Unfractionated Mixture Research Articles

Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses.

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.

Adapting an Isobaric Tag-Labeled Yeast Peptide Standard to Develop Targeted Proteomics Assays.

Targeted proteomics strategies present a streamlined hypothesis-driven approach to analyze specific sets of pathways or disease related proteins. goDig is a quantitative, targeted tandem mass tag (TMT)-based assay that can measure the relative abundance differences for hundreds of proteins directly from unfractionated mixtures. Specific protein groups or entire pathways of up to 200 proteins can be selected for quantitative profiling, while leveraging sample multiplexing permits the simultaneous analysis of up to 18 samples. Despite these benefits, implementing goDig is not without challenges, as it requires access to an instrument application programming interface (iAPI), an elution order and spectral library, a web-based method builder, and dedicated companion software. In addition, the absence of an example test assay may dissuade researchers from testing or implementing goDig. Here, we repurpose the TKO11 standard─which is commercially available but may also be assembled in-lab─and establish it as a de facto test assay for goDig. We build a proteome-wide goDig yeast library, quantify protein expression across several gene ontology (GO) categories, and compare these results to a fully fractionated yeast gold-standard data set. Essentially, we provide a guide detailing the goDig-based quantification of TKO11, which can also be used as a template for user-defined assays in other species.

Oxidation of M252 but not M428 in hu-IgG1 is responsible for decreased binding to and activation of hu-FcγRIIa (His131)

Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa.

I14 West meets east: A systems biology approach to the mechanistic understanding of traditional medicines

Many traditional medicines are of plant origin and are made up of extracts from multiple plants. As a result, identifying the key bioactives in these mixtures and their mode of action is a difficult process. Generally, the approach to this problem has been one of fractionation and testing of individual compounds from these mixtures. However, very often, this standard purification and testing approach fails to recapitulate the full bioactivity of the original complex mixture. Presumably, this is because the compounds in these mixtures do not act individually, but rather in combination with other compounds. We have elected to tackle this problem using a systems biology approach that relies on identifying pathways that are perturbed by unfractionated complex mixtures, and using that information to infer a mode of action for the mixture in question. Our primary tool for this approach is transcriptome profiling using next generation sequencing. An illustration of the value of this approach is the work we have carried out on Compound Kushen Injection (CKI), an injectable Traditional Chinese Medicine based on extracts from the roots of Sophora flavescens and Smilax glabra, used in oncology in China, that contains over 21 compounds based on HPLC fractionation [1] . We have extended this work to additional cancer cell lines identifying core genes/pathways with common expression changes, confirming pathways that affect cell cycle as principal targets of this injection.

Neutrophil extracellular traps promote deep vein thrombosis in mice

Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be prothrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). To explore the source and role of extracellular chromatin in DVT. We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). We demonstrate that the levels of extracellular DNA increase in plasma after 6 h IVC stenosis, compared with sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs' structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6 h and also 48 h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development.

