Betaine distribution in Angiosperms

Over 140 Angiosperm species, included in 45 of the 62 orders listed by Engler, have been investigated for the presence of betaines, which were detected in 86% of the species examined and in 43 of the orders. Moreover, betaines were reported earlier in a further seven of the orders. Thus, it can be concluded that betaines are very widely distributed in Angiosperms. The most commonly detected betaines in the study were glycinebetaine and trigonelline, although others, such as prolinebetaine, trans-4-hydroxyprolinebetaine and pipecolatebetaine were found, although with a very restricted distribution. In the large majority of species tested, betaine levels were low (below 0.1%, dry weight).     

Genotypic Variation for Glycinebetaine among Public Inbreds of Maize

Screening of a range of public maize (Zea mays L.) inbred lines for glycinebetaine (betaine) content over two growing seasons (1987 and 1988), using fast atom bombardment mass spectrometry, has identified 19 public inbred lines which all exhibit low betaine levels (<100 nanomoles per gram fresh weight). These include common inbreds such as A188, A619, B37, H95, N6, and Oh43. Several inbreds exhibit high betaine levels (3000 to 10000 nanomoles per gram fresh weight); in these strongly betaine-positive inbreds, betaine levels tended to be, on average, 1.38-fold greater in the 1988 growing season presumably in part due to field water deficits experienced during the drought of 1988. Where several different sources of the same inbred line were available (including cytoplasmic male sterile and restored lines of A632, B37, B73, Oh43, and WF9), betaine levels were found to be similar when the inbreds were tested in the same environment. Because W22-R/r-X1 was found to be strongly betaine-positive, it should be possible to map the putative recessive gene(s) determining betaine deficiency to specific chromosome(s) from monosomics resulting from crosses between W22-R/r-X1 and betaine-deficient lines.     

Rapid MALDI-TOF Mass Spectrometry Strain Typing during a Large Outbreak of Shiga-Toxigenic Escherichia coli

Background

In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification.

Methods

Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak.

Results

Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates.

Conclusions

MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.     

Nutritional quality and yield of seedling alfalfa established with a barley companion crop and weeds

Alfalfa (Medicago sativa L.) was established with barley (Hordeum vulgare L.) and weeds to determine the effect of weed management treatments on yield and nutritional quality of alfalfa and companion crop and weeds. Alfalfa+barley and alfalfa+wild oat (Avena fatua L.) had much higher yields in August of the first year than alfalfa alone. Total yields for the first three harvests were similar for alfalfa established weed free, with broad leaf weeds, wild oat or green foxtail (Setaria viridis L.). Protein contents in forage from the August harvest were 228, 196, 117 and 181 g/kg dry matter (DM) in weed free alfalfa, alfalfa plus broadleaf weeds, alfalfa plus wild oat, and alfalfa plus green foxtail, respectively. Alfalfa+barley had higher total in vitro gas production than pure alfalfa, which is correlated with the digestibility of the forage. Total in vitro gas production indicated that alfalfa+barley+weeds had slightly lower digestibility than weed free alfalfa+barley. However, alfalfa with a high percentage of redroot pigweed (Amaranthus retroflexus L.), wild oat or green foxtail had similar or higher in vitro gas production than pure alfalfa. The results from this experiment support a previous economic study that indicated herbicide application for weed control in seedling alfalfa was not necessary.     

Allelopathic interference of alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) genotypes to annual ryegrass (<Emphasis Type="Italic">Lolium rigidum</Emphasis>)

Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.     

New crop residues and forages for Western Canada: Assessment of feeding value in vitro and response to ammonia treatment

