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PubMed2006

Sparse encoding of natural scents.

Natural scents are mixtures of tens to hundreds of individual chemical compounds. While previous studies have investigated how the olfactory bulb responds to simple odorants, how the nose and the brain respond to and interpret complex mixtures is less clear. In a paper in this issue of Neuron, Lin et al. combined gas chromatography and intrinsic signal imaging to examine the responses of individual olfactory bulb glomeruli in the mouse to natural odors and their component parts.

Animals↗
PubMed2000

Simultaneous analysis of butene isomer mixtures using process mass spectrometry.

The feasibility of simultaneous analysis of mixtures containing two to four butene isomers and up to six total components using process mass spectrometry is assessed. As for typical (nonisomeric) applications of process mass spectrometry, simultaneous analysis is based on the assumption that the electron ionization mass spectra of mixtures are linear combinations of the spectra of the individual constituents. Limits of detection for binary isomer mixtures are on the order of 0.1% to 10%, limited by the ability to distinguish small differences between similar spectra. As spectral and mixture complexity increase, both accuracy and precision decrease. Not surprisingly the similarity of the spectra of stereoisomers cis- and trans-2-butene is greater than that of the other (nonstereoisomeric) isomer pairs, and mixtures containing both cis- and trans-2-butene are the most difficult to quantitate. However, even for mixtures of all four butenes, accuracy (root-mean-square error = 2.43%), precision (average coefficient of variation = 6.72%), and linearity (correlation coefficient of a plot of measured versus actual concentration r2 = 0.985 +/- 0.002) are reasonably good.

Journal Article↗
PubMed1995

Evaluation of a possible association of urban air toxics and asthma.

The prevalence of asthma, measured either as the frequency of hospital admissions or number of deaths attributed to asthma, has increased over the last 15 to 20 years. Rapid increases in disease prevalence are more likely to be attributable to environmental than genetic factors. Inferring from past associations between air pollution and asthma, it is feasible that changes in the ambient environment could contribute to this increase in morbidity and mortality. Scientific evaluation of the links between air pollution and the exacerbation of asthma is incomplete, however. Currently, criteria pollutants [SOx, NOx, O3, CO, Pb, particulate matter (PM10)] and other risk factors (exposure to environmental tobacco smoke, volatile organic compounds, etc.) are constantly being evaluated as to their possible contributions to this situation. Data from these studies suggest that increases in respiratory disease are associated with exposures to ambient concentrations of particulate and gaseous pollutants. Similarly, exposure to environmental tobacco smoke, also a mixture of particulate and gaseous air toxics, has been associated with an increase in asthma among children. In addition, current associations of adverse health effects with existing pollution measurements are often noted at concentrations below those that produce effects in controlled animal and human exposures to each pollutant alone. These findings imply that adverse responses are augmented when persons are exposed to irritant mixtures of particles and gases and that current measurements of air pollution are, in part, indirect in that the concentrations of criteria pollutants are acting as surrogates of our exposure to a complex mixture. Other irritant air pollutants, including certain urban air toxics, are associated with asthma in occupational settings and may interact with criteria pollutants in ambient air to exacerbate asthma. An evaluation of dose-response information for urban air toxics and biological feasibility as possible contributors to asthma is therefore needed. However, this evaluation is compounded by a lack of information on the concentrations of these compounds in the ambient air and their effects on asthma morbidity and mortality. Through an initial review of the current toxicological literature, we propose a tentative list of 30 compounds that could have the highest impact on asthma and respiratory health. These compounds were selected based on their ability to induce or exacerbate asthma in occupational and nonoccupational settings, their allergic potential and ability to react with biological macromolecules, and lastly, their ability to irritate the respiratory passages. We recommend better documentation of exposure to these compounds through routine air sampling and evaluation of total exposure and further evaluation of biological mechanisms through laboratory and epidemiological studies directed specifically at the role these substances play in the induction and exacerbation of asthma.

Air Pollutants↗
PubMed2001

Biofiltration of a mixture of volatile organic emissions.

