Detection of Molecular Ions of Cerebroside by Gas Chromatography—Mass Spectrometry

Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX-53. The molecular weight of the enzyme was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH 4.7 by isoelectric focusing. This enzyme contained 1.12 atoms of manganese and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzyme was 9.5 mM for oxalic acid at the optimal pH 4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I^–, Br^–, semicarbazide, and hydroxylamine.     

Study of Caprine β-casein using Reversed-phase High-performance Liquid Chromatography and Mass Spectroscopy: Identification of a New Genetic Variant

The genotypes and the main phosphorylation levels of β-casein of goat milk were studied using RP-HPLC/ESI-MS. A new variant of caprine β-casein named E has been characterized using RP-HPLC/ESI-MS, MALDI-MS and NanoESI MS/MS methods. Its sequence differed from that of variant A in the mono amino acid substitution D47 → Y47, which resulted in a 48 Da experimental mass difference between them. The calculated molecular mass of the new variant E 6 P was estimated as 23,869 Da. Its phosphorylation pattern was similar to that of variant A, the most abundant types being those with 5 and 6 P in similar quantities.     

Isolation and characterization of microsatellite loci in Peganum harmala (Peganaceae), an important resist-drought and medicinal plant

Peganum harmala is a herb grows spontaneously in arid and rocky areas. From ancient time, it has been claimed to be an important medicinal plant in rough environment. In this study, we developed 12 microsatellite loci from P. harmala by the combining biotin capture method for the first time. A total of 31 microsatellite sequences were recovered through screening the library and 12 of them are polymorphic. The number of alleles per locus in 36 sampled individuals ranged from 3 to 8, expected heterozygosity and observed heterozygosity ranged from 0.1381 to 0.6821 and from 0.3573 to 0.8739, respectively. In addition, all markers have been crossly checked in the other congeneric species. These microsatellite markers would provide a useful tool for investigating the genetic diversity and study the population genetic structure in detail.     

Combined Liquid Chromatography–Tandem Mass Spectrometry Analysis of Progesterone Metabolites

Progesterone has a number of important functions throughout the human body. While the roles of progesterone are well known, the possible actions and implications of progesterone metabolites in different tissues remain to be determined. There is a growing body of evidence that these metabolites are not inactive, but can have significant biological effects, as anesthetics, anxiolytics and anticonvulsants. Furthermore, they can facilitate synthesis of myelin components in the peripheral nervous system, have effects on human pregnancy and onset of labour, and have a neuroprotective role. For a better understanding of the functions of progesterone metabolites, improved analytical methods are essential. We have developed a combined liquid chromatography—tandem mass spectrometry (LC-MS/MS) method for detection and quantification of progesterone and 16 progesterone metabolites that has femtomolar sensitivity and good reproducibility in a single chromatographic run. MS/MS analyses were performed in positive mode and under constant electrospray ionization conditions. To increase the sensitivity, all of the transitions were recorded using the Scheduled MRM algorithm. This LC-MS/MS method requires small sample volumes and minimal sample preparation, and there is no need for derivatization. Here, we show the application of this method for evaluation of progesterone metabolism in the HES endometrial cell line. In HES cells, the metabolism of progesterone proceeds mainly to (20S)-20-hydroxy-pregn-4-ene-3-one, (20S)-20-hydroxy-5α-pregnane-3-one and (20S)-5α-pregnane-3α,20-diol. The investigation of possible biological effects of these metabolites on the endometrium is currently undergoing.     

Fatty acid–genotype interactions and cardiovascular risk

Cardiovascular disease (CVD) risk and rate of progression is determined by genetic, environmental and behavioural factors. Majority of genotype–diet–CVD phenotype research till date has focussed on the interactive impact of single nucleotide polymorphisms (SNP) and dietary fat composition, on blood lipids levels, with strong evidence of the existence of hypo- and hyper-responders. However, a recognised concern in the field of nutrigenetics is a lack of consistency between findings of different studies. This apparent lack of consistency is likely to be attributable to the impact of factors such as ethnicity and gender on the ‘size’ of nutrigenetic interactions, a clear understanding of which needs to be gained. Although not yet ready for widespread use, in the future a greater use of genetic profiling is likely to enhance current strategies of CVD prediction, and improve the design of more personalised approaches to minimise risk in the individual.     

The Identification of 5′-Deoxy-5′-Methylthioguanosine in Human Urine by Gas Chromatography/Mass Spectrometry

Abstract

5′-Deoxy-5′-methylthioguanosine (MTG) has been identified in human urine by combined gas chromatography/mass spectrometry. Preliminary identification of MTG in the urine of a lung cancer patient was based upon mass spectral comparisons of the trimethylsilylated (TMS) urinary component to both MTA-(TMS)₃ and guanosine-(TMS)₅. Structural confirmation was obtained by comparing the mass spectral and chromatographic characteristics of authentic MTG to those of the urinary component.     