A survey was conducted on the feed value for ruminants of a variety of crop residues and forages, with particular emphasis on crop residues and forages not currently grown extensively in Western Canada. Crop residues with feed values significantly higher than wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) straw were investigated, and the response to ammonia treatment was evaluated, to determine which materials could be appreciably improved in feed value by this technique. Some of the crop residues evaluated by digestibility in vitro and crude-protein content had a significantly higher feed value, without chemical processing, than the cereal straws often given to cattle in Western Canada. Ammonia treatment improved both the organic matter digestibility in vitro and crude-protein content of some residues significantly, while other materials showed only an improvement in crude protein. The organic matter digestibility in vitro of sunflower (Helianthus annus L.) crop residue and Jerusalem-artichoke (Helianthus tuberosis L.) forage and residue was initially higher than that of alfalfa (Medicago sativa L.) or sweet clover [Melilotus officinalus (L.) Lam] and did not increase appreciably with ammonia treatment. Faba-bean (Vicia faba L.) residue was only slightly inferior to these materials and showed a moderate improvement after treatment with ammonia. Untreated bullrush-millet [Pennisetum americanum (L.) Schum] whole-crop forage was equivalent to alfalfa and sweet clover in digestibility in vitro and responded quite well to ammonia treatment. Kochia (Kochia scoparia Shrad) forage had a relatively high digestibility in vitro and crude-protein content when harvested at the flowering stage. Mature kochia had a digestibility in vitro equivalent to barley straw and did not respond well to ammonia treatment.     

Utilization of Whole-Cell MALDI-TOF Mass Spectrometry to Differentiate Burkholderia pseudomallei Wild-Type and Constructed Mutants

Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (whole-cell MALDI-TOF MS) has been widely adopted as a useful technology in the identification and typing of microorganisms. This study employed the whole-cell MALDI-TOF MS to identify and differentiate wild-type and mutants containing constructed single gene mutations of Burkholderia pseudomallei, a pathogenic bacterium causing melioidosis disease in both humans and animals. Candidate biomarkers for the B. pseudomallei mutants, including rpoS, ppk, and bpsI isolates, were determined. Taxon-specific and clinical isolate-specific biomarkers of B. pseudomallei were consistently found and conserved across all average mass spectra. Cluster analysis of MALDI spectra of all isolates exhibited separate distribution. A total of twelve potential mass peaks discriminating between wild-type and mutant isolates were identified using ClinProTools analysis. Two peaks (m/z 2721 and 2748 Da) were specific for the rpoS isolate, three (m/z 3150, 3378, and 7994 Da) for ppk, and seven (m/z 3420, 3520, 3587, 3688, 4623, 4708, and 5450 Da) for bpsI. Our findings demonstrated that the rapid, accurate, and reproducible mass profiling technology could have new implications in laboratory-based rapid differentiation of extensive libraries of genetically altered bacteria.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.     

Regioisomer separation and identification of triacylglycerols containing vaccenic and oleic acids,and α- and γ-linolenic acids,in thermophilic cyanobacteria Mastigocladus laminosus and Tolypothrix sp.

Reversed phase liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/APCI-MS) was used for direct analysis of triacylglycerols (TAGs) from different strains of the cyanobacteria Mastigocladus laminosus, Tolypothrix cf. tenuis and Tolypothrix distorta. This technique enabled us to identify and quantify the specific molecular species of TAGs directly from lipid extracts of the cyanobacteria. The regioisomeric series of TAGs having α-linolenic and γ-linolenic and also oleic and cis-vaccenic acids were separated by RP-HPLC and identified by APCI-MS. M. laminosus produced only a few molecular species of TAGs, including both isomers of octadecenoic (oleic and vaccenic) acid, while T. distorta contained tens of molecular species of TAGs having FAs with up to four double bonds (stearidonic acid and including also its positional isomer, i.e. 3,6,9,12-octadecatetraenoic acid) and both positional isomers (α and γ) of linolenic acids. Individual strains of both cyanobacteria exhibited different contents of polyunsaturated fatty acids (Tolypothrix sp.) and different distribution of positional isomers of monoenoic fatty acids in TAGs (M. laminosus).     

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species'' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.     

Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [²H₄]IAA (m/z 194, 134) or [²H₅]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [²H₃]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.     

Water Transfer in an Alfalfa/Maize Association : Survival of Maize during Drought

We investigated the possibility of interspecific water transfer in an alfalfa (Medicago sativa L.) and maize (Zea mays L.) association. An alfalfa plant was grown through two vertically stacked plastic tubes. A 5 centimeter air gap between tubes was bridged by alfalfa roots. Five-week old maize plants with roots confined to the top tube were not watered, while associated alfalfa roots had free access to water in the bottom tube (the −/+ treatment). Additional treatments included: top and bottom tubes watered (+/+), top and bottom tubes droughted (−/−), and top tube droughted after removal of alfalfa root bridges and routine removal of alfalfa tillers (−^*). Predawn leaf water potential of maize in the −/+ treatment fell to −1.5 megapascals 13 days after the start of drought; thereafter, predawn and midday potentials were maintained near −1.9 megapascals. Leaf water potentials of maize in the −/− and −^* treatments declined steadily; all plants in these treatments were completely desiccated before day 50. High levels of tritium activity were detected in water extracted from both alfalfa and maize leaves after ³H₂O was injected into the bottom −/+ tube at day 70 or later. Maize in the −/+ treatment was able to survive an otherwise lethal period of drought by utilizing water lost by alfalfa roots.     