Air biofiltration is now under active consideration for the removal of the volatile organic compounds (VOCs) from polluted airstreams. To optimize this emerging environmental technology and to understand compound removal mechanisms, a biofilter packed with peat was developed to treat a complex mixture of VOCs: oxygenated, aromatic, and chlorinated compounds. The removal efficiency of this process was high. The maximum elimination capacity (ECmax) obtained was approximately 120 g VOCs/m3 peat/hr. Referring to each of the mixture's components, the ECmax showed the limits in terms of biodegradability of VOCs, especially for the halogenated compounds and xylene. A stratification of biodegradation was observed in the reactor. The oxygenated compounds were metabolized before the aromatic and halogenated ones. Two assumptions are suggested. There was a competition between bacterial communities. Different communities colonized the peat-based biofilter, one specialized for the elimination of oxygenated compounds, the others more specialized for elimination of aromatic and halogenated compounds. There was also substrate competition. Bacterial communities were the same over the height of the column, but the more easily biodegradable compounds were used first for the microorganism metabolism when they were present in the gaseous effluent.

Air Pollutants↗
PubMed2005

Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture.

Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin-nucleic acid-probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA-specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 10(2). The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA.

Bacteria↗
PubMed2005

Mass mapping of cancer cell lysates using two-dimensional liquid separations, electrospray-time of flight-mass spectrometry, and automated data processing.

Intact protein masses from immortal, nontransformed MCF10A, a human breast epithelial cell line, and its malignant derivative MCF10CA1a.cl1 have been mapped using a combination of all-liquid separations and automated data interpretation. Preparative liquid isoelectric focusing combined with nonporous silica reverse-phase high-performance liquid chromatography allows efficient separation of a large number of proteins in complex mixtures such as whole-cell lysates. Molecular weight determination of these proteins is achieved using electrospray-time of flight-mass spectrometry, however, manual data analysis for these separations is both complex and time-consuming. Protein mass mapping can be significantly enhanced by automating deconvolution functions typically performed manually, with resulting reductions in hands-on analysis time from 20-30 h per chromatogram to approximately 15 min. This reduction in analysis time allows for rapid screening of cancer cell lines for potential biomarkers over a wider pI range than would otherwise be possible.

Biomarkers, Tumor↗
PubMed2004

Self-association of the yeast nucleosome assembly protein 1.

The self-association properties of the yeast nucleosome assembly protein 1 (yNAP1) have been investigated using biochemical and biophysical methods. Protein cross-linking and calibrated gel filtration chromatography of yNAP1 indicate the protein exists as a complex mixture of species at physiologic ionic strength (75-150 mM). Sedimentation velocity reveals a distribution of species of 4.5-12 Svedbergs (S) over a 50-fold range of concentrations. The solution-state complexity is reduced at higher ionic strength, allowing for examination of the fundamental oligomer. Sedimentation equilibrium of a homogeneous 4.5 S population at 500 mM sodium chloride reveals these species to be yNAP1 dimers. These dimers self-associate to form higher order oligomers at more moderate ionic strength. Titration of guanidine hydrochloride converts the higher order oligomers to the homogeneous 4.5 S dimer and then converts the 4.5 S dimers to 2.5 S monomers. Circular dichroism shows that guanidine-mediated dissociation of higher order oligomers into yNAP1 dimers is accompanied by only slight changes in secondary structure. Dissociation of the dimer requires a nearly complete denaturation event.

Cell Cycle Proteins↗
PubMed1997

Synthesis and two-dimensional electrophoretic analysis of mixed populations of circular and linear RNAs.

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Base Sequence↗
PubMed1992

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.

Benzene Derivatives↗
PubMed1995

Methods for the analysis of triacylglycerols.