Validation of Biomarkers for Distinguishing Mycobacterium tuberculosis from Non-Tuberculous Mycobacteria Using Gas Chromatography?Mass Spectrometry and Chemometrics

Tuberculosis (TB) remains a major international health problem. Rapid differentiation of Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM) is critical for decisions regarding patient management and choice of therapeutic regimen. Recently we developed a 20-compound model to distinguish between MTB and NTM. It is based on thermally assisted hydrolysis and methylation gas chromatography-mass spectrometry and partial least square discriminant analysis. Here we report the validation of this model with two independent sample sets, one consisting of 39 MTB and 17 NTM isolates from the Netherlands, the other comprising 103 isolates (91 MTB and 12 NTM) from Stellenbosch, Cape Town, South Africa. All the MTB strains in the 56 Dutch samples were correctly identified and the model had a sensitivity of 100% and a specificity of 94%. For the South African samples the model had a sensitivity of 88% and specificity of 100%. Based on our model, we have developed a new decision-tree that allows the differentiation of MTB from NTM with 100% accuracy. Encouraged by these findings we will proceed with the development of a simple, rapid, affordable, high-throughput test to identify MTB directly in sputum.     

Fatty acid composition of odonate’s eyes

We have studied the fatty acid composition of eyes of amphibiotic insects, namely, the odonate Sympetrum flaveolum. The main polyunsaturated fatty acid of odonate’s eyes has been found to be 20:5n-3 (eicosapentaenoic fatty acid, EPA) rather than 18:2n-6 and 18:3n-3, which usually dominate in eyes of terrestrial insects, or 22:6n-3, which dominates in eyes of vertebrates. The prevalence of EPA in odonate’s eyes probably provides a more effective transmission of light signal in this animal compared to terrestrial insects. It is important for odonates because vision plays a decisive role in finding and catching prey.     

Effects of long-term simulated acid rain on a plant–herbivore interaction

Anthropogenic pollution causes oxidative stress in plants and reactive oxygen species (ROS) are diminished by antioxidative enzymes and small molecular antioxidants. Pollution may also affect the performance of plant-eating animals by increasing or decreasing their performance. The effects of pollution cannot be fully understood without knowledge of how pollution affects the interactions with the third trophic level, namely natural enemies and diseases of herbivores. In this study, we examined how long-term (19 yr) acid rain pollution affects (i) the oxidative responses in mountain birch foliage and (ii) the growth and immune responses of autumnal moth larvae. We found that pollution caused a 50% increase (p<0.05) in the peroxidase activities (PODs) in birch leaves whereas polyphenoloxidase (PPO) or catalase (CAT) activities were not affected, suggesting that PODs play an important role in the quenching of the oxidative stress in birches. In polluted trees, phenoloxidases probably acted as antioxidative not prooxidative enzymes, which was shown as positive relations between enzyme activities (PPO, CAT) and larval performance (pupal weights). Although acid rain pollution did not have any direct effect on either pupal weight or the length of larval period, the stronger acid rain treatment reduced slightly (6% in females) the encapsulation response of pupae. A decrease of this magnitude might be too small to have measurable effects on the incidence of moth outbreaks.     

Long-Chain Free Fatty Acid Profiling Analysis by Liquid Chromatography–Mass Spectrometry in Mouse Treated with Peroxisome Proliferator-Activated Receptor α Agonist

A change in the free fatty acid (FFA) profile reflects an alteration in the lipid metabolism of peripheral tissue. A high-throughput quantitative analysis method for individual FFAs therefore needs to be established. We report here an optimized LC-MS assay for a high-throughput and high-sensitivity analysis of the 10 major long-chain FFAs in mouse plasma and liver. This assay enables quantification of individual FFAs by using trace amounts of samples (2 µL of plasma and 10 mg of liver tissue). We apply this method to analyze the FFA profile of plasma and liver samples from an obese mouse model treated with bezafibrate, the peroxisome proliferator-activated receptor α (PPARα) agonist, and show a change in the FFA profile, particularly in the palmitoleic and oleic acid contents. This assay is useful for quantifying individual FFAs and helpful for monitoring the condition of lipid metabolism.     