Comparison of chorismate mutase isozyme patterns in selected plants

A wide variety of plants have been assayed to determine if they contain three isozymes of chorismate mutase (EC 5.4.99.5) as does alfalfa (Medicago sativa L.) or two isozymes, as does mung bean (Phaseolus aureus). The isozymes were separated by disc electrophoresis. All anthophyta with the exception of some closely related Leguminosae contained three isozymes of chorismate mutase. The one coniferophyta (a pine), and pterophyta (a fern) and one microphyllophyta (a Selaginella) assayed contained two isozymes of chorismate mutase. All plants assayed contained measurable chorismate mutase levels and at least two isozymes of chorismate mutase.     

Characterization of Rhizobia from Ineffective Alfalfa Nodules: Ability to Nodulate Bean Plants [Phaseolus vulgaris (L.) Savi.]

This study was initiated to characterize Rhizobium isolates obtained from root nodules of ineffectively nodulated, field-grown alfalfa (Medicago sativa L.) plants. The purpose was to determine if these isolates possessed characteristics which would explain either their ineffectiveness in N₂ fixation or their apparent ability to tolerate the moderately acid soil conditions from which they originated. Isolates were characterized by analysis of growth rate, 39°C tolerance, acid production on conventional media, and symbiotic performance. All isolates were ineffective in N₂ fixation on alfalfa, and they contained one or more anomalous characteristics. These included either slow growth rate, lack of 39°C tolerance, or lack of acid production on conventional media. Infectiveness tests on a broad range of legumes revealed that the isolates formed root nodules on M. sativa, Medicago lupulina L., and Phaseolus vulgaris (L.) Savi. (common bean). These results provide evidence that, in some situations, ineffective nodulation of M. sativa in the field may be due to the presence of promiscuous, native Rhizobium species.     

Oxygen fixation into hydroxyproline in etiolated maize seedlings : verification by tandem mass spectrometry

Etiolated maize (Zea mays L.) seedlings were grown in the dark for 5 days in an atmosphere enriched with 10.0 atom% ¹⁸O₂. Hydroxyproline was isolated from root and shoot tissues, purified, and methylated. It was not possible to determine ¹⁸O incorporation into hydroxyproline by conventional mass spectrometry because the final product was not sufficiently pure. The final product was analyzed successfully by tandem mass spectrometry. The ¹⁸O content of the hydroxyl oxygen atom was 10 ± 0.7 atom%. This result demonstrates that the hydroxyl oxygen atom in hydroxyproline was derived exclusively from molecular oxygen.     

Mass spectrometric analysis of cytokinins in plant tissue VII. Quantification of cytokinin bases by negative ion mass spectrometry

A study of derivatives of N⁶-(isopent-2-enyl)adenine formed by substitution at N-9 indicated that sensitivity of detection by chemical ionization mass spectrometry was maximized by a pentafluorobenzyl substituent and negative ion monitoring. O-t-Butyldimethylsilyl-9-pentafluorobenzyl derivatives of zeatin (Z),cis-zeatin (cis-Z), and dihydrozeatin (DZ) were characterized by mass spectrometry. A procedure was based on these stable derivatives and negative ion chemical ionization mass spectrometry for quantification of zeatin and dihydrozeatin in plant tissue.     

