable reverse osmosis (RO) system. The humic acid (HA), fulvic acid (FA) and hydrophilic (HyI) fractions were isolated and purified by the XAD-8 resin combined with the cation exchange resin method. The FA fractions predominated in natural waters and accounted for 54-68% of the total amount of dissolved organic carbon (DOC), whereas the HA and HyI fractions constituted, respectively, 13-29 and 9-30% of the total DOC. The effluent of wastewater was almost devoid of HA and the HyI fraction exceeded FA. The elemental compositions of HA and FA were in the ranges typical for natural humic materials, but the HyI fractions did not exhibit humic character. 1H NMR spectra revealed that the HyI fractions were almost devoid of aromatic protons and the aliphatic region featured more sharp signals than HA and FA fractions, indicating that HyI fractions were consisted of more simple compounds and less complex mixtures. The aliphatic functional groups in these fractions of DOM samples followed the order HA < FA HyI. The rate of Cu complexation with the HyI fraction was faster than the rate with the HA or FA fraction of the Suwannee River DOM, implying that copper reacted with relatively weak ligands faster than with strong ligands.

Benzopyrans↗
PubMed1999

Selection of antibodies for intracellular function using a two-hybrid in vivo system.

Expression of antibodies inside cells has been used successfully to ablate protein function. This finding suggests that the technology should have an impact on disease treatment and in functional genomics where proteins of unknown function are predicted from genomic sequences. A major hindrance is the paucity of antibodies that function in eukaryotic cells, presumably because the antibodies fold incorrectly in the cytoplasm. To overcome this problem, we have developed an in vivo assay for functional intracellular antibodies using a two-hybrid approach. In this assay, antibody, as single-chain Fv (scFv) linked to a transcriptional transactivation domain, can interact with a target antigen, linked to a LexA-DNA binding domain, and thereby activate a reporter gene. We find that several characterized antibodies can bind their target antigen in eukaryotic cells in this two-hybrid format, and we have been able to isolate intracellular binders from among sets of scFv that can bind antigen in vitro. Furthermore, we show a model selection in which a single scFv was isolated from a mixture of half a million clones, indicating that this is a robust procedure that should facilitate capture of antibody specificities from complex mixtures. The approach can provide the basis for de novo selection of intracellular scFv from libraries, such as those made from spleen RNA after immunization with antigen, for intracellular analysis of protein function based only on genomic or cDNA sequences.

Animals↗
PubMed2004

Utilisation of proteomics datasets generated via multidimensional protein identification technology (MudPIT).

Technological developments in proteomics have had a dramatic impact on biology in recent years. One of these developments--named multidimensional protein identification technology (MudPIT)--couples two-dimensional chromatography of peptides in mass spectrometry-compatible solutions directly to tandem mass spectrometry, allowing for the identification of proteins from highly complex mixtures. Since the initial descriptions of MudPIT, this approach has been implemented in the analysis of whole proteomes, organelles and protein complexes. Key aspects of many of the analyses are the validation of MudPIT datasets with alternate strategies and the integration of MudPIT datasets with other biochemical, cell biology or molecular biology approaches. This paper presents strategies for validating MudPIT datasets and incorporating these datasets into biologically driven experimental design.

Automation↗
PubMed1990

Approaches used by the US National Toxicology Program in assessing the toxicity of chemical mixtures.

The US National Toxicology Program has made a definite commitment towards improving the understanding and predictability of the toxicity of chemical mixtures. The commitment has consisted of a thorough review by the National Research Council of the state of the art of complex mixture testing and strategy for in vivo tests as well as a series of laboratory studies to better characterize the toxicity of selected chemical mixtures and research programmes to improve our ability to predict the toxicological properties of other mixtures.

Animals↗
PubMed1994

Perceptual characteristics of binary, trinary, and quaternary odor mixtures consisting of unpleasant constituents.

