[Show abstract][Hide abstract] ABSTRACT: No large group of recently extinct placental mammals remains as evolutionarily cryptic as the approximately 280 genera grouped as 'South American native ungulates'. To Charles Darwin 1,2 , who first collected their remains, they included perhaps the 'strangest animal[s] ever discovered'. Today, much like 180 years ago, it is no clearer whether they had one origin or several, arose before or after the Cretaceous/Palaeogene transition 66.2 million years ago 3 , or are more likely to belong with the elephants and sirenians of superorder Afrotheria than with the euungulates (cattle, horses, and allies) of superorder Laurasiatheria 4–6. Morphology-based analyses have proved unconvincing because convergences are pervasive among unrelated ungulate-like placentals. Approaches using ancient DNA have also been unsuccessful, probably because of rapid DNA degradation in semitropical and temperate deposits. Here we apply proteomic analysis to screen bone samples of the Late Quaternary South American native ungulate taxa Toxodon (Notoungulata) and Macrauchenia (Litopterna) for phylogenetically informative protein sequences. For each ungulate, we obtain approximately 90% direct sequence coverage of type I collagen a1-and a2-chains, representing approximately 900 of 1,140 amino-acid residues for each subunit. A phylogeny is estimated from an alignment of these fossil sequences with collagen (I) gene transcripts from available mammalian genomes or mass spectrometrically derived sequence data obtained for this study. The resulting consensus tree agrees well with recent higher-level mammalian phylogenies
[Show abstract][Hide abstract] ABSTRACT: Metabolomics is one omics approach that can be used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include analysis of a wide range of chemical species with diverse physical properties, from ionic inorganic compounds to biochemically derived hydrophilic carbohydrates, organic and amino acids, and a range of hydrophobic lipid-related compounds. This complexitiy brings huge challenges to the analytical technologies employed in current plant metabolomics programs, and powerful analytical tools are required for the separation and characterization of this extremely high compound diversity present in biological sample matrices. The use of mass spectrometry (MS)-based analytical platforms to profile stress-responsive metabolites that allow some plants to adapt to adverse environmental conditions is fundamental in current plant biotechnology research programs for the understanding and development of stress-tolerant plants. In this review, we describe recent applications of metabolomics and emphasize its increasing application to study plant responses to environmental (stress-) factors, including drought, salt, low oxygen caused by waterlogging or flooding of the soil, temperature, light and oxidative stress (or a combination of them). Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance.
Mass Spectrometry Reviews 01/2015; DOI:10.1002/mas.21449 · 8.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
The species Neorhizobium galegae comprises two symbiovars that induce nodules on Galega plants. Strains of both symbiovars, orientalis and officinalis, induce nodules on the same plant species, but fix nitrogen only in their own host species. The mechanism behind this strict host specificity is not yet known. In this study, genome sequences of representatives of the two symbiovars were produced, providing new material for studying properties of N. galegae, with a special interest in genomic differences that may play a role in host specificity.
The genome sequences confirmed that the two representative strains are much alike at a whole-genome level. Analysis of orthologous genes showed that N. galegae has a higher number of orthologs shared with Rhizobium than with Agrobacterium. The symbiosis plasmid of strain HAMBI 1141 was shown to transfer by conjugation under optimal conditions. In addition, both sequenced strains have an acetyltransferase gene which was shown to modify the Nod factor on the residue adjacent to the non-reducing-terminal residue. The working hypothesis that this gene is of major importance in directing host specificity of N. galegae could not, however, be confirmed.
Strains of N. galegae have many genes differentiating them from strains of Agrobacterium, Rhizobium and Sinorhizobium. However, the mechanism behind their ecological difference is not evident. Although the final determinant for the strict host specificity of N. galegae remains to be identified, the gene responsible for the species-specific acetylation of the Nod factors was identified in this study. We propose the name noeT for this gene to reflect its role in symbiosis.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-500) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Pharmaceuticals present a potential threat to soil organisms, yet our understanding of their fate and uptake in soil systems is limited. This study therefore investigated fate and uptake of 14C labelled carbamazepine, diclofenac, fluoxetine and orlistat in soil-earthworm systems. Sorption coefficients increased in the order of carbamazepine < diclofenac < fluoxetine < orlistat. Dissipation of 14C varied by compound and, for orlistat, there was evidence of formation of non-extractable residues. Uptake of 14C was seen for all compounds. Depuration studies showed complete elimination of 14C for carbamazepine and fluoxetine treatments and partial elimination for orlistat and diclofenac, with >30% of the 14C remaining in the tissue at the end experiment. Pore-water based bioconcentration factors, based on uptake and elimination of 14C, increased in the order carbamazepine < diclofenac < fluoxetine and orlistat. LC-MS/MS and LC-FTMS indicated that the observed uptake in the fluoxetine and carbamazepine treatments was due to the parent compound but that diclofenac was degraded in the test system so uptake was due to unidentifiable transformation products. Comparison of our data with outputs of quantitative structure-activity relationship for estimating BCFs in worms showed that these models tend to overestimate pharmaceutical BCFs so new models are needed.