NMR in Metabolomics and Natural Products Research: Two Sides of the Same Coin

Small molecules are central to biology, mediating critical phenomena such as metabolism, signal transduction, mating attraction, and chemical defense. The traditional categories that define small molecules, such as metabolite, secondary metabolite, pheromone, hormone, and so forth, often overlap, and a single compound can appear under more than one functional heading. Therefore, we favor a unifying term, biogenic small molecules (BSMs), to describe any small molecule from a biological source.In a similar vein, two major fields of chemical research,natural products chemistry and metabolomics, have as their goal the identification of BSMs, either as a purified active compound (natural products chemistry) or as a biomarker of a particular biological state (metabolomics). Natural products chemistry has a long tradition of sophisticated techniques that allow identification of complex BSMs, but it often fails when dealing with complex mixtures. Metabolomics thrives with mixtures and uses the power of statistical analysis to isolate the proverbial “needle from a haystack”, but it is often limited in the identification of active BSMs. We argue that the two fields of natural products chemistry and metabolomics have largely overlapping objectives: the identification of structures and functions of BSMs, which in nature almost inevitably occur as complex mixtures.Nuclear magnetic resonance (NMR) spectroscopy is a central analytical technique common to most areas of BSM research. In this Account, we highlight several different NMR approaches to mixture analysis that illustrate the commonalities between traditional natural products chemistry and metabolomics. The primary focus here is two-dimensional (2D) NMR; because of space limitations, we do not discuss several other important techniques, including hyphenated methods that combine NMR with mass spectrometry and chromatography.We first describe the simplest approach of analyzing 2D NMR spectra of unfractionated mixtures to identify BSMs that are unstable to chemical isolation. We then show how the statistical method of covariance can be used to enhance the resolution of 2D NMR spectra and facilitate the semi-automated identification of individual components in a complex mixture. Comparative studies can be used with two or more samples, such as active vs inactive, diseased vs healthy, treated vs untreated, wild type vs mutant, and so on. We present two overall approaches to comparative studies: a simple but powerful method for comparing two 2D NMR spectra and a full statistical approach using multiple samples. The major bottleneck in all of these techniques is the rapid and reliable identification of unknown BSMs; the solution will require all the traditional approaches of both natural products chemistry and metabolomics as well as improved analytical methods, databases, and statistical tools.

NMR-based stable isotope resolved metabolomics in systems biochemistry

An important goal of metabolomics is to characterize the changes in metabolic networks in cells or various tissues of an organism in response to external perturbations or pathologies. The profiling of metabolites and their steady state concentrations does not directly provide information regarding the architecture and fluxes through metabolic networks. This requires tracer approaches. NMR is especially powerful as it can be used not only to identify and quantify metabolites in an unfractionated mixture such as biofluids or crude cell/tissue extracts, but also determine the positional isotopomer distributions of metabolites derived from a precursor enriched in stable isotopes such as (13)C and (15)N via metabolic transformations. In this article we demonstrate the application of a variety of 2-D NMR editing experiments to define the positional isotopomers of compounds present in polar and non-polar extracts of human lung cancer cells grown in either [U-(13)C]-glucose or [U-(13)C,(15)N]-glutamine as source tracers. The information provided by such experiments enabled unambiguous reconstruction of metabolic pathways, which is the foundation for further metabolic flux modeling.

Characterizing the Reproducibility of a Protein Profiling Method for the Analysis of Mouse Bronchoalveolar Lavage Fluid

The detection of biomarkers in biological fluids has been advanced by the introduction of mass spectrometry screening methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), which enables the detection of the presence and the molecular mass of proteins in unfractionated mixtures. The generation of reproducible mass spectra over the course of an experiment is vital in obtaining data in which differences in protein profiles between diseased and healthy states can be assessed correctly. We have developed a protocol to automate the collection of protein profiling data from a large number of samples using MALDI-TOFMS, and we used these samples to characterize the technical reproducibility of the method. This protocol has been used for the analysis of proteins found in bronchoalveolar lavage fluid samples from mice with the ultimate goal of enabling the discovery of differential expression patterns predictive of the development of chronic obstructive pulmonary disease. Samples were purified using magnetic bead-based technology and analyzed on an AnchorChip target plate. Our results demonstrate that the number of peaks detected reproducibly decreases significantly as sample size increases, which motivates the need for technical replicates to be explicitly included in the analysis of MALDI-TOF-based protein profiling studies.

Identification and Validation of Mannose 6-Phosphate Glycoproteins in Human Plasma Reveal a Wide Range of Lysosomal and Non-lysosomal Proteins

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.