Among the most obnoxious stimuli that the population at large is exposed to during everyday life are odorous emissions from sewage treatment plants. Such emissions are complex and contain many different types of odorants that vary in quantity depending upon the contents and efficiency of treatment processes. Because little is known about how individual odorants in complex mixtures affect the perception of each other, it is difficult to develop mathematical models that can predict the pleasantness, strength, and quality characteristics of an emission at different distances from a source. In the present study, the interactions of the four major types of odorants emitted by treatment plants worldwide, namely, hydrogen sulphide, isovaleric acid, butanethiol, and skatole, were investigated by measuring the perceived intensity of individual odorants alone and in mixtures, and the overall perceived intensity, unpleasantness, and qualities of mixtures. In addition, models for predicting odor strength were investigated. The results indicated that (i) the perceived odor intensity (odor strength) of mixtures of the odorants was equal or greater than that of any of the individual constituents, but less than the sum of their intensities. However, as the number of components in a mixture increased, the intensity of the most dominant component provided a good approximation of the intensity of the mixture. (ii) The vector model of intensity summation also satisfactorily predicted the odor intensity of mixtures containing two, three, or four of the odorants investigated. (iii) In no instance was the intensity of one odorant enhanced by another, i.e., no synergistic interactions occurred; the greater the number of odorants in a mixture, the greater was the degree of suppression of the individual constituents. (iv) The greater the number of constituents in a mixture the more difficult it became to identify individual constituents. (v) Hydrogen sulphide was the least frequently suppressed constituent, and isovaleric acid and skatole were the most frequently suppressed constituents in mixtures. (vi) The unpleasantness of mixtures was usually greater than that of the individual constituents, indicating that models used for predicting complaint levels in communities affected by sewage odor and based on assumptions related to a single odorant, e.g., hydrogen sulphide, will underestimate the number of complaints. Even mixtures with low but above threshold concentrations of these odorants are likely to generate complaints.

Adult↗
PubMed2006

Combined use of gas chromatography and selected ion flow tube mass spectrometry for absolute trace gas quantification.

The value of the gas chromatography (GC) and selected ion flow tube mass spectrometry (SIFT-MS) combination for the analysis of trace gases is demonstrated by the quantification of acetone in air samples using the three precursor ions available to SIFT-MS, viz. H3O+, NO+ and O2+, and by the separation of the isomers 1-propanol and 2-propanol, and their analysis using H3O+ precursor ions. It is shown that the GC/SIFT-MS combination allows for accurate trace gas quantification obviating the regular, time-consuming calibrations that are usually required for the more commonly used detectors of GC systems, and the positive identification of isomers in mixtures that is often challenging using SIFT-MS alone. Thus, the GC/SIFT-MS combination paves the way to more confident analyses of complex mixtures such as exhaled breath.

Journal Article↗
PubMed1992

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.

Arachidonic Acid↗
PubMed1978

[Preparative isolation of deoxyriboadenylic acids from hydrolysates of oxidized herring sperm DNA (author's transl)].

The preparative-scale chemical degradation of DNA from herring sperm to mixtures of purine oligonucleotides or deoxyriboadenylic acids is described. The deoxyriboadenylic acid mixture was separated into six fractions according to increasing ionic charge by column chromatography on QAE-Sephadex A-25. Of the first fraction, 75% was deoxyriboadenosine monophosphate; of the second, 99% consisted of a mixture of p(dA)2 and (dA)2p, while of the third 93% was pdAp. The remaining fractions contained more or less complex mixtures of longer-chain oligonucleotides which could be further separated by subsequent re-chromatography on QAE-Sephadex at pH 9.6. By application of paper chromatography to the fractions obtained from column chromatography, the pure nucleotide phosphates pdAp, p(dA)2p, p(dA)3p, p(dA)4p and the mixtures of sequence isomers p(dA)2, (dA)2p; p(dA)3, (dA)3p; and p(dA)6, (dA)6p could be isolated preparatively. The nucleotide phosphates were converted to (dA)2, (dA)3 or (dA)4 and the mixtures of sequence isomers to (dA)2, (dA)3 or (dA)6 by treatment with alkaline phosphatase. By this means dephosphorylated, paper chromatographically pure oligodeoxyriboadenylic acid may be obtained in a preparative scale from the hydrolysates. The structures of the nucleotides thus isolated were deduced from the absorbtion characteristics, the RF values and the results of enzymatic degradation.

Animals↗
PubMed1993

Optimization of capillary electrophoresis of mixtures of basic peptides and comparison with HPLC.