[Show abstract][Hide abstract] ABSTRACT: We have developed a simple method for the release and isolation of glycoprotein N-glycans from whole cell lysates using less than a million cells, for subsequent implementation with mass spectrometric analysis. Cellular protein extracts prepared using SDS solubilisation were sequentially treated in a membrane filter device to ultimately release glycans enzymatically using PNGase F in the volatile buffer ammonium bicarbonate. The released glycans are recovered in the filtrate following centrifugation, and typically permethylated prior to mass spectrometric analysis. We call our method 'filter-aided N-glycan separation' (FANGS), and have successfully applied it to investigate N-glycan profiles of wild type (WT) and mutant Chinese hamster ovary (CHO) cells. This method is readily multiplexed, and, due to the small numbers of cells needed, is compatible with the analysis of replicate samples to assess the true nature of glycan variability in tissue culture samples.
Journal of Proteome Research 01/2014; 13(3). DOI:10.1021/pr401043r · 5.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study reports the development of calcium alginate-based highly mesoporous carbon spheres (AMCS). These materials exhibit significant potential in chromatographic applications and demonstrated similar efficiency to commercially available porous graphitic carbon (PGC) and are superior to Starbon (R) materials in separation of polar carbohydrate analytes. The AMCS exhibit a narrow particle size distribution, are highly spherical and exhibit extensive mesoporous networks (up to ca. 90% mesoporosity). It is shown how calcium content can significantly influence the textural properties of the resulting AMCS materials. The AMCS production process requires no additional templating agents and temperatures of only 800 degrees C. In contrast, PGC are manufactured through an energy-intensive manufacture process, which requires a hard-templated mesoporous carbon intermediate to be pyrolysed to over 2000 degrees C under argon. Thus AMCS demonstrate promise as a green alternative to PGC in chromatographic applications.
[Show abstract][Hide abstract] ABSTRACT: We have investigated the microstructure and bonding of two biomass-based porous carbon chromatographic stationary phase materials (alginic acid-derived Starbon â and calcium alginate-derived mesoporous carbon spheres (AMCS)) and a commercial porous graphitic carbon (PGC), using high resolution transmission electron microscopy, electron energy loss spectroscopy (EELS), N 2 porosimetry and X-ray photoelectron spectroscopy (XPS). The pla-nar carbon sp 2 -content of all three material types is similar to that of traditional non-graphitizing carbon although, both biomass-based carbon types contain a greater percent-age of fullerene character (i.e. curved graphene sheets) than a non-graphitizing carbon pyrolyzed at the same temperature. This is thought to arise during the pyrolytic breakdown of hexauronic acid residues into C5 intermediates. Energy dispersive X-ray and XPS analy-sis reveals a homogeneous distribution of calcium in the AMCS and a calcium catalysis mechanism is discussed. That both Starbon â and AMCS, with high-fullerene character, show chromatographic properties similar to those of a commercial PGC material with extended graphitic stacks, suggests that, for separations at the molecular level, curved ful-lerene-like and planar graphitic sheets are equivalent in PGC chromatography. In addition, variation in the number of graphitic layers suggests that stack depth has minimal effect on the retention mechanism in PGC chromatography.
[Show abstract][Hide abstract] ABSTRACT: The facile racemization of protein-bound amino acids plays an important role in the ageing and pathologies of living tissues, and can be exploited for protein geochronological studies in sub-fossil biominerals. However, the in-chain degradation pathways of amino acids are complex and difficult to elucidate. Serine has proven to be particularly elusive and its ability to racemize as a peptide-bound residue (like asparagine and aspartic acid) has not been demonstrated. This study investigates the patterns of degradation of a model peptide (WNSVWAW) at elevated temperatures, quantifying the extent of racemization and peptide bond hydrolysis using RP-HPLC and tracking the presence of degradation products by MALDI-MS. We provide direct evidence that under these experimental conditions both serine and asparagine are able to undergo racemization as internally-bound residues, which shows their potential for initiating protein breakdown and provides an explanation for the presence of d-enantiomers in living mammalian tissues.