Identification of Sites of Mannose 6-Phosphorylation on Lysosomal Proteins

Most newly synthesized soluble lysosomal proteins contain mannose 6-phosphate (Man-6-P), a specific carbohydrate modification that is recognized by Man-6-P receptors (MPRs) that direct targeting to the lysosome. A number of proteomic studies have focused on lysosomal proteins, exploiting the fact that Man-6-P-containing forms can be purified by affinity chromatography on immobilized MPRs. These studies have identified many known lysosomal proteins as well as many proteins not previously classified as lysosomal. The latter are of considerable biological interest with potential implications for lysosomal function and as candidates for lysosomal storage diseases of unknown etiology. However, a significant problem in interpreting the biological relevance of such proteins has been in distinguishing true Man-6-P glycoproteins from simple contaminants and from proteins associated with true Man-6-P glycoproteins (e.g. protease inhibitors and lectins). In this report, we describe a mass spectrometric approach to the verification of Man-6-phosphorylation based upon LC-MS of MPR-purified proteolytic glycopeptides. This provided a useful tool in validating novel MPR-purified proteins as true Man-6-P glycoproteins and also allowed identification of low abundance components not observed in the analysis of the total Man-6-P glycoprotein mixture. In addition, this approach allowed the global mapping of 99 Man-6-phosphorylation sites from 44 known lysosomal proteins purified from mouse and human brain. This information is likely to provide useful insights into protein determinants for this modification and may be of significant value in protein engineering approaches designed to optimize protein delivery to the lysosome in therapeutic applications such as gene and enzyme replacement therapies.

The human brain mannose 6-phosphate glycoproteome: A complex mixture composed of multiple isoforms of many soluble lysosomal proteins

The lysosome is a membrane delimited cytoplasmic organelle that contains at least 50 hydrolytic enzymes and associated cofactors. The biomedical importance of these enzymes is highlighted by the many lysosomal storage disorders that are associated with mutations in genes encoding lysosomal proteins, and there is also evidence that lysosomal activities may be involved in more widespread human diseases. The aim of this study was to characterize the human brain lysosomal proteome with the goal of establishing a reference map to investigate human diseases of unknown etiology and to gain insights into the cellular function of the lysosome. Proteins containing mannose 6-phosphate (Man6-P), a carbohydrate modification used for targeting resident soluble lysosomal proteins to the lysosome, were affinity-purified using immobilized Man6-P receptor. Fractionation by two-dimensional electrophoresis resolved a complex mixture comprising approximately 800 spots. Constituent proteins in each spot were identified using a combination of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (both peptide mass fingerprinting and tandem mass spectrometry) [corrected] on in-gel tryptic digests and N-terminal sequencing. In a complementary analysis, we also analyzed a tryptic digest of the unfractionated mixture by liquid chromatography MS/MS. In total, 61 different proteins were identified. Seven were likely contaminants associated with true Man6-P glycoproteins. Forty-one were known lysosomal proteins of which 11 have not previously been reported to contain Man6-P. An additional nine proteins were either uncharacterized or proteins not previously reported to have lysosomal function. We found that the human brain Man6-P-containing lysosomal proteome is highly complex and contains more proteins with a much greater number of individual isoforms than found in previous studies of Man6-P glycoproteomes.

Approaching complete peroxisome characterization by gas-phase fractionation.

We examined the utility of gas-phase fractionation (GPF) in the m/z dimension to increase proteome coverage and reproducibility of peptide ion selection by direct microliquid chromatography/electrospray ionization-tandem mass spectrometry (microLC/ESI-MS/MS) analysis of the peptides produced by proteolytic digestion of unfractionated proteins from a yeast whole-cell lysate and in a peroxisomal membrane protein fraction derived from isolated yeast peroxisomes. We also investigated GPF in the relative ion intensity dimension and propose denoting the two types of GPF as GPF(m/z) and GPF(RI). Comparison of results of direct nuLC/ESI-MS/MS analysis of the unfractionated mixture of peptides from proteolysis of a yeast whole cell lysate by DD ion selection from 400-1800 m/z in triplicate and GPF(m/z) from 400-800, 800-1200 and 1200-1800 produced the following results: (i) 1.3 x more proteins were identified by GPF(m/z) for an equal amount of effort (i.e., 3 microLC/ESI-MS/MS) and (ii) proteins identified by GPF(m/z) had a lower average codon bias value. Use of GPF(RI) identified more proteins per m/z unit scanned than GPF(m/z) or triplicate analysis over a wide m/z range. After tryptic digestion of all the proteins from a discontinuous Nycodenz gradient fraction known to be enriched with yeast peroxisomal membrane proteins we detected 93% (38/41) of known peroxisomal proteins using GPF(m/z), but only 73% using a standard wide m/z range survey scan.