Fourteen synthetic, strongly basic hexadeca- to octadecapeptides, which differ only in one amino acid residue, were synthesized and characterized. In order to investigate the application of capillary electrophoresis for the separation of mixtures of closely related basic peptides, seven different mixtures, which represent possible byproducts during solid-phase synthesis, were analyzed by HPLC and different techniques of capillary electrophoresis. Peaks within the mixture were identified by single peak injection or on-line CE/MS. Utilization of micellar electrokinetic capillary electrophoresis offered the separation of complex mixtures. Comparing the electrophoretic mobilities with calculated correlations, we found that the separation of peptides of this length is influenced not only by the different size and charge but also by hydrophobicity and the orientation of the charge due to different conformations.

Amino Acid Sequence↗
PubMed1997

Evaluation of temperature effects on selectivity in RPLC separations using polybutadiene-coated zirconia.

The effect of temperature on selectivity in RPLC method development has been evaluated on polybutadiene-coated zirconia. We find that the influence of temperature on selectivity depends strongly on solute type. For solutes of similar structure such as polyaromatic hydrocarbons, temperature has almost no effect on selectivity; however, for solutes with very different functional groups such as chlorophenols, temperature changes did significantly affect selectivity. We feel that simple mixtures with one dominant retention mechanism-e.g., solvophobic retention-will not be helped appreciably by adjusting temperature. However, in complex mixtures with polar and ionizable solutes, optimization by varying the temperature may well be fruitful.

Acrylonitrile↗
PubMed2003

Quantitative detection of probiotic Bifidobacterium strains in bacterial mixtures by using real-time PCR.

Strain-specific rRNA-targeted primers were designed for the quantitative detection of Bifidobacterium infantis Y1, B. breve Y8 and B. longum Y10 used in a pharmaceutical probiotic product (VSL-3). PCR and real-time PCR techniques with the selected primers were employed for the direct enumeration of the bifidobacteria in the probiotic preparation and for studying their kinetic characteristics in batch cultures. These analysis revealed that B. infantis Y1 was the predominant strain in the probiotic product and that its growth rate was the highest. Since B. infantis Y1, B. breve Y8 and B. longum Y10 are co-cultured during the industrial production of VSL-3, the kinetic characteristics of these strains can explain their different concentrations in the probiotic preparation. A validation of the PCR quantification method was performed by identifying a representative number of isolates from the bacterial mixtures with automated ribotyping. The methodology described represents a useful tool for the specific quantitative detection of bacterial strains and species in complex mixtures such as pharmaceutical preparations, dairy starter cultures, faecal samples and biopsies.

Bacterial Typing Techniques↗
PubMed1975

Lysosomal enzymes as agents of turnover of soluble cytoplasmic proteins.

The degradation of cytosol proteins in vitro by purified cathepsin D and cathepsin B1 and by mixtures of lysosomal enzymes, was studied. By means of a double-labelling method, it was shown that the relative rates of degradation of cytosol proteins by the purified enzymes and by mixtures of enzymes under a wide range of conditions in vitro correlated well with their relative rates of turnover in vivo. The complex mixture of cytosol proteins was degraded less rapidly after denaturation than in the native state, both by the purified proteases and by the mixture of lysosomal enzymes. This contrasts with previous results on proteolysis of single purified proteins. The possible role of lysosomal enzymes in turnover in vivo was discussed.

Animals↗
PubMed1987

Inactivation of C-1 inhibitor by proteases: demonstration by a monoclonal antibody of a neodeterminant on inactivated, non-complexed C-1 inhibitor.

Monoclonal antibodies were raised against kallikrein-C-1 inhibitor and factor XIIa-C-1 inhibitor complexes. One of the monoclonal antibodies (KII) appeared to react predominantly with C-1 inhibitor complexes in an ELISA. However, the apparent binding of KII to C-1 inhibitor complexes was probably due to the presence of proteolytically inactivated C-1 inhibitor in the complex mixture used for the coating:KII did not bind either kallikrein-C-1 inhibitor or factor XIIa-C-1 inhibitor complexes generated in plasma by dextran sulphate. SDS-PAGE analysis of C-1 inhibitor incubated with proteases revealed that KII-reactive C-1 inhibitor has a lower molecular weight than native C-1 inhibitor. We propose that the determinant that reacts with KII is exposed after cleavage of C-1 inhibitor in its reactive site. The monoclonal antibody KII will enable us to study the inactivation of C-1 inhibitor in human inflammatory disease.

Antibodies, Monoclonal↗
PubMed1999

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.