[Show abstract][Hide abstract] ABSTRACT: The nod(ulation) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (nM -microM range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod(ulation) factors, which can induce root nodule primordia and pseudo-nodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin (Estévez et al., 2009). At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin) some Nod factor-like spots were also observed on the thin layer plates used to detect incorporation of radiolabelled glucosamine into newly synthesised Nod factors. This phenomenon was further investigated with thin layer plates, fusions of nod genes to the lacZ gene, HPLC, mass spectrometry and the formation of pseudo-nodules on bean roots. Here we report that in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.
[Show abstract][Hide abstract] ABSTRACT: Haberlea rhodopensis is a resurrection plant with remarkable tolerance to desiccation. Haberlea exposed to drought stress, desiccation, and subsequent rehydration showed no signs of damage or severe oxidative stress compared to untreated control plants. Transcriptome analysis by next-generation sequencing revealed a drought-induced reprogramming, which redirected resources from growth towards cell protection. Repression of photosynthetic and growth-related genes during water deficiency was concomitant with induction of transcription factors (members of the NAC, NF-YA, MADS box, HSF, GRAS, and WRKY families) presumably acting as master switches of the genetic reprogramming, as well as with an upregulation of genes related to sugar metabolism, signaling, and genes encoding early light-inducible (ELIP), late embryogenesis abundant (LEA), and heat shock (HSP) proteins. At the same time, genes encoding other LEA, HSP, and stress protective proteins were constitutively expressed at high levels even in unstressed controls. Genes normally involved in tolerance to salinity, chilling, and pathogens were also highly induced, suggesting a possible cross-tolerance against a number of abiotic and biotic stress factors. A notable percentage of the genes highly regulated in dehydration and subsequent rehydration were novel, with no sequence homology to genes from other plant genomes. Additionally, an extensive antioxidant gene network was identified with several gene families possessing a greater number of antioxidant genes than most other species with sequenced genomes. Two of the transcripts most abundant during all conditions encoded catalases and five more catalases were induced in water-deficient samples. Using the pharmacological inhibitor 3-aminotriazole (AT) to compromise catalase activity resulted in increased sensitivity to desiccation. Metabolome analysis by GC or LC-MS revealed accumulation of sucrose, verbascose, spermidine, and γ-aminobutyric acid during drought, as well as particular secondary metabolites accumulating during rehydration. This observation, together with the complex antioxidant system and the constitutive expression of stress protective genes suggests that both constitutive and inducible mechanisms contribute to the extreme desiccation tolerance of H. rhodopensis.
Cellular and Molecular Life Sciences CMLS 09/2012; 70(4). DOI:10.1007/s00018-012-1155-6 · 5.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genes for mannitol-metabolizing enzymes, mannitol phosphate dehydrogenase (MPDH) and mannitol dehydrogenase (MDH), have been recently identified in the genome of Acanthamoeba castellanii and their potential role in stress tolerance was proposed. Using qRT-PCR, comparison has been made of mRNA levels of the enzymes for mannitol metabolism at various time intervals during the stress defence reactions of encystation and pseudocyst formation. Gradual decrease of both enzymes during encystation and slight increases at the beginning of pseudocyst formation were observed. Detailed analysis of mRNA sequences of the two genes revealed similarities with various alcohol dehydrogenases rather than mannitol dehydrogenases. Our results indicate there is probably no protective role for mannitol in Acanthamoeba as no mannitol was detected using HILIC ESI MS, in any Acanthamoeba life cycle stage. Possible misinterpretation of previously published sequences as encoding enzymes of the mannitol metabolic pathway is discussed.
[Show abstract][Hide abstract] ABSTRACT: We have explored two divinylbenzene cross-linked polystyrene supports for use in a solid-supported N-terminal peptide tagging strategy. Resin-bound tags designed to be cleaved in a single step at the N-terminus of peptides have been devised and explored as peptide N-terminal tagging reagents (constructs) for subsequent mass spectrometric analysis. While the brominated tagging approach shows promise, the use of these specific solid supports has drawbacks, in terms of tagging reaction scale, for real applications in proteomics.
[Show abstract][Hide abstract] ABSTRACT: N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (β/α)(8) barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.