Gas chromatography–mass spectrometry analysis of fatty acid profiles of Antarctic and non-Antarctic yeasts

The fatty acid profiles of Antarctic (n = 7) and non-Antarctic yeasts (n = 7) grown at different temperatures were analysed by gas chromatography–mass spectrometry. The Antarctic yeasts were enriched in oleic 18:1 (20–60 %), linoleic 18:2 (20–50 %) and linolenic 18:3 (5–40 %) acids with lesser amounts of palmitic 16:0 (<15 %) and palmitoleic 16:1 (<10 %) acids. The non-Antarctic yeasts (n = 4) were enriched in 18:1 (20–55 %, with R. mucilaginosa at 75–80 %) and 18:2 (10–40 %) with lesser amounts of 16:0 (<20 %), 16:1 (<20 %) and stearic 18:0 (<10 %) acids. By contrast, Saccharomyces cerevisiae strains (n = 3) were enriched in 16:1 (30–50 %) and 18:1 (20–40 %) with lesser amounts of 16:0 (10–25 %) and 18:0 (5–10 %) acids. Principal component analysis grouped the yeasts into three clusters, one belonging to the S. cerevisiae strains (enriched in 16:0, 16:1 and 18:1), one to the other non-Antarctic yeasts (enriched in 18:1 and 18:2) and the third to the Antarctic yeasts (enriched in 18:2 and 18:3).     

Are feeding preferences of white-tailed deer related to plant constituents?

Controlled feeding experiments can provide valuable insights into food selection of herbivores. We conducted cafeteria trials on captive yearling white-tailed deer (Odocoileus virginianus) during 2 years to determine feeding preferences in relation to plant chemical constituents, i.e., nitrogen and fibers. We simultaneously offered 8 species of cultivated and wild plants in monthly foraging trials conducted from June to October. We predicted that species preferences would be positively related to protein content from June to August and to digestible energy in September and October. As predicted, crude protein (CP) was positively related to feeding preferences, particularly as summer progressed. Feeding preferences were also negatively related to fiber content, especially in early summer. Our results indicate high protein needs over the complete growing season for yearling deer but a decrease in overall plant selectivity as summer progresses. Our results also suggest that deer browsing on cultivated plants might be due to higher CP content of cultivated plants than wild plants. To prevent deer impact on crops, managers should favor regeneration of plants rich in CP content in forests. © 2011 The Wildlife Society.     

Significant Variation for Bio-oil Compounds After Pyrolysis/Gas Chromatography–Mass Spectrometry of Cobs and Stover Among Five Near-Isogenic Brown Midrib Hybrids in Maize

We analyzed five near-isogenic brown midrib hybrids in maize via pyrolysis/gas chromatography–mass spectrometry (Py/GC-MS) in order to determine how differing lignin composition and structure impacts individual bio-oil compounds. Twenty-six compounds were analyzed for differences among the five hybrids and between cob and stover materials. We found statistically significant differences for 9 compounds, when comparing the 5 hybrids, and 17 significant differences when comparing maize cobs with stover. Our data indicate that it may be possible to predict phenolic compounds within bio-oil based on cell wall lignin composition. The genetic variation observed in this study suggests that bio-oil quality can be improved by plant breeding.     

One man’s quest for plant constituents of therapeutic value

Distinguished Economic Botanist’s address delivered 13 June 1989 at the 30th Annual Meeting of the Society for Economic Botany, University of Tennessee, Knoxville.     

Indole-3-acetic acid in plant–microbe interactions

Indole-3-acetic acid (IAA) is an important phytohormone with the capacity to control plant development in both beneficial and deleterious ways. The ability to synthesize IAA is an attribute that many bacteria including both plant growth-promoters and phytopathogens possess. There are three main pathways through which IAA is synthesized; the indole-3-pyruvic acid, indole-3-acetamide and indole-3-acetonitrile pathways. This chapter reviews the factors that effect the production of this phytohormone, the role of IAA in bacterial physiology and in plant–microbe interactions including phytostimulation and phytopathogenesis.     

Are tissue cultures of Peganum harmala a useful model system for studying how to manipulate the formation of secondary metabolites?

This article reviews our present knowledge on the formation of tryptophan derived secondary metabolites in tissue cultures of Peganum harmala. With the presence of -carboline alkaloids and serotonin, P. harmala contains two rather simple, interrelated biosynthetic pathways. The long term disadvantage of low and unstable productivity of P. harmala suspension culture has recently been overcome by establishing highly productive hairy root cultures. The first -carboline alkaloid biosynthetic enzymes, specific for the O-methylation of harmalol and harmol as well as for the oxidation of harmaline to harmine, have been detected in these cultures, and they should thus provide a suitable source for studying the yet unknown initial two enzymatic steps of -carboline alkaloid biosynthesis. Seedlings of P. harmala have also been successfully transformed with constructed strains of Agrobacterium, as demonstrated by the overexpression of a tryptophan decarboxylase gene from Catharanthus roseus in cultures of P. harmala. In such transgenic cultures a large overproduction of serotonin was observed. The relative simplicity of these pathways and the rather easy handling of the cultures could make P. harmala a useful and attractive model system for studying the interaction, regulation and manipulation of secondary pathways in cultured cells.Abbreviations TDC tryptophan decarboxylase - tdc gene of tryptophan decarboxylase