This article discusses the methods most commonly employed in the analysis of the triacylglycerols (TAGs) in natural fats and considers the main advantages and disadvantages of each and the techniques for optimising analytical conditions. Complete analysis of the composition of a natural fat calls for a method of extracting and purifying the triglyceride fraction, normally by preparatory thin-layer and column chromatography. Determination of the individual components of triglyceride mixtures still entails certain difficulties, namely, the separation and identification of the TAGs in natural fats. High-performance liquid chromatography (HPLC) offers significant advantages over gas and thin-layer chromatography. Many workers have developed non-aqueous, reversed-phase HPLC systems capable of successfully resolving complex mixtures of TAGs, and combining reversed-phase (RP) HPLC and argentation chromatography may improve the results. Identification of the TAGs separated by HPLC becomes an extremely complex task if many different fatty acids are involved and if the sn-stereoscopic positions on the glycerol are to be determined. Enzymatic analysis and chiral-phase chromatography are capable of localising fatty acids on the TAG molecule. In closing, some of the most interesting biomedical applications of TAG analysis are summarised.

Chromatography, Gas↗
PubMed2006

Plant bioassays for an in situ monitoring of air near an industrial area and a municipal solid waste: Zilina (Slovakia).

The process of a bioindication of genotoxic effects of complex mixtures on the environment using higher plants is very appropriate and effective. We present the results of an in situ indication of the genotoxic effects of polluted environment near Zilina city. For a more complex monitoring we used: the Tradescantia micronucleus (Trad-MCN) assay, the Tradescantia microspore test and an evaluation of the abortivity of the pollen grains of native plant species. We found significant differences in the frequency of the micronuclei when using the Trad-MCN test in local of Duben. The Tradescantia pollen abortivity test showed significant differences in the frequency of the abortive pollen grains between the exposed groups and the control group. By using native plant species in the pollen abortivity test we found significant differences in both of the two locations for the four following species during two consecutive years: Artemisia vulgaris, Melilotus albus, Trifolium pratense, Typha latifolia.

Air Pollutants↗
PubMed2026

Pilot study of allele-specific multi-InDel markers for the detection of extremely unbalanced DNA mixtures.

Mixtures are common in forensic casework, and they represent one of the most challenging types of biological evidence. Traditional short tandem repeat analyses are often associated with limitations when dealing with extremely unbalanced mixtures because alleles from minor contributors can easily be masked by those of major contributors. Consequently, researchers have developed new technologies and methods for improving the analysis of mixtures, spanning upstream DNA extraction and downstream software analysis. Among these, strategies combining allele-specific amplification with compound markers have drawn particular interest because of their ability to selectively detect minor contributors in complex mixtures. In this study, we screened multi-InDels across the entire genome, designed allele-specific primers compatible with the capillary electrophoresis platform, and further explored their potential in unbalanced DNA mixtures and cell-free fetal DNA (cffDNA). Ultimately, a set comprising 10 multi-InDels was developed, and this included two groups of primers that separately amplified the long alleles (L primer set) and short alleles (S primer set). The results demonstrated that each primer pair could detect the minor component at a 1:1000 mixture ratio, whereas the L and S primer sets successfully detected the minor contributors at mixture ratios of 1:200 and 1:500, respectively. Furthermore, in the cffDNA analysis, 60 of 78 informative markers were successfully detected, with the complete detection of all informative markers achieved in 18 mother-child reference pairs. Overall, allele-specific amplification-based multi-InDel markers enabled the sensitive detection of minor contributors, providing a potential strategy for the analysis of unbalanced two-person mixtures.

Allelic-specific amplification↗
PubMed1988

Mechanisms of antigen processing.