Proceedings of the National Academy of Sciences 01/2012; 109(3):781-6. DOI:10.1073/pnas.1111482109 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The early (2-4 d) effects of slowly imposed soil water deficit on Lupinus albus photosynthetic performance, carbon metabolism, and hormonal balance in different organs (leaf blade, stem stele, stem cortex, and root) were evaluated on 23-d-old plants (growth chamber assay). Our work shows that several metabolic adjustments occurred prior to alteration of the plant water status, implying that water deficit is perceived before the change in plant water status. The slow, progressive decline in soil water content started to be visible 3 d after withholding water (3 DAW). The earliest plant changes were associated with organ-specific metabolic responses (particularly in the leaves) and with leaf conductance and only later with plant water status and photosynthetic rate (4 DAW) or photosynthetic capacity (according to the Farquhar model; 6 DAW). Principal component analysis (PCA) of the physiological parameters, the carbohydrate and the hormone levels and their relative values, as well as leaf water-soluble metabolites full scan data (LC-MS/MS), showed separation of the different sampling dates. At 6 DAW classically described stress responses are observed, with plant water status, ABA level, and root hormonal balance contributing to the separation of these samples. Discrimination of earlier stress stages (3 and 4 DAW) is only achieved when the relative levels of indole-3-acetic acid (IAA), cytokinins (Cks), and carbon metabolism (glucose, sucrose, raffinose, and starch levels) are taken into account. Our working hypothesis is that, in addition to single responses (e.g. ABA increase), the combined alterations in hormone and carbohydrate levels play an important role in the stress response mechanism. Response to more advanced stress appears to be associated with a combination of cumulative changes, occurring in several plant organs. The carbohydrate and hormonal balance in the leaf (IAA to bioactive-Cks; soluble sugars to IAA and starch to IAA; relative abundances of the different soluble sugars) flag the initial responses to the slight decrease in soil water availability (10-15% decrease). Further alterations in sucrose to ABA and in raffinose to ABA relative values (in all organs) indicate that soil water availability continues to decrease. Such alterations when associated with changes in the root hormone balance indicate that the stress response is initiated. It is concluded that metabolic balance (e.g. IAA/bioactive Cks, carbohydrates/IAA, sucrose/ABA, raffinose/ABA, ABA/IAA) is relevant in triggering adjustment mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Archaeological work in advance of construction at a site on the edge of York, UK, yielded human remains of prehistoric to Romano-British date. Amongst these was a mandible and cranium, the intra-cranial space of which contained shrunken but macroscopically recognizable remains of a brain. Although the distinctive surface morphology of the organ is preserved, little recognizable brain histology survives. Though rare, the survival of brain tissue in otherwise skeletalised human remains from wet burial environments is not unique. A survey of the literature shows that similar brain masses have been previously reported in diverse circumstances. We argue for a greater awareness of these brain masses and for more attention to be paid to their detection and identification in order to improve the reporting rate and to allow a more comprehensive study of this rare archaeological survival.
[Show abstract][Hide abstract] ABSTRACT: Burkholderia cenocepacia is an important opportunistic pathogen, and one of the most striking features of the Burkholderia genus is the collection of polar lipids present in its membrane, including phosphatidylethanolamine (PE) and ornithine-containing lipids (OLs), as well as the 2-hydroxylated derivatives of PE and OLs (2-OH-PE and 2-OH-OLs, respectively), which differ from the standard versions by virtue of the presence of a hydroxyl group at C2 (2-OH) of an esterified fatty acyl residue. Similarly, a lipid A-esterified myristoyl group from Salmonella typhimurium can have a 2-hydroxy modification that is due to the LpxO enzyme. We thus postulated that 2-hydroxylation of 2-OH-OLs might be catalyzed by a novel dioxygenase homologue of LpxO. In B. cenocepacia, we have now identified two open reading frames (BCAM1214 and BCAM2401) homologous to LpxO from S. typhimurium. The introduction of bcam2401 (designated olsD) into Sinorhizobium meliloti leads to the formation of one new lipid and in B. cenocepacia of two new lipids. Surprisingly, the lipid modifications on OLs due to OlsD occur on the amide-linked fatty acyl chain. This is the first report of a hydroxyl modification of OLs on the amide-linked fatty acyl moiety. Formation of hydroxylated OLs occurs only when the biosynthesis pathway for nonmodified standard OLs is intact. The hydroxyl modification of OLs on the amide-linked fatty acyl moiety occurs only under acid stress conditions. An assay has been developed for the OlsD dioxygenase, and an initial characterization of the enzyme is presented.