Algorithms↗
PubMed2003

Degradation of pesticides in biobeds: the effect of concentration and pesticide mixtures.

Biobeds aim to create an environment whereby any pesticide spills are retained and then degraded, thus reducing the potential for surface or groundwater contamination. Biobeds may receive high concentrations of relatively complex mixtures of pesticides. The effects of concentration and pesticide interaction on degradation rate were therefore investigated. At concentrations up to 20 times the maximum recommended application rate for isoproturon and chlorothalonil, the rate of degradation in topsoil and biomix decreased with increasing concentration. With the exception of isoproturon at concentrations above 11 mg kg(-1), degradation was quicker in biomix (a composted mixture of topsoil, compost, and wheat straw) than in topsoil. One possible explanation for faster isoproturon degradation in topsoil as compared to biomix may be that previous treatments of isoproturon applied to the field soil as part of normal agricultural practices had resulted in proliferation of microbial communities specifically adapted to use isoproturon as an energy source. Such microbial adaptation could enhance the performance of a biobed. Studies with a mixture of isoproturon and chlorothalonil showed that interactions between pesticides are possible. In biomix, the degradation of either isoproturon or chlorothalonil was unaffected by the presence of the other pesticide, whereas in topsoil, isoproturon DT(50) values increased from 18.5 to 71.5 days in the presence of chlorothalonil. These studies suggest that biobeds appear capable of treating high concentrations of more than one pesticide.

Biodegradation, Environmental↗
PubMed2006

Assessment of the toxicity of mixtures of copper, 9,10-phenanthrenequinone, and phenanthrene to Daphnia magna: evidence for a reactive oxygen mechanism.

Polycyclic aromatic hydrocarbons and their derivatives are ubiquitous environmental contaminants. They are commonly present in complex mixtures with other contaminants, such as metals. The toxicities of phenanthrene (PHE) and 9,10-phenanthrenequinone (PHQ) with or without Cu were determined using Daphnia magna. Copper was the most toxic among the three chemicals tested, followed by PHQ and then PHE, with 48-h median effective concentrations (EC50s) of 0.96, 1.72, and 5.33 microM, respectively. Copper at 0.31 microM, or approximately the 5% effective concentration, decreased the EC50 of PHQ from 1.72 to 0.28 microM. Likewise, PHQ at 1.2 microM, or approximately the 10% effective concentration, significantly lowered the EC50 of Cu from 0.96 to 0.30 microM. This synergistic effect was not observed, however, in mixtures of Cu and PHE based on the response addition model. Assimilation of Cu wasfound to be similar with or without PHQ at increasing external concentrations of Cu, indicating that the increased toxicity of their mixtures is physiologically based. The ability of Cu plus PHQ to generate reactive oxygen species (ROS) was measured as well. Copper alone caused elevated ROS levels at a low concentration (0.63 microM). With PHQ present, however, this elevation in ROS occurred at an even lower Cu level (0.31 microM). Possible attenuation effects of ascorbic acid (vitamin C) on toxicity and ROS production induced by Cu, PHQ, and their mixtures were then examined. Ascorbic acid protected against Cu and Cu-plus-PHQ mixture-mediated toxicity but did not affect PHQ toxicity. Ascorbic acid also lowered ROS levels in the presence of Cu and Cu plus PHQ. We conclude that there exist potential toxic interactions between metals and modified PAHs and that these interactions can involve ROS formation.

Animals↗
PubMed1991

Fractionation of the human recombinant tissue plasminogen activator (rtPA) glycoforms by high-performance capillary zone electrophoresis and capillary isoelectric focusing.

This paper reports the fractionation of recombinant human tissue plasminogen activator (rtPA) glycoforms, a complex mixture to demonstrate the high resolving power of capillary zone electrophoresis (CZE) and capillary isoelectric focusing (cIEF). rtPA is a glycoprotein with a complex carbohydrate structure. The electropherograms and IEF patterns have been discussed in light of the known carbohydrate structures of rtPA. rtPA was treated with neuraminidase which removes the sialic acids from the carbohydrate chains. The desialylated rtPA was analyzed by both CZE and IEF and the results were compared to those of untreated rtPA. The usefulness of CZE and cIEF in the characterization of glycoproteins proteins is also discussed.

Amino Acid Sequence↗