This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the ␦-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid ␦-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% ␦-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulationspecific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid ␦-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of ␦-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed. Dormant bacterial endospores formed by the genera Bacillus and Clostridium are the most resistant living structures known and are able to survive thousands if not millions of years (9). During the quiescent state, spores exhibit high-level resistance to many treatments, including heat, UV light, desiccation, and the action of deleterious chemicals. As a result of their resistance properties, spores are able to survive many food preservation and pasteurization procedures and thus cause huge problems to the food industry (11).Endospores are characterized by a relatively dehydrated protoplast encased in integument layers (20). The most prominent of the integuments are the spore coat layers which determine the physical properties of the spore surface and are responsible for resistance to enzymatic assault (56). However, the spore coats are not involved in the maintenance of dormancy and heat resistance (56). Between the spore coats and the protoplast membrane is a thick layer of bacterial peptidoglycan, consisting of two sublayers. Innermost is the ...

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The content of zearalenone and its metabolites in urine and tissue samples from pigs fed zearalenone-contaminated oats was established by analytical methods combining solid-phase extraction cleanup of the samples with highly selective liquid chromatography-mass spectrometry (LC-MS)/MS detection. Investigation of the urine samples revealed that approximately 60% of zearalenone was transformed in vivo to alpha-zearalenol and its epimer beta-zearalenol in a mean ratio of 3:1. Zeranol and taleranol as further metabolites could only be detected in trace amounts. Zearalanone was identified at considerable concentrations, though only in a couple of samples. In contrast, liver samples contained predominantly alpha-zearalenol, and to a minor extent beta-zearalenol and zearalenone, with a mean ratio of alpha-/beta-zearalenol of 2.5:1, while zeranol, taleranol, or zearalanone could not be identified in any of the investigated samples. The degree of glucoronidation was established for zearalenone as 27% in urine and 62% in liver; for alpha-zearalenol as 88% in urine and 77% in liver; and for beta-zearalenol as 94% in urine and 29% in liver. Analyses of muscle tissue revealed relatively high amounts of nonglucuronidated zeranol and alpha-zearalenol together with traces of taleranol and zearalenone, indicating that the metabolism of zearalenone and its metabolites is not restricted to hepatic and gastrointestinal metabolic pathways.

Further research with the aim of identifying the gene or genes responsible for conferring metabolic resistance to HPPD inhibitors should focus on cytochrome P450s. Such research is important because non-target-site-based resistance (NTSR) poses the threat of cross resistance to other chemical classes of HPPD inhibitors, other herbicide modes of action, or even unknown herbicides. © 2017 Society of Chemical Industry.

A group of five heifers were fed for 84 days with 2 kg of zearalenone-contaminated oats (1370 microg/kg) resulting in an average daily intake of 2740 microg of zearalenone per animal. In a parallel experiment five heifers were implanted with two 25 mg zeranol pellets, one at the beginning of the study and one after 42 days, and fed with 2 kg of "blank" control oats (79 microg/kg, daily intake = 158 microg). A third group of five animals were also fed with 2 kg of "blank" oats and served as control. Urine samples of all animals were collected every 5-6 days during the whole period of the study. Animals of all three groups were killed 84 days after the beginning of the feeding study. Tissue samples (back, femoral region, liver, and residues of implanted pellets) were taken during post-mortem investigations. The content of zearalenone and zeranol and their metabolites in urine and tissue samples was established by an analytical method combining solid-phase extraction and high-performance liquid chromatography-tandem mass spectrometry. Urinary excretion rates of zeralenone and zeranol were calculated from these results.

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