Using MAb and monovalent Fab probes and saponin permeabilization we have demonstrated that PEC and TA3 B lymphoma-hybridoma cells contain a significant intracellular pool of Ia. At least in TA3 cells, this intracellular pool was independent of protein synthesis. In PEC, adherence caused redistribution of Ia with disappearance of the intracellular pool. Endocytosis of Ia occurred in both TA3 and PEC, and internalized Ia reached a plateau level corresponding in size to the total intracellular Ia pool revealed by saponin treatment. These results suggest that intracellular Ia is largely in a recycling pool derived from the plasma membrane by endocytosis. Subcellular fractionation studies suggest that Ia processing occurs in endosomes similar to those involved in transferrin processing. Antigen processing by TA3 cells was found to be unaffected by cycloheximide. In contrast, antigen processing by adherent PEC was markedly inhibited by cycloheximide, despite the fact that they maintained surface Ia and were still capable of presenting antigen peptides. This suggests that an important intracellular Ia processing step or antigen processing step was blocked in these cells. Adherent PEC may contain less recycling Ia, making protein synthesis the major source for intracellular Ia and the availability of intracellular Ia sensitive to cycloheximide. Alternatively, the inhibition of antigen processing by cycloheximide in PEC may reflect depletion of enzymes or other factors involved in antigen processing. Proteins and polysaccharides may interfere with the events that result in the formation of an immunogenic Ia-peptide complex. We had previously documented that peptides compete for the binding site of Ia molecules. We discussed here a second form of interference by polysaccharides and microbial products. These materials did not compete or interfere with the binding and presentation of processed peptides by Ia. Rather, their presence inside the macrophage inhibited MHC-dependent presentation of immunogenic proteins by inhibiting intracellular steps in antigen processing. This intracellular interference with antigen presentation can be of major importance in the presentation of complex mixtures of protein and carbohydrates.

Antigen-Presenting Cells↗
PubMed2004

Interaction between halogenated aromatic compounds in the Ah receptor signal transduction pathway.

Many toxic and biochemical responses to halogenated aromatic compounds (HACs) such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs) are mediated through the aryl hydrocarbon receptor (AhR), which is an intracellular cytosolic target for HACs. Environmental exposure to HACs almost always involves complex mixtures of congeners, some of which can antagonize the action of potent HACs such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this work we studied TCDD and representative PCB congeners, alone and in mixture, for their effect on CYP1A gene transcription and protein levels in primary rat hepatocytes. Together with our previous work, our results suggest that formation of the Ah receptor-ligand-DRE (dioxin response element) complex is the principal point of divergence in the mechanism between an AhR agonist and an AhR antagonist. The coplanar PCBs 77 and 126 and the mono-ortho PCB 156 were full agonists toward CYP1A1 gene transcription and CYP1A protein levels, showing typical additive behavior with TCDD to the target molecule AhR. In contrast, the nonplanar PCB 153 antagonized the action of TCDD, even at concentrations that occupied a significant fraction of AhR molecules. Competitive inhibition explains the commonly reported decrease of ethoxyresorufin-O-deethylase (EROD) activity when PCBs are present in high concentrations and the antagonism of PCBs to the EROD activity of TCDD. The result is that Western blotting offers a much more reliable measure of CYP1A protein concentration than does the EROD assay, despite the greater convenience of the latter.

Animals↗
PubMed2005

Extracting single genomes from heterogenous DNA samples: a test case with Carsonella ruddii, the bacterial symbiont of psyllids (Insecta).

Analysis of many bacterial genomes is impeded by the inability to separate individual species from complex mixtures of cells or to propagate cells in pure culture. This problem is an obstacle to the study of many bacterial symbionts that live intracellularly in insects and other animals. To recover bacterial DNA from complex samples, we devised a method that facilitates the cloning of DNA fragments of distinctive G+C contents in order to generate shotgun DNA libraries enriched in inserts having a specific base composition. DNA preparations are first treated with a restriction enzyme having a common cleavage site in a particular genome and then shotgun cloned following size-fractionation. This method was applied to whole bacteriomes of the psyllid, Pachypsylla venusta, which harbors the bacterial symbiont Candidatus Carsonella ruddii. The resulting libraries were highly enriched in bacterial sequences. Through the use of alternate enzymes and partial digests, this technique can be adapted to yield virtually pure DNA libraries for individual bacterial species.

Animals↗
PubMed2000

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Acyltransferases↗
PubMed1998

Genomic structure of three phenotypically different isolates of peach latent mosaic viroid: implications of the existence of constraints limiting the heterogeneity of viroid quasispecies.

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.

Base Sequence↗