A novel activation of Ca(2+)-activated Cl(-) channel in Xenopus oocytes by Ginseng saponins: evidence for the involvement of phospholipase C and intracellular Ca(2+) mobilization.

1. The signal transduction mechanism of ginsenosides, the active ingredients of ginseng, was studied in Xenopus oocytes using two-electrode voltage-clamp technique. Ginseng total saponin (GTS), i.e., an unfractionated mixture of ginsenosides produced a large outward current at membrane potentials more positive than -20 mV when it was applied to the exterior of oocytes, but not when injected intracellularly. The effect of GTS was concentration-dependent (EC(50): 4.4 microg ml(-1)) and reversible. 2. Certain fractionated ginsenosides (Rb(1), Rb(2), Rc, Rf, Rg(2) and Ro) also produced an outward current in a concentration-dependent manner with the order of potency of Rf>Ro>Rb(1)=Rb(2)>Rg(2)>Rc. Other ginsenosides (Rd, Re and Rg(1)) had little or no effect. 3. The GTS effect was completely blocked by bath application of the Ca(2+)-activated Cl(-) channel blocker niflumic acid and by intracellular injection of the calcium chelator BAPTA or the IP(3) receptor antagonist heparin. Also, the effect was partially blocked by bath-applied U-73122, a phospholipase C (PLC) inhibitor and by intracellularly injected GTP gamma S, a non-hydrolyzable GTP analogue. Whereas, it was not altered by pertussin toxin (PTX) pretreatment. 4. These results indicate that: (1) interaction of ginsenosides with membrane component(s) at the extracellular side leads to Ca(2+)-activated Cl(-) channel opening in Xenopus oocyte membrane; and (2) this process involves PLC activation, the release of Ca(2+) from the IP(3)-sensitive intracellular store and PTX-insensitive G protein activation.

RNA-transfected dendritic cells in cancer immunotherapy.

The field of cancer immunotherapy has been recently invigorated by the discovery that vaccination with dendritic cells (DCs) pulsed with tumor antigens is a potent strategy to elicit protective immunity in tumor-bearing animals. The recognition that the cellular arm of the immune response is best equipped to recognize tumor cells as foreign and to lead to their eradication has shifted the emphasis in vaccine development. Vaccines that induce cellular responses, especially by the CD8+ cytotoxic T lymphocyte (CTL) arm of the immune system, are now favored over those that activate humoral immunity. At the same time, DCs have emerged as the most potent antigen-presenting cells (APCs) for eliciting antitumor CTLs. DCs can be generated from cancer patients by culturing adherent PBMCs from the patients for 5–7 days in the presence of cytokines (1). The major research effort in many labs involves the choice of tumor antigen with which to load these DCs. The issues being addressed are, first, the composition of the antigen, whether a defined tumor antigen or an unfractionated mixture of tumor-derived antigens; and second, the form in which the antigen should be presented, whether as a polypeptide or a nucleic acid. Here, we focus on the use of RNA-transfected DCs in cancer immunotherapy, reviewing current data on the transfection of DCs with RNAs encoding either defined or unfractionated tumor antigens. We also consider the advantages and disadvantages of employing RNA transfection in loading DCs with tumor antigens and the merits of RNA transfection in situations where a low level of transiently expressed protein is sufficient to allow antigen presentation.

Antibody binding characteristics of geometrical isomers of testosterone 3-(O-carboxymethyl)oxime

Geometrical isomers of testosterone 3-(O-carboxymethyl)oxime and their histamine derivatives were purified on reverse-phase high pressure liquid chromatography, and their antibody binding characteristics were studied. Using a competitive testosterone enzyme immunoassay, the unfractionated mixture of the oximes showed 75% cross-reactivity with respect to testosterone, whereas the isolated 3 Z- and 3 E-isomers showed 124% and 26% cross-reactivity, respectively. The cross-reactivity was increased in the histamine derivatives, but the difference in cross-reactivity of the two isomers was reduced. Suppression of the ionization of the carboxyl group by lowering the pH of the incubation mixture in the antigen-antibody binding step raised the cross-reactivity of the mixture of free oximes to 128%, at pH 4.0. Thus, the geometry and ionization state of the carboxymethyl oxime group has a profound effect on the affinity of the isomers for the antibody.

Deconvolution of Combinatorial Oligonucleotide Libraries by Electrospray Ionization Tandem Mass Spectrometry

Studies were undertaken to explore the application of tandem mass spectrometry for the structure analysis of unfractionated mixtures of oligonucleotides. Limited combinatorial libraries were constructed of mixtures of 8-mers (NGACACNG; nine compounds) and 12-mers (NGACTNAGACNG; 27 compounds), where N is any of the 2‘-deoxyribonucleotides of uracil, thymine, or 5-[N-(aminoethyl)-3-acrylimido]uracil. Molecular mass measurements of the mixture components (single mass analyzer) in the simplest cases or acquisition of collision-induced dissociation mass spectra (tandem mass analyzers) for mixtures of sequence isomers of the same molecular mass were used to establish guidelines for structure assignments. Although the construction of mass sequencing ladders from gas phase backbone cleavage reactions is notably more complex in the case of isomeric mixtures than for the single isomer case, assignment ambiguities are reduced by the presence of nonrandomized sequence positions and by recognition of likely fragment ion relative abundances. The approaches described offer a significant advance toward solving problems common to the development of oligonucleotide therapeutics using combinatorial synthesis techniques.

Effects of xylooligosaccharides on suspension-cultured cells and protoplasts of Pinus radiata

The effects of xylooligosaccharides isolated from the cell walls of Betula platyphylla var. japonica on cells and protoplasts of Pinus radiata were examined. The addition of a semi-purified mixture of xylooligosaccharides at a concentration of 5µg.ml−1 promoted elongation of cultured cells, whereas the neutral fraction of this mixture had no effect; a similar effect was seen in the presence of conditioned medium. The unfractionated mixture of xylooligosaccharides was also found to enhance the viability of protoplasts prepared from cell cultures of Pinus radiata in a concentration dependent manner, highly similar to the effect provided by addition of medium conditioned by pine cells. Such effects are considered to be due to the addition of components that play a structural role in the cell wall of pines. It is inferred that the acidic components of the xylooligosaccharide mixture derived from t Betula are responsible for this effect in the distant pine species. It is speculated that acidic xylooligosaccharides operate either by replacing, or mimicking, the natural cell wall components required for growth and development of pine cultured cells.

The preferential cytolytic T lymphocyte response to immunodominant minor histocompatibility antigen peptides.

C57BL/6 mice preferentially generate cytolytic T lymphocytes (CTL) to a limited number of immunodominant minor antigens and associated immunogenic peptides when primed with H2-matched Balb.B spleen cells despite multiple minor histocompatibility (H) antigen differences. We have examined the complexity of dominant H antigens recognized by these CTLs to estimate the number of peptides associated with single antigens. Peptides eluted from Kb molecules of lymphoblasts from Balb.B and CXB recombinant inbred (RI) strains were tested for sensitization of RMA-S cells for lysis by short-term C57BL/6 CTL lines specific for Balb.B and CXB strains. Anti-Balb.B CTLs recognized four Kb-bound peptides; subsets of these peptides were recognized by anti-CXB CTLs when tested with peptides from the respective CXB strains. Single peptides segregated independently among the CXB strains, confirming that single peptides were encoded by independently segregating alleles. These peptides were expressed in diverse inbred mouse strains and were recognized preferentially by C57BL/6 CTLs stimulated by different inbred mouse strains. This set of peptides was subclassified by their capacity to sensitize targets when presented in unfractionated mixtures of Kb-bound peptides. The peptide associated with the previously classified dominant CTT-2 antigen was the only peptide to strongly sensitize RMA-S cells for lysis under these conditions. These results suggest that dominant peptides have a wide strain distribution and may have a distinct advantage over dominated peptides in binding to class I molecules and/or in presentation to CTLs.

Effects of calcium‐binding proteins (S‐100a o , S‐100a, S‐100b) on desmin assembly in vitro

S-100a(o), the alpha alpha isoform of a subfamily of Ca(2+)-binding proteins of the EF-hand type expressed in cardiac and skeletal muscle cells, is reported to inhibit the assembly of the intermediate filament subunit desmin and to stimulate the disassembly of desmin intermediate filaments in the presence of micromolar levels of free Ca(2+). These effects are dose-dependent with respect to the S-100a(o) concentration and maximal at a desmin/S-100a(o) (dimer) molar ratio of approximately 2. Other members of the S-100 subfamily [S-100a (alpha beta) and S-100b (beta beta) and the unfractionated mixture of S-100a plus S-100b produce qualitatively similar effects on desmin assembly, with a potency that depends on the fraction of S-100alpha subunit (the most potent) or S-100beta subunit (the least potent) present in the S-100 isoforms tested. A binding stoichiometry of 2 mol of desmin/mol of S-100a(o) (dimer) and an affinity in the submicromolar range are calculated. The S-100beta subunit also interacts with desmin, but with a lower affinity compared with S-100alpha. By contrast, the S-100-like proteins calcyclin and p11 neither interact with desmin nor affect desmin assembly. The present data suggest that S-100a(o) might play a role in the regulation of the state of assembly of desmin intermediate filaments.

Mutation spectra of chemical fractions of a complex mixture: role of nitroarenes in the mutagenic specificity of municipal waste incinerator emissions

Using an ion-exchange procedure coupled to a microsuspension Salmonella assay, we fractionated the dichloromethane-extractable particulate organics emitted by a municipal waste incinerator. Most (80–95%) of the mutagenic activity resided in the neutral/base fraction; however, the polar neutral fraction accounted for 12% of the direct-acting mutagenic activity. The mutagenic potencies of the whole extract and the various fractions were 4–15 times greater in the absence than in the presence of S9. Results with strains deficient in classical nitroreductase (TA98NR) and transacetylase (TA98/1,8-DNP 6) indicated that a majority of the direct-acting mutagenicity was due to nitroarenes. This was confirmed by bioassay-directed subfractionation of the neutral/base faction by a cyanopropyl/HPLC method. The mutations in −3,000 revertants (∼ 400 each induced in TA98 by the whole extract, the neutral/base and polar neutral fractions from the ion-exchange column and 3 of the neutral/base subfractions from the HPLC column; along with 200 revertants each induced by the model nitroarene 1-nitropyrene (1NP) in strains TA98, TA1538 and TA100) were analyzed by probe hybridization and PCR DNA sequence analysis. The results indicated that nitroarenes such as 1NP that eluted in the neutral/base fraction accounted for at least 50% of the direct-acting mutagenicity and induced only a hotspot 2-base deletion in the sequence (CG) 4 in TA98. In contrast, most of the complex frameshifts (a frameshift with a flanking base substitution) induced by the whole extract were induced by nitroarenes other than 1NP that were activated by transacetylation and that eluted in the polar neutral fraction. This study (1) identifies nitroarenes as an important contributor to the mutagenic activity of the emissions from municipal waste incinerators; (2) confirms our previous conclusion that the mutation spectrum of a complex mixture reflects the dominance of particular classes of chemical mutagens within the mixture; and (3) demonstrates the possibility of isolating certain chemical fractions of a complex mixture that induce certain classes of mutations produced by the whole, unfractionated mixture.