Oxidative stress: Determination of 4-hydroxy-2-nonenal by gas chromatography/mass spectrometry in human and rat plasma

Abstract

The lipid peroxidation product 4-hydroxynonenal (HNE) is a biomarker of oxidative stress which is essentially involved in the pathophysiology of many diseases. The analysis of HNE is challenging because this aldehyde is extremely reactive and thus unstable. Hence, we adopted a gas chromatography–mass spectrometry (GC–MS) method based on derivatization of HNE with pentafluorobenzylhydroxylamine–HCl followed by trimethylsilylation to trimethylsilyl ethers. Ions representative for a negative ion chemical ionization mode were recorded at m/z = 152 for HNE and at m/z = 162 for the deuterated analogon (HNE-d₁₁) as internal standard. This excellent stable and precise GC–MS method was carefully validated for HNE, and showed good linearity (r² = 0.998), and high specificity and sensitivity. Within-day precisions were 4.4–6.1% and between-day precisions were 5.2–10.2%. Accuracies were between 99% and 104% for the whole calibration range (2.5–250 nmol/L) of HNE.

To examine the versatility of this modified GC–MS method, we analyzed HNE in ethylenediaminetetraacetic acid (EDTA) plasma in a well-defined collective of migraine patients; recently published. The results underline our former observations that women with migraine are afflicted with increased levels of HNE. Patients with thyroidal dysfunction showed no significant HNE alterations. This was confirmed by normal HNE EDTA plasma levels in hyper- und hypothyroid Sprague-Dawley rats.

Taken together, the GC–MS method presented herein is of excellent quality to record oxidative stress-related bioactive HNE levels. This is important for a reorientation of oxidative stress analytics in other human diseases first of atherosclerosis and cancer.     

Selective inhibitors of germination of legume seeds in activated sludge compost

Water extracts of the compost produced from activated sludge and coffee residue were found to be selectively inhibitory to seed germination of some legumes. Germination rate of white clover (Trifolium repens L.), red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) seeds were reduced to 2, 29 and 73% of the control, respectively, by water extracts of the compost (20 g l^–1). However, the extracts did not show any inhibition to seed germination of sorghum (Sorghum bicolor Moench), African millet (Eleusine coracana Gaertn.), and Komatsuna (Brassica rapa L.) at the same concentration. The inhibitors in the compost extracts were separated by ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC) and the inhibitory activities of seed germination were tested with white clover seeds. Five inhibitors were isolated and identified as 3,4-dichlorophenylacetic acid (3,4-DCP), 3,4-dichlorobenzoic acid (3,4-DCB), 3,4,5-trichlorophenylacetic acid, 3,4,5-trichlorobenzoic acid and mono-2-ethylhexylphthalate by ¹H-, ¹³C-NMR spectroscopy and mass spectrometry. The inhibitory activities of some authentic chemicals of the inhibitors and the related compounds were compared. The results indicated that the main inhibitor in the compost could be 3,4-DCB, which was contained at the concentration of 6.58 mg kg^–1 compost and showed the strongest inhibitory effect on seed germination of white clover among the tested compounds.     

Fine Mapping the Spatial Distribution and Concentration of Unlabeled Drugs within Tissue Micro-Compartments Using Imaging Mass Spectrometry

Readouts that define the physiological distributions of drugs in tissues are an unmet challenge and at best imprecise, but are needed in order to understand both the pharmacokinetic and pharmacodynamic properties associated with efficacy. Here we demonstrate that it is feasible to follow the in vivo transport of unlabeled drugs within specific organ and tissue compartments on a platform that applies MALDI imaging mass spectrometry to tissue sections characterized with high definition histology. We have tracked and quantified the distribution of an inhaled reference compound, tiotropium, within the lungs of dosed rats, using systematic point by point MS and MS/MS sampling at 200 µm intervals. By comparing drug ion distribution patterns in adjacent tissue sections, we observed that within 15 min following exposure, tiotropium parent MS ions (mass-to-charge; m/z 392.1) and fragmented daughter MS/MS ions (m/z 170.1 and 152.1) were dispersed in a concentration gradient (80 fmol-5 pmol) away from the central airways into the lung parenchyma and pleura. These drug levels agreed well with amounts detected in lung compartments by chemical extraction. Moreover, the simultaneous global definition of molecular ion signatures localized within 2-D tissue space provides accurate assignment of ion identities within histological landmarks, providing context to dynamic biological processes occurring at sites of drug presence. Our results highlight an important emerging technology allowing specific high resolution identification of unlabeled drugs at sites of in vivo uptake and retention.     

Defense mechanisms of conifers : relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content

Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue.