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Sample records for acidic fens reveal

  1. Microarray and Functional Gene Analyses of Sulfate-Reducing Prokaryotes in Low-Sulfate, Acidic Fens Reveal Cooccurrence of Recognized Genera and Novel Lineages

    PubMed Central

    Loy, Alexander; Küsel, Kirsten; Lehner, Angelika; Drake, Harold L.; Wagner, Michael

    2004-01-01

    Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats. PMID:15574893

  2. Dynamics of organic and inorganic arsenic in the solution phase of an acidic fen in Germany

    NASA Astrophysics Data System (ADS)

    Huang, J.-H.; Matzner, E.

    2006-04-01

    Wetland soils play a key role for the transformation of heavy metals in forested watersheds, influencing their mobility, and ecotoxicity. Our goal was to investigate the mechanisms of release from solid to solution phase, the mobility, and the transformation of arsenic species in a fen soil. In methanol-water extracts, monomethylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, arsenobetaine, and two unknown organic arsenic species were found with concentrations up to 14 ng As g -1 at the surface horizon. Arsenate is the dominant species at the 0-30 cm depth, whereas arsenite predominated at the 30-70 cm depth. Only up to 2.2% of total arsenic in fen was extractable with methanol-water. In porewaters, depth gradient spatial variation of arsenic species, pH, redox potentials, and the other chemical parameters along the profile was observed in June together with high proportion of organic arsenic species (up to 1.2 μg As L -1, 70% of total arsenic). Tetramethylarsonium ion and an unknown organic arsenic species were additionally detected in porewaters at deeper horizons. In comparison, the arsenic speciation in porewaters in April was homogeneous with depth and no organic arsenic species were found. Thus, the occurrence of microbial methylation of arsenic in fen was demonstrated for the first time. The 10 times elevated total arsenic concentrations in porewaters in June compared to April were accompanied by elevated concentrations of total iron, lower concentrations of sulfate and the presence of ammonium and phosphate. The low proportion of methanol-water extractable total arsenic suggests a generally low mobility of arsenic in fen soils. The release of arsenic from solid to solution phases in fen is dominantly controlled by dissolution of iron oxides, redox transformation, and methylation of arsenic, driven by microbial activity in the growing season. As a result, increased concentrations of total arsenic and potentially toxic arsenic species in fen

  3. Drying-Rewetting and Flooding Impact Denitrifier Activity Rather than Community Structure in a Moderately Acidic Fen

    PubMed Central

    Palmer, Katharina; Köpp, Julia; Gebauer, Gerhard; Horn, Marcus A.

    2016-01-01

    Wetlands represent sources or sinks of the greenhouse gas nitrous oxide (N2O). The acidic fen Schlöppnerbrunnen emits denitrification derived N2O and is also capable of N2O consumption. Global warming is predicted to cause more extreme weather events in future years, including prolonged drought periods as well as heavy rainfall events, which may result in flooding. Thus, the effects of prolonged drought and flooding events on the abundance, community composition, and activity of fen denitrifiers were investigated in manipulation experiments. The water table in the fen was experimentally lowered for 8 weeks in 2008 and raised for 5.5 months in 2009 on three treatment plots, while three plots were left untreated and served as controls. In situ N2O fluxes were rather unaffected by the drought treatment and were marginally increased by the flooding treatment. Samples were taken before and after treatment in both years. The structural gene markers narG and nosZ were used to assess possible changes in the nitrate reducer and denitrifier community in response to water table manipulations. Detected copy numbers of narG and nosZ were essentially unaffected by the experimental drought and flooding. Terminal restriction fragment length polymorphism (TRFLP) patterns of narG and nosZ were similar before and after experimental drought or experimental flooding, indicating a stable nitrate reducer and denitrifier community in the fen. However, certain TRFs of narG and nosZ transcripts responded to experimental drought or flooding. Nitrate-dependent Michaelis-Menten kinetics were assessed in anoxic microcosms with peat samples taken before and 6 months after the onset of experimental flooding. Maximal reaction velocities vmax were higher after than before flooding in samples from treament but not in those from control plots taken at the same time. The ratio of N2O to N2O + N2 was lower in soil from treatment plots after flooding than in soil from control plots, suggesting

  4. Drying-Rewetting and Flooding Impact Denitrifier Activity Rather than Community Structure in a Moderately Acidic Fen.

    PubMed

    Palmer, Katharina; Köpp, Julia; Gebauer, Gerhard; Horn, Marcus A

    2016-01-01

    Wetlands represent sources or sinks of the greenhouse gas nitrous oxide (N2O). The acidic fen Schlöppnerbrunnen emits denitrification derived N2O and is also capable of N2O consumption. Global warming is predicted to cause more extreme weather events in future years, including prolonged drought periods as well as heavy rainfall events, which may result in flooding. Thus, the effects of prolonged drought and flooding events on the abundance, community composition, and activity of fen denitrifiers were investigated in manipulation experiments. The water table in the fen was experimentally lowered for 8 weeks in 2008 and raised for 5.5 months in 2009 on three treatment plots, while three plots were left untreated and served as controls. In situ N2O fluxes were rather unaffected by the drought treatment and were marginally increased by the flooding treatment. Samples were taken before and after treatment in both years. The structural gene markers narG and nosZ were used to assess possible changes in the nitrate reducer and denitrifier community in response to water table manipulations. Detected copy numbers of narG and nosZ were essentially unaffected by the experimental drought and flooding. Terminal restriction fragment length polymorphism (TRFLP) patterns of narG and nosZ were similar before and after experimental drought or experimental flooding, indicating a stable nitrate reducer and denitrifier community in the fen. However, certain TRFs of narG and nosZ transcripts responded to experimental drought or flooding. Nitrate-dependent Michaelis-Menten kinetics were assessed in anoxic microcosms with peat samples taken before and 6 months after the onset of experimental flooding. Maximal reaction velocities v max were higher after than before flooding in samples from treament but not in those from control plots taken at the same time. The ratio of N2O to N2O + N2 was lower in soil from treatment plots after flooding than in soil from control plots, suggesting

  5. Genomic and protein expression analysis reveals flap endonuclease 1 (FEN1) as a key biomarker in breast and ovarian cancer.

    PubMed

    Abdel-Fatah, Tarek M A; Russell, Roslin; Albarakati, Nada; Maloney, David J; Dorjsuren, Dorjbal; Rueda, Oscar M; Moseley, Paul; Mohan, Vivek; Sun, Hongmao; Abbotts, Rachel; Mukherjee, Abhik; Agarwal, Devika; Illuzzi, Jennifer L; Jadhav, Ajit; Simeonov, Anton; Ball, Graham; Chan, Stephen; Caldas, Carlos; Ellis, Ian O; Wilson, David M; Madhusudan, Srinivasan

    2014-10-01

    FEN1 has key roles in Okazaki fragment maturation during replication, long patch base excision repair, rescue of stalled replication forks, maintenance of telomere stability and apoptosis. FEN1 may be dysregulated in breast and ovarian cancers and have clinicopathological significance in patients. We comprehensively investigated FEN1 mRNA expression in multiple cohorts of breast cancer [training set (128), test set (249), external validation (1952)]. FEN1 protein expression was evaluated in 568 oestrogen receptor (ER) negative breast cancers, 894 ER positive breast cancers and 156 ovarian epithelial cancers. FEN1 mRNA overexpression was highly significantly associated with high grade (p = 4.89 × 10(-57)), high mitotic index (p = 5.25 × 10(-28)), pleomorphism (p = 6.31 × 10(-19)), ER negative (p = 9.02 × 10(-35)), PR negative (p = 9.24 × 10(-24)), triple negative phenotype (p = 6.67 × 10(-21)), PAM50.Her2 (p = 5.19 × 10(-13)), PAM50. Basal (p = 2.7 × 10(-41)), PAM50.LumB (p = 1.56 × 10(-26)), integrative molecular cluster 1 (intClust.1) (p = 7.47 × 10(-12)), intClust.5 (p = 4.05 × 10(-12)) and intClust. 10 (p = 7.59 × 10(-38)) breast cancers. FEN1 mRNA overexpression is associated with poor breast cancer specific survival in univariate (p = 4.4 × 10(-16)) and multivariate analysis (p = 9.19 × 10(-7)). At the protein level, in ER positive tumours, FEN1 overexpression remains significantly linked to high grade, high mitotic index and pleomorphism (ps < 0.01). In ER negative tumours, high FEN1 is significantly associated with pleomorphism, tumour type, lymphovascular invasion, triple negative phenotype, EGFR and HER2 expression (ps < 0.05). In ER positive as well as in ER negative tumours, FEN1 protein overexpression is associated with poor survival in univariate and multivariate analysis (ps < 0.01). In ovarian epithelial cancers, similarly, FEN1 overexpression is associated with high grade, high stage and poor survival (ps < 0.05). We conclude that FEN1

  6. Micropepsia pineolensis gen. nov., sp. nov., an acid-tolerant alphaproteobacterium isolated from a poor fen, and proposal of Micropepsiaceae fam. nov. within Micropepsiales ord. nov.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel, obligately anaerobic, acid-tolerant, fermentative alphaproteobacterium, designated strain CS4T, was isolated from an acidic, oligotrophic (nutrient-poor) poor fen located near Pineola, NC, USA. Cultures contained Gram-negative, slightly curved, non-motile, non-spore forming, non-prosthecat...

  7. Acid-tolerant microaerophilic Fe(II)-oxidizing bacteria promote Fe(III)-accumulation in a fen.

    PubMed

    Lüdecke, Claudia; Reiche, Marco; Eusterhues, Karin; Nietzsche, Sandor; Küsel, Kirsten

    2010-10-01

    The ecological importance of Fe(II)-oxidizing bacteria (FeOB) at circumneutral pH is often masked in the presence of O(2) where rapid chemical oxidation of Fe(II) predominates. This study addresses the abundance, diversity and activity of microaerophilic FeOB in an acidic fen (pH ∼ 5) located in northern Bavaria, Germany. Mean O(2) penetration depth reached 16 cm where the highest dissolved Fe(II) concentrations (up to 140 µM) were present in soil water. Acid-tolerant FeOB cultivated in gradient tubes were most abundant (10(6) cells g(-1) peat) at the 10-20 cm depth interval. A stable enrichment culture was active at up to 29% O(2) saturation and Fe(III) accumulated 1.6 times faster than in abiotic controls. An acid-tolerant, microaerophilic isolate (strain CL21) was obtained which was closely related to the neutrophilic, lithoautotrophic FeOB Sideroxydans lithotrophicus strain LD-1. CL21 oxidized Fe(II) between pH 4 and 6.0, and produced nanoscale-goethites with a clearly lower mean coherence length (7 nm) perpendicular to the (110) plane than those formed abiotically (10 nm). Our results suggest that an acid-tolerant population of FeOB is thriving at redox interfaces formed by diffusion-limited O(2) transport in acidic peatlands. Furthermore, this well-adapted population is successfully competing with chemical oxidation and thereby playing an important role in the microbial iron cycle.

  8. Satellites reveal an increase in gross primary production in a greenlandic high arctic fen 1992-2008

    NASA Astrophysics Data System (ADS)

    Tagesson, T.; Mastepanov, M.; Tamstorf, M. P.; Eklundh, L.; Schubert, P.; Ekberg, A.; Sigsgaard, C.; Christensen, T. R.; Ström, L.

    2010-02-01

    Arctic wetlands play a key role in the terrestrial carbon cycle. Recent studies have shown a greening trend and indicated an increase in CO2 uptake in boreal and sub- to low-arctic areas. Our aim was to combine satellite-based normalized difference vegetation index (NDVI) with ground-based flux measurements of CO2 to investigate a possible greening trend and potential changes in gross primary production (GPP) between 1992 and 2008 in a high arctic fen area. The study took place in Rylekaerene in the Zackenberg Research Area (74°28' N 20°34' W), located in the National park of North Eastern Greenland. We estimated the light use efficiency (ɛ) for the dominant vegetation types from field measured fractions of photosynthetic active radiation (FAPAR) and ground-based flux measurements of GPP. Measured FAPAR were correlated to satellite-based NDVI. The FAPAR-NDVI relationship in combination with ɛ was applied to satellite data to model GPP 1992-2008. The model was evaluated against field measured GPP. The model was a useful tool for up-scaling GPP and all basic requirements for the model were well met, e.g., FAPAR was well correlated to NDVI and modeled GPP was well correlated to field measurements. The studied high arctic fen area has experienced a strong increase in GPP between 1992 and 2008. The area has during this period also experienced a substantial increase in local air temperature. Consequently, the observed greening trend is most likely due to ongoing climatic change possibly in combination with CO2 fertilization, due to increasing atmospheric concentrations of CO2.

  9. Seed dispersal in fens

    USGS Publications Warehouse

    Middleton, B.; Van Diggelen, R.; Jensen, K.

    2006-01-01

    Question: How does seed dispersal reduce fen isolation and contribute to biodiversity? Location: European and North American fens. Methods: This paper reviews the literature on seed dispersal to fens. Results: Landscape fragmentation may reduce dispersal opportunities thereby isolating fens and reducing genetic exchange. Species in fragmented wetlands may have lower reproductive success, which can lead to biodiversity loss. While fens may have always been relatively isolated from each other, they have become increasingly fragmented in modern times within agricultural and urban landscapes in both Europe and North America. Dispersal by water, animals and wind has been hampered by changes related to development in landscapes surrounding fens. Because the seeds of certain species are long-lived in the seed bank, frequent episodes of dispersal are not always necessary to maintain the biodiversity of fens. However, of particular concern to restoration is that some dominant species, such as the tussock sedge Carex stricta, may not disperse readily between fens. Conclusions: Knowledge of seed dispersal can be used to maintain and restore the biodiversity of fens in fragmented landscapes. Given that development has fragmented landscapes and that this situation is not likely to change, the dispersal of seeds might be enhanced by moving hay or cattle from fens to damaged sites, or by reestablishing lost hydrological connections. ?? IAVS; Opulus Press.

  10. EXPRESSION AND BIOCHEMICAL CHARACTERIZATION OF THE PLASMODIUM FALCIPARUM DNA REPAIR ENZYME, FLAP ENDONUCLEASE-1 (PfFEN-1)

    PubMed Central

    Casta, Louis J.; Buguliskis, Jeffery S.; Matsumoto, Yoshihiro; Taraschi, Theodore F.

    2009-01-01

    Flap Endonuclease-1 (FEN-1) is a structure-specific endonuclease that is critical for the resolution of single-stranded DNA flap intermediates that form during long patch DNA Base Excision Repair (BER). This investigation reports that Plasmodium species encode FEN-1 homologs. Protein sequence analysis revealed the N and I domains of Plasmodium falciparum (PfFEN-1) and Plasmodium yoelii (PyFEN-1) to be homologous to FEN-1 from other species. However, each possessed an extended C domain which had limited homology to apicomplexan FEN-1s and no homology to eukaryotic FEN-1s. A conserved Proliferating Cell Nuclear Antigen (PCNA) binding site was identified at an internal location rather than the extreme C-terminal location typically seen in FEN-1 from other organisms. The endonuclease and exonuclease activities of PfFEN-1 and PyFEN-1 were investigated using recombinant protein produced in Escherichia coli. Pf and PyFEN-1 possessed DNA structure-specific flap endonuclease and 5′→3′ exonuclease activities, similar to FEN-1’s from other species. Endonuclease activity was stimulated by Mg+2 or Mn+2 and inhibited by monovalent ions (>20.0 mM). A PfFEN-1 C-terminal truncation mutant lacking the terminal 250 amino acids (PfFEN-1ΔC) had endonuclease activity that was ~130-fold greater (kcat= 1.2x10−1) than full-length PfFEN-1 (kcat= 9.1x10−4) or ~240-fold greater than PyFEN-1 (kcat= 4.9x10−4) in vitro. PfFEN-1 generated a nicked DNA substrate that was ligated by recombinant Pf DNA Ligase I (PfLigI) using an in vitro DNA repair assay. Plasmodium FEN-1s have enzymatic activities similar to other species but contain extended C-termini and a more internally located PCNA binding site. PMID:17928073

  11. Ecoenzymatic stoichiometry and microbial processing of organic matter in northern bogs and fens reveals a common P limitation among peatland types

    EPA Science Inventory

    We compared C, N, and P concentrations in atmospheric deposition, runoff, and soil standing stocks with microbial respiration (DHA) and ecoenzyme activity (EEA) in an ombrotrophic bog (S2) and a minerotrophic fen (S3) to investigate the environmental drivers of biogeochemical cyc...

  12. Fatty acids profiling reveals potential candidate markers of semen quality.

    PubMed

    Zerbinati, C; Caponecchia, L; Rago, R; Leoncini, E; Bottaccioli, A G; Ciacciarelli, M; Pacelli, A; Salacone, P; Sebastianelli, A; Pastore, A; Palleschi, G; Boccia, S; Carbone, A; Iuliano, L

    2016-11-01

    Previous reports showed altered fatty acid content in subjects with altered sperm parameters compared to normozoospermic individuals. However, these studies focused on a limited number of fatty acids, included a short number of subjects and results varied widely. We conducted a case-control study involving 155 patients allocated into four groups, including normozoospermia (n = 33), oligoasthenoteratozoospermia (n = 32), asthenozoospermia (n = 25), and varicocoele (n = 44). Fatty acid profiling, including 30 species, was analyzed by a validated gas chromatography (GC) method on the whole seminal fluid sample. Multinomial logistic regression modeling was used to identify the associations between fatty acids and the four groups. Specimens from 15 normozoospermic subjects were also analyzed for fatty acids content in the seminal plasma and spermatozoa to study the distribution in the two compartments. Fatty acids lipidome varied markedly between the four groups. Multinomial logistic regression modeling revealed that high levels of palmitic acid, behenic acid, oleic acid, and docosahexaenoic acid (DHA) confer a low risk to stay out of the normozoospermic group. In the whole population, seminal fluid stearic acid was negatively correlated (r = -0.53), and DHA was positively correlated (r = 0.65) with sperm motility. Some fatty acids were preferentially accumulated in spermatozoa and the highest difference was observed for DHA, which was 6.2 times higher in spermatozoa than in seminal plasma. The results of this study highlight complete fatty acids profile in patients with different semen parameters. Given the easy-to-follow and rapid method of analysis, fatty acid profiling by GC method can be used for therapeutic purposes and to measure compliance in infertility trials using fatty acids supplements.

  13. Fens As Ecohydrologic Gauges of Climate Change in California

    NASA Astrophysics Data System (ADS)

    Drexler, J. Z.; Flint, L. E.; Flint, A. L.; Knifong, D. L.

    2014-12-01

    The purpose of this study was to demonstrate the use of montane groundwater-fed peatlands called fens as gauges for changes in groundwater recharge through time. Due to their consistent groundwater flows, fens remain saturated during the growing season, thus serving as ecological refugia for a range of sensitive biota including the federally threatened Yosemite toad (Anaxyrus canorus). In this project, five fens in the Sierra Nevada and two in the southern Cascade Mountains were studied over a 50-80 year period using historic aerial photography and GIS analysis. Fen areas were delineated using vegetation distributions and site wetness as visual keys to fen areal extent. In addition, a climate analysis of precipitation, snowpack, evapotranspiration, and mean minimum monthly temperature (Tmin) was carried out for the years 1950 - 2010 using a distributed parameter water-balance model called the Basin Characterization Model. Over the study period, all five Sierra fens decreased by 10-15% in delineated area, but little change occurred in the Cascade fens. The climate analysis revealed major changes through time in Tmin and the frequency of June months with zero snowpack (snowpack longevity) at the five Sierra sites. At the Cascade sites, increases in Tmin occurred more recently and decreases in snowpack longevity were more subtle. The different behavior of fens in the Cascades versus the Sierra Nevada suggests that underlying geology, particularly hydraulic conductivity, rock porosity, and size of groundwater reservoirs, may be an important buffer to change. These results show that fens in the Sierra Nevada are highly responsive ecohydrologic gauges that can be used to track reductions in groundwater recharge due to climate change. Further studies using isotopic and/or geochemical approaches are needed to quantify the changes in groundwater recharge that were identified with fens. Because fens provide critical habitat for sensitive species, major changes in hydrology

  14. Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure

    PubMed Central

    Lewis, Amanda L.; Desa, Nolan; Hansen, Elizabeth E.; Knirel, Yuriy A.; Gordon, Jeffrey I.; Gagneux, Pascal; Nizet, Victor; Varki, Ajit

    2009-01-01

    Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phylogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of ≈1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the “unique” Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars. PMID:19666579

  15. Biodiversity management of fens and fen meadows by grazing, cutting and burning

    USGS Publications Warehouse

    Middleton, B.A.; Holsten, B.; Van Diggelen, R.

    2006-01-01

    Question: Can the biodiversity of fens in Europe and North America be maintained through the use of grazing (especially cattle grazing), fire, and/or cutting? Location: European and North American fens. Methods: This paper is a review of the literature on the effects of grazing, fire and cutting on fens, to explore the relationship between management and biodiversity in fens. Results: A reduction of cattle grazing, mowing and burning in fens has led to a reduction in biodiversity in fens. The vegetation of abandoned fens shifts to trees and shrubs after 10-15 years, which shade the smaller and rarer species of these wetlands. While careful use of fire is used to manage fens in North America, it is not widely used in European fens, perhaps because the peat of drained fens may catch fire. Cattle grazing cannot be considered a natural disturbance in North America, since cattle did not evolve on that continent. In Europe, cattle do not generally graze in unaltered fens, but they do use slightly drained fen meadows. Conclusions: Three approaches have been used to control the dominance of tall woody and herbaceous species in abandoned fens, including the re-introduction of cattle, mowing, and burning. Overgrazing results in a permanent reduction in biodiversity, therefore cattle re-introduction must be approached cautiously. In Europe, but not in North America, mowing has been an important management tool, and mowing has been successful in maintaining species richness, particularly in fens that have been mowed annually for centuries. Fire has been the most common and successful management tool in North America although it is not effective in removing shrubs that have become large. Because the problems and solutions are similar, the literature of both European and North American fen management can be analyzed to better assess the management of fens on both continents. Many management questions require further study and these are listed in the paper. ?? IAVS; Opulus Press.

  16. Emissions of biogenic sulfur gases from northern bogs and fens

    NASA Technical Reports Server (NTRS)

    Demello, William Zamboni; Hines, Mark E.; Bayley, Suzanne E.

    1992-01-01

    Sulfur gases are important components of the global cycle of S. They contribute to the acidity of precipitation and they influence global radiation balance and climate. The role of terrestrial sources of biogenic S and their effect on atmospheric chemistry remain as major unanswered questions in our understanding of the natural S cycle. The role of northern wetlands as sources and sinks of gaseous S by measuring rates of S gas exchange as a function of season, hydrologic conditions, and gradients in tropic status was investigated. Experiments were conducted in wetlands in New Hampshire, particularly a poor fen, and in Mire 239, a poor fen at the Experimental Lakes Area (ELA) in Ontario. Emissions were determined using Teflon enclosures, gas cryotrapping methods and gas chromatography (GC) with flame photometric detection. Dynamic (sweep flow) and static enclosures were employed which yielded similar results. Dissolved S gases and methane were determined by gas stripping followed by GC.

  17. Geochemistry and hydrology of a calcareous fen within the Savage Fen wetlands complex, Minnesota, USA

    USGS Publications Warehouse

    Komor, S.C.

    1994-01-01

    Savage Fen is a wetlands complex at the base of north-facing bluffs in the Minnesota River Valley. The complex includes 27.8 hectares of calcareous fen that host rare calciphile plants whose populations are declining in Minnesota. Water and sediment compositions in the calcareous fen were studied to gain a better understanding of the hydrologie System that sustains the rare vegetation. Groundwater in the fen is a calcium-magnesium-bicarbonate type with circumneutral pH values. The groundwater composition is the resuit of interactions among water, dissolved and gaseous carbon species, carbonates, and ion exchangers. Shallow groundwater is distinguished from deep groundwater by smaller concentrations of chloride, sulfate, magnesium, and sodium, and larger concentrations of calcium, bicarbonate, hydrogen sulfide, and ammonium. Magnesian calcite is the prevalent carbonate in unconsolidated sedimentary fill beneath the fen and is an important source and sink for dissolved calcium, magnesium, and inorganic carbon. Calcite concentrations just below the water table are small because aerobic and anaerobic oxidation of organic matter increase the partial pressure of carbon dioxide (PCO2), decrease pH, and cause calcite to dissolve. Thick calcite accumulations just above the water table, in the root zone of calciphile plants, result from water table fluctuations and attendant changes in PCO2. Groundwater beneath Savage Fen recharges in lakes and ponds south of the fen and upwells to the surface within the fen. Water at the water table is a mixture of upwelling groundwater and water near the surface that flows downslope from higher elevations in the fen. Changes in oxygen and hydrogen isotope compositions of shallow groundwater indicate that the proportion of upwelling groundwater in shallow groundwater decreases downgradient in the calcareous fen. Encroachment of reed grasses into the calcareous fen may reflect human-caused disturbances in the recharge area.

  18. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  19. Structural Conservation of Ligand Binding Reveals a Bile Acid-like Signaling Pathway in Nematodes*

    PubMed Central

    Zhi, Xiaoyong; Zhou, X. Edward; Melcher, Karsten; Motola, Daniel L.; Gelmedin, Verena; Hawdon, John; Kliewer, Steven A.; Mangelsdorf, David J.; Xu, H. Eric

    2012-01-01

    Bile acid-like molecules named dafachronic acids (DAs) control the dauer formation program in Caenorhabditis elegans through the nuclear receptor DAF-12. This mechanism is conserved in parasitic nematodes to regulate their dauer-like infective larval stage, and as such, the DAF-12 ligand binding domain has been identified as an important therapeutic target in human parasitic hookworm species that infect more than 600 million people worldwide. Here, we report two x-ray crystal structures of the hookworm Ancylostoma ceylanicum DAF-12 ligand binding domain in complex with DA and cholestenoic acid (a bile acid-like metabolite), respectively. Structure analysis and functional studies reveal key residues responsible for species-specific ligand responses of DAF-12. Furthermore, DA binds to DAF-12 mechanistically and is structurally similar to bile acids binding to the mammalian bile acid receptor farnesoid X receptor. Activation of DAF-12 by cholestenoic acid and the cholestenoic acid complex structure suggest that bile acid-like signaling pathways have been conserved in nematodes and mammals. Together, these results reveal the molecular mechanism for the interplay between parasite and host, provide a structural framework for DAF-12 as a promising target in treating nematode parasitism, and provide insight into the evolution of gut parasite hormone-signaling pathways. PMID:22170062

  20. Urinary metabolomics in Fxr-null mice reveals activated adaptive metabolic pathways upon bile acid challenge.

    PubMed

    Cho, Joo-Youn; Matsubara, Tsutomu; Kang, Dong Wook; Ahn, Sung-Hoon; Krausz, Kristopher W; Idle, Jeffrey R; Luecke, Hans; Gonzalez, Frank J

    2010-05-01

    Farnesoid X receptor (FXR) is a nuclear receptor that regulates genes involved in synthesis, metabolism, and transport of bile acids and thus plays a major role in maintaining bile acid homeostasis. In this study, metabolomic responses were investigated in urine of wild-type and Fxr-null mice fed cholic acid, an FXR ligand, using ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS). Multivariate data analysis between wild-type and Fxr-null mice on a cholic acid diet revealed that the most increased ions were metabolites of p-cresol (4-methylphenol), corticosterone, and cholic acid in Fxr-null mice. The structural identities of the above metabolites were confirmed by chemical synthesis and by comparing retention time (RT) and/or tandem mass fragmentation patterns of the urinary metabolites with the authentic standards. Tauro-3alpha,6,7alpha,12alpha-tetrol (3alpha,6,7alpha,12alpha-tetrahydroxy-5beta-cholestan-26-oyltaurine), one of the most increased metabolites in Fxr-null mice on a CA diet, is a marker for efficient hydroxylation of toxic bile acids possibly through induction of Cyp3a11. A cholestatic model induced by lithocholic acid revealed that enhanced expression of Cyp3a11 is the major defense mechanism to detoxify cholestatic bile acids in Fxr-null mice. These results will be useful for identification of biomarkers for cholestasis and for determination of adaptive molecular mechanisms in cholestasis.

  1. Analysis of putative nonulosonic acid biosynthesis pathways in Archaea reveals a complex evolutionary history.

    PubMed

    Kandiba, Lina; Eichler, Jerry

    2013-08-01

    Sialic acids and the other nonulosonic acid sugars, legionaminic acid and pseudaminic acid, are nine carbon-containing sugars that can be detected as components of the glycans decorating proteins and other molecules in Eukarya and Bacteria. Yet, despite the prevalence of N-glycosylation in Archaea and the variety of sugars recruited for the archaeal version of this post-translational modification, only a single report of a nonulosonic acid sugar in an archaeal N-linked glycan has appeared. Hence, to obtain a clearer picture of nonulosonic acid sugar biosynthesis capability in Archaea, 122 sequenced genomes were scanned for the presence of genes involved in the biogenesis of these sugars. The results reveal that while Archaea and Bacteria share a common route of sialic acid biosynthesis, numerous archaeal nonulosonic acid sugar biosynthesis pathway components were acquired from elsewhere via various routes. Still, the limited number of Archaea encoding components involved in the synthesis of nonulosonic acid sugars implies that such saccharides are not major components of glycans in this domain.

  2. A Siglec-like sialic-acid-binding motif revealed in an adenovirus capsid protein

    PubMed Central

    Rademacher, Christoph; Bru, Thierry; McBride, Ryan; Robison, Elizabeth; Nycholat, Corwin M; Kremer, Eric J; Paulson, James C

    2012-01-01

    Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are a family of transmembrane receptors that are well documented to play roles in regulation of innate and adaptive immune responses. To see whether the features that define the molecular recognition of sialic acid were found in other sialic-acid-binding proteins, we analyzed 127 structures with bound sialic acids found in the Protein Data Bank database. Of these, the canine adenovirus 2-fiber knob protein showed close local structural relationship to Siglecs despite low sequence similarity. The fiber knob harbors a noncanonical sialic-acid recognition site, which was then explored for detailed specificity using a custom glycan microarray comprising 58 diverse sialosides. It was found that the adenoviral protein preferentially recognizes the epitope Neu5Acα2-3[6S]Galβ1-4GlcNAc, a structure previously identified as the preferred ligand for Siglec-8 in humans and Siglec-F in mice. Comparison of the Siglec and fiber knob sialic-acid-binding sites reveal conserved structural elements that are not clearly identifiable from the primary amino acid sequence, suggesting a Siglec-like sialic-acid-binding motif that comprises the consensus features of these proteins in complex with sialic acid. PMID:22522600

  3. Complexity of gastric acid secretion revealed by targeted gene disruption in mice.

    PubMed

    Chen, Duan; Zhao, Chun-Mei

    2010-01-01

    Physiology of gastric acid secretion is one of the earliest subjects in medical research and education. Gastric acid secretion has been sometimes inadequately expressed as pH value rather than amount of gastric H(+) secreted per unit time. Gastric acid secretion is regulated by endocrine, paracrine and neurocrine signals via at least three messenger pathways: gastrin-histamine, CCK-somatostatin, and neural network. These pathways have been largely validated and further characterized by phenotyping a series of knockout mouse models. The complexity of gastric acid secretion is illustrated by both expected and unexpected phenotypes of altered acid secretion. For examples, in comparison with wild-type mice, gastrin and CCK double knockout and SSTR(2) knockout mice displayed a shift in the regulation of ECL cells from somatostatin-SSTR(2) pathway to galanin-Gal1 receptor pathway; a shift in the regulation of parietal cells from gastrin-histamine pathway to vagal pathway; and a shift in the CCK(2) receptors on parietal cells from functional silence to activation. The biological function of glycine-extended gastrin in synergizing gastrin-17 has been revealed in gastrin knockout mice. The roles of gastric acid secretion in tumorigenesis and ulceration have not been fully understood. Transgenic hypergastrinemic INS-GAS mice developed a spontaneous gastric cancer, which was associated with an impaired acid secretion. Gastrin knockout mice were still able to produce acid in response to vagal stimulation, especially after H. pylori infection. Taken together, phenotyping of a series of genetically engineered mouse models reveals a high degree of complexity of gastric acid secretion in both physiological and pathophysiological conditions.

  4. Dietary intake and plasma metabolomic analysis of polyunsaturated fatty acids in bipolar subjects reveal dysregulation of linoleic acid metabolism.

    PubMed

    Evans, Simon J; Ringrose, Rachel N; Harrington, Gloria J; Mancuso, Peter; Burant, Charles F; McInnis, Melvin G

    2014-10-01

    Polyunsaturated fatty acids (PUFA) profiles associate with risk for mood disorders. This poses the hypothesis of metabolic differences between patients and unaffected healthy controls that relate to the primary illness or are secondary to medication use or dietary intake. However, dietary manipulation or supplementation studies show equivocal results improving mental health outcomes. This study investigates dietary patterns and metabolic profiles relevant to PUFA metabolism, in bipolar I individuals compared to non-psychiatric controls. We collected seven-day diet records and performed metabolomic analysis of fasted plasma collected immediately after diet recording. Regression analyses adjusted for age, gender and energy intake found that bipolar individuals had significantly lower intake of selenium and PUFAs, including eicosapentaenoic acid (EPA) (n-3), docosahexaenoic acid (DHA) (n-3), arachidonic acid (AA) (n-6) and docosapentaenoic acid (DPA) (n-3/n-6 mix); and significantly increased intake of the saturated fats, eicosanoic and docosanoic acid. Regression analysis of metabolomic data derived from plasma samples, correcting for age, gender, BMI, psychiatric medication use and dietary PUFA intake, revealed that bipolar individuals had reduced 13S-HpODE, a major peroxidation product of the n-6, linoleic acid (LA), reduced eicosadienoic acid (EDA), an elongation product of LA; reduced prostaglandins G2, F2 alpha and E1, synthesized from n-6 PUFA; and reduced EPA. These observations remained significant or near significant after Bonferroni correction and are consistent with metabolic variances between bipolar and control individuals with regard to PUFA metabolism. These findings suggest that specific dietary interventions aimed towards correcting these metabolic disparities may impact health outcomes for individuals with bipolar disorder.

  5. RECQ1 interacts with FEN-1 and promotes binding of FEN-1 to telomeric chromatin

    PubMed Central

    Sami, Furqan; Lu, Xing; Parvathaneni, Swetha; Roy, Rabindra; Gary, Ronald K.; Sharma, Sudha

    2015-01-01

    RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in replication restart mechanisms. Cellular phenotypes of RECQ1 deficiency are indicative of aberrant repair of stalled replication forks, but the molecular functions of RECQ1, the most abundant of the five known human RecQ homologs, have remained poorly understood. We show that RECQ1 associates with FEN-1 in nuclear extracts and exhibits direct protein interaction in vitro. Recombinant RECQ1 significantly stimulated FEN-1 endonucleolytic cleavage of 5’-flap DNA substrates containing nontelomeric or telomeric repeat sequence. RECQ1 and FEN-1 were constitutively present at telomeres and their binding to the telomeric chromatin was enhanced following DNA damage. Telomere residence of FEN-1 was dependent on RECQ1 since depletion of RECQ1 reduced FEN-1 binding to telomeres in unperturbed cycling cells. Our results confirm a conserved collaboration of human RecQ helicases with FEN-1, and suggest both overlapping and specialized roles of RECQ1 in the processing of DNA structure intermediates proposed to arise during replication, repair and recombination. PMID:25774876

  6. Novel fen ecosystems in western North Carolina

    NASA Astrophysics Data System (ADS)

    Wilcox, J. D.

    2012-12-01

    Western North Carolina is mountainous, and groundwater flows from hillslope recharge zones to valley stream and spring discharge zones. Depending on surface topography and geologic conditions, the water table may approach or intersect the ground surface to form seepage wetlands, or fens. Fen ecosystems can be very sensitive to changes in land use, groundwater pumping, and upslope development. This presentation will focus on two sites where historical land use and human activity played important roles in creating or preserving fen ecosystems. Both sites now support—and are being managed to protect—federally endangered flora and fauna. The first site is home to Sarracenia oreophilia, an endangered pitcher plant that thrives on saturated soils with low nutrient content. The site's early history includes tree clearing, drain tile installation, and cattle grazing, while more recent management activities have included drain tile excavation, manual invasive removal, and prescribed burns. A 15-year water-level record indicates seasonal artesian conditions wet a 3m clay unit (K=2E-5 cm/sec) beneath the site, which is able to retain moisture during drier periods. Shorter "clay wetting periods" during drought years (1999-2000; 2007-2008) correspond to reduced clump counts in pitcher-plant surveys. The second site is a former aggregate quarry that now supports over 60 bog turtles (Clemmys muhlenbergii). The biggest threat to this site is encroachment of non-native and invasive multiflora rose (Rosa multiflora) and other large woody species. Management activities include manual removal and prescribed goat herbivory. Current efforts to characterize the springs, water-table, and surface-water flows will be used to detect changes in the future to the hydrologic regime in the fen.

  7. Regional scale hydrological and biogeochemical processes controlling high biodiversity of a groundwater fed alkaline fen

    NASA Astrophysics Data System (ADS)

    van der Zee, Sjoerd E. A. T. M.; (D. G.) Cirkel, Gijsbert; (J. P. M) witte, Flip

    2014-05-01

    The high floral biodiversity of groundwater fed fens and mesotrophic grasslands depends on the different chemical signatures of the shallow rainwater fed topsoil water and the slightly deeper geochemically affected groundwater. The relatively abrupt gradients between these two layers of groundwater enable the close proximity of plants that require quite different site factors and have different rooting depths. However, sulphur inflow into such botanically interesting areas is generally perceived as a major threat to biodiversity. Although in Europe atmospheric deposition of sulphur has decreased considerably over the last decades, groundwater pollution by sulphate may still continue due to pyrite oxidation in soil as a result of excessive fertilisation. Inflowing groundwater rich in sulphate can change biogeochemical cycling in nutrient-poor wetland ecosystems because of 'so called' internal eutrophication as well as the accumulation of dissolved sulphide, which is phytotoxic. Complementary to conventions, we propose that upwelling sulphate rich groundwater may, in fact, promote the conservation of rare and threatened alkaline fens: excessive fertilisation and pyrite oxidation also produces acidity, which invokes calcite dissolution, and increased alkalinity and hardness of the inflowing groundwater. For a very species-rich wetland nature reserve, we show that sulphate is reduced and effectively precipitated as iron sulphides, when this calcareous and sulphate rich groundwater flows upward through the organic soil of the investigated nature reserve. Also, we show that sulphate reduction occurs simultaneously with an increase in alkalinity production, which in our case results in active calcite precipitation in the soil. In spite of the occurring sulphate reduction, we found no evidence for internal eutrophication. Extremely low phosphorous concentration in the pore water could be attributed to a high C:P ratio of soil organic matter and co-precipitation with

  8. Functionalised carboxylic acids in atmospheric particles: An annual cycle revealing seasonal trends and possible sources

    NASA Astrophysics Data System (ADS)

    Teich, Monique; van Pinxteren, Dominik; Herrmann, Hartmut

    2013-04-01

    acids. The high concentrations in summer could lead to the conclusion that these acids are mostly formed during photochemical processes in the atmosphere. However, the concentrations in autumn were often exceeded by the ones in winter. Therefore probably other sources beside photochemical processes have to be considered. The second group consists of aromatic compounds. Because of the high concentrations in winter it can be concluded that photochemical formation plays a minor role and primary emission sources e.g., wood combustion are likely. Further evidence in determining sources of the carboxylic acids could be obtained from the air mass origin. In general, air masses transported from East have a more anthropogenic influence than the air mass inflow from West. For all aromatic carboxylic acids higher concentrations were determined during eastern inflow, indicating anthropogenic sources. This presumption is supported by high correlations with the elemental carbon (EC). Regarding the aliphatic carboxylic there is one group with higher concentrations when the air mass is transported from West and one with higher concentrations when air mass is transported from East. In summary the findings of this study reveal a clear difference in the seasonal trends of the single target acids indicating a variety of different sources.

  9. Fenretinide mediated retinoic acid receptor signalling and inhibition of ceramide biosynthesis regulates adipogenesis, lipid accumulation, mitochondrial function and nutrient stress signalling in adipocytes and adipose tissue

    PubMed Central

    Mcilroy, George D.; Tammireddy, Seshu R.; Maskrey, Benjamin H.; Grant, Louise; Doherty, Mary K.; Watson, David G.; Delibegović, Mirela; Whitfield, Phillip D.; Mody, Nimesh

    2016-01-01

    Fenretinide (FEN) is a synthetic retinoid that inhibits obesity and insulin resistance in high-fat diet (HFD)-fed mice and completely prevents 3T3-L1 pre-adipocyte differentiation. The aim of this study was to determine the mechanism(s) of FEN action in 3T3-L1 adipocytes and in mice. We used the 3T3-L1 model of adipogenesis, fully differentiated 3T3-L1 adipocytes and adipose tissue from HFD-induced obese mice to investigate the mechanisms of FEN action. We measured expression of adipogenic and retinoid genes by qPCR and activation of nutrient-signalling pathways by western blotting. Global lipid and metabolite analysis was performed and specific ceramide lipid species measured by liquid chromatography-mass spectrometry. We provide direct evidence that FEN inhibits 3T3-L1 adipogenesis via RA-receptor (RAR)-dependent signaling. However, RARα antagonism did not prevent FEN-induced decreases in lipid levels in mature 3T3-L1 adipocytes, suggesting an RAR-independent mechanism. Lipidomics analysis revealed that FEN increased dihydroceramide lipid species 5- to 16-fold in adipocytes, indicating an inhibition of the final step of ceramide biosynthesis. A similar blockade in adipose tissue from FEN-treated obese mice was associated with a complete normalisation of impaired mitochondrial β-oxidation and tricarboxylic acid cycle flux. The FEN catabolite, 4-oxo-N-(4-hydroxyphenyl)retinamide (4-OXO), also decreased lipid accumulation without affecting adipogenesis. FEN and 4-OXO (but not RA) treatment additionally led to the activation of p38-MAPK, peIF2α and autophagy markers in adipocytes. Overall our data reveals FEN utilises both RAR-dependent and -independent pathways to regulate adipocyte biology, both of which may be required for FEN to prevent obesity and insulin resistance in vivo. PMID:26592777

  10. Fatty acid profiling reveals seasonal and spatial shifts in zooplankton diet in a temperate estuary

    NASA Astrophysics Data System (ADS)

    Gonçalves, A. M. M.; Azeiteiro, U. M.; Pardal, M. A.; De Troch, M.

    2012-08-01

    Fatty acids composition of copepod and cladoceran species and their possible food sources was investigated in the Mondego estuary (southern Europe) in order to explain the seasonal variation of the small copepods Acartia clausi, Acartia tonsa, Copidodiaptomus numidicus, Temora longicornis and the freshwater cladoceran Daphnia longispina. A total of 12 zooplankton species (7 marine, 2 estuarine and 3 freshwater species) were studied. A multivariate analysis revealed a clear seasonal distribution of zooplankton species in terms of fatty acids composition and abundance, with winter and spring zooplankton species showing maximal concentrations and diversity of total fatty acids. These findings underline the role of lipids as storage during the colder seasons in a highly variable environment like an estuary. Estuarine and freshwater species showed a more diverse array of saturated and unsaturated fatty acids rather than marine species, except for Centropages typicus. Fatty acids markers of trophic position indicated the presence of two trophic levels: copepod species were primarily omnivorous, whereas cladocerans showed to be herbivorous. Our results suggest that feeding patterns of plankton change spatially and temporally, reflecting the shifts in dominance between diatoms and flagellates as well as between dinoflagellates/diatoms and small animals.

  11. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    PubMed Central

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  12. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

  13. Critical role for CaFEN1 and CaFEN12 of Candida albicans in cell wall integrity and biofilm formation

    PubMed Central

    Alfatah, Md.; Bari, Vinay K.; Nahar, Anubhav S.; Bijlani, Swati; Ganesan, K.

    2017-01-01

    Sphingolipids are involved in several cellular functions, including maintenance of cell wall integrity. To gain insight into the role of individual genes of sphingolipid biosynthetic pathway, we have screened Saccharomyces cerevisiae strains deleted in these genes for sensitivity to cell wall perturbing agents calcofluor white and congo red. Only deletants of FEN1 and SUR4 genes were found to be sensitive to both these agents. Candida albicans strains deleted in their orthologs, CaFEN1 and CaFEN12, respectively, also showed comparable phenotypes, and a strain deleted for both these genes was extremely sensitive to cell wall perturbing agents. Deletion of these genes was reported earlier to sensitise cells to amphotericin B (AmB), which is a polyene drug that kills the cells mainly by binding and sequestering ergosterol from the plasma membrane. Here we show that their AmB sensitivity is likely due to their cell wall defect. Further, we show that double deletant of C. albicans is defective in hyphae formation as well as biofilm development. Together this study reveals that deletion of FEN1 and SUR4 orthologs of C. albicans leads to impaired cell wall integrity and biofilm formation, which in turn sensitise cells to AmB. PMID:28079132

  14. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

    PubMed Central

    Wang, Lijie; Zhang, Tong; Watson, David G.; Silva, Ana Marta; Coombs, Graham H.

    2015-01-01

    Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts. PMID:26368322

  15. Lipidomic analysis reveals a radiosensitizing role of gamma-linolenic acid in glioma cells.

    PubMed

    Antal, Otilia; Péter, Mária; Hackler, László; Mán, Imola; Szebeni, Gábor; Ayaydin, Ferhan; Hideghéty, Katalin; Vigh, László; Kitajka, Klára; Balogh, Gábor; Puskás, Laszló G

    2015-09-01

    Previous studies have demonstrated that gamma-linolenic acid (GLA) is effective against glioma cells under both in vitro and in vivo conditions. In the present study we determined how GLA alone or in combination with irradiation alters the fatty acid (FA) and lipid profiles, the lipid droplet (LD) content, the lipid biosynthetic gene expression and the apoptosis of glioma cells. In GLA-treated cells direct correlations were found between the levels of various FAs and the expression of the corresponding FA biosynthetic genes. The total levels of saturated and monosaturated FAs decreased in concert with the down-regulation of FASN and SCD1 gene expression. Similarly, decreased FADS1 gene expression was paralleled by lowered arachidonic acid (20:4 n-6) and eicosapentaenoic acid (20:5 n-3) contents, while the down-regulation of FADS2 expression was accompanied by a diminished docosahexaenoic acid (22:6 n-3) content. Detailed mass spectrometric analyses revealed that individual treatments gave rise to distinct lipidomic fingerprints. Following uptake, GLA was subjected to elongation, resulting in dihomo-gamma-linolenic acid (20:3 n-6, DGLA), which was used for the synthesis of the LD constituent triacylglycerols and cholesteryl esters. Accordingly, an increased number of LDs were observed in response to GLA administration after irradiation. GLA increased the radioresponsiveness of U87 MG cells, as demonstrated by an increase in the number of apoptotic cells determined by FACS analysis. In conclusion, treatment with GLA increased the apoptosis of irradiated glioma cells, and GLA might therefore increase the therapeutic efficacy of irradiation in the treatment of gliomas.

  16. Decreased eicosapentaenoic acid levels in acne vulgaris reveals the presence of a proinflammatory state.

    PubMed

    Aslan, İbrahim; Özcan, Filiz; Karaarslan, Taner; Kıraç, Ebru; Aslan, Mutay

    2017-01-01

    This study aimed to determine circulating levels of polyunsaturated fatty acids (PUFAs), secretory phospholipase A2 (sPLA2), lipoprotein lipase (LPL) and measure circulating protein levels of angiopoietin-like protein 3 (ANGPTL3), ANGPTL4, cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in patients with acne vulgaris. Serum from 21 control subjects and 31 acne vulgaris patients were evaluated for levels of arachidonic acid (AA, C20:4n- 6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3). PUFA levels were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods Serum EPA levels were significantly decreased while AA/EPA and DGLA/EPA ratio were significantly increased in acne vulgaris patients compared to controls. Serum levels of AA, DGLA and DHA showed no significant difference while activity of sPLA2 and LPL were significantly increased in acne vulgaris compared to controls. Results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly decreased serum EPA levels and increased activity of sPLA2, AA/EPA and DGLA/EPA ratio. Increased LPL activity in the serum of acne vulgaris patients can be protective through its anti-dyslipidemic actions. This is the first study reporting altered EPA levels and increased sPLA2 activity in acne vulgaris and supports the use of omega-3 fatty acids as adjuvant treatment for acne patients.

  17. Modelling Seasonal Carbon Dynamics on Fen Peatlands

    NASA Astrophysics Data System (ADS)

    Giebels, Michael; Beyer, Madlen; Augustin, Jürgen; Roppel, Mario; Juszczak, Radoszlav; Serba, Tomasz

    2010-05-01

    In Germany more than 99 % of fens have lost their carbon and nutrient sink function due to heavy drainage and agricultural land use especially during the last decades and thus resulted in compression and heavy peat loss (CHARMAN 2002; JOOSTEN & CLARKE 2002; SUCCOW & JOOSTEN 2001; AUGUSTIN et al. 1996; KUNTZE 1993). Therefore fen peatlands play an important part (4-5 %) in the national anthropogenic trace gas budget. But only a small part of drained and agricultural used fens in NE Germany can be restored. Knowledge of the influence of land use to trace gas exchange is important for mitigation of the climate impact of the anthropogenic peatland use. We study carbon exchanges between soil and atmosphere on several fen peatland use areas at different sites in NE-Germany. Our research covers peatlands of supposed strongly climate forcing land use (cornfield and intensive pasture) and of probably less forcing, alternative types (meadow and extensive pasture) as well as rewetted (formerly drained) areas and near-natural sites like a low-degraded fen and a wetted alder woodland. We measured trace gas fluxes with manual and automatic chambers in periodic routines since spring 2007. The used chamber technique bases on DROESLER (2005). In total we now do research at 22 sites situated in 5 different locations covering agricultural, varying states of rewetted and near-natural treatments. We present results of at least 2 years of measurements on our site of varying types of agricultural land use. There we found significant differences in the annual carbon balances depending on the genesis of the observed sites and the seasonal dynamics. Annual balances were constructed by applying single respiration and photosynthesis CO2 models for each measurement campaign. These models were based on LLOYD-TAYLOR (1994) and Michaelis-Menten-Kinetics respectively. Crosswise comparison of different site treatments combined with the seasonal environmental observations give good hints for the

  18. Fire management in fens and wet grasslands grazed by cattle

    USGS Publications Warehouse

    Middleton, Beth A.

    2006-01-01

    Managers grapple with the problem of shrub invasion in fens and wet grasslands, and the invasion of shrubs is a particular problem in newly acquired natural areas that were once grazed by cattle. The specific management for any particular fen or wet grassland depends greatly on its previous land-use history. Managers should have a clear understanding of the grazing and drainage history of newly acquired fens and wet grasslands so that well-informed management decisions can be made.

  19. FEN1 protein with and without DNA.mov

    SciTech Connect

    2011-01-01

    The FEN1 protein is especially important during replication of the DNA lagging strand, where it is needed to trim "flaps" of single-strand DNA and prepare them for rejoining. FEN1 and its superfamily are also important in many forms of DNA repair. How FEN1 works only became apparent from crystallography of its structure bound to DNA. The presence of DNA appears to induce a transition from disorder to order that precisely positions and prepares the junction.

  20. Site directed mutagenesis of StSUT1 reveals target amino acids of regulation and stability.

    PubMed

    Krügel, Undine; Wiederhold, Elena; Pustogowa, Jelena; Hackel, Aleksandra; Grimm, Bernhard; Kühn, Christina

    2013-11-01

    Plant sucrose transporters (SUTs) are functional as sucrose-proton-cotransporters with an optimal transport activity in the acidic pH range. Recently, the pH optimum of the Solanum tuberosum sucrose transporter StSUT1 was experimentally determined to range at an unexpectedly low pH of 3 or even below. Various research groups have confirmed these surprising findings independently and in different organisms. Here we provide further experimental evidence for a pH optimum at physiological extrema. Site directed mutagenesis provides information about functional amino acids, which are highly conserved and responsible for this extraordinary increase in transport capacity under extreme pH conditions. Redox-dependent dimerization of the StSUT1 protein was described earlier. Here the ability of StSUT1 to form homodimers was demonstrated by heterologous expression in Lactococcus lactis and Xenopus leavis using Western blots, and in plants by bimolecular fluorescence complementation. Mutagenesis of highly conserved cysteine residues revealed their importance in protein stability. The accessibility of regulatory amino acid residues in the light of StSUT1's compartmentalization in membrane microdomains is discussed.

  1. Antiparasitic evaluation of betulinic acid derivatives reveals effective and selective anti-Trypanosoma cruzi inhibitors.

    PubMed

    Meira, Cássio Santana; Barbosa-Filho, José Maria; Lanfredi-Rangel, Adriana; Guimarães, Elisalva Teixeira; Moreira, Diogo Rodrigo Magalhães; Soares, Milena Botelho Pereira

    2016-07-01

    Betulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent.

  2. [Acute fatty liver in pregnancy: revealing fetal fatty acid oxidation disorders].

    PubMed

    Lamireau, D; Feghali, H; Redonnet-Vernhet, I; Mesli, S; Carles, D; Brissaud, O

    2012-03-01

    Acute fatty liver of pregnancy (AFLP) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome are serious maternal illnesses occurring in the third trimester of pregnancy with significant perinatal and maternal mortality. AFLP may result from mitochondrial defects in the beta-oxidation of fatty acids, in particular a deficiency of the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) in the fetus. Clinical findings in AFLP vary and its diagnosis is complicated by a significant overlap in clinical and biochemical features with HELLP syndrome. We report the case of 2 siblings who died, the first one in the neonatal period of asphyxia with multivisceral presentation and the second one from sudden death at 7 months. Autopsy of the latter infant revealed hepatic steatosis associated with cardiomyopathy, which led to suspicion of a fatty acid oxidation deficiency. Mutation analysis demonstrated that both children were homozygous for the common mutation c.1528G>C and the parents were heterozygous for this same mutation. This case demonstrates the importance of screening mothers with acute fatty liver disease of pregnancy and their children at birth for a metabolic disease. This article proposes several metabolic tests for mother and child suspected of having beta-oxidation of a fatty acid disorder.

  3. Water flow and solute transport in floating fen root mats

    NASA Astrophysics Data System (ADS)

    Stofberg, Sija F.; EATM van der Zee, Sjoerd

    2015-04-01

    Floating fens are valuable wetlands, found in North-Western Europe, that are formed by floating root mats when old turf ponds are colonized by plants. These terrestrialization ecosystems are known for their biodiversity and the presence of rare plant species, and the root mats reveal different vegetation zones at a small scale. The vegetation zones are a result of strong gradients in abiotic conditions, including groundwater dynamics, nutrients and pH. To prevent irreversible drought effects such as land subsidence and mineralization of peat, water management involves import of water from elsewhere to maintain constant surface water levels. Imported water may have elevated levels of salinity during dry summers, and salt exposure may threaten the vegetation. To assess the risk of exposure of the rare plant species to salinity, the hydrology of such root mats must be understood. Physical properties of root mats have scarcely been investigated. We have measured soil characteristics, hydraulic conductivity, vertical root mat movement and groundwater dynamics in a floating root mat in the nature reserve Nieuwkoopse Plassen, in the Netherlands. The root mat mostly consists of roots and organic material, in which the soil has a high saturated water content, and strongly varies in its stage of decomposition. We have found a distinct negative correlation between degree of decomposition and hydraulic conductivity, similar to observations for bogs in the literature. Our results show that the relatively young, thin edge of the root mat that colonizes the surface water has a high hydraulic conductivity and floats in the surface water, resulting in very small groundwater fluctuations within the root mat. The older part of the root mat, that is connected to the deeper peat layers is hydrologically more isolated and the material has a lower conductivity. Here, the groundwater fluctuates strongly with atmospheric forcing. The zones of hydraulic properties and vegetation, appear to

  4. Cation–Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting

    PubMed Central

    Gebala, Magdalena; Giambasu, George M.; Lipfert, Jan; Bisaria, Namita; Bonilla, Steve; Li, Guangchao; York, Darrin M.; Herschlag, Daniel

    2016-01-01

    The ion atmosphere is a critical structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Experimental methods that “count” the number of ions thermodynamically associated with the ion atmosphere allow dissection of energetic properties of the ion atmosphere, and thus provide direct comparison to theoretical results. Previous experiments have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atmosphere. Herein, we have systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, we find, surprisingly, that the extent of anion exclusion and cation inclusion significantly depends on salt identity. The differences are prominent at higher concentrations and mirror trends in mean activity coefficients of the electrolyte solutions. Salts with lower activity coefficients exhibit greater accumulation of both cations and anions within the ion atmosphere, strongly suggesting that cation–anion correlation effects are present in the ion atmosphere and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation–anion correlations extend to nucleic acid kinetics and thermodynamics, we followed the folding of P4–P6, a domain of the Tetrahymena group I ribozyme, via single-molecule fluorescence resonance energy transfer in solutions with different salts. Solutions of identical concentration but lower activity gave slower and less favorable folding. Our results reveal hitherto unknown properties of the ion atmosphere and suggest possible roles of oriented ion pairs or anion-bridged cations in the ion atmosphere for electrolyte solutions of salts with reduced activity. Consideration of these new

  5. Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid.

    PubMed

    Hollstein, M; Moriya, M; Grollman, A P; Olivier, M

    2013-01-01

    Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the pieces of evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterized by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbor human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk.

  6. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    PubMed Central

    2010-01-01

    Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should

  7. Fens as whole-ecosystem gauges of groundwater recharge under climate change

    USGS Publications Warehouse

    Drexler, Judith Z.; Knifong, Donna L.; JayLee Tuil,; Flint, Lorraine E.; Flint, Alan L.

    2013-01-01

    Currently, little is known about the impact of climate change on groundwater recharge in the Sierra Nevada and southern Cascade Range of California or other mountainous regions of the world. The purpose of this study was to determine whether small alpine peat lands called fens can be used as whole-ecosystem gauges of groundwater recharge through time. Fens are sustained by groundwater discharge and are highly sensitive to changes in groundwater flow due to hydrologic disturbance including climate change. Seven fens in the Sierra Nevada and southern Cascade Range were studied over a 50-80 year period using historic aerial photography. In each aerial photograph, fen areas were identified as open lawn and partially treed areas that exhibited (1) dark brownish-green coloring or various shades of gray and black in black and white imagery and (2) mottling of colors and clustering of vegetation, which signified a distinct moss canopy with overlying clumped sedge vegetation. In addition to the aerial photography study, a climate analysis for the study sites was carried out using both measured data (U.S. Department of Agriculture Natural Resources Conservation Service SNOwpack TELemetry system) and modeled data (a downscaled version of the Parameter-elevation Regressions on Independent Slopes Model) for the period from 1951 to 2010. Over the study period, the five fens in the Sierra Nevada were found to be decreasing between 10% and 16% in delineated area. The climate analysis revealed significant increases through time in annual mean minimum temperature (Tmin) between 1951-1980 and 1981-2010. In addition, April 1 snow water equivalent and snowpack longevity also decreased between 1951-1980 and 1981-2010. For the fens in the Cascade Range, there were no discernible changes in delineated area. At these sites, increases in Tmin occurred only within the past 20-25 years and decreases in snowpack longevity were more subtle. A conceptual model is presented, which illustrates

  8. Fens as whole-ecosystem gauges of groundwater recharge under climate change

    NASA Astrophysics Data System (ADS)

    Drexler, Judith Z.; Knifong, Donna; Tuil, JayLee; Flint, Lorraine E.; Flint, Alan L.

    2013-02-01

    SummaryCurrently, little is known about the impact of climate change on groundwater recharge in the Sierra Nevada and southern Cascade Range of California or other mountainous regions of the world. The purpose of this study was to determine whether small alpine peatlands called fens can be used as whole-ecosystem gauges of groundwater recharge through time. Fens are sustained by groundwater discharge and are highly sensitive to changes in groundwater flow due to hydrologic disturbance including climate change. Seven fens in the Sierra Nevada and southern Cascade Range were studied over a 50-80 year period using historic aerial photography. In each aerial photograph, fen areas were identified as open lawn and partially treed areas that exhibited (1) dark brownish-green coloring or various shades of gray and black in black and white imagery and (2) mottling of colors and clustering of vegetation, which signified a distinct moss canopy with overlying clumped sedge vegetation. In addition to the aerial photography study, a climate analysis for the study sites was carried out using both measured data (U.S. Department of Agriculture Natural Resources Conservation Service SNOwpack TELemetry system) and modeled data (a downscaled version of the Parameter-elevation Regressions on Independent Slopes Model) for the period from 1951 to 2010. Over the study period, the five fens in the Sierra Nevada were found to be decreasing between 10% and 16% in delineated area. The climate analysis revealed significant increases through time in annual mean minimum temperature (Tmin) between 1951-1980 and 1981-2010. In addition, April 1 snow water equivalent and snowpack longevity also decreased between 1951-1980 and 1981-2010. For the fens in the Cascade Range, there were no discernible changes in delineated area. At these sites, increases in Tmin occurred only within the past 20-25 years and decreases in snowpack longevity were more subtle. A conceptual model is presented, which

  9. Acidic Properties and Structure-Activity Correlations of Solid Acid Catalysts Revealed by Solid-State NMR Spectroscopy.

    PubMed

    Zheng, Anmin; Li, Shenhui; Liu, Shang-Bin; Deng, Feng

    2016-04-19

    Solid acid materials with tunable structural and acidic properties are promising heterogeneous catalysts for manipulating and/or emulating the activity and selectivity of industrially important catalytic reactions. On the other hand, the performances of acid-catalyzed reactions are mostly dictated by the acidic features, namely, type (Brønsted vs Lewis acidity), amount, strength, and local environment of acid sites. The latter is relevant to their location (intra- vs extracrystalline), and possible confinement and Brønsted-Lewis acid synergy effects that may strongly affect the host-guest interactions, reaction mechanism, and shape selectivity of the catalytic system. This account aims to highlight some important applications of state-of-the-art solid-state NMR (SSNMR) techniques for exploring the structural and acidic properties of solid acid catalysts as well as their catalytic performances and relevant reaction pathway invoked. In addition, density functional theory (DFT) calculations may be exploited in conjunction with experimental SSNMR studies to verify the structure-activity correlations of the catalytic system at a microscopic scale. We describe in this Account the developments and applications of advanced ex situ and/or in situ SSNMR techniques, such as two-dimensional (2D) double-quantum magic-angle spinning (DQ MAS) homonuclear correlation spectroscopy for structural investigation of solid acids as well as study of their acidic properties. Moreover, the energies and electronic structures of the catalysts and detailed catalytic reaction processes, including the identification of reaction species, elucidation of reaction mechanism, and verification of structure-activity correlations, made available by DFT theoretical calculations were also discussed. Relevant discussions will focus primarily on results obtained from our laboratories in the past decade, including (i) quantitative and qualitative acidity characterization utilizing assorted probe molecules

  10. Short-Term Summer Inundation as a Measure to Counteract Acidification in Rich Fens.

    PubMed

    Mettrop, Ivan S; Cusell, Casper; Kooijman, Annemieke M; Lamers, Leon P M

    2015-01-01

    In regions with intensive agriculture, water level fluctuation in wetlands has generally become constricted within narrow limits. Water authorities are, however, considering the re-establishment of fluctuating water levels as a management tool in biodiverse, base-rich fens ('rich fens'). This includes temporary inundation with surface water from ditches, which may play an important role in counteracting acidification in order to conserve and restore biodiversity. Inundation may result in an increased acid neutralizing capacity (ANC) for two reasons: infiltration of base-rich inundation water into peat soils, and microbial alkalinity generation under anaerobic conditions. The main objectives of this study were to test whether short-term (2 weeks) summer inundation is more effective than short-term winter inundation to restore the ANC in the upper 10 cm of non-floating peat soils, and to explain potential differences. Large-scale field experiments were conducted for five years in base-rich fens and Sphagnum-dominated poor fens. Winter inundation did not result in increased porewater ANC, because infiltration was inhibited in the waterlogged peat and evapotranspiration rates were relatively low. Also, low temperatures limit microbial alkalinity generation. In summer, however, when temperature and evapotranspiration rates are higher, inundation resulted in increased porewater Ca and HCO3- concentrations, but only in areas with characteristic rich fen bryophytes. This increase was not only due to stronger infiltration into the soil, but also to higher microbial alkalinity generation under anaerobic conditions. In contrast, porewater ANC did not increase in Sphagnum-plots as a result of the ability of Sphagnum spp. to acidify their environment. In both rich and poor fens, flooding-induced P-mobilization remained sufficiently low to safeguard P-limited vegetation. NO3(-) and NH4(+) dynamics showed no considerable changes either. In conclusion, short-term summer inundation

  11. Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking*

    PubMed Central

    Wong, Shenq-Huey; King, Chih-Yen

    2015-01-01

    Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1–61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1–61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1–61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of 14N- and 15N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains. PMID:26265470

  12. Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking.

    PubMed

    Wong, Shenq-Huey; King, Chih-Yen

    2015-10-09

    Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of (14)N- and (15)N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains.

  13. Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid

    PubMed Central

    1978-01-01

    Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle. PMID:83321

  14. Mollusc and plant assemblages controlled by different ecological gradients at Eastern European fens

    NASA Astrophysics Data System (ADS)

    Schenková, Veronika; Horsák, Michal; Hájek, Michal; Plesková, Zuzana; Dítě, Daniel; Pawlikowski, Paweł

    2014-04-01

    Ecological patterns of mollusc assemblages and vegetation in relation to water chemistry, water regime, nutrient availability and climate were studied in eastern Polish lowland fens. Our goal was to examine if major compositional changes differ for molluscs and vegetation under the joint influence of multiple ecological gradients. Altogether 32 fen sites were investigated in 2010-2011, and analyzed using metric multidimensional scaling, cluster analysis and generalized additive models. Two major gradients driving the differences in mollusc species composition were revealed. The main direction of compositional changes was associated with the water table gradient, governing a species turnover from inundated and strongly water-logged sites occupied mostly by aquatic mollusc species, to moderately wet sites with the predominance of fen and meadow species. The second most important gradient for molluscs was that of mineral richness. For vegetation, three major gradients explained the changes in species composition. The highest importance was assigned to the nitrogen-to-phosphorus availability gradient (defined as a shift from N-limited to P-limited vegetation), followed by the water table gradient, and the mineral richness gradient. Our results demonstrate that the impact of mineral richness gradient, which has been often reported as the major determinant of compositional changes of fen molluscs and vegetation, can be exceeded by other ecological gradients of comparable variation. We also document for the first time that the main species turnover of fen vegetation is not accompanied by the analogous change in species composition of mollusc assemblages, due to a different sensitivity of these taxa to particular environmental factors (i.e. water level dynamics and type of nutrient limitation).

  15. Microbial Response to Soil Liming of Damaged Ecosystems Revealed by Pyrosequencing and Phospholipid Fatty Acid Analyses

    PubMed Central

    Narendrula-Kotha, Ramya; Nkongolo, Kabwe K.

    2017-01-01

    Aims To assess the effects of dolomitic limestone applications on soil microbial communities’ dynamics and bacterial and fungal biomass, relative abundance, and diversity in metal reclaimed regions. Methods and Results The study was conducted in reclaimed mining sites and metal uncontaminated areas. The limestone applications were performed over 35 years ago. Total microbial biomass was determined by Phospholipid fatty acids. Bacterial and fungal relative abundance and diversity were assessed using 454 pyrosequencing. There was a significant increase of total microbial biomass in limed sites (342 ng/g) compared to unlimed areas (149 ng/g). Chao1 estimates followed the same trend. But the total number of OTUs (Operational Taxonomic Units) in limed (463 OTUs) and unlimed (473 OTUs) soil samples for bacteria were similar. For fungi, OTUs were 96 and 81 for limed and unlimed soil samples, respectively. Likewise, Simpson and Shannon diversity indices revealed no significant differences between limed and unlimed sites. Bacterial and fungal groups specific to either limed or unlimed sites were identified. Five major bacterial phyla including Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, and Proteobacteria were found. The latter was the most prevalent phylum in all the samples with a relative abundance of 50%. Bradyrhizobiaceae family with 12 genera including the nitrogen fixing Bradirhizobium genus was more abundant in limed sites compared to unlimed areas. For fungi, Ascomycota was the most predominant phylum in unlimed soils (46%) while Basidiomycota phylum represented 86% of all fungi in the limed areas. Conclusion Detailed analysis of the data revealed that although soil liming increases significantly the amount of microbial biomass, the level of species diversity remain statistically unchanged even though the microbial compositions of the damaged and restored sites are different. Significance and Impact of the study Soil liming still have a significant

  16. BOREAS TF-11 SSA-Fen Leaf Gas Exchange Data

    NASA Technical Reports Server (NTRS)

    Arkebauer, Timothy J.; Hall, Forrest G. (Editor); Knapp, David E. (Editor)

    2000-01-01

    The BOREAS TF-11 team gathered a variety of data to complement its tower flux measurements collected at the SSA-Fen site. This data set contains single-leaf gas exchange data from the SSA-Fen site during 1994 and 1995. These leaf gas exchange properties were measured for the dominant vascular plants using portable gas exchange systems. The data are stored in tabular ASCII files.

  17. Transcriptome analysis reveals specific modulation of abscisic acid signaling by ROP10 small GTPase in Arabidopsis.

    PubMed

    Xin, Zeyu; Zhao, Yihong; Zheng, Zhi-Liang

    2005-11-01

    Abscisic acid (ABA) is a hormone that modulates a variety of agronomically important growth and developmental processes and various stresses responses, but its signal transduction pathways remain poorly understood. ROP10, a member of ROP small GTPases in Arabidopsis (Arabidopsis thaliana), is a plasma membrane-associated protein specifically involved in negative regulation of ABA responses. To dissect the ROP10-mediated ABA signaling, we carried out transcriptome analysis using the Arabidopsis full-genome chip. Our analysis revealed a total of 262 and 125 genes that were, respectively, up- and down-regulated (> or =2-fold cutoff) by 1 mum ABA in wild type (Wassilewskija [Ws]); 42 up-regulated and 38 down-regulated genes have not been identified in other studies. Consistent with the nonpleiotropic phenotypes of rop10-1, only three genes were altered in rop10-1 in the absence of ABA treatment. In response to 1 microm ABA, 341 and 127 genes were, respectively, activated and repressed in rop10-1. Interestingly, a particular subset of 21 genes that were not altered by 1 microm ABA in Ws but only activated in rop10-1 was identified. Reverse transcription-polymerase chain reaction analysis revealed the existence of three distinct categories of ABA dose-response patterns. One novel category is characterized by their ABA unresponsiveness in Ws and activation in rop10-1 at 1 microm but not 10 and 100 microm of ABA. This indicates that ROP10 gates the expression of genes that are specific to low concentrations of ABA. Furthermore, almost all of these 21 genes are known to be highly induced by various biotic and abiotic stresses. Consequently, we found that rop10-1 enhanced the sensitivity of seed germination inhibition to mannitol and sodium chloride. Our results suggest that ROP10 negatively regulates ABA responses by specifically and differentially modulating the ABA sensitivity of a subset of genes including protein kinases and zinc-finger family proteins.

  18. Optimized Jasmonic Acid Production by Lasiodiplodia theobromae Reveals Formation of Valuable Plant Secondary Metabolites

    PubMed Central

    Eng, Felipe; Haroth, Sven; Feussner, Kirstin; Meldau, Dorothea; Rekhter, Dmitrij; Ischebeck, Till; Brodhun, Florian

    2016-01-01

    Jasmonic acid is a plant hormone that can be produced by the fungus Lasiodiplodia theobromae via submerged fermentation. From a biotechnological perspective jasmonic acid is a valuable feedstock as its derivatives serve as important ingredients in different cosmetic products and in the future it may be used for pharmaceutical applications. The objective of this work was to improve the production of jasmonic acid by L. theobromae strain 2334. We observed that jasmonic acid formation is dependent on the culture volume. Moreover, cultures grown in medium containing potassium nitrate as nitrogen source produced higher amounts of jasmonic acid than analogous cultures supplemented with ammonium nitrate. When cultivated under optimal conditions for jasmonic acid production, L. theobromae secreted several secondary metabolites known from plants into the medium. Among those we found 3-oxo-2-(pent-2-enyl)-cyclopentane-1-butanoic acid (OPC-4) and hydroxy-jasmonic acid derivatives, respectively, suggesting that fungal jasmonate metabolism may involve similar reaction steps as that of plants. To characterize fungal growth and jasmonic acid-formation, we established a mathematical model describing both processes. This model may form the basis of industrial upscaling attempts. Importantly, it showed that jasmonic acid-formation is not associated to fungal growth. Therefore, this finding suggests that jasmonic acid, despite its enormous amount being produced upon fungal development, serves merely as secondary metabolite. PMID:27907207

  19. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics

    SciTech Connect

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick SG; Huang, Li-Nan; Shu, Wen-Sheng

    2014-11-07

    Here we report that high-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a ‘divide and conquer’ strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We report the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Finally, our study demonstrates the potential of the ‘divide and conquer’ strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.

  20. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics

    DOE PAGES

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; ...

    2014-11-07

    Here we report that high-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a ‘divide and conquer’ strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We reportmore » the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Finally, our study demonstrates the potential of the ‘divide and conquer’ strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.« less

  1. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics.

    PubMed

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick S G; Huang, Li-Nan; Shu, Wen-Sheng

    2015-06-01

    High-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a 'divide and conquer' strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We report the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Our study demonstrates the potential of the 'divide and conquer' strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.

  2. Temperate zone fens of the glaciated Midwestern USA

    USGS Publications Warehouse

    Amon, J.P.; Thompson, C.A.; Carpenter, Q.J.; Miner, J.

    2002-01-01

    A study of more than 70 fens in the Midwestern United States and a review of the literature indicates that these temperate zone wetlands may differ from fens of the boreal zone and are not adequately differentiated from them by present classification systems. Fens of the Midwestern temperate zone 1) are wetlands with high botanical diversity, 2) are supported in part by ground water with conductivity > 100mS/ cm and circumneutral pH, 3) contain water in the root zone during most of the growing season yet are not usually inundated, and 4) accumulate organic and/or carbonate substrates. Individually, none of these descriptors is adequate to distinguish fens from other wetland communities of the Midwest such as marshes, sedge meadows, and wet prairies; yet, when they are taken together, such discrimination is possible. While fens of this zone share many species, our study does not support using indicator species because too few are both faithfully represented and geographically widespread. Midwestern temperate fens are sustained by forces of climate, landscape, and geology, which permit ground water to seep continuously into the root zone in a focused location. Since water availability in the temperate Midwest is less than in the boreal zone, continuous discharge is needed to maintain the saturation conducive to peat formation. ?? 2002, The Society of Wetland Scientists.

  3. Short-Term Summer Inundation as a Measure to Counteract Acidification in Rich Fens

    PubMed Central

    Mettrop, Ivan S.; Cusell, Casper; Kooijman, Annemieke M.; Lamers, Leon P. M.

    2015-01-01

    In regions with intensive agriculture, water level fluctuation in wetlands has generally become constricted within narrow limits. Water authorities are, however, considering the re-establishment of fluctuating water levels as a management tool in biodiverse, base-rich fens (‘rich fens’). This includes temporary inundation with surface water from ditches, which may play an important role in counteracting acidification in order to conserve and restore biodiversity. Inundation may result in an increased acid neutralizing capacity (ANC) for two reasons: infiltration of base-rich inundation water into peat soils, and microbial alkalinity generation under anaerobic conditions. The main objectives of this study were to test whether short-term (2 weeks) summer inundation is more effective than short-term winter inundation to restore the ANC in the upper 10 cm of non-floating peat soils, and to explain potential differences. Large-scale field experiments were conducted for five years in base-rich fens and Sphagnum-dominated poor fens. Winter inundation did not result in increased porewater ANC, because infiltration was inhibited in the waterlogged peat and evapotranspiration rates were relatively low. Also, low temperatures limit microbial alkalinity generation. In summer, however, when temperature and evapotranspiration rates are higher, inundation resulted in increased porewater Ca and HCO3- concentrations, but only in areas with characteristic rich fen bryophytes. This increase was not only due to stronger infiltration into the soil, but also to higher microbial alkalinity generation under anaerobic conditions. In contrast, porewater ANC did not increase in Sphagnum-plots as a result of the ability of Sphagnum spp. to acidify their environment. In both rich and poor fens, flooding-induced P-mobilization remained sufficiently low to safeguard P-limited vegetation. NO3- and NH4+ dynamics showed no considerable changes either. In conclusion, short-term summer inundation

  4. Lipidomic Profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii Reveals Critical Changes in Lipid Composition in Response to Acetic Acid Stress

    PubMed Central

    Riezman, Howard; Olsson, Lisbeth; Bettiga, Maurizio

    2013-01-01

    When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L−1, while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L−1 acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large

  5. Lipidomic profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii reveals critical changes in lipid composition in response to acetic acid stress.

    PubMed

    Lindberg, Lina; Santos, Aline Xs; Riezman, Howard; Olsson, Lisbeth; Bettiga, Maurizio

    2013-01-01

    When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L(-1), while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L(-1) acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large

  6. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

    PubMed Central

    Junager, Nina P. L.; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. PMID:27472344

  7. Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

    PubMed Central

    Bera, Asim K; Aukema, Kelly G.; Elias, Mikael; Wackett, Lawrence P.

    2017-01-01

    Cyanuric acid hydrolases are of industrial importance because of their use in aquatic recreational facilities to remove cyanuric acid, a stabilizer for the chlorine. Degradation of excess cyanuric acid is necessary to maintain chlorine disinfection in the waters. Cyanuric acid hydrolase opens the cyanuric acid ring hydrolytically and subsequent decarboxylation produces carbon dioxide and biuret. In the present study, we report the X-ray structure of TrzD, a cyanuric acid hydrolase from Acidovorax citrulli. The crystal structure at 2.19 Å resolution shows a large displacement of the catalytic lysine (Lys163) in domain 2 away from the active site core, whereas the two other active site lysines from the two other domains are not able to move. The lysine displacement is proposed here to open up a channel for product release. Consistent with that, the structure also showed two molecules of the co-product, carbon dioxide, one in the active site and another trapped in the proposed exit channel. Previous data indicated that the domain 2 lysine residue plays a role in activating an adjacent serine residue carrying out nucleophilic attack, opening the cyanuric acid ring, and the mobile lysine guides products through the exit channel. PMID:28345631

  8. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  9. Chemoproteomic Profiling of Acetanilide Herbicides Reveals Their Role in Inhibiting Fatty Acid Oxidation.

    PubMed

    Counihan, Jessica L; Duckering, Megan; Dalvie, Esha; Ku, Wan-Min; Bateman, Leslie A; Fisher, Karl J; Nomura, Daniel K

    2017-03-17

    Acetanilide herbicides are among the most widely used pesticides in the United States, but their toxicological potential and mechanisms remain poorly understood. Here, we have used chemoproteomic platforms to map proteome-wide cysteine reactivity of acetochlor (AC), the most widely used acetanilide herbicide, in vivo in mice. We show that AC directly reacts with >20 protein targets in vivo in mouse liver, including the catalytic cysteines of several thiolase enzymes involved in mitochondrial and peroxisomal fatty acid oxidation. We show that the fatty acids that are not oxidized, due to impaired fatty acid oxidation, are instead diverted into other lipid pathways, resulting in heightened free fatty acids, triglycerides, cholesteryl esters, and other lipid species in the liver. Our findings show the utility of chemoproteomic approaches for identifying novel mechanisms of toxicity associated with environmental chemicals like acetanilide herbicides.

  10. Natural variation among Arabidopsis accessions reveals malic acid as a key mediator of Nickel (Ni) tolerance.

    PubMed

    Agrawal, Bhavana; Lakshmanan, Venkatachalam; Kaushik, Shail; Bais, Harsh P

    2012-08-01

    Plants have evolved various mechanisms for detoxification that are specific to the plant species as well as the metal ion chemical properties. Malic acid, which is commonly found in plants, participates in a number of physiological processes including metal chelation. Using natural variation among Arabidopsis accessions, we investigated the function of malic acid in Nickel (Ni) tolerance and detoxification. The Ni-induced production of reactive oxygen species was found to be modulated by intracellular malic acid, indicating its crucial role in Ni detoxification. Ni tolerance in Arabidopsis may actively involve malic acid and/or complexes of Ni and malic acid. Investigation of malic acid content in roots among tolerant ecotypes suggested that a complex of Ni and malic acid may be involved in translocation of Ni from roots to leaves. The exudation of malic acid from roots in response to Ni treatment in either susceptible or tolerant plant species was found to be partially dependent on AtALMT1 expression. A lower concentration of Ni (10 µM) treatment induced AtALMT1 expression in the Ni-tolerant Arabidopsis ecotypes. We found that the ecotype Santa Clara (S.C.) not only tolerated Ni but also accumulated more Ni in leaves compared to other ecotypes. Thus, the ecotype S.C. can be used as a model system to delineate the biochemical and genetic basis of Ni tolerance, accumulation, and detoxification in plants. The evolution of Ni hyperaccumulators, which are found in serpentine soils, is an interesting corollary to the fact that S.C. is also native to serpentine soils.

  11. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  12. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-07

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.

  13. Warming increases isoprene emissions from an arctic fen.

    PubMed

    Lindwall, Frida; Svendsen, Sophie Sylvest; Nielsen, Cecilie Skov; Michelsen, Anders; Rinnan, Riikka

    2016-05-15

    Emissions of biogenic volatile organic compounds (BVOCs) from dry ecosystems at high latitudes respond strongly to small increases in temperature, and warm canopy surface temperatures drive emissions to higher levels than expected. However, it is not known whether emissions from wetlands, cooled by through-flowing water and higher evapotranspiration show similar response to warming as in drier ecosystems. Climate change will cause parts of the Arctic to experience increased snow fall, which delays the start of the growing season, insulates soil from low temperatures in winter, and increases soil moisture and possibly nutrient availability. Currently the effects of increasing snow depth on BVOC emissions are unknown. BVOC emissions were measured in situ across the growing season in a climate experiment, which used open top chambers to increase temperature and snow fences to increase winter snow depth. The treatments were arranged in a full factorial design. Measurements took place during two growing seasons in a fen ecosystem in west Greenland. BVOC samples collected by an enclosure technique in adsorbent cartridges were analysed using gas chromatography-mass spectrometry. Gross ecosystem production (GEP) was measured with a closed chamber technique, to reveal any immediate effect of treatments on photosynthesis, which could further influence BVOC emissions. Isoprene made up 84-92% of the emitted BVOCs. Isoprene emission increased 240 and 340% due to an increase in temperature of 1.3 and 1.6°C in 2014 and 2015, respectively. Isoprene emissions were 25 times higher in 2015 than in 2014 most likely due to a 2.4°C higher canopy air temperature during sampling in 2015. Snow addition had no significant effect on isoprene emissions even though GEP was increased by 24%. Arctic BVOC emissions respond strongly to rising temperatures in wet ecosystems, suggesting a large increase in arctic emissions in a future warmer climate.

  14. Tertiary structure of human alpha1-acid glycoprotein (orosomucoid). Straightforward fluorescence experiments revealing the presence of a binding pocket.

    PubMed

    Albani, Jihad R

    2004-02-25

    Binding of hemin to alpha1-acid glycoprotein has been investigated. Hemin binds to the hydrophobic pocket of hemoproteins. The fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonate (TNS) binds to a hydrophobic domain in alpha1-acid glycoprotein with a dissociation constant equal to 60 microM. Addition of hemin to an alpha1-acid glycoprotein-TNS complex induces the displacement of TNS from its binding site. At saturation (1 hemin for 1 protein) all the TNS has been displaced from its binding site. The dissociation constant of hemin-alpha1-acid glycoprotein was found equal to 2 microM. Thus, TNS and hemin bind to the same hydrophobic site: the pocket of alpha1-acid glycoprotein. Energy-transfer studies performed between the Trp residues of alpha1-acid glycoprotein and hemin indicated that efficiency (E) of Trp fluorescence quenching was equal to 80% and the Förster distance, R0 at which the efficiency of energy transfer is 50% was calculated to be 26 A, revealing a very high energy transfer.

  15. Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

    PubMed

    Patella, Francesca; Schug, Zachary T; Persi, Erez; Neilson, Lisa J; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-03-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability.

  16. Comparative Transcriptomics Reveals Discrete Survival Responses of S. aureus and S. epidermidis to Sapienic Acid

    PubMed Central

    Moran, Josephine C.; Alorabi, Jamal A.; Horsburgh, Malcolm J.

    2017-01-01

    Staphylococcal colonization of human skin is ubiquitous, with particular species more frequent at different body sites. Whereas Staphylococcus epidermidis can be isolated from the skin of every individual tested, Staphylococcus aureus is isolated from <5% of healthy individuals. The factors that drive staphylococcal speciation and niche selection on skin are incompletely defined. Here we show that S. aureus is inhibited to a greater extent than S. epidermidis by the sebaceous lipid sapienic acid, supporting a role for this skin antimicrobial in selection of skin staphylococci. We used RNA-Seq and comparative transcriptomics to identify the sapienic acid survival responses of S. aureus and S. epidermidis. Consistent with the membrane depolarization mode of action of sapienic acid, both species shared a common transcriptional response to counteract disruption of metabolism and transport. The species differed in their regulation of SaeRS and VraRS regulons. While S. aureus upregulated urease operon transcription, S. epidermidis upregulated arginine deiminase, the oxygen-responsive NreABC nitrogen regulation system and the nitrate and nitrite reduction pathways. The role of S. aureus ACME and chromosomal arginine deiminase pathways in sapienic acid resistance was determined through mutational studies. We speculate that ammonia production could contribute to sapienic acid resistance in staphylococci. PMID:28179897

  17. Comparative Transcriptomics Reveals Discrete Survival Responses of S. aureus and S. epidermidis to Sapienic Acid.

    PubMed

    Moran, Josephine C; Alorabi, Jamal A; Horsburgh, Malcolm J

    2017-01-01

    Staphylococcal colonization of human skin is ubiquitous, with particular species more frequent at different body sites. Whereas Staphylococcus epidermidis can be isolated from the skin of every individual tested, Staphylococcus aureus is isolated from <5% of healthy individuals. The factors that drive staphylococcal speciation and niche selection on skin are incompletely defined. Here we show that S. aureus is inhibited to a greater extent than S. epidermidis by the sebaceous lipid sapienic acid, supporting a role for this skin antimicrobial in selection of skin staphylococci. We used RNA-Seq and comparative transcriptomics to identify the sapienic acid survival responses of S. aureus and S. epidermidis. Consistent with the membrane depolarization mode of action of sapienic acid, both species shared a common transcriptional response to counteract disruption of metabolism and transport. The species differed in their regulation of SaeRS and VraRS regulons. While S. aureus upregulated urease operon transcription, S. epidermidis upregulated arginine deiminase, the oxygen-responsive NreABC nitrogen regulation system and the nitrate and nitrite reduction pathways. The role of S. aureus ACME and chromosomal arginine deiminase pathways in sapienic acid resistance was determined through mutational studies. We speculate that ammonia production could contribute to sapienic acid resistance in staphylococci.

  18. Conversion of Fe-NH2 to Fe-N2 with release of NH3

    PubMed Central

    Anderson, John S.; Moret, Marc-Etienne; Peters, Jonas C.

    2013-01-01

    Tris(phosphine)borane ligated Fe(I) centers featuring N2H4, NH3, NH2, and OH ligands are described. Conversion of Fe-NH2 to Fe-NH3+ by addition of acid, and subsequent reductive release of NH3 to generate Fe-N2, is demonstrated. This sequence models the final steps of proposed Fe-mediated nitrogen fixation pathways. The five-coordinate trigonal bipyramidal complexes described are unusual in that they adopt S = 3/2 ground states and are prepared from a four-coordinate, S = 3/2 trigonal pyramidal precursor. PMID:23259776

  19. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs.

  20. Six Tissue Transcriptomics Reveals Specific Immune Suppression in Spleen by Dietary Polyunsaturated Fatty Acids

    PubMed Central

    Gabrielsson, Britt G.; Peris, Eduard; Nookaew, Intawat; Grahnemo, Louise; Sandberg, Ann-Sofie; Wernstedt Asterholm, Ingrid; Jansson, John-Olov; Nielsen, Jens

    2016-01-01

    Dietary polyunsaturated fatty acids (PUFA) are suggested to modulate immune function, but the effects of dietary fatty acids composition on gene expression patterns in immune organs have not been fully characterized. In the current study we investigated how dietary fatty acids composition affects the total transcriptome profile, and especially, immune related genes in two immune organs, spleen (SPL) and bone marrow cells (BMC). Four tissues with metabolic function, skeletal muscle (SKM), white adipose tissue (WAT), brown adipose tissue (BAT), and liver (LIV), were investigated as a comparison. Following 8 weeks on low fat diet (LFD), high fat diet (HFD) rich in saturated fatty acids (HFD-S), or HFD rich in PUFA (HFD-P), tissue transcriptomics were analyzed by microarray and metabolic health assessed by fasting blood glucose level, HOMA-IR index, oral glucose tolerance test as well as quantification of crown-like structures in WAT. HFD-P corrected the metabolic phenotype induced by HFD-S. Interestingly, SKM and BMC were relatively inert to the diets, whereas the two adipose tissues (WAT and BAT) were mainly affected by HFD per se (both HFD-S and HFD-P). In particular, WAT gene expression was driven closer to that of the immune organs SPL and BMC by HFDs. The LIV exhibited different responses to both of the HFDs. Surprisingly, the spleen showed a major response to HFD-P (82 genes differed from LFD, mostly immune genes), while it was not affected at all by HFD-S (0 genes differed from LFD). In conclusion, the quantity and composition of dietary fatty acids affected the transcriptome in distinct manners in different organs. Remarkably, dietary PUFA, but not saturated fat, prompted a specific regulation of immune related genes in the spleen, opening the possibility that PUFA can regulate immune function by influencing gene expression in this organ. PMID:27166587

  1. Sulphur Kβ emission spectra reveal protonation states of aqueous sulfuric acid

    PubMed Central

    Niskanen, Johannes; Sahle, Christoph J.; Ruotsalainen, Kari O.; Müller, Harald; Kavčič, Matjaž; Žitnik, Matjaž; Bučar, Klemen; Petric, Marko; Hakala, Mikko; Huotari, Simo

    2016-01-01

    In this paper we report an X-ray emission study of bulk aqueous sulfuric acid. Throughout the range of molarities from 1 M to 18 M the sulfur Kβ emission spectra from H2SO4 (aq) depend on the molar fractions and related deprotonation of H2SO4. We compare the experimental results with results from emission spectrum calculations based on atomic structures of single molecules and structures from ab initio molecular dynamics simulations. We show that the S Kβ emission spectrum is a sensitive probe of the protonation state of the acid molecules. Using non-negative matrix factorization we are able to extract the fractions of different protonation states in the spectra, and the results are in good agreement with the simulation for the higher part of the concentration range. PMID:26888159

  2. beta-Keratins in crocodiles reveal amino acid homology with avian keratins.

    PubMed

    Ye, Changjiang; Wu, Xiaobing; Yan, Peng; Amato, George

    2010-03-01

    The DNA sequences encoding beta-keratin have been obtained from Marsh Mugger (Crocodylus palustris) and Orinoco Crocodiles (Crocodylus intermedius). Through the deduced amino acid sequence, these proteins are rich in glycine, proline and serine. The central region of the proteins are composed of two beta-folded regions and show a high degree of identity with beta-keratins of aves and squamates. This central part is thought to be the site of polymerization to build the framework of beta-keratin filaments. It is believed that the beta-keratins in reptiles and birds share a common ancestry. Near the C-terminal, these beta-keratins contain a peptide rich in glycine-X and glycine-X-X, and the distinctive feature of the region is some 12-amino acid repeats, which are similar to the 13-amino acid repeats in chick scale keratin but absent from avian feather keratin. From our phylogenetic analysis, the beta-keratins in crocodile have a closer relationship with avian keratins than the other keratins in reptiles.

  3. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

    PubMed

    Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

    2012-07-31

    Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

  4. Proteomic Stable Isotope Probing Reveals Taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

    PubMed Central

    Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; Hettich, Robert L.; Mayali, Xavier; Pan, Chongle

    2016-01-01

    ABSTRACT Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual primary production, yet defining how substrate utilization preferences and resource partitioning structure microbial communities remains a challenge. In this study, proteomic stable isotope probing (proteomic SIP) was used to characterize population-specific assimilation of dissolved free amino acids (DFAAs), a major source of dissolved organic carbon for bacterial secondary production in aquatic environments. Microcosms of seawater collected from Newport, Oregon, and Monterey Bay, California, were incubated with 1 µM 13C-labeled amino acids for 15 and 32 h. The taxonomic compositions of microcosm metaproteomes were highly similar to those of the sampled natural communities, with Rhodobacteriales, SAR11, and Flavobacteriales representing the dominant taxa. Analysis of 13C incorporation into protein biomass allowed for quantification of the isotopic enrichment of identified proteins and subsequent determination of differential amino acid assimilation patterns between specific bacterioplankton populations. Proteins associated with Rhodobacterales tended to have a significantly high frequency of 13C-enriched peptides, opposite the trend for Flavobacteriales and SAR11 proteins. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time point 2. Alteromonadales proteins also had a significantly high frequency of 13C-enriched peptides, particularly within ribosomal proteins, demonstrating their rapid growth during incubations. Overall, proteomic SIP facilitated quantitative comparisons of DFAA assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population level metabolic responses to resource acquisition in complex microbial communities

  5. Proteomic Stable Isotope Probing Reveals Taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton.

    PubMed

    Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; Hettich, Robert L; Mayali, Xavier; Pan, Chongle; Mueller, Ryan S

    2016-01-01

    Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual primary production, yet defining how substrate utilization preferences and resource partitioning structure microbial communities remains a challenge. In this study, proteomic stable isotope probing (proteomic SIP) was used to characterize population-specific assimilation of dissolved free amino acids (DFAAs), a major source of dissolved organic carbon for bacterial secondary production in aquatic environments. Microcosms of seawater collected from Newport, Oregon, and Monterey Bay, California, were incubated with 1 µM (13)C-labeled amino acids for 15 and 32 h. The taxonomic compositions of microcosm metaproteomes were highly similar to those of the sampled natural communities, with Rhodobacteriales, SAR11, and Flavobacteriales representing the dominant taxa. Analysis of (13)C incorporation into protein biomass allowed for quantification of the isotopic enrichment of identified proteins and subsequent determination of differential amino acid assimilation patterns between specific bacterioplankton populations. Proteins associated with Rhodobacterales tended to have a significantly high frequency of (13)C-enriched peptides, opposite the trend for Flavobacteriales and SAR11 proteins. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of (13)C-enriched spectra at time point 2. Alteromonadales proteins also had a significantly high frequency of (13)C-enriched peptides, particularly within ribosomal proteins, demonstrating their rapid growth during incubations. Overall, proteomic SIP facilitated quantitative comparisons of DFAA assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population level metabolic responses to resource acquisition in complex microbial communities

  6. Mutagenesis of conserved amino acids of Helicobacter pylori fur reveals residues important for function.

    PubMed

    Carpenter, Beth M; Gancz, Hanan; Benoit, Stéphane L; Evans, Sarah; Olsen, Cara H; Michel, Sarah L J; Maier, Robert J; Merrell, D Scott

    2010-10-01

    The ferric uptake regulator (Fur) of the medically important pathogen Helicobacter pylori is unique in that it has been shown to function as a repressor both in the presence of an Fe2+ cofactor and in its apo (non-Fe2+-bound) form. However, virtually nothing is known concerning the amino acid residues that are important for Fur functioning. Therefore, mutations in six conserved amino acid residues of H. pylori Fur were constructed and analyzed for their impact on both iron-bound and apo repression. In addition, accumulation of the mutant proteins, protein secondary structure, DNA binding ability, iron binding capacity, and the ability to form higher-order structures were also examined for each mutant protein. While none of the mutated residues completely abrogated the function of Fur, we were able to identify residues that were critical for both iron-bound and apo-Fur repression. One mutation, V64A, did not alter regulation of any target genes. However, each of the five remaining mutations showed an effect on either iron-bound or apo regulation. Of these, H96A, E110A, and E117A mutations altered iron-bound Fur regulation and were all shown to influence iron binding to different extents. Additionally, the H96A mutation was shown to alter Fur oligomerization, and the E110A mutation was shown to impact oligomerization and DNA binding. Conversely, the H134A mutant exhibited changes in apo-Fur regulation that were the result of alterations in DNA binding. Although the E90A mutant exhibited alterations in apo-Fur regulation, this mutation did not affect any of the assessed protein functions. This study is the first for H. pylori to analyze the roles of specific amino acid residues of Fur in function and continues to highlight the complexity of Fur regulation in this organism.

  7. Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure.

    PubMed

    Stockand, James D; Staruschenko, Alexander; Pochynyuk, Oleh; Booth, Rachell E; Silverthorn, Dee U

    2008-09-01

    The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.

  8. Contrasting species-environment relationships in communities of testate amoebae, bryophytes and vascular plants along the fen-bog gradient.

    PubMed

    Lamentowicz, Mariusz; Lamentowicz, Lukasz; van der Knaap, Willem O; Gabka, Maciej; Mitchell, Edward A D

    2010-04-01

    We studied the vegetation, testate amoebae and abiotic variables (depth of the water table, pH, electrical conductivity, Ca and Mg concentrations of water extracted from mosses) along the bog to extremely rich fen gradient in sub-alpine peatlands of the Upper Engadine (Swiss Alps). Testate amoeba diversity was correlated to that of mosses but not of vascular plants. Diversity peaked in rich fen for testate amoebae and in extremely rich fen for mosses, while for testate amoebae and mosses it was lowest in bog but for vascular plants in extremely rich fen. Multiple factor and redundancy analyses (RDA) revealed a stronger correlation of testate amoebae than of vegetation to water table and hydrochemical variables and relatively strong correlation between testate amoeba and moss community data. In RDA, hydrochemical variables explained a higher proportion of the testate amoeba and moss data than water table depth. Abiotic variables explained a higher percentage of the species data for testate amoebae (30.3% or 19.5% for binary data) than for mosses (13.4%) and vascular plants (10%). These results show that (1) vascular plant, moss and testate amoeba communities respond differently to ecological gradients in peatlands and (2) testate amoebae are more strongly related than vascular plants to the abiotic factors at the mire surface. These differences are related to vertical trophic gradients and associated niche differentiation.

  9. Chemical genetics reveals negative regulation of abscisic acid signaling by a plant immune response pathway.

    PubMed

    Kim, Tae-Houn; Hauser, Felix; Ha, Tracy; Xue, Shaowu; Böhmer, Maik; Nishimura, Noriyuki; Munemasa, Shintaro; Hubbard, Katharine; Peine, Nora; Lee, Byeong-Ha; Lee, Stephen; Robert, Nadia; Parker, Jane E; Schroeder, Julian I

    2011-06-07

    Coordinated regulation of protection mechanisms against environmental abiotic stress and pathogen attack is essential for plant adaptation and survival. Initial abiotic stress can interfere with disease-resistance signaling [1-6]. Conversely, initial plant immune signaling may interrupt subsequent abscisic acid (ABA) signal transduction [7, 8]. However, the processes involved in this crosstalk between these signaling networks have not been determined. By screening a 9600-compound chemical library, we identified a small molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that rapidly downregulates ABA-dependent gene expression and also inhibits ABA-induced stomatal closure. Transcriptome analyses show that DFPM also stimulates expression of plant defense-related genes. Major early regulators of pathogen-resistance responses, including EDS1, PAD4, RAR1, and SGT1b, are required for DFPM-and notably also for Pseudomonas-interference with ABA signal transduction, whereas salicylic acid, EDS16, and NPR1 are not necessary. Although DFPM does not interfere with early ABA perception by PYR/RCAR receptors or ABA activation of SnRK2 kinases, it disrupts cytosolic Ca(2+) signaling and downstream anion channel activation in a PAD4-dependent manner. Our findings provide evidence that activation of EDS1/PAD4-dependent plant immune responses rapidly disrupts ABA signal transduction and that this occurs at the level of Ca(2+) signaling, illuminating how the initial biotic stress pathway interferes with ABA signaling.

  10. Revealing the molecular structural transformation of hardwood and softwood in dilute acid flowthrough pretreatment

    DOE PAGES

    Zhang, Libing; Pu, Yunqiao; Univ. of Tennessee, Knoxville, TN; ...

    2016-10-03

    To understand better the intrinsic recalcitrance of lignocellulosic biomass, the main hurdle to its efficient deconstruction, the effects of dilute acid flowthrough pretreatment on the dissolution chemistry of hemicellulose, cellulose, and lignin for both hardwood (e.g., poplar wood) and softwood (e.g., lodgepole pine wood) were investigated at temperatures of 200 to 270 °C and a flow rate of 25 mL/min with 0.05% (w/w) H2SO4. Results suggested that the softwood cellulose was more readily degraded into monomeric sugars than that of hardwood under same pretreatment conditions. However, while the hardwood lignin was completely removed into hydrolysate, ~30% of the softwood ligninmore » remained as solid residues under identical conditions, which was plausibly caused by vigorous C5-active recondensation reactions (C–C5). As a result, effects of molecular structural features (i.e., lignin molecular weight, cellulose crystallinity, and condensed lignin structures) on the recalcitrance of hardwood and softwood to dilute acid pretreatment were identified for the first time in this study, providing important insights to establish the effective biomass pretreatment.« less

  11. Revealing the molecular structural transformation of hardwood and softwood in dilute acid flowthrough pretreatment

    SciTech Connect

    Zhang, Libing; Pu, Yunqiao; Cort, John R.; Ragauskas, Arthur J.; Yang, Bin

    2016-10-03

    To understand better the intrinsic recalcitrance of lignocellulosic biomass, the main hurdle to its efficient deconstruction, the effects of dilute acid flowthrough pretreatment on the dissolution chemistry of hemicellulose, cellulose, and lignin for both hardwood (e.g., poplar wood) and softwood (e.g., lodgepole pine wood) were investigated at temperatures of 200 to 270 °C and a flow rate of 25 mL/min with 0.05% (w/w) H2SO4. Results suggested that the softwood cellulose was more readily degraded into monomeric sugars than that of hardwood under same pretreatment conditions. However, while the hardwood lignin was completely removed into hydrolysate, ~30% of the softwood lignin remained as solid residues under identical conditions, which was plausibly caused by vigorous C5-active recondensation reactions (C–C5). As a result, effects of molecular structural features (i.e., lignin molecular weight, cellulose crystallinity, and condensed lignin structures) on the recalcitrance of hardwood and softwood to dilute acid pretreatment were identified for the first time in this study, providing important insights to establish the effective biomass pretreatment.

  12. Stable nitrogen isotopic composition of amino acids reveals food web structure in stream ecosystems.

    PubMed

    Ishikawa, Naoto F; Kato, Yoshikazu; Togashi, Hiroyuki; Yoshimura, Mayumi; Yoshimizu, Chikage; Okuda, Noboru; Tayasu, Ichiro

    2014-07-01

    The stable N isotopic composition of individual amino acids (SIAA) has recently been used to estimate trophic positions (TPs) of animals in several simple food chain systems. However, it is unknown whether the SIAA is applicable to more complex food web analysis. In this study we measured the SIAA of stream macroinvertebrates, fishes, and their potential food sources (periphyton and leaf litter of terrestrial C3 plants) collected from upper and lower sites in two streams having contrasting riparian landscapes. The stable N isotope ratios of glutamic acid and phenylalanine confirmed that for primary producers (periphyton and C3 litter) the TP was 1, and for primary consumers (e.g., mayfly and caddisfly larvae) it was 2. We built a two-source mixing model to estimate the relative contributions of aquatic and terrestrial sources to secondary and higher consumers (e.g., stonefly larva and fishes) prior to the TP calculation. The estimated TPs (2.3-3.5) roughly corresponded to their omnivorous and carnivorous feeding habits, respectively. We found that the SIAA method offers substantial advantages over traditional bulk method for food web analysis because it defines the food web structure based on the metabolic pathway of amino groups, and can be used to estimate food web structure under conditions where the bulk method cannot be used. Our result provides evidence that the SIAA method is applicable to the analysis of complex food webs, where heterogeneous resources are mixed.

  13. Revealing the Molecular Structural Transformation of Hardwood and Softwood in Dilute Acid Flowthrough Pretreatment

    SciTech Connect

    Zhang, Libing; Pu, Yunqiao; Cort, John R.; Ragauskas, Arthur J.; Yang, Bin

    2016-12-05

    To better understand the intrinsic recalcitrance of lignocellulosic biomass, the main hurdle to its efficient deconstruction, the effects of dilute acid flowthrough pretreatment on the dissolution chemistry of hemicellulose, cellulose, and lignin for both hardwood (e.g. poplar wood) and softwood (e.g. lodgepole pine wood) were investigated at temperatures of 200 °C to 270 °C and a flow rate of 25 mL/minute with 0.05% (w/w) H2SO4. Results suggested that the softwood cellulose was more readily to be degraded into monomeric sugars than that of hardwood under same pretreatment conditions. However, while the hardwood lignin was completely removed into hydrolysate, ~30% of the softwood lignin remained as solid residues under identical conditions, which was plausibly caused by vigorous C5-active recondensation reactions (C-C5). Unique molecular structural features that pronounced the specific recalcitrance of hardwood and softwood to dilute acid pretreatment were identified for the first time in this study, providing important insights to establish the effective biomass pretreatment.

  14. A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

    PubMed

    Liu, Ting-Wu; Niu, Li; Fu, Bin; Chen, Juan; Wu, Fei-Hua; Chen, Juan; Wang, Wen-Hua; Hu, Wen-Jun; He, Jun-Xian; Zheng, Hai-Lei

    2013-01-01

    Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress.

  15. Down-regulation of the Caffeic acid O-methyltransferase Gene in Switchgrass Reveals a Novel Monolignol Analog

    SciTech Connect

    Tschaplinski, Timothy J; Standaert, Robert F; Engle, Nancy L; Martin, Madhavi Z; Sangha, Amandeep K; Parks, Jerry M; Smith, Jeremy C; Samuel, Reichel; Pu, Yunqiao; Ragauskas, A J; Hamilton, Choo Yieng; Fu, Chunxiang; Wang, Zeng-Yu; Davison, Brian H; Dixon, Richard A; Mielenz, Jonathan R

    2012-01-01

    Down-regulation of the caffeic acid 3-O-methyltransferase (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography-mass spectrometry-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors, confirming the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. Although there was no indication that iso-sinapyl alcohol was integrated into the cell wall, diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth.

  16. In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator, Euglena mutabilis.

    PubMed

    Halter, David; Goulhen-Chollet, Florence; Gallien, Sébastien; Casiot, Corinne; Hamelin, Jérôme; Gilard, Françoise; Heintz, Dimitri; Schaeffer, Christine; Carapito, Christine; Van Dorsselaer, Alain; Tcherkez, Guillaume; Arsène-Ploetze, Florence; Bertin, Philippe N

    2012-07-01

    Euglena mutabilis is a photosynthetic protist found in acidic aquatic environments such as peat bogs, volcanic lakes and acid mine drainages (AMDs). Through its photosynthetic metabolism, this protist is supposed to have an important role in primary production in such oligotrophic ecosystems. Nevertheless, the exact contribution of E. mutabilis in organic matter synthesis remains unclear and no evidence of metabolite secretion by this protist has been established so far. Here we combined in situ proteo-metabolomic approaches to determine the nature of the metabolites accumulated by this protist or potentially secreted into an AMD. Our results revealed that the secreted metabolites are represented by a large number of amino acids, polyamine compounds, urea and some sugars but no fatty acids, suggesting a selective organic matter contribution in this ecosystem. Such a production may have a crucial impact on the bacterial community present on the study site, as it has been suggested previously that prokaryotes transport and recycle in situ most of the metabolites secreted by E. mutabilis. Consequently, this protist may have an indirect but important role in AMD ecosystems but also in other ecological niches often described as nitrogen-limited.

  17. Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes

    SciTech Connect

    Lowden, Michael J.; Skorupski, Karen; Pellegrini, Maria; Chiorazzo, Michael G.; Taylor, Ronald K.; Kull, F. Jon

    2010-03-04

    Cholera is an acute intestinal infection caused by the bacterium Vibrio cholerae. In order for V. cholerae to cause disease, it must produce two virulence factors, the toxin-coregulated pilus (TCP) and cholera toxin (CT), whose expression is controlled by a transcriptional cascade culminating with the expression of the AraC-family regulator, ToxT. We have solved the 1.9 {angstrom} resolution crystal structure of ToxT, which reveals folds in the N- and C-terminal domains that share a number of features in common with AraC, MarA, and Rob as well as the unexpected presence of a buried 16-carbon fatty acid, cis-palmitoleate. The finding that cis-palmitoleic acid reduces TCP and CT expression in V. cholerae and prevents ToxT from binding to DNA in vitro provides a direct link between the host environment of V. cholerae and regulation of virulence gene expression.

  18. Pretreatment of Gymnema sylvestre revealed the protection against acetic acid-induced ulcerative colitis in rats

    PubMed Central

    2014-01-01

    Background Overproduction of free radicals and decreased antioxidant capacity are well-known risk factors for inflammatory bowel diseases. Gymnema sylvestre (GS) leaves extract is distinguished for its anti-diabetic, antioxidant and anti-inflammatory properties. Present study is designed to evaluate the preventative activities of GS against acetic acid (AA)-induced ulcerative colitis in Wistar rats. Methods Experimentally ulcerative colitis (UC) was induced by AA in animals pretreated with three different doses of GS leaves extract (50, 100, 200 mg/kg/day) and a single dose of mesalazine (MES, 300 mg/kg/day) for seven days. Twenty four hours later, animals were sacrificed and the colonic tissues were collected. Colonic mucus content was determined using Alcian blue dye binding technique. Levels of thiobarbituric acid reactive substances (TBARS), total glutathione sulfhydryl group (T-GSH) and non-protein sulfhydryl group (NPSH) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were estimated in colon tissues. Colonic nucleic acids (DNA and RNA) and total protein (TP) concentrations were also determined. Levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) as well as prostaglandin E2 (PGE2) and nitric oxide (NO) were estimated in colonic tissues. The histopathological changes of the colonic tissues were also observed. Results In AA administered group TBARS levels were increased, while colonic mucus content, T-GSH and NP-SH, SOD and CAT were reduced in colon. Pretreatment with GS inhibited TBARS elevation as well as mucus content, T-GSH and NP-SH reduction. Enzymatic activities of SOD and CAT were brought back to their normal levels in GS pretreated group. A significant reduction in DNA, RNA and TP levels was seen following AA administration and this inhibition was significantly eliminated by GS treatment. GS pretreatment also inhibited

  19. Analysis of Known Bacterial Protein Vaccine Antigens Reveals Biased Physical Properties and Amino Acid Composition

    PubMed Central

    Mayers, Carl; Rowe, Sonya; Miller, Julie; Lingard, Bryan; Hayward, Sarah; Titball, Richard W.

    2003-01-01

    Many vaccines have been developed from live attenuated forms of bacterial pathogens or from killed bacterial cells. However, an increased awareness of the potential for transient side-effects following vaccination has prompted an increased emphasis on the use of sub-unit vaccines, rather than those based on whole bacterial cells. The identification of vaccine sub-units is often a lengthy process and bioinformatics approaches have recently been used to identify candidate protein vaccine antigens. Such methods ultimately offer the promise of a more rapid advance towards preclinical studies with vaccines. We have compared the properties of known bacterial vaccine antigens against randomly selected proteins and identified differences in the make-up of these two groups. A computer algorithm that exploits these differences allows the identification of potential vaccine antigen candidates from pathogenic bacteria on the basis of their amino acid composition, a property inherently associated with sub-cellular location. PMID:18629010

  20. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors.

    PubMed

    Hopf, Thomas A; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S; Benton, Richard

    2015-01-13

    Insect odorant receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection.

  1. Stable isotope tracer reveals that viviparous snakes transport amino acids to offspring during gestation.

    PubMed

    Van Dyke, James U; Beaupre, Steven J

    2012-03-01

    Viviparity and placentation have evolved from oviparity over 100 times in squamate reptiles (lizards and snakes). The independent origins of placentation have resulted in a variety of placental morphologies in different taxa, ranging from simple apposition of fetal and maternal tissues to endotheliochorial implantation that is homoplasious with mammalian placentation. Because the eggs of oviparous squamates transport gases and water from the environment and calcium from the eggshell, the placentae of viviparous squamates are thought to have initially evolved to accomplish these functions from within the maternal oviduct. Species with complex placentae have also been shown to rely substantially, or even primarily, on placental transport of organic nutrients for embryonic nutrition. However, it is unclear whether species with only simple placentae are also capable of transporting organic nutrients to offspring. Among viviparous squamates, all of the snakes that have been studied thus far have been shown to have simple placentae. However, most studies of snake placentation are limited to a single lineage, the North American Natricinae. We tested the abilities of four species of viviparous snakes - Agkistrodon contortrix (Viperidae), Boa constrictor (Boidae), Nerodia sipedon (Colubridae: Natricinae) and Thamnophis sirtalis (Colubridae: Natricinae) - to transport diet-derived amino acids to offspring during gestation. We fed [(15)N]leucine to pregnant snakes, and compared offspring (15)N content with that of unlabeled controls. Labeled females allocated significantly more (15)N to offspring than did controls, but (15)N allocation did not differ among species. Our results indicate that viviparous snakes are capable of transporting diet-derived amino acids to their offspring during gestation, possibly via placentation.

  2. Proteomics reveals the effects of salicylic acid on growth and tolerance to subsequent drought stress in wheat.

    PubMed

    Kang, Guozhang; Li, Gezi; Xu, Wei; Peng, Xiaoqi; Han, Qiaoxia; Zhu, Yunji; Guo, Tiancai

    2012-12-07

    Pretreatment with 0.5 mM salicylic acid (SA) for 3 days significantly enhanced the growth and tolerance to subsequent drought stress (PEG-6000, 15%) in wheat seedlings, manifesting as increased shoot and root dry weights, and decreased lipid peroxidation. Total proteins from wheat leaves exposed to (i) 0.5 mM SA pretreatment, (ii) drought stress, and (iii) 0.5 mM SA treatment plus drought-stress treatments were analyzed using a proteomics method. Eighty-two stress-responsive protein spots showed significant changes, of which 76 were successfully identified by MALDI-TOF-TOF. Analysis of protein expression patterns revealed that proteins associated with signal transduction, stress defense, photosynthesis, carbohydrate metabolism, protein metabolism, and energy production could by involved in SA-induced growth and drought tolerance in wheat seedlings. Furthermore, the SA-responsive protein interaction network revealed 35 key proteins, suggesting that these proteins are critical for SA-induced tolerance.

  3. Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK.

    PubMed

    Kim, S I; Andaya, C B; Newman, J W; Goyal, S S; Tai, T H

    2008-11-01

    Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations, which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexakisphosphate). High-performance liquid chromatography and GC-MS analysis of seed extracts from N15-186 indicated that, in addition to phytic acid, inositol monophosphate was significantly reduced whereas inorganic phosphorus and myo-inositol were greatly increased when compared with wild-type. The changes observed in N15-186 resemble those previously described for the maize lpa3 mutant. Analysis of N15-375 revealed changes similar to those observed in previously characterized rice lpa1 mutants (i.e. significant reduction in phytic acid and corresponding increase in inorganic phosphorus with little or no change in inositol phosphate intermediates or myo-inositol). Further genetic analysis of the N15-186 mutant indicated that the mutation, designated lpa N15-186, was located in a region on chromosome 3 between the microsatellite markers RM15875 and RM15907. The rice orthologue of maize lpa3, which encodes a myo-inositol kinase, is in this interval. Sequence analysis of the N15-186 allele of this orthologue (Os03g52760) revealed a single base pair change (C/G to T/A) in the first exon of the gene, which results in a nonsense mutation. Our results indicate that lpa N15-186 is a mutant allele of the rice myo-inositol kinase (OsMIK) gene. Identification and characterization of lpa mutants, such as N15-186, will facilitate studies on the regulation of phytic acid biosynthesis and accumulation and help address questions concerning the contribution of the inositol lipid-dependent and independent biosynthetic pathways to the production of seed phytic acid.

  4. Quantitative Proteomics Reveals the Flooding-Tolerance Mechanism in Mutant and Abscisic Acid-Treated Soybean.

    PubMed

    Yin, Xiaojian; Nishimura, Minoru; Hajika, Makita; Komatsu, Setsuko

    2016-06-03

    Flooding negatively affects the growth of soybean, and several flooding-specific stress responses have been identified; however, the mechanisms underlying flooding tolerance in soybean remain unclear. To explore the initial flooding tolerance mechanisms in soybean, flooding-tolerant mutant and abscisic acid (ABA)-treated plants were analyzed. In the mutant and ABA-treated soybeans, 146 proteins were commonly changed at the initial flooding stress. Among the identified proteins, protein synthesis-related proteins, including nascent polypeptide-associated complex and chaperonin 20, and RNA regulation-related proteins were increased in abundance both at protein and mRNA expression. However, these proteins identified at the initial flooding stress were not significantly changed during survival stages under continuous flooding. Cluster analysis indicated that glycolysis- and cell wall-related proteins, such as enolase and polygalacturonase inhibiting protein, were increased in abundance during survival stages. Furthermore, lignification of root tissue was improved even under flooding stress. Taken together, these results suggest that protein synthesis- and RNA regulation-related proteins play a key role in triggering tolerance to the initial flooding stress in soybean. Furthermore, the integrity of cell wall and balance of glycolysis might be important factors for promoting tolerance of soybean root to flooding stress during survival stages.

  5. Competing mechanisms for perfluoroalkyl acid accumulation in plants revealed using an Arabidopsis model system.

    PubMed

    Müller, Claudia E; LeFevre, Gregory H; Timofte, Anca E; Hussain, Fatima A; Sattely, Elizabeth S; Luthy, Richard G

    2016-05-01

    Perfluoroalkyl acids (PFAAs) bioaccumulate in plants, presenting a human exposure route if present in irrigation water. Curiously, accumulation of PFAAs in plant tissues is greatest for both the short-chain and long-chain PFAAs, generating a U-shaped relationship with chain length. In the present study, the authors decouple competing mechanisms of PFAA accumulation using a hydroponic model plant system (Arabidopsis thaliana) exposed to a suite of 10 PFAAs to determine uptake, depuration, and translocation kinetics. Rapid saturation of root concentrations occurred for all PFAAs except perfluorobutanoate, the least-sorptive (shortest-chain) PFAA. Shoot concentrations increased continuously, indicating that PFAAs are efficiently transported and accumulate in shoots. Tissue concentrations of PFAAs during depuration rapidly declined in roots but remained constant in shoots, demonstrating irreversibility of the translocation process. Root and shoot concentration factors followed the U-shaped trend with perfluoroalkyl chain length; however, when normalized to dead-tissue sorption, this relationship linearized. The authors therefore introduce a novel term, the "sorption normalized concentration factor," to describe PFAA accumulation in plants; because of their hydrophobicity, sorption is the determining factor for long-chain PFAAs, whereas the shortest-chain PFAAs are most effectively transported in the plant. The present study provides a mechanistic explanation for previously unexplained PFAA accumulation trends in plants and suggests that shorter-chained PFAAs may bioaccumulate more readily in edible portions.

  6. A systems study reveals concurrent activation of AMPK and mTOR by amino acids

    PubMed Central

    Pezze, Piero Dalle; Ruf, Stefanie; Sonntag, Annika G.; Langelaar-Makkinje, Miriam; Hall, Philip; Heberle, Alexander M.; Navas, Patricia Razquin; van Eunen, Karen; Tölle, Regine C.; Schwarz, Jennifer J.; Wiese, Heike; Warscheid, Bettina; Deitersen, Jana; Stork, Björn; Fäßler, Erik; Schäuble, Sascha; Hahn, Udo; Horvatovich, Peter; Shanley, Daryl P.; Thedieck, Kathrin

    2016-01-01

    Amino acids (aa) are not only building blocks for proteins, but also signalling molecules, with the mammalian target of rapamycin complex 1 (mTORC1) acting as a key mediator. However, little is known about whether aa, independently of mTORC1, activate other kinases of the mTOR signalling network. To delineate aa-stimulated mTOR network dynamics, we here combine a computational–experimental approach with text mining-enhanced quantitative proteomics. We report that AMP-activated protein kinase (AMPK), phosphatidylinositide 3-kinase (PI3K) and mTOR complex 2 (mTORC2) are acutely activated by aa-readdition in an mTORC1-independent manner. AMPK activation by aa is mediated by Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). In response, AMPK impinges on the autophagy regulators Unc-51-like kinase-1 (ULK1) and c-Jun. AMPK is widely recognized as an mTORC1 antagonist that is activated by starvation. We find that aa acutely activate AMPK concurrently with mTOR. We show that AMPK under aa sufficiency acts to sustain autophagy. This may be required to maintain protein homoeostasis and deliver metabolite intermediates for biosynthetic processes. PMID:27869123

  7. Transcriptomic Analysis Reveals the Metabolic Mechanism of L-Ascorbic Acid in Ziziphus jujuba Mill.

    PubMed Central

    Zhang, Chunmei; Huang, Jian; Li, Xingang

    2016-01-01

    Chinese jujube (Ziziphus jujuba Mill.) is the most economically important member of the Rhamnaceae family and contains a high concentration of ascorbic acid (AsA). To explore the metabolic mechanism of AsA accumulation, we investigated the abundance of AsA in the fruit development stages, the leaf and flower of Z. jujuba cv Junzao, and the mature fruit of one type of wild jujube (Z. jujuba var. spinosa Hu, Yanchuan sour jujube). And the expression patterns of genes involved in AsA biosynthesis, degradation, and recycling were analyzed. The result showed that AsA biosynthesis during early fruit development (the enlargement stage) is the main reason for jujube high accumulation. The L-galactose pathway plays a predominant role in the biosynthesis of AsA during jujube fruit development, and the genes GMP1, GME1, GGP, and GaLDH involved in the determination of AsA concentration during fruit development and in different genotypes; the myo-inositol pathway along with the genes GME2 and GMP2 in the L-galactose pathway play a compensatory role in maintaining AsA accumulation during the ripening stage. These findings enhance our understanding of the molecular mechanism in regulating AsA accumulation for jujube. PMID:26913041

  8. Transcriptomics of Environmental Enrichment Reveals a Role for Retinoic Acid Signaling in Addiction

    PubMed Central

    Zhang, Yafang; Kong, Fanping; Crofton, Elizabeth J.; Dragosljvich, Steven N.; Sinha, Mala; Li, Dingge; Fan, Xiuzhen; Koshy, Shyny; Hommel, Jonathan D.; Spratt, Heidi M.; Luxon, Bruce A.; Green, Thomas A.

    2016-01-01

    There exists much variability in susceptibility/resilience to addiction in humans. The environmental enrichment paradigm is a rat model of resilience to addiction-like behavior, and understanding the molecular mechanisms underlying this protective phenotype may lead to novel targets for pharmacotherapeutics to treat cocaine addiction. We investigated the differential regulation of transcript levels using RNA sequencing of the rat nucleus accumbens after environmental enrichment/isolation and cocaine/saline self-administration. Ingenuity Pathways Analysis and Gene Set Enrichment Analysis of 14,309 transcripts demonstrated that many biofunctions and pathways were differentially regulated. New functional pathways were also identified for cocaine modulation (e.g., Rho GTPase signaling) and environmental enrichment (e.g., signaling of EIF2, mTOR, ephrin). However, one novel pathway stood out above the others, the retinoic acid (RA) signaling pathway. The RA signaling pathway was identified as one likely mediator of the protective enrichment addiction phenotype, an interesting result given that nine RA signaling-related genes are expressed selectively and at high levels in the nucleus accumbens shell (NAcSh). Subsequent knockdown of Cyp26b1 (an RA degradation enzyme) in the NAcSh of rats confirmed this role by increasing cocaine self-administration as well as cocaine seeking. These results provide a comprehensive account of enrichment effects on the transcriptome and identify RA signaling as a contributing factor for cocaine addiction. PMID:27899881

  9. Structural analysis of fructans produced by acetic acid bacteria reveals a relation to hydrocolloid function.

    PubMed

    Jakob, Frank; Pfaff, Andre; Novoa-Carballal, Ramon; Rübsam, Heinrich; Becker, Thomas; Vogel, Rudi F

    2013-02-15

    Some strains of acetic acid bacteria (Gluconobacter frateurii TMW 2.767, Gluconobacter cerinus DSM 9533T, Neoasaia chiangmaiensis NBRC 101099, Kozakia baliensis DSM 14400) produce high amounts of fructans, which can be exploited in food applications as previously demonstrated empirically for dough systems. In order to get insight into the structure and functionality of these polymers, we investigated the fructans isolated from these strains with respect to their linkage types and molecular weights/shapes using NMR spectroscopy and AF4-MALS-RI. Each fructan was identified as levan. The isolated levan fractions were highly similar according to their basic linearity and linkage types, but differed significantly in terms of their individual molecular weight distributions. In aqueous solutions the size of levan molecules present in all isolated levans continuously increased with their molecular weight and they tended to adopt a more compact molecular shape. Our data suggest that the increasing molecular weight of a levan particle enforces intramolecular interactions to reach the structural compactness of a microgel with hydrocolloid properties.

  10. Mechanisms controlling Cu, Fe, Mn, and Co profiles in peat of the Filson Creek Fen, northeastern Minnesota

    USGS Publications Warehouse

    Walton-Day, K.; Filipek, L.H.; Papp, C.S.E.

    1990-01-01

    Filson Creek Fen, located in northeastern Minnesota, overlies a Cu-Ni sulfide deposit. A site in the fen was studied to evaluate the hydrogeochemical mechanisms governing the development of Fe, Mn, Co, and Cu profiles in the peat. At the study site, surface peat approximately 1 m thick is separated from the underlying mineralized bedrock by a 6-12 m thickness of lake and glaciofluvial sediments and till. Concentrations of Fe, Mn, Co, and Cu in peat and major elements in pore water delineate a shallow, relatively oxidized, Cu-rich zone overlying a deeper, reduced, Fe-, Mn-, and Co-rich zone within the peat. Sequential metal extractions from peat samples reveal that 40-55% of the Cu in the shallow zone is associated with organic material, whereas the remaining Cu is distributed between iron-oxide, sulfide, and residual fractions. Sixty to seventy percent of the Fe, Mn, and Co concentrated in the deeper zone occur in the residual phase. The metal profiles and associations probably result from non-steady-state input of metals and detritus into the fen during formation of the peat column. The enrichment of organic-associated Cu in the upper, oxidized zone represents a combination of Cu transported into the fen with detrital plant fragments and soluble Cu, derived from weathering of outcrop and subcrop of the mineral deposit, transported into the fen, and fixed onto organic matter in the peat. The variable stratigraphy of the peat indicates that weathering processes and surface vegetation have changed through time in the fen. The Fe, Mn, and Co maxima at the base of the peat are associated with a maximum in detrital matter content of the peat resulting from a transition between the underlying inorganic sedimentary environment to an organic sedimentary environment. The chemistry of sediments and ground water collected beneath the peat indicate that mobilization of metals from sulfide minerals in the buried mineral deposit or glacial deposits is minimal. Therefore, the

  11. Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant

    PubMed Central

    Dümig, Michaela; O'Keeffe, Gráinne; Binder, Jasmin; Doyle, Sean; Beilhack, Andreas

    2016-01-01

    Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements of A. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection. PMID:26787716

  12. Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

    PubMed Central

    2011-01-01

    Background Retinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined. Results Treatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA. Conclusions These data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor

  13. Genomic Profiling Reveals an Alternate Mechanism for Hepatic Tumor Promotion by Perfluorooctanoic Acid in Rainbow Trout

    PubMed Central

    Tilton, Susan C.; Orner, Gayle A.; Benninghoff, Abby D.; Carpenter, Hillary M.; Hendricks, Jerry D.; Pereira, Cliff B.; Williams, David E.

    2008-01-01

    Background Perfluorooctanoic acid (PFOA) is a potent hepatocarcinogen and peroxisome proliferator (PP) in rodents. Humans are not susceptible to peroxisome proliferation and are considered refractory to carcinogenesis by PPs. Previous studies with rainbow trout indicate they are also insensitive to peroxisome proliferation by the PP dehydroepiandrosterone (DHEA), but are still susceptible to enhanced hepatocarcinogenesis after chronic exposure. Objectives In this study, we used trout as a unique in vivo tumor model to study the potential for PFOA carcinogenesis in the absence of peroxisome proliferation compared with the structurally diverse PPs clofibrate (CLOF) and DHEA. Mechanisms of carcinogenesis were identified from hepatic gene expression profiles phenotypically anchored to tumor outcome. Methods We fed aflatoxin B1 or sham-initiated animals 200–1,800 ppm PFOA in the diet for 30 weeks for tumor analysis. We subsequently examined gene expression by cDNA array in animals fed PFOA, DHEA, CLOF, or 5 ppm 17β-estradiol (E2, a known tumor promoter) in the diet for 14 days. Results PFOA (1,800 ppm or 50 mg/kg/day) and DHEA treatments resulted in enhanced liver tumor incidence and multiplicity (p < 0.0001), whereas CLOF showed no effect. Carcinogenesis was independent of peroxisome proliferation, measured by lack of peroxisomal β-oxidation and catalase activity. Alternately, both tumor promoters, PFOA and DHEA, resulted in estrogenic gene signatures with strong correlation to E2 by Pearson correlation (R = 0.81 and 0.78, respectively), whereas CLOF regulated no genes in common with E2. Conclusions These data suggest that the tumor-promoting activities of PFOA in trout are due to novel mechanisms involving estrogenic signaling and are independent of peroxisome proliferation. PMID:18709148

  14. DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Brown, W. Ted; Junaid, Mohammed A.

    2016-01-01

    It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases. PMID:27199632

  15. FeN foils by nitrogen ion-implantation

    SciTech Connect

    Jiang, Yanfeng; Wang, Jian-Ping; Al Mehedi, Md; Fu, Engang; Wang, Yongqiang

    2014-05-07

    Iron nitride samples in foil shape (free standing, 500 nm in thickness) were prepared by a nitrogen ion-implantation method. To facilitate phase transformation, the samples were bonded on the substrate followed by a post-annealing step. By using two different substrates, single crystal Si and GaAs, structural and magnetic properties of iron nitride foil samples prepared with different nitrogen ion fluences were characterized. α″-Fe{sub 16}N{sub 2} phase in iron nitride foil samples was obtained and confirmed by the proposed approach. A hard magnetic property with coercivity up to 780 Oe was achieved for the FeN foil samples bonded on Si substrate. The feasibility of using nitrogen ion implantation techniques to prepare FeN foil samples up to 500 nm thickness with a stable martensitic phase under high ion fluences has been demonstrated. A possible mechanism was proposed to explain this result. This proposed method could potentially be an alternative route to prepare rare-earth-free FeN bulk magnets by stacking and pressing multiple free-standing thick α″-Fe{sub 16}N{sub 2} foils together.

  16. Lipidomics Analysis Reveals Efficient Storage of Hepatic Triacylglycerides Enriched in Unsaturated Fatty Acids after One Bout of Exercise in Mice

    PubMed Central

    Zhao, Xinjie; Häring, Hans U.; Schleicher, Erwin; Lehmann, Rainer; Han, Xianlin; Xu, Guowang; Weigert, Cora

    2010-01-01

    Background Endurance exercise induces lipolysis, increases circulating concentrations of free fatty acids (FFA) and the uptake and oxidation of fatty acids in the working muscle. Less is known about the regulation of lipid metabolism in the liver during and post-exercise. Methodology/Principal Findings We performed an ultra fast liquid chromatography-mass spectrometry (UFLC-MS) based lipidomics analysis of liver tissue samples obtained from C57Bl/6J mice immediately after a 60 min treadmill run of moderate intensity, and after 3 h of recovery. The PLS-DA scores plot for 115 quantified lipid molecular species revealed a clear separation of the hepatic lipid profile of sedentary from recovering mice, but not from mice immediately after running. 21 lipid species were considered to be most responsible for the difference in the hepatic lipid profiles, including 17 triacylglycerides (TG), one lysophosphatidylcholine (LPC) and three phosphatidylcholines (PC). TG species were found to be more abundant in the recovery phase, while PC species were decreased. The degree of accumulation of individual TG species correlated well with the amount of theoretical energy stored whereas no increase was found for TG species containing only saturated or one monounsaturated fatty acid. Total liver TG content as assayed by an enzymatic method was increased to 163% in the recovery phase, while it was significantly decreased in skeletal muscle by the exercise bout and remained less in the recovery phase. Results from fasted and refed mice indicate that fasting-induced lipolysis was associated with a pronounced accumulation of hepatic TG, which is reversed by refeeding for 5 h. Thus food intake per se did not elevate hepatic TG. Conclusion These data indicate that high availability of FFA induced by endurance exercise or fasting resulted in a transient hepatic TG accumulation, while muscle TG content was decreased during exercise presumably due to increased muscle fatty acid oxidation. PMID

  17. Impacts of oil sands process water on fen plants: implications for plant selection in required reclamation projects.

    PubMed

    Pouliot, Rémy; Rochefort, Line; Graf, Martha D

    2012-08-01

    Fen plant growth in peat contaminated with groundwater discharges of oil sands process water (OSPW) was assessed in a greenhouse over two growing seasons. Three treatments (non-diluted OSPW, diluted OSPW and rainwater) were tested on five vascular plants and four mosses. All vascular plants tested can grow in salinity and naphthenic acids levels currently produced by oil sands activity in northwestern Canada. No stress sign was observed after both seasons. Because of plant characteristics, Carex species (C. atherodes and C. utriculata) and Triglochin maritima would be more useful for rapidly restoring vegetation and creating a new peat-accumulating system. Groundwater discharge of OSPW proved detrimental to mosses under dry conditions and ensuring adequate water levels would be crucial in fen creation following oil sands exploitation. Campylium stellatum would be the best choice to grow in contaminated areas and Bryum pseudotriquetrum might be interesting as it has spontaneously regenerated in all treatments.

  18. Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

    PubMed Central

    2012-01-01

    Background Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. Results GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. Conclusions Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl

  19. Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, QM/MM Simulation, and Mutagenesis

    PubMed Central

    2014-01-01

    N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C

  20. Bacterial and fungal diversity in the starter production process of Fen liquor, a traditional Chinese liquor.

    PubMed

    Li, Xiao-Ran; Ma, En-Bo; Yan, Liang-Zhen; Meng, Han; Du, Xiao-Wei; Quan, Zhe-Xue

    2013-08-01

    Fermented foods and beverages are important parts of human diet. Fen liquor, a Chinese liquor is a fermented beverage that uses a traditional fermentation process. Starters are the main microbial source and also provide nutrients for microorganisms during fermentation. In this study, starters of Fen liquor were produced through a complex traditional fermentation process. To investigate the community structure and the composition of microorganisms in the starter production process, bacterial 16S rRNA and fungal internal transcribed spacer (ITS) regions were sequenced using clone libraries and pyrosequencing, respectively. There was much higher diversity among the bacteria than among the fungi in the starter production process. Bacteria on the surface of the starters belonged mostly to the Lactobacillaceae family, while members of the Bacillacae family were dominant in the interior of the samples that lacked access to air and water. In the fungi population, diversity was high only in the raw material. In all other samples, nearly all of the fungal sequences were from Pichia kudriavzevii, a member of the Saccharomycetaceae family. Nearly all samples showed similar fungal community structures, indicating that there was little change in the fungal community. To the best of our knowledge, this is the first report to reveal the whole process of the starter production of Chinese traditional liquor. The findings obtained in this study provide new insights into understanding the composition of the microbial community during the traditional Chinese liquor starter production process and information about the production process control and monitoring.

  1. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling.

    PubMed

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-09-15

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system.

  2. Metabonomics Reveals Drastic Changes in Anti-Inflammatory/Pro-Resolving Polyunsaturated Fatty Acids-Derived Lipid Mediators in Leprosy Disease

    PubMed Central

    Amaral, Julio J.; Antunes, Luis Caetano M.; de Macedo, Cristiana S.; Mattos, Katherine A.; Han, Jun; Pan, Jingxi; Candéa, André L. P.; Henriques, Maria das Graças M. O.; Ribeiro-Alves, Marcelo; Borchers, Christoph H.; Sarno, Euzenir N.; Bozza, Patrícia T.; Finlay, B. Brett; Pessolani, Maria Cristina V.

    2013-01-01

    Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases. PMID:23967366

  3. 1H NMR-based metabolic profiling reveals the effects of fluoxetine on lipid and amino acid metabolism in astrocytes.

    PubMed

    Bai, Shunjie; Zhou, Chanjuan; Cheng, Pengfei; Fu, Yuying; Fang, Liang; Huang, Wen; Yu, Jia; Shao, Weihua; Wang, Xinfa; Liu, Meiling; Zhou, Jingjing; Xie, Peng

    2015-04-15

    Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is a prescribed and effective antidepressant and generally used for the treatment of depression. Previous studies have revealed that the antidepressant mechanism of fluoxetine was related to astrocytes. However, the therapeutic mechanism underlying its mode of action in astrocytes remains largely unclear. In this study, primary astrocytes were exposed to 10 µM fluoxetine; 24 h post-treatment, a high-resolution proton nuclear magnetic resonance (1H NMR)-based metabolomic approach coupled with multivariate statistical analysis was used to characterize the metabolic variations of intracellular metabolites. The orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots of the spectra demonstrated that the fluoxetine-treated astrocytes were significantly distinguished from the untreated controls. In total, 17 differential metabolites were identified to discriminate the two groups. These key metabolites were mainly involved in lipids, lipid metabolism-related molecules and amino acids. This is the first study to indicate that fluoxetine may exert antidepressant action by regulating the astrocyte's lipid and amino acid metabolism. These findings should aid our understanding of the biological mechanisms underlying fluoxetine therapy.

  4. Salts of hexamethylenetetramine with organic acids: Enhanced anomeric interactions with a lowering of molecular symmetry revealed by crystal structures

    NASA Astrophysics Data System (ADS)

    Chandrasekhar, Sosale; Mukherjee, Somnath

    2015-02-01

    The hexamethylenetetramine (HMT) framework displays interesting stereoelectronic interactions of the anomeric type. In the highly symmetrical parent system, the nitrogen centres act as both donors and acceptors. Protonation lowers symmetry and also leads to an enhancement of the anomeric interaction around the protonated centre. X-ray diffraction crystal structures of four derivatives of HMT - with succinic, (DL)-malic, phthalic and 4-hydroxybenzoic acids - reveal significant trends. (The first three form well-defined salts, 4-hydroxybenzoic acid forming a co-crystalline compound.) Each molecular structure is essentially characterised by a major anomeric interaction involving the protonated centre as acceptor. In two cases (succinic and 4-hydroxybenzoic), secondary protonation leads to a weaker anomeric interaction site that apparently competes with the dominant one. Bond length changes indicate that the anomeric interaction decreases as malic > phthalic > succinic > 4-hydroxybenzoic, which correlates with the degree of proton transfer to the nitrogen centre. Along with other bond length and angle changes, the results offer insight into the applicability of the antiperiplanar lone pair hypothesis (ALPH) in a rigid system.

  5. Carbon and Nitrogen Isotopes from Top Predator Amino Acids Reveal Rapidly Shifting Ocean Biochemistry in the Outer California Current

    PubMed Central

    Ruiz-Cooley, Rocio I.; Koch, Paul L.; Fiedler, Paul C.; McCarthy, Matthew D.

    2014-01-01

    Climatic variation alters biochemical and ecological processes, but it is difficult both to quantify the magnitude of such changes, and to differentiate long-term shifts from inter-annual variability. Here, we simultaneously quantify decade-scale isotopic variability at the lowest and highest trophic positions in the offshore California Current System (CCS) by measuring δ15N and δ13C values of amino acids in a top predator, the sperm whale (Physeter macrocephalus). Using a time series of skin tissue samples as a biological archive, isotopic records from individual amino acids (AAs) can reveal the proximate factors driving a temporal decline we observed in bulk isotope values (a decline of ≥1 ‰) by decoupling changes in primary producer isotope values from those linked to the trophic position of this toothed whale. A continuous decline in baseline (i.e., primary producer) δ15N and δ13C values was observed from 1993 to 2005 (a decrease of ∼4‰ for δ15N source-AAs and 3‰ for δ13C essential-AAs), while the trophic position of whales was variable over time and it did not exhibit directional trends. The baseline δ15N and δ13C shifts suggest rapid ongoing changes in the carbon and nitrogen biogeochemical cycling in the offshore CCS, potentially occurring at faster rates than long-term shifts observed elsewhere in the Pacific. While the mechanisms forcing these biogeochemical shifts remain to be determined, our data suggest possible links to natural climate variability, and also corresponding shifts in surface nutrient availability. Our study demonstrates that isotopic analysis of individual amino acids from a top marine mammal predator can be a powerful new approach to reconstructing temporal variation in both biochemical cycling and trophic structure. PMID:25329915

  6. Sulfur and carbon isotope biogeochemistry of a rewetted brackish fen

    NASA Astrophysics Data System (ADS)

    Koebsch, Franziska; Gehre, Matthias; Winkel, Matthias; Koehler, Stefan; Koch, Marian; Jurasinski, Gerald; Spitzy, Alejandro; Liebner, Susanne; Sachs, Torsten; Schmiedinger, Iris; Kretzschmann, Lisett; Saborowski, Anke; Böttcher, Michael E.

    2015-04-01

    Coastal wetlands are at the interface between terrestrial freshwater and marine and exhibit very specific biogeochemical conditions. Intermittent sea water intrusion affects metabolic pathways, i. e. anaerobic carbon metabolism is progressively dominated by sulfate reduction with lower contribution of methanogenesis whilst methane production is increasingly shifted from acetoclastic to hydrogenotrophic. Due to expanding anthropogenic impact a large proportion of coastal ecosystems is degraded with severe implications for the biogeochemical processes. We use concentration patterns and stable isotope signatures of water, sulfate, dissolved carbonate, and methane (δ2H, δ13C, δ18O, δ34S) to investigate the S and C metabolic cycle in a rewetted fen close to the southern Baltic Sea border. Such studies are crucial to better predict dynamic ecosystem feedback to global change like organic matter (OM) decomposition or greenhouse gas emissions. Yet, little is known about the metabolic pathways in such environments. The study site is part of the TERENO Observatory "Northeastern German Lowlands' and measurements of methane emissions have run since 2009. High methane fluxes up to 800 mg m-2 hr-1 indicate that methanogenesis is the dominant C metabolism pathway despite of high sulfate concentrations (up to 37 mM). The presented data are part of a comprehensive biogeochemical investigation that we conducted in autumn 2014 and that comprises 4 pore water profiles and sediment samples within a transect of 300-1500 m distance to the Baltic Sea. Depth of organic layers ranged from 25 to 140 cm with high OM contents (up to 90 dwt.%). Sulfate/chloride ratios in the pore waters were lower than in the Baltic Sea for most sites and sediment depths indicated a substantial net sulfate loss. Sulfide concentrations were negligible at the top and increased parallel to the sulfate concentrations with depth to values of up to 0.3 mM. One pore water profiles situated 1150 m from the Baltic

  7. Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) β-Lactamase

    PubMed Central

    Winkler, Marisa L.; Rodkey, Elizabeth A.; Taracila, Magdalena A.; Drawz, Sarah M.; Bethel, Christopher R.; Papp-Wallace, Krisztina M.; Smith, Kerri M.; Xu, Yan; Dwulit-Smith, Jeffrey R.; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; van den Akker, Focco; Bonomo, Robert A.

    2014-01-01

    Inhibitor resistant (IR) class A β-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV β-lactamase, from Klebsiella pneumoniae, results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting β-lactamases revealed that only –Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM Ki for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR β-lactamases. PMID:23252553

  8. Distinct Anaerobic Bacterial Consumers of Cellobiose-Derived Carbon in Boreal Fens with Different CO2/CH4 Production Ratios.

    PubMed

    Juottonen, Heli; Eiler, Alexander; Biasi, Christina; Tuittila, Eeva-Stiina; Yrjälä, Kim; Fritze, Hannu

    2017-02-15

    Northern peatlands in general have high methane (CH4) emissions, but individual peatlands show considerable variation as CH4 sources. Particularly in nutrient-poor peatlands, CH4 production can be low and exceeded by carbon dioxide (CO2) production from unresolved anaerobic processes. To clarify the role anaerobic bacterial degraders play in this variation, we compared consumers of cellobiose-derived carbon in two fens differing in nutrient status and the ratio of CO2 to CH4 produced. After [(13)C]cellobiose amendment, the mesotrophic fen produced equal amounts of CH4 and CO2 The oligotrophic fen had lower CH4 production but produced 3 to 59 times more CO2 than CH4 RNA stable-isotope probing revealed that in the mesotrophic fen with higher CH4 production, cellobiose-derived carbon was mainly assimilated by various recognized fermenters of Firmicutes and by Proteobacteria The oligotrophic peat with excess CO2 production revealed a wider variety of cellobiose-C consumers, including Firmicutes and Proteobacteria, but also more unconventional degraders, such as Telmatobacter-related Acidobacteria and subphylum 3 of Verrucomicrobia Prominent and potentially fermentative Planctomycetes and Chloroflexi did not appear to process cellobiose-C. Our results show that anaerobic degradation resulting in different levels of CH4 production can involve distinct sets of bacterial degraders. By distinguishing cellobiose degraders from the total community, this study contributes to defining anaerobic bacteria that process cellulose-derived carbon in peat. Several of the identified degraders, particularly fermenters and potential Fe(III) or humic substance reducers in the oligotrophic peat, represent promising candidates for resolving the origin of excess CO2 production in peatlands.

  9. Revealing biogenic sulfuric acid corrosion in sludge digesters: detection of sulfur-oxidizing bacteria within full-scale digesters.

    PubMed

    Huber, B; Drewes, J E; Lin, K C; König, R; Müller, E

    2014-01-01

    Biogenic sulfuric acid corrosion (BSA) is a costly problem affecting both sewerage infrastructure and sludge handling facilities such as digesters. The aim of this study was to verify BSA in full-scale digesters by identifying the microorganisms involved in the concrete corrosion process, that is, sulfate-reducing (SRB) and sulfur-oxidizing bacteria (SOB). To investigate the SRB and SOB communities, digester sludge and biofilm samples were collected. SRB diversity within digester sludge was studied by applying polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the dsrB-gene (dissimilatory sulfite reductase beta subunit). To reveal SOB diversity, cultivation dependent and independent techniques were applied. The SRB diversity studies revealed different uncultured SRB, confirming SRB activity and H2S production. Comparable DGGE profiles were obtained from the different sludges, demonstrating the presence of similar SRB species. By cultivation, three pure SOB strains from the digester headspace were obtained including Acidithiobacillus thiooxidans, Thiomonas intermedia and Thiomonas perometabolis. These organisms were also detected with PCR-DGGE in addition to two new SOB: Thiobacillus thioparus and Paracoccus solventivorans. The SRB and SOB responsible for BSA were identified within five different digesters, demonstrating that BSA is a problem occurring not only in sewer systems but also in sludge digesters. In addition, the presence of different SOB species was successfully associated with the progression of microbial corrosion.

  10. BOREAS TF-11 Biomass Data over the SSA-Fen

    NASA Technical Reports Server (NTRS)

    Valentine, David W.; Hall, Forrest G. (Editor); Conrad, Sara (Editor)

    2000-01-01

    The BOREAS TF-11 team collected several data sets in its efforts to fully describe the flux and site characteristics at the SSA-Fen site. This data set contains plant cover, standing crop of plant biomass, and estimated net primary productivity at each chamber site at the end of the 1994 field season. The measurements were conducted as part of a 2 x 2 factorial experiment in which we added carbon (300 g/sq m as wheat straw) and nitrogen (6 g/sq m as urea) to four replicate locations in the vicinity of the TF-11 tower. The data are stored in tabular ASCII files.

  11. BOREAS TF-11 Decomposition Data over the SSA-Fen

    NASA Technical Reports Server (NTRS)

    Valentine, David W.; Hall, Forrest G. (Editor); Conrad, Sara (Editor)

    2000-01-01

    The BOREAS TF-11 team collected several data sets in its efforts to fully describe the flux and site characteristics at the SSA-Fen site. This data set contains decomposition rates of a standard substrate (wheat straw) across treatments. The measurements were conducted in 1994 as part of a 2 x 2 factorial experiment in which we added carbon (300 g/sq m as wheat straw) and nitrogen (6 g/sq m as urea) to four replicate locations in the vicinity of the TF-11 tower. The data are stored in tabular ASCII files.

  12. Structure and specificity of FEN-1 from Methanopyrus kandleri

    DOE PAGES

    Shah, Santosh; Dunten, Pete; Stiteler, Amanda; ...

    2014-11-18

    DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Ultimately, we found both to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.

  13. Understanding the Groundwater Hydrology of a Geographically-Isolated Prairie Fen: Implications for Conservation

    PubMed Central

    Sampath, Prasanna Venkatesh; Liao, Hua-Sheng; Curtis, Zachary Kristopher; Doran, Patrick J.; Herbert, Matthew E.; May, Christopher A.; Li, Shu-Guang

    2015-01-01

    The sources of water and corresponding delivery mechanisms to groundwater-fed fens are not well understood due to the multi-scale geo-morphologic variability of the glacial landscape in which they occur. This lack of understanding limits the ability to effectively conserve these systems and the ecosystem services they provide, including biodiversity and water provisioning. While fens tend to occur in clusters around regional groundwater mounds, Ives Road Fen in southern Michigan is an example of a geographically-isolated fen. In this paper, we apply a multi-scale groundwater modeling approach to understand the groundwater sources for Ives Road fen. We apply Transition Probability geo-statistics on more than 3000 well logs from a state-wide water well database to characterize the complex geology using conditional simulations. We subsequently implement a 3-dimensional reverse particle tracking to delineate groundwater contribution areas to the fen. The fen receives water from multiple sources: local recharge, regional recharge from an extensive till plain, a regional groundwater mound, and a nearby pond. The regional sources deliver water through a tortuous, 3-dimensional “pipeline” consisting of a confined aquifer lying beneath an extensive clay layer. Water in this pipeline reaches the fen by upwelling through openings in the clay layer. The pipeline connects the geographically-isolated fen to the same regional mound that provides water to other fen clusters in southern Michigan. The major implication of these findings is that fen conservation efforts must be expanded from focusing on individual fens and their immediate surroundings, to studying the much larger and inter-connected hydrologic network that sustains multiple fens. PMID:26452279

  14. Understanding the Groundwater Hydrology of a Geographically-Isolated Prairie Fen: Implications for Conservation.

    PubMed

    Sampath, Prasanna Venkatesh; Liao, Hua-Sheng; Curtis, Zachary Kristopher; Doran, Patrick J; Herbert, Matthew E; May, Christopher A; Li, Shu-Guang

    2015-01-01

    The sources of water and corresponding delivery mechanisms to groundwater-fed fens are not well understood due to the multi-scale geo-morphologic variability of the glacial landscape in which they occur. This lack of understanding limits the ability to effectively conserve these systems and the ecosystem services they provide, including biodiversity and water provisioning. While fens tend to occur in clusters around regional groundwater mounds, Ives Road Fen in southern Michigan is an example of a geographically-isolated fen. In this paper, we apply a multi-scale groundwater modeling approach to understand the groundwater sources for Ives Road fen. We apply Transition Probability geo-statistics on more than 3000 well logs from a state-wide water well database to characterize the complex geology using conditional simulations. We subsequently implement a 3-dimensional reverse particle tracking to delineate groundwater contribution areas to the fen. The fen receives water from multiple sources: local recharge, regional recharge from an extensive till plain, a regional groundwater mound, and a nearby pond. The regional sources deliver water through a tortuous, 3-dimensional "pipeline" consisting of a confined aquifer lying beneath an extensive clay layer. Water in this pipeline reaches the fen by upwelling through openings in the clay layer. The pipeline connects the geographically-isolated fen to the same regional mound that provides water to other fen clusters in southern Michigan. The major implication of these findings is that fen conservation efforts must be expanded from focusing on individual fens and their immediate surroundings, to studying the much larger and inter-connected hydrologic network that sustains multiple fens.

  15. Holocene Vegetation and Climate Shifts from Sutherland Fen, Black Rock Forest, New York - Plant Macrofossils, Charcoal, and Carbon

    NASA Astrophysics Data System (ADS)

    Peteet, D. M.; Guilderson, T.

    2008-12-01

    Sutherland Fen formed about 12,600 C-14 years ago (15,000 calendar years), the same time as adjacent Sutherland Pond and regional deglaciation. High-resolution (2 cm) analysis of the 3.2 m fen core indicates three major macrofossils zones indicative of climate shifts. These climate shifts were defined over fifty years ago through pollen stratigraphy of the regional northeastern US, but macrofossils provide new details concerning hydrological and ecological shifts. The lowest (SUB-1) dated to the late-glacial, is indicative of a shallow pond characterized by Najas, Nuphar, and Potamogeton seeds and containing Salix (willow) buds, a Rubus (berry) seed, and Picea glauca (white spruce) needles and sterigmata from the surrounding upland. Sedimention rates are highest in this boreal environmental zone. The overlying zone (SUB-2) beginning at 11,500 years ago (Holocene) indicates a continuing pond environment with aquatics such as Najas, Nuphar, and Brasenia, but Picea disappears and Pinus strobus (white pine) dominates the lower section of the zone. A warmer, drier climate produces sustained charcoal in the record at the Holocene boundary. Pinus strobus needles and seeds subsequently disappear and are replaced from 9000 to 7500 years ago by Pinus rigida (pitch pine), Betula populifolia/papyrifera (grey/paper birch), and emergent wetland plants such as Decodon, Cladium, and Cephalanthus, as well as Dulichium, Eleocharis, and Carex, suggesting a shallowing pond and a drier climate. Chara oospores indicate probably groundwater influx into the fen. About 4000 years ago, charcoal again is present. In the subsequent late Holocene a more acidic, moist, fen environment is characterized by Sphagnum, Rubus, Hypericum, Viola, Chamaedaphne, and Carex, though Brasenia and Potamogeton (pond indicators) are occasionally present. The continued presence of Sphagnum led to high carbon accumulation because of less decomposition. This increase in Sphagnum in recent millennia with aquatics

  16. Seed longevity and germination characteristics of six fen plant species.

    PubMed

    Tatár, S

    2010-01-01

    Fens are among the most threatened habitats in Europe as their area has decreased considerably in the last centuries. For successful management and restoration conservationists need detailed knowledge about seed bank formation and seed longevity of plants, as these features are closely related to successional and vegetation dynamical processes. I analysed seed longevity and the germination characteristics of six fen plant species by seed burial experiments. Based on seed weight, seed bank was expected for long-term persistent for the light-seeded Schoenus nigricans, Carex appropinquata, C. pseudocyperus, C. davalliana and Peucedanum palustre and also that for the medium-seeded Cicuta virosa. It was proved that, the latter two species have short-term persistent seed banks, while Carex pseudocyperus has a transient seed bank, therefore these species may only have a limited role in restoration from seed banks. It was found that Schoenus nigricans, Carex appropinquata and C. davalliana have persistent seed banks, because some of their four-year-old seeds have emerged. Fresh seeds had low germination rate in all studied species and majority of seeds emerged after winter, except for Carex pseudocyperus. After the germination peak in spring, the majority of the ungerminated seeds of Schoenus nigricans, Peucedanum palustre, Carex appropinquata, C. davalliana and Cicuta virosa entered a secondary dormancy phase that was broken in autumn. I found the seasonal emergence of the latter three species highly similar.

  17. Effects of elevated CO2 on fen peat biogeochemistry.

    PubMed

    Kang, H; Freeman, C; Ashendon, T W

    2001-11-12

    Effects of elevated atmospheric CO2 concentration on northern peatland biogeochemistry was studied in a short-term experiment. Eight intact soil cores (11-cm diameter x 40-cm depth) with Juncus and Festuca spp. were collected from a calcareous fen in north Wales. Half of the cores were incubated under 350 ppm CO2 concentration, whilst the other four cores were maintained at 700 ppm CO2. After a 4-month incubation, significantly higher biomass (root + shoot + algal mat) was determined under elevated CO2 conditions. Higher emissions of N2O and CO2, and higher concentration of pore-water DOC (dissolved organic carbon) were also observed under elevated CO2. However, no significant differences were found in CH4 emission or soil enzyme activities (beta-glucosidase, phosphatase, and N-acetylglucosaminidase) in the bulk soil. Overall, the results suggest that elevated CO2 would increase the primary productivity of the fen vegetation, and stimulate N2O and CO2 emissions as a consequence of an enhanced DOC supply from the vegetation to the soil microbes.

  18. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  19. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGES

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; ...

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  20. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  1. Novel and Unexpected Microbial Diversity in Acid Mine Drainage in Svalbard (78° N), Revealed by Culture-Independent Approaches.

    PubMed

    García-Moyano, Antonio; Austnes, Andreas Erling; Lanzén, Anders; González-Toril, Elena; Aguilera, Ángeles; Øvreås, Lise

    2015-10-13

    Svalbard, situated in the high Arctic, is an important past and present coal mining area. Dozens of abandoned waste rock piles can be found in the proximity of Longyearbyen. This environment offers a unique opportunity for studying the biological control over the weathering of sulphide rocks at low temperatures. Although the extension and impact of acid mine drainage (AMD) in this area is known, the native microbial communities involved in this process are still scarcely studied and uncharacterized. Several abandoned mining areas were explored in the search for active AMD and a culture-independent approach was applied with samples from two different runoffs for the identification and quantification of the native microbial communities. The results obtained revealed two distinct microbial communities. One of the runoffs was more extreme with regards to pH and higher concentration of soluble iron and heavy metals. These conditions favored the development of algal-dominated microbial mats. Typical AMD microorganisms related to known iron-oxidizing bacteria (Acidithiobacillus ferrivorans, Acidobacteria and Actinobacteria) dominated the bacterial community although some unexpected populations related to Chloroflexi were also significant. No microbial mats were found in the second area. The geochemistry here showed less extreme drainage, most likely in direct contact with the ore under the waste pile. Large deposits of secondary minerals were found and the presence of iron stalks was revealed by microscopy analysis. Although typical AMD microorganisms were also detected here, the microbial community was dominated by other populations, some of them new to this type of system (Saccharibacteria, Gallionellaceae). These were absent or lowered in numbers the farther from the spring source and they could represent native populations involved in the oxidation of sulphide rocks within the waste rock pile. This environment appears thus as a highly interesting field of potential

  2. Novel and Unexpected Microbial Diversity in Acid Mine Drainage in Svalbard (78° N), Revealed by Culture-Independent Approaches

    PubMed Central

    García-Moyano, Antonio; Austnes, Andreas Erling; Lanzén, Anders; González-Toril, Elena; Aguilera, Ángeles; Øvreås, Lise

    2015-01-01

    Svalbard, situated in the high Arctic, is an important past and present coal mining area. Dozens of abandoned waste rock piles can be found in the proximity of Longyearbyen. This environment offers a unique opportunity for studying the biological control over the weathering of sulphide rocks at low temperatures. Although the extension and impact of acid mine drainage (AMD) in this area is known, the native microbial communities involved in this process are still scarcely studied and uncharacterized. Several abandoned mining areas were explored in the search for active AMD and a culture-independent approach was applied with samples from two different runoffs for the identification and quantification of the native microbial communities. The results obtained revealed two distinct microbial communities. One of the runoffs was more extreme with regards to pH and higher concentration of soluble iron and heavy metals. These conditions favored the development of algal-dominated microbial mats. Typical AMD microorganisms related to known iron-oxidizing bacteria (Acidithiobacillus ferrivorans, Acidobacteria and Actinobacteria) dominated the bacterial community although some unexpected populations related to Chloroflexi were also significant. No microbial mats were found in the second area. The geochemistry here showed less extreme drainage, most likely in direct contact with the ore under the waste pile. Large deposits of secondary minerals were found and the presence of iron stalks was revealed by microscopy analysis. Although typical AMD microorganisms were also detected here, the microbial community was dominated by other populations, some of them new to this type of system (Saccharibacteria, Gallionellaceae). These were absent or lowered in numbers the farther from the spring source and they could represent native populations involved in the oxidation of sulphide rocks within the waste rock pile. This environment appears thus as a highly interesting field of potential

  3. A phylogenetic analysis of the boreal lichen Mycoblastus sanguinarius (Mycoblastaceae, lichenized Ascomycota) reveals cryptic clades correlated with fatty acid profiles

    PubMed Central

    Spribille, Toby; Klug, Barbara; Mayrhofer, Helmut

    2011-01-01

    Lichens are a prominent feature of northern conifer forests and a large number of species are thought to be circumboreal. Whether or not circumboreal lichen species really constitute monophyletic groups has seldom been tested. We investigated molecular phylogenetic patterns in the mycobiont of Mycoblastus sanguinarius, a well known epiphytic lichen species of the boreal forest, based on material collected from across the high latitude northern hemisphere. A three-locus dataset of internal transcribed spacer rDNA, translation elongation factor 1-α and replication licensing factor Mcm7 DNA sequences revealed that material treated until now as belonging to M. sanguinarius does indeed form a monophyletic group within the genus and is distinct from a strongly supported Mycoblastus affinis. The M. sanguinarius complex appears closely related to the rare Mycoblastus glabrescens, which is currently known only from the Pacific Northwest and was rediscovered during the present study. However, within M. sanguinarius s.lat. in the northern hemisphere, two deeply divergent and morphologically coherent species can be recovered, one of which matches the southern hemisphere species Mycoblastus sanguinarioides and turns out to be widespread in North America and Asia, and one of which corresponds to M. sanguinarius s.str. Both M. sanguinarius and M. sanguinarioides exhibit additional low-level genetic differentiation into geographically structured clades, the most prominent of which are distributed in East Asia/eastern North America and western North America/Europe, respectively. Individuals from these lowest-level clades are morphologically indistinguishable but chemical analyses by thin layer chromatography revealed that each clade possesses its own fatty acid profile, suggesting that chemical differentiation precedes morphological differentiation and may be a precursor to speciation. PMID:21443957

  4. Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

    PubMed Central

    Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

    2015-01-01

    Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

  5. Comparative Proteomic Analysis Reveals the Effects of Exogenous Calcium against Acid Rain Stress in Liquidambar formosana Hance Leaves.

    PubMed

    Hu, Wen-Jun; Wu, Qian; Liu, Xiang; Shen, Zhi-Jun; Chen, Juan; Liu, Ting-Wu; Chen, Juan; Zhu, Chun-Quan; Wu, Fei-Hua; Chen, Lin; Wei, Jia; Qiu, Xiao-Yun; Shen, Guo-Xin; Zheng, Hai-Lei

    2016-01-04

    Acid rain (AR) impacts forest health by leaching calcium (Ca) away from soils and plants. Ca is an essential element and participates in various plant physiological responses. In the present study, the protective role of exogenous Ca in alleviating AR stress in Liquidambar formosana Hance at the physiological and proteomic levels was examined. Our results showed that low Ca condition resulted in the chlorophyll content and photosynthesis decreasing significantly in L. formosana leaves; however, these effects could be reversed by high Ca supplementation. Further proteomic analyses successfully identified 81 differentially expressed proteins in AR-treated L. formosana under different Ca levels. In particular, some of the proteins are involved in primary metabolism, photosynthesis, energy production, antioxidant defense, transcription, and translation. Moreover, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that low Ca significantly increased the expression level of the investigated Ca-related genes, which can be reversed by high Ca supplementation under AR stress. Further, Western blotting analysis revealed that exogenous Ca supply reduced AR damage by elevating the expression of proteins involved in the Calvin cycle, reactive oxygen species (ROS) scavenging system. These findings allowed us to better understand how woody plants respond to AR stress at various Ca levels and the protective role of exogenous Ca against AR stress in forest tree species.

  6. Transcript profile analysis reveals important roles of jasmonic acid signalling pathway in the response of sweet potato to salt stress.

    PubMed

    Zhang, Huan; Zhang, Qian; Zhai, Hong; Li, Yan; Wang, Xiangfeng; Liu, Qingchang; He, Shaozhen

    2017-01-13

    Sweet potato is an important food and bio-energy crop, and investigating the mechanisms underlying salt tolerance will provide information for salt-tolerant breeding of this crop. Here, the root transcriptomes of the salt-sensitive variety Lizixiang and the salt-tolerant line ND98 were compared to identify the genes and pathways involved in salt stress responses. In total, 8,744 and 10,413 differentially expressed genes (DEGs) in Lizixiang and ND98, respectively, were involved in salt responses. A lower DNA methylation level was detected in ND98 than in Lizixiang. In both genotypes, the DEGs, which function in phytohormone synthesis and signalling and ion homeostasis, may underlie the different degrees of salt tolerance. Significant up-regulations of the genes involved in the jasmonic acid (JA) biosynthesis and signalling pathways and ion transport, more accumulation of JA, a higher degree of stomatal closure and a lower level of Na(+) were found in ND98 compared to Lizixiang. This is the first report on transcriptome responses to salt tolerance in sweet potato. These results reveal that the JA signalling pathway plays important roles in the response of sweet potato to salt stress. This study provides insights into the mechanisms and genes involved in the salt tolerance of sweet potato.

  7. Transcript profile analysis reveals important roles of jasmonic acid signalling pathway in the response of sweet potato to salt stress

    PubMed Central

    Zhang, Huan; Zhang, Qian; Zhai, Hong; Li, Yan; Wang, Xiangfeng; Liu, Qingchang; He, Shaozhen

    2017-01-01

    Sweet potato is an important food and bio-energy crop, and investigating the mechanisms underlying salt tolerance will provide information for salt-tolerant breeding of this crop. Here, the root transcriptomes of the salt-sensitive variety Lizixiang and the salt-tolerant line ND98 were compared to identify the genes and pathways involved in salt stress responses. In total, 8,744 and 10,413 differentially expressed genes (DEGs) in Lizixiang and ND98, respectively, were involved in salt responses. A lower DNA methylation level was detected in ND98 than in Lizixiang. In both genotypes, the DEGs, which function in phytohormone synthesis and signalling and ion homeostasis, may underlie the different degrees of salt tolerance. Significant up-regulations of the genes involved in the jasmonic acid (JA) biosynthesis and signalling pathways and ion transport, more accumulation of JA, a higher degree of stomatal closure and a lower level of Na+ were found in ND98 compared to Lizixiang. This is the first report on transcriptome responses to salt tolerance in sweet potato. These results reveal that the JA signalling pathway plays important roles in the response of sweet potato to salt stress. This study provides insights into the mechanisms and genes involved in the salt tolerance of sweet potato. PMID:28084460

  8. Exploratory Metabolomics Profiling in the Kainic Acid Rat Model Reveals Depletion of 25-Hydroxyvitamin D3 during Epileptogenesis

    PubMed Central

    Heischmann, Svenja; Quinn, Kevin; Cruickshank-Quinn, Charmion; Liang, Li-Ping; Reisdorph, Rick; Reisdorph, Nichole; Patel, Manisha

    2016-01-01

    Currently, no reliable markers are available to evaluate the epileptogenic potential of a brain injury. The electroencephalogram is the standard method of diagnosis of epilepsy; however, it is not used to predict the risk of developing epilepsy. Biomarkers that indicate an individual’s risk to develop epilepsy, especially those measurable in the periphery are urgently needed. Temporal lobe epilepsy (TLE), the most common form of acquired epilepsy, is characterized by spontaneous recurrent seizures following brain injury and a seizure-free “latent” period. Elucidation of mechanisms at play during epilepsy development (epileptogenesis) in animal models of TLE could enable the identification of predictive biomarkers. Our pilot study using liquid chromatography-mass spectrometry metabolomics analysis revealed changes (p-value ≤ 0.05, ≥1.5-fold change) in lipid, purine, and sterol metabolism in rat plasma and hippocampus during epileptogenesis and chronic epilepsy in the kainic acid model of TLE. Notably, disease development was associated with dysregulation of vitamin D3 metabolism at all stages and plasma 25-hydroxyvitamin D3 depletion in the acute and latent phase of injury-induced epileptogenesis. These data suggest that plasma VD3 metabolites reflect the severity of an epileptogenic insult and that a panel of plasma VD3 metabolites may be able to serve as a marker of epileptogenesis. PMID:27526857

  9. Zinc inhibition of γ-aminobutyric acid transporter 4 (GAT4) reveals a link between excitatory and inhibitory neurotransmission

    PubMed Central

    Cohen-Kfir, Einav; Lee, William; Eskandari, Sepehr; Nelson, Nathan

    2005-01-01

    γ-Aminobutyric acid (GABA) transporters (GATs) play an important role in inhibitory neurotransmission by clearing synaptically released GABA and by maintaining low resting levels of GABA in synaptic and extrasynaptic regions. In certain brain regions, vesicular zinc is colocalized and coreleased with glutamate and modulates the behavior of a number of channels, receptors, and transporters. We examined the effect of zinc on expressed GATs (GAT1, GAT2, GAT3, and GAT4) in Xenopus laevis oocytes by using tracer flux and electrophysiological methods. We show that zinc is a potent inhibitor of GAT4 (Ki of 3 μM). Immunolocalization of GAT4 in the hippocampus revealed dense localization in the CA1 and CA3 regions of the hippocampus, regions which are known to be heavily populated by zinc-containing glutamatergic neurons. The results suggest a physiological role of synaptically released zinc in the hippocampus, because zinc released from hyperactive glutamatergic neurons may simultaneously bring about elevated GABAergic inhibition. Therefore, this mode of zinc function signifies a link between excitatory and inhibitory neurotransmission and may play a neuroprotective role against glutamate-induced excitotoxicity. PMID:15829583

  10. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis

    PubMed Central

    Li, Ling; Bonneton, François; Tohme, Marie; Bernard, Laure; Chen, Xiao Yong; Laudet, Vincent

    2016-01-01

    The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo. PMID:26900852

  11. Microbial community structure and activity linked to contrasting biogeochemical gradients in bog and fen environments of the Glacial Lake Agassiz Peatland.

    PubMed

    Lin, X; Green, S; Tfaily, M M; Prakash, O; Konstantinidis, K T; Corbett, J E; Chanton, J P; Cooper, W T; Kostka, J E

    2012-10-01

    The abundances, compositions, and activities of microbial communities were investigated at bog and fen sites in the Glacial Lake Agassiz Peatland of northwestern Minnesota. These sites contrast in the reactivity of dissolved organic matter (DOM) and the presence or absence of groundwater inputs. Microbial community composition was characterized using pyrosequencing and clone library construction of phylogenetic marker genes. Microbial distribution patterns were linked to pH, concentrations of dissolved organic carbon and nitrogen, C/N ratios, optical properties of DOM, and activities of laccase and peroxidase enzymes. Both bacterial and archaeal richness and rRNA gene abundance were >2 times higher on average in the fen than in the bog, in agreement with a higher pH, labile DOM content, and enhanced enzyme activities in the fen. Fungi were equivalent to an average of 1.4% of total prokaryotes in gene abundance assayed by quantitative PCR. Results revealed statistically distinct spatial patterns between bacterial and fungal communities. Fungal distribution did not covary with pH and DOM optical properties and was vertically stratified, with a prevalence of Ascomycota and Basidiomycota near the surface and much higher representation of Zygomycota in the subsurface. In contrast, bacterial community composition largely varied between environments, with the bog dominated by Acidobacteria (61% of total sequences), while the Firmicutes (52%) dominated in the fen. Acetoclastic Methanosarcinales showed a much higher relative abundance in the bog, in contrast to the dominance of diverse hydrogenotrophic methanogens in the fen. This is the first quantitative and compositional analysis of three microbial domains in peatlands and demonstrates that the microbial abundance, diversity, and activity parallel with the pronounced differences in environmental variables between bog and fen sites.

  12. Stable isotope studies reveal pathways for the incorporation of non-essential amino acids in Acyrthosiphon pisum (pea aphids).

    PubMed

    Haribal, Meena; Jander, Georg

    2015-12-01

    Plant roots incorporate inorganic nitrogen into the amino acids glutamine, glutamic acid, asparagine and aspartic acid, which together serve as the primary metabolites of nitrogen transport to other tissues. Given the preponderance of these four amino acids, phloem sap is a nutritionally unbalanced diet for phloem-feeding insects. Therefore, aphids and other phloem feeders typically rely on microbial symbionts for the synthesis of essential amino acids. To investigate the metabolism of the four main transport amino acids by the pea aphid (Acyrthosiphon pisum), and its Buchnera aphidicola endosymbionts, aphids were fed defined diets with stable isotope-labeled glutamine, glutamic acid, asparagine or aspartic acid (U-(13)C, U-(15)N; U-(15)N; α-(15)N; or γ-(15)N). The metabolic fate of the dietary (15)N and (13)C was traced using gas chromatography-mass spectrometry (GC-MS). Nitrogen was the major contributor to the observed amino acid isotopomers with one additional unit mass (M+1). However, there was differential incorporation, with the amine nitrogen of asparagine being incorporated into other amino acids more efficiently than the amide nitrogen. Higher isotopomers (M+2, M+3 and M+4) indicated the incorporation of varying numbers of (13)C atoms into essential amino acids. GC-MS assays also showed that, even with an excess of dietary labeled glutamine, glutamic acid, asparagine or aspartic acid, the overall content of these amino acids in aphid bodies was mostly the product of catabolism of dietary amino acids and subsequent re-synthesis within the aphids. Thus, these predominant dietary amino acids are not passed directly to Buchnera endosymbionts for synthesis of essential amino acids, but are rather are produced de novo, most likely by endogenous aphid enzymes.

  13. Key plant species and succession patterns associated to past fen-bog transitions - perspective to future

    NASA Astrophysics Data System (ADS)

    Väliranta, Minna; Luoto, Miska; Juutinen, Sari; Korhola, Atte; Tuittila, Eeva-stiina

    2016-04-01

    Minerotrophic fens and ombrotrophic bogs differ in their hydrology, vegetation and carbon dynamics and their geographical distribution seems to be linked to certain climate parameters, such as temperature and effective precipitation. Currently bogs dominate the southern boreal zone but the climate warming with altered temperature and effective precipitation may shift the distribution of bog zone northwards. In this study, we first used plant macrofossil method and radiocarbon analysis to identify and date past fen-bog transitions. These transitions were compared to major Holocene climate phases. Subsequently, palaeoecological data were associated to ecological and environmental data collected along the current fen-bog ecotone in Finland. We identified three successional phases 1) initial minerotrophic fen phase 2) Eriophorum vaginatum-dominated oligotrophic fen phase which was followed by 3) ombrorophic bog phase. Duration of these phases varied but late Holocene timing of fen-bog transition showed some consistency. Based on palaeoecological data 57 % of the modern ecotone peatlands were classified to be in a fen phase, 10 % were in an Eriophorum-dominated phase and 33 % were going through a transition from fen to bog. The study showed that regime shifts are driven by autogenic succession and climate but also fires may efficiently control succession pathways. Our results support the hypothesis that climate change can promote the ombrotrophication process in the southern border of the fen-bog ecotone due to changes in hydrology balance.

  14. Nuevos fenómenos en erupciones cometarias

    NASA Astrophysics Data System (ADS)

    Silva, A.

    Se discuten aquí tres procesos físicos novedosos encontrados en la actividad de cometas: 1) El rol de una distribución de granos de hielo como fuente extendida de H2O en la coma, 2) El efecto de una discontinuidad en el plasma cometario, llamada Cometopausa, sobre la excitación del radical OH , y 3) La actividad por erupciones a grandes distancias heliocéntricas (r > 5 AU). Con respecto a 1) y 2), se presentan modelos que ajustan bien con las observaciones. En cuanto a 3), se presentan explicaciones posibles al fenómeno, y se trata el interesante caso de Chirón 2060, basándose en observaciones propias tomadas desde el CASLEO y datos anteriores.

  15. A fully validated GC-TOF-MS method for the quantification of fatty acids revealed alterations in the metabolic profile of fatty acids after smoking cessation.

    PubMed

    Goettel, Michael; Niessner, Reinhard; Pluym, Nikola; Scherer, Gerhard; Scherer, Max

    2017-01-15

    We developed and validated an efficient and robust method for the simultaneous quantification of 44 fatty acid species in human plasma via GC-TOF-MS. The method is characterized by its robustness, accuracy and precision covering a wide range of fatty acid species with various saturation degrees including short chain fatty acids (beginning with FA 4:0) and long chain fatty acids (up to FA 32:0). The fatty acids were methylated prior to analyses and subsequently detected as fatty acid methyl esters by means of GC-TOF-MS. A highly substituted polar column allowed the separation of geometrical and positional isomers of fatty acid species. The method was applied to plasma samples of a strictly diet controlled clinical smoking cessation study including 39 smokers followed over the course of three months after having quit. Statistical significant alterations within the fatty acid profile were observed when comparing the baseline (subjects still smoking) with one week, one month and three months of smoking cessation. After 3 months of smoking cessation, a partial recovery of alterations in the fatty acid profile evoked by smoking was observed. In conclusion, the developed fatty acid profiling method using GC-TOF-MS has proven as a reliable tool for the quantitative determination of 44 individual fatty acid species within clinical studies.

  16. Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

    PubMed Central

    2013-01-01

    Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that

  17. Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.; Cravatt, Benjamin F.; Boger, Dale L.; Stevens, Raymond C.

    2010-08-17

    The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

  18. Relationship between acid tolerance and cell membrane in Bifidobacterium, revealed by comparative analysis of acid-resistant derivatives and their parental strains grown in medium with and without Tween 80.

    PubMed

    Yang, Xu; Hang, Xiaomin; Zhang, Min; Liu, Xianglong; Yang, Hong

    2015-06-01

    The acid tolerance is particularly important for bifidobacteria to function as probiotics because they usually encounter acidic environments in food products and gastrointestinal tract passage. In this study, two acid-resistant derivatives Bifidobacterium longum JDY1017dpH and Bifidobacterium breve BB8dpH, which displayed a stable acid-resistant phenotype, were generated. The relationship between acid tolerance and cell membrane was investigated by comparing the two acid-resistant derivatives and their parental strains grown in medium with and without Tween 80. The fold increase in acid tolerance of the two acid-resistant derivatives relative to their parental strains was much higher when cells were grown in medium with Tween 80 (10(4) ~ 10(5)-fold) than without Tween 80 (181- and 245-fold). Moreover, when cells were grown in medium with Tween 80, the two acid-resistant derivatives exhibited more C18:1 and cycC19:0, higher mean fatty acid chain length, lower membrane fluidity, and higher expression of cfa gene encoding cyclopropane fatty acid synthase than their parental strains. No significant differences in cell membrane were observed between the two acid-resistant derivatives and their parental strains when cells were grown in medium without Tween 80. The present study revealed that, when cells were grown in medium with Tween 80, the significant fold increase in acid tolerance of the two acid-resistant derivatives was mainly ascribed to the pronounced changes in cell membrane compared with their parental strains. Results presented here could provide a basis for developing new strategies of cell membrane modification to enhance acid tolerance in bifidobacteria.

  19. Denitrification Activity of a Remarkably Diverse Fen Denitrifier Community in Finnish Lapland Is N-Oxide Limited

    PubMed Central

    Palmer, Katharina; Horn, Marcus A.

    2015-01-01

    Peatlands cover more than 30% of the Finnish land area and impact N2O fluxes. Denitrifiers release N2O as an intermediate or end product. In situ N2O emissions of a near pH neutral pristine fen soil in Finnish Lapland were marginal during gas chamber measurements. However, nitrate and ammonium fertilization significantly stimulated in situ N2O emissions. Stimulation with nitrate was stronger than with ammonium. N2O was produced and subsequently consumed in gas chambers. In unsupplemented anoxic microcosms, fen soil produced N2O only when acetylene was added to block nitrous oxide reductase, suggesting complete denitrification. Nitrate and nitrite stimulated denitrification in fen soil, and maximal reaction velocities (vmax) of nitrate or nitrite dependent denitrification where 18 and 52 nmol N2O h-1 gDW-1, respectively. N2O was below 30% of total produced N gases in fen soil when concentrations of nitrate and nitrite were <500 μM. vmax for N2O consumption was up to 36 nmol N2O h-1 gDW-1. Denitrifier diversity was assessed by analyses of narG, nirK/nirS, and nosZ (encoding nitrate-, nitrite-, and nitrous oxide reductases, respectively) by barcoded amplicon pyrosequencing. Analyses of ~14,000 quality filtered sequences indicated up to 25 species-level operational taxonomic units (OTUs), and up to 359 OTUs at 97% sequence similarity, suggesting diverse denitrifiers. Phylogenetic analyses revealed clusters distantly related to publicly available sequences, suggesting hitherto unknown denitrifiers. Representatives of species-level OTUs were affiliated with sequences of unknown soil bacteria and Actinobacterial, Alpha-, Beta-, Gamma-, and Delta-Proteobacterial sequences. Comparison of the 4 gene markers at 97% similarity indicated a higher diversity of narG than for the other gene markers based on Shannon indices and observed number of OTUs. The collective data indicate (i) a high denitrification and N2O consumption potential, and (ii) a highly diverse, nitrate limited

  20. Fine-scale horizontal and vertical micro-distribution patterns of testate amoebae along a narrow Fen/Bog gradient.

    PubMed

    Jassey, Vincent E J; Chiapusio, Geneviève; Mitchell, Edward A D; Binet, Philippe; Toussaint, Marie-Laure; Gilbert, Daniel

    2011-02-01

    The ecology of peatland testate amoebae is well studied along broad gradient from very wet (pool) to dry (hummock) micro-sites where testate amoebae are often found to respond primarily to the depth to water table (DWT). Much less is known on their responses to finer-scale gradients, and nothing is known of their possible response to phenolic compounds, which play a key role in carbon storage in peatlands. We studied the vertical (0-3, 3-6, and 6-9 cm sampling depths) micro-distribution patterns of testate amoebae in the same microhabitat (Sphagnum fallax lawn) along a narrow ecological gradient between a poor fen with an almost flat and homogeneous Sphagnum carpet (fen) and a "young bog" (bog) with more marked micro-topography and mosaic of poor fen and bog vegetation. We analyzed the relationships between the testate amoeba data and three sets of variables (1) "chemical" (pH, Eh potential, and conductivity), (2) "physical" (water temperature, altitude, i.e., Sphagnum mat micro-topography, and DWT), and (3) phenolic compounds in/from Sphagnum (water-soluble and primarily bound phenolics) as well as the habitat (fen/bog) and the sampling depth. Testate amoeba Shannon H' diversity, equitability J of communities, and total density peaked in lower parts of Sphagnum, but the patterns differed between the fen and bog micro-sites. Redundancy analyses revealed that testate amoeba communities differed significantly in relation to Eh, conductivity, water temperature, altitude, water-soluble phenolics, habitat, and sampling depth, but not to DWT, pH, or primarily bound phenolics. The sensitivity of testate amoebae to weak environmental gradients makes them particularly good integrators of micro-environmental variations and has implications for their use in paleoecology and environmental monitoring. The correlation between testate amoeba communities and the concentration of water-soluble phenolic suggests direct (e.g., physiological) and/or indirect (e.g., through impact on

  1. Development of hydraulic properties and nitrate turnover processes in minerotrophic fen soil on differnet scales

    NASA Astrophysics Data System (ADS)

    Kleimeier, Christian; Lennartz, Bernd

    2014-05-01

    Generally, it is recommended to remove the uppermost highly degraded peat layer from fens prior to rewetting to eliminate a potential source of organic pollutants for downstream water bodies. We investigated this material as a potential medium for denitrifying filters to further use the organic material. We are aiming to remove nitrate from tile drainage runoff at the outlet drainage dominated catchments to fullfill the requirements of the European Water Framework Directive. In a lysimeter scale long term mesocosm experiments we were aiming to reveal the peats behavior after disturbing and rewetting under constant flow conditions. Tracer experiments revealed a restructuring of the peat ending up at 20/80 percentage of mobile immobile pore volume. Additionally we observed the nitrate turnover. The turnover rate was determined by the hydraulic load. Absolute turnover rates were equal at lower and higher concentrations as well as flow rates, whereas the turnover reached higher percentages at lower concentrations. To further reveal the nitrate turnover processes flow through rector experiments were conducted in an anaerobic environment. We found that strongly reducing conditions can be created in peat even at the presence of nitrate. Thus we can conclude that the minerotrophic peat with its high iron and sulfur concentrations also enables autotrophic denitrification oxidizing iron and sulfur. While the conditions are favorable to re-reduce iron and sulfur,thus an electron shuttling system developed transporting electrons from the organic material as initial e- donor to nitrate as terminal e- acceptor.

  2. Pseudomonas lini Strain ZBG1 Revealed Carboxylic Acid Utilization and Copper Resistance Features Required for Adaptation to Vineyard Soil Environment: A Draft Genome Analysis

    PubMed Central

    Chan, Kok-Gan; Chong, Teik-Min; Adrian, Tan-Guan-Sheng; Kher, Heng Leong; Grandclément, Catherine; Faure, Denis; Yin, Wai-Fong; Dessaux, Yves; Hong, Kar-Wai

    2016-01-01

    Pseudomonas lini strain ZBG1 was isolated from the soil of vineyard in Zellenberg, France and the draft genome was reported in this study. Bioinformatics analyses of the genome revealed presence of genes encoding tartaric and malic acid utilization as well as copper resistance that correspond to the adaptation this strain in vineyard soil environment. PMID:27512520

  3. Iron sources used by the nonpathogenic lactic acid bacterium Lactobacillus sakei as revealed by electron energy loss spectroscopy and secondary-ion mass spectrometry.

    PubMed

    Duhutrel, Philippe; Bordat, Christian; Wu, Ting-Di; Zagorec, Monique; Guerquin-Kern, Jean-Luc; Champomier-Vergès, Marie-Christine

    2010-01-01

    Lactobacillus sakei is a lactic acid bacterium naturally found on meat. Although it is generally acknowledged that lactic acid bacteria are rare species in the microbial world which do not have iron requirements, the genome sequence of L. sakei 23K has revealed quite complete genetic equipment dedicated to transport and use of this metal. Here, we aimed to investigate which iron sources could be used by this species as well as their role in the bacterium's physiology. Therefore, we developed a microscopy approach based on electron energy loss spectroscopy (EELS) analysis and nano-scale secondary-ion mass spectrometry (SIMS) in order to analyze the iron content of L. sakei cells. This revealed that L. sakei can use iron sources found in its natural ecosystem, myoglobin, hemoglobin, hematin, and transferrin, to ensure long-term survival during stationary phase. This study reveals that analytical image methods (EELS and SIMS) are powerful complementary tools for investigation of metal utilization by bacteria.

  4. Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize mik.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexaki...

  5. Adaptive response to acetic acid in the highly resistant yeast species Zygosaccharomyces bailii revealed by quantitative proteomics.

    PubMed

    Guerreiro, Joana F; Mira, Nuno P; Sá-Correia, Isabel

    2012-08-01

    Zygosaccharomyces bailii is the most tolerant yeast species to acetic acid-induced toxicity, being able to grow in the presence of concentrations of this food preservative close to the legal limits. For this reason, Z. bailii is the most important microbial contaminant of acidic food products but the mechanisms behind this intrinsic resistance to acetic acid are very poorly characterized. To gain insights into the adaptive response and tolerance to acetic acid in Z. bailii, we explored an expression proteomics approach, based on quantitative 2DE, to identify alterations occurring in the protein content in response to sudden exposure or balanced growth in the presence of an inhibitory but nonlethal concentration of this weak acid. A coordinate increase in the content of proteins involved in cellular metabolism, in particular, in carbohydrate metabolism (Mdh1p, Aco1p, Cit1p, Idh2p, and Lpd1p) and energy generation (Atp1p and Atp2p), as well as in general and oxidative stress response (Sod2p, Dak2p, Omp2p) was registered. Results reinforce the concept that glucose and acetic acid are coconsumed in Z. bailii, with acetate being channeled into the tricarboxylic acid cycle. When acetic acid is the sole carbon source, results suggest the activation of gluconeogenic and pentose phosphate pathways, based on the increased content of several proteins of these pathways after glucose exhaustion.

  6. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  7. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    PubMed Central

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  8. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms.

    PubMed

    Widdows, Kate L; Panitchob, Nuttanont; Crocker, Ian P; Please, Colin P; Hanson, Mark A; Sibley, Colin P; Johnstone, Edward D; Sengers, Bram G; Lewis, Rohan M; Glazier, Jocelyn D

    2015-06-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [(14)C]L-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [(14)C]L-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with L-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.

  9. Metabolic analysis revealed altered amino acid profiles in Lupinus albus organs as a result of boron deficiency.

    PubMed

    Alves, Marta; Chicau, Paula; Matias, Helena; Passarinho, José; Pinheiro, Carla; Ricardo, Cândido Pinto

    2011-07-01

    We analysed the changes in the metabolites of Lupinus albus organs (leaf-blades, petioles, apexes, hypocotyls and roots) as a consequence of B deficiency. The deficiency did not affect malate concentration and induced only minor changes in the sugar content, suggesting that the carbohydrate metabolism is little affected by the deficiency. Contrarily, marked changes in the content of free amino acids were observed, with some specific variations associated with the different organs. These changes indicate that various aspects of metabolism implicated in the amino acid accumulation were affected by B deficiency. Most of the detected changes appear to have implications with some stress responses or signalling processes. Asparagine and proline that increase in many stresses also accumulated in petioles, apexes and hypocotyls. Accumulation of γ-aminobutyric acid shunt amino acids, indicative of production of reactive oxygen species, occurs in the same three organs and also the roots. The increase in the branched-chain amino acids, observed in all organs, suggests the involvement of B with the cytoskeleton, whereas glycine decrease in leaf-blades and active growing organs (apexes and roots) could be associated with the proposed role of this amino acids in plant signalling in processes that might be associated with the decreased growth rates observed in B deficiency. Despite the admitted importance of free amino acids in plant metabolism, the available information on this matter is scarce. So our results bring new information concerning the effects of B deficiency in the metabolism of the several L. albus organs.

  10. Urinary loss of tricarboxylic acid cycle intermediates as revealed by metabolomics studies: an underlying mechanism to reduce lipid accretion by whey protein ingestion?

    PubMed

    Lillefosse, Haldis H; Clausen, Morten R; Yde, Christian C; Ditlev, Ditte B; Zhang, Xumin; Du, Zhen-Yu; Bertram, Hanne C; Madsen, Lise; Kristiansen, Karsten; Liaset, Bjørn

    2014-05-02

    Whey protein intake is associated with the modulation of energy metabolism and altered body composition both in human subjects and in animals, but the underlying mechanisms are not yet elucidated. We fed obesity-prone C57BL/6J mice high-fat diets with either casein (HF casein) or whey (HF whey) for 6 weeks. At equal energy intake and apparent fat and nitrogen digestibility, mice fed HF whey stored less energy as lipids, evident both as lower white adipose tissue mass and as reduced liver lipids, compared with HF-casein-fed mice. Explorative analyses of 48 h urine, both by (1)H NMR and LC-MS metabolomic platforms, demonstrated higher urinary excretion of tricarboxylic acid (TCA) cycle intermediates citric acid and succinic acid (identified by both platforms), and cis-aconitic acid and isocitric acid (identified by LC-MS platform) in the HF whey, relative to in the HF-casein-fed mice. Targeted LC-MS analyses revealed higher citric acid and cis-aconitic acid concentrations in fed state plasma, but not in liver of HF-whey-fed mice. We propose that enhanced urinary loss of TCA cycle metabolites drain available substrates for anabolic processes, such as lipogenesis, thereby leading to reduced lipid accretion in HF-whey-fed compared to HF-casein-fed mice.

  11. BOREAS TE-1 SSA-Fen Soil Profile Nutrient Data

    NASA Technical Reports Server (NTRS)

    Papagno, Andrea; Anderson, Darwin; Newcomer, Jeffrey A. (Editor); Hall, Forrest G. (Editor)

    2000-01-01

    The BOREAS TE-1 team collected various data to characterize the soil-plant systems in the BOREAS SSA. Particular emphasis was placed on nutrient biochemistry, the stores and transfers of organic carbon, and how the characteristics were related to measured methane fluxes. The overall traniect in the Prince Albert National Park (Saskatchewan, Canada) included the major plant communities and related soils that occurred in that section of the boreal forest. Soil physical, chemical, and biological measurements along the transect were used to characterize the static environment, which allowed them to be related to methane fluxes. Chamber techniques were used to provide a measure of methane production/uptake. Chamber measurements coupled with flask sampling were used to determine the seasonality of methane fluxes. This particular data set contains soil profile measurements of various nutrients at the SSA-Fen site. The data were collected from 23-May to 21-Oct- 1994. The data are stored in tabular ASCII files. The data files are available on a CD-ROM (see document number 20010000884), or from the Oak Ridge National Laboratory (ORNL) Distributed Active Archive Center (DAAC).

  12. Targeted metabolomic analysis reveals the association between the postprandial change in palmitic acid, branched-chain amino acids and insulin resistance in young obese subjects.

    PubMed

    Liu, Liyan; Feng, Rennan; Guo, Fuchuan; Li, Ying; Jiao, Jundong; Sun, Changhao

    2015-04-01

    Obesity is the result of a positive energy balance and often leads to difficulties in maintaining normal postprandial metabolism. The changes in postprandial metabolites after an oral glucose tolerance test (OGTT) in young obese Chinese men are unclear. In this work, the aim is to investigate the complex metabolic alterations in obesity provoked by an OGTT using targeted metabolomics. We used gas chromatography-mass spectrometry and ultra high performance liquid chromatography-triple quadrupole mass spectrometry to analyze serum fatty acids, amino acids and biogenic amines profiles from 15 control and 15 obese subjects at 0, 30, 60, 90 and 120 min during an OGTT. Metabolite profiles from 30 obese subjects as independent samples were detected in order to validate the change of metabolites. There were the decreased levels of fatty acid, amino acids and biogenic amines after OGTT in obesity. At 120 min, percent change of 20 metabolites in obesity has statistical significance when comparing with the controls. The obese parameters was positively associated with changes in arginine and histidine (P<0.05) and the postprandial change in palmitic acid (PA), branched-chain amino acids (BCAAs) and phenylalanine between 1 and 120 min were positively associated with fasting insulin and HOMA-IR (all P<0.05) in the obese group. The postprandial metabolite of PA and BCAAs may play important role in the development and onset of insulin resistance in obesity. Our findings offer new insights in the complex physiological regulation of the metabolism during an OGTT in obesity.

  13. Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses.

    PubMed

    King, Thea; Lucchini, Sacha; Hinton, Jay C D; Gobius, Kari

    2010-10-01

    The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing.

  14. Integrated Transcriptome and Metabolic Analyses Reveals Novel Insights into Free Amino Acid Metabolism in Huangjinya Tea Cultivar

    PubMed Central

    Zhang, Qunfeng; Liu, Meiya; Ruan, Jianyun

    2017-01-01

    The chlorotic tea variety Huangjinya, a natural mutant, contains enhanced levels of free amino acids in its leaves, which improves the drinking quality of its brewed tea. Consequently, this chlorotic mutant has a higher economic value than the non-chlorotic varieties. However, the molecular mechanisms behind the increased levels of free amino acids in this mutant are mostly unknown, as are the possible effects of this mutation on the overall metabolome and biosynthetic pathways in tea leaves. To gain further insight into the effects of chlorosis on the global metabolome and biosynthetic pathways in this mutant, Huangjinya plants were grown under normal and reduced sunlight, resulting in chlorotic and non-chlorotic leaves, respectively; their leaves were analyzed using transcriptomics as well as targeted and untargeted metabolomics. Approximately 5,000 genes (8.5% of the total analyzed) and ca. 300 metabolites (14.5% of the total detected) were significantly differentially regulated, thus indicating the occurrence of marked effects of light on the biosynthetic pathways in this mutant plant. Considering primary metabolism, including that of sugars, amino acids, and organic acids, significant changes were observed in the expression of genes involved in both nitrogen (N) and carbon metabolism. The suite of changes not only generated an increase in amino acids, including glutamic acid, glutamine, and theanine, but it also elevated the levels of free ammonium, citrate, and α-ketoglutarate, and lowered the levels of mono- and di-saccharides and of caffeine as compared with the non-chlorotic leaves. Taken together, our results suggest that the increased levels of amino acids in the chlorotic vs. non-chlorotic leaves are likely due to increased protein catabolism and/or decreased glycolysis and diminished biosynthesis of nitrogen-containing compounds other than amino acids, including chlorophyll, purines, nucleotides, and alkaloids. PMID:28321230

  15. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted.

  16. Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

    PubMed

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

    2014-06-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)-CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.

  17. Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

    PubMed

    Yuan, Feng-Jie; Zhu, Dan-Hua; Tan, Yuan-Yuan; Dong, De-Kun; Fu, Xu-Jun; Zhu, Shen-Long; Li, Bai-Quan; Shu, Qing-Yao

    2012-11-01

    Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

  18. The Dependence of Peat Soil Hydraulic Conductivity on Dominant Vegetation Type in Mountain Fens

    NASA Astrophysics Data System (ADS)

    Crockett, A. C.; Ronayne, M. J.; Cooper, D. J.

    2014-12-01

    The peat soil within fen wetlands provides water storage that can substantially influence the hydrology of mountain watersheds. In this study, we investigated the relationship between hydraulic conductivity and vegetation type for fens occurring in Rocky Mountain National Park (RMNP), Colorado, USA. Vegetation in RMNP fens can be dominated by woody plants and shrubs, such as willows; by mosses; or by herbaceous plants such as sedges. Fens dominated by each vegetation type were selected for study. Six fens were investigated, all of which are in the Colorado River watershed on the west side of RMNP. For each site, soil hydraulic conductivity was measured at multiple locations using a single-ring infiltrometer. As a result of the shallow water table in these fens (the water table was always within 10 cm of the surface), horizontal hydraulic gradients were produced during the field tests. The measured infiltration rates were analyzed using the numerical model HYDRUS. In order to determine the hydraulic conductivity, a parameter estimation problem was solved using HYDRUS as the forward simulator. Horizontal flow was explicitly accounted for in the model. This approach produced more accurate estimates of hydraulic conductivity than would be obtained using an analytical solution that assumes strictly vertical flow. Significant differences in hydraulic properties between fens appear to result at least in part from the effects of different dominant vegetation types on peat soil formation.

  19. Omics-based approaches reveal phospholipids remodeling of Rhizopus oryzae responding to furfural stress for fumaric acid-production from xylose.

    PubMed

    Pan, Xinrong; Liu, Huanhuan; Liu, Jiao; Wang, Cheng; Wen, Jianping

    2016-12-01

    In order to relieve the toxicity of furfural on Rhizopus oryzae fermentation, the molecular mechanism of R. oryzae responding to furfural stress for fumaric acid-production was investigated by omics-based approaches. In metabolomics analysis, 29 metabolites including amino acid, sugars, polyols and fatty acids showed significant changes for maintaining the basic cell metabolism at the cost of lowering fumaric acid production. To further uncover the survival mechanism, lipidomics was carried out, revealing that phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R. oryzae's adapting to furfural stress. Based on the above omics analysis, lecithin, inositol and soybean oil were exogenously supplemented separately with an optimized concentration in the presence of furfural, which increased fumaric acid titer from 5.78g/L to 10.03g/L, 10.05g/L and 12.13g/L (increased by 73.5%, 73.8% and 110%, respectively). These findings provide a methodological guidance for hemicellulose-fumaric acid development.

  20. Proteomic-based stable isotope probing reveals taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

    DOE PAGES

    Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; ...

    2016-04-26

    Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual global primary production, yet defining how substrate utilization preferences and resource partitioning structure these microbial communities remains a challenge. In this study, we utilized proteomics-based stable isotope probing (proteomic SIP) to characterize the assimilation of amino acids by coastal marine bacterioplankton populations. We incubated microcosms of seawater collected from Newport, OR and Monterey Bay, CA with 1 M 13C-amino acids for 15 and 32 hours. Subsequent analysis of 13C incorporation into protein biomass quantified the frequency and extent of isotope enrichment for identifiedmore » proteins. Using these metrics we tested whether amino acid assimilation patterns were different for specific bacterioplankton populations. Proteins associated with Rhodobacterales and Alteromonadales tended to have a significantly high number of tandem mass spectra from 13C-enriched peptides, while Flavobacteriales and SAR11 proteins generally had significantly low numbers of 13C-enriched spectra. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time-point 2, while Alteromonadales ribosomal proteins were 13C- enriched across time-points. Overall, proteomic SIP facilitated quantitative comparisons of dissolved free amino acids assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population-level metabolic responses to resource acquisition in complex microbial communities.« less

  1. Transcriptomics and proteomic studies reveal acaricidal mechanism of octadecanoic acid-3, 4 - tetrahydrofuran diester against Sarcoptes scabiei var. cuniculi

    PubMed Central

    Song, Xu; Chen, Zhenzhen; Jia, Renyong; Cao, Mei; Zou, Yuanfeng; Li, Lixia; Liang, Xiaoxia; Yin, Lizi; He, Changliang; Yue, Guizhou; Yin, Zhongqiong

    2017-01-01

    In our previous study, a new compound, octadecanoic acid-3, 4-tetrahydrofuran diester, possessing potent acaricidal activity was obtained from neem oil. This study performed RNA-seq transcriptomics and iTRAQ proteomics to uncover the acaricidal mechanism of the compound against Sarcoptes scabiei var. cuniculi. The results of transcriptomics indicated that after treatment with octadecanoic acid-3, 4-tetrahydrofuran diester, genes related to “Energy metabolism” were significantly up-/down-regulated, including citrate cycle, oxidative phosphorylation pathway and fatty acid metabolism. Proteomics analysis showed accordant changes of proteins related to oxidative phosphorylation pathway. The target proteins of the compound were NADH dehydrogenase, Ubiquinol-cytochrome c reductase, Cytochrome c oxidase, ATP synthase, enolase and superoxide dismutase. In transcriptomics-proteomics correlation analysis, the concordance rate between protein abundances and their corresponding mRNAs was 57%, while others (43%) were discordant changes, suggesting divergent regulating effects of octadecanoic acid-3, 4-tetrahydrofuran diester. These results suggested that the acaricidal mechanism of octadecanoic acid-3, 4-tetrahydrofuran diester attributed to interference with energy metabolism, especially oxidative phosphorylation pathway. PMID:28361965

  2. Comparisons of soil nitrogen mass balances for an ombrotrophic bog and a minerotrophic fen in northern Minnesota

    EPA Science Inventory

    We compared the N budgets of an ombrotrophic bog and a minerotrophic fen to quantify the importance of denitrification in peatlands and their watersheds. We also compared the watershed upland mineral soils to bog/fen peat; lagg and transition zone peat to central bog/fen peat; an...

  3. Regional and local hydrogeology of calcareous fens in the Minnesota river basin, USA

    USGS Publications Warehouse

    Almendinger, J.E.; Leete, J.H.

    1998-01-01

    Six calcareous fens in the Minnesota River Basin, USA are in regional hydrogeologic settings with large discharges of calcareous ground water. These settings juxtapose topographically high areas of ground-water recharge with fens in lower areas of discharge, thus creating steep upward hydraulic gradients at the fens. Coarse glacial deposits with high permeability connect recharge areas to discharge areas and transmit large amounts of ground water to the fens. Calcareous fens in the Minnesota River Basin are associated with two regional landforms, river terraces and glacial moraines. The calcareous drift is the likely source of carbonate for the fens; carbonate bedrock is not required. Five of the calcareous fens form peat aprons over broad areas of diffuse ground-water discharge on river terraces. One of the calcareous fens is a peat dome over an aquifer window, a relatively small area (about 15-m radius) of localized ground-water discharge through a breach in the clayey confining layer of the underlying aquifer. Carbonate content of calcareous fen peat averaged about 27% (calcium carbonate equivalent, dry weight basis) in the surface layer, which commonly overlies a carbonate-depleted zone with a carbonate content of 10% or less. Hydraulic conductivity (K) of calcareous fen peat determined from slug tests ranged from 2.7×10−7 to 9.8×10−5 m s−1 and had a geometric mean of 3.8×10−6 m s−1. These values likely underestimate the true horizontal hydraulic conductivity (Kh) and overestimate the true vertical hydraulic conductivity (Kv) because of errors in assumptions commonly used in slug-test analyses. Median (over time) hydraulic heads in wells screened below the base of the peat ranged from about 25 to 69 cm above the peat surface. Upward vertical gradients (dimensionless) through the peat ranged from 0.040 to 0.209. Vertical ground-water discharge was calculated by Darey’s Law and ranged from 2 to 172 L m−2 d−1. Because of bias in estimating

  4. Controls on evapotranspiration at a subarctic sedge fen

    NASA Astrophysics Data System (ADS)

    Eaton, Andrea K.; Rouse, Wayne R.

    2001-12-01

    In this study, 10 years (1990-99) of summertime data collected at a representative sedge fen in the Hudson Bay Lowland (HBL) are used to investigate the energy and water balance dynamics of subarctic wetlands. The summertime climatic characteristics at the study site during the 10 year study period are also examined. It is shown that mean cumulative summertime precipitation Pavg for the study decade closely approximates the 30 year mean Pavg. However, the mean summertime air temperature Tavg for the study decade is 1 °C higher than the 30 year mean Tavg.To examine the energy and water balance dynamics at the study site, the variation in each of their respective components throughout the study decade is considered. Little variation is observed in cumulative summertime net radiation and cumulative summertime ground heat flux QGcum; however, substantial year-to-year variation is evident in cumulative summertime water deficit WDcum, cumulative summertime precipitation P

  5. Mapping of Saccharomyces cerevisiae metabolites in fermenting wheat straight-dough reveals succinic acid as pH-determining factor.

    PubMed

    Jayaram, Vinay B; Cuyvers, Sven; Lagrain, Bert; Verstrepen, Kevin J; Delcour, Jan A; Courtin, Christophe M

    2013-01-15

    Fermenting yeast does not merely cause dough leavening, but also contributes to the bread aroma and might alter dough rheology. Here, the yeast carbon metabolism was mapped during bread straight-dough fermentation. The concentration of most metabolites changed quasi linearly as a function of fermentation time. Ethanol and carbon dioxide concentrations reached up to 60 mmol/100g flour. Interestingly, high levels of glycerol (up to 10 mmol/100g flour) and succinic acid (up to 1.6 mmol/100g flour) were produced during dough fermentation. Further tests showed that, contrary to current belief, the pH decrease in fermenting dough is primarily caused by the production of succinic acid by the yeast instead of carbon dioxide dissolution or bacterial organic acids. Together, our results provide a comprehensive overview of metabolite production during dough fermentation and yield insight into the importance of some of these metabolites for dough properties.

  6. Transcriptomic analysis of Shiga-toxigenic bacteriophage carriage reveals a profound regulatory effect on acid resistance in Escherichia coli.

    PubMed

    Veses-Garcia, Marta; Liu, Xuan; Rigden, Daniel J; Kenny, John G; McCarthy, Alan J; Allison, Heather E

    2015-12-01

    Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli.

  7. Factors affecting the interactions between beta-lactoglobulin and fatty acids as revealed in molecular dynamics simulations.

    PubMed

    Yi, Changhong; Wambo, Thierry O

    2015-09-21

    Beta-lactoglobulin (BLG), a bovine dairy protein, is a promiscuously interacting protein that can bind multiple hydrophobic ligands. Fatty acids (FAs), common hydrophobic molecules bound to BLG, are important sources of fuel for life because they yield large quantities of ATP when metabolized. The binding affinity increases with the length of the ligands, indicating the importance of the van der Waals (vdW) interactions between the hydrocarbon tail and the hydrophobic calyx of BLG. An exception to this rule is caprylic acid (OCA) which is two-carbon shorter but has a stronger binding affinity than capric acid. Theoretical calculations in the current literature are not accurate enough to shed light on the underlying physics of this exception. The computed affinity values are greater for longer fatty acids without respect for the caprylic exception and those values are generally several orders of magnitude away from the experimental data. In this work, we used hybrid steered molecular dynamics to accurately compute the binding free energies between BLG and the five saturated FAs of 8 to 16 carbon atoms. The computed binding free energies agree well with experimental data not only in rank but also in absolute values. We gained insights into the exceptional behavior of caprylic acid in the computed values of entropy and electrostatic interactions. We found that the electrostatic interaction between the carboxyl group of caprylic acid and the two amino groups of K60/69 in BLG is much stronger than the vdW force between the OCA's hydrophobic tail and the BLG calyx. This pulls OCA to the top of the beta barrel where it is easier to fluctuate, giving rise to greater entropy of OCA at the binding site.

  8. Surface-enhanced Raman scattering and fluorescence spectroscopy reveal molecular interactions of all-trans retinoic acid and RAR gamma ligand-binding domain.

    PubMed

    Morjani, H; Beljebbar, A; Sockalingum, G D; Mattioli, T A; Bonnier, D; Gronemeyer, H; Manfait, M

    1998-01-01

    Surface-enhanced Raman scattering and fluorescence were used to investigate the interactions of all-trans retinoic acid with the gamma-type retinoic acid receptor. Raman data revealed a significant attenuation in intensity of the bands originating from the retinoic acid polyenic chain upon receptor binding, with the spectrum being dominantly that of the beta-ionone ring. Fluorescence measurements supported the hydrophobic character of the ligand binding. These novel spectroscopic results are fully consistent with the published X-ray crystallographic data and suggest that these techniques may be valuable additional tools to characterize the interactions of agonists and antagonists with residues in the ligand-binding pockets of retinoid receptor homo- and heterodimers.

  9. Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis.

    PubMed

    Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D

    2016-03-14

    Nanoparticles entering the human body instantly become coated with a "protein corona" that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an "organic corona" containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.

  10. Metabolism of Seriola lalandi during Starvation as Revealed by Fatty Acid Analysis and Compound-Specific Analysis of Stable Isotopes within Amino Acids

    PubMed Central

    Barreto-Curiel, Fernando; Focken, Ulfert; D’Abramo, Louis R.

    2017-01-01

    Fish starvation is defined as food deprivation for a long period of time, such that physiological processes become confined to basal metabolism. Starvation provides insights in physiological processes without interference from unknown factors in digestion and nutrient absorption occurring in fed state. Juveniles of amberjack Seriola lalandi were isotopically equilibrated to a formulated diet for 60 days. One treatment consisted of fish that continued to be fed and fish in the other treatment were not fed for 35 days. The isotopic signatures prior to the beginning of and after the starvation period, for fish in the starvation and control treatments, were analysed for lipid content, fatty acid composition and isotopic analysis of bulk (EA-IRMS) and of amino acids (compound specific isotope analysis, CSIA). There were three replicates for the starvation group. Fatty acid content in muscle and liver tissue before and after starvation was determined to calculate percent change. Results showed that crude lipid was the most used source of energy in most cases; the PUFAs and LC-PUFAs were highly conserved. According to the protein signature in bulk (δ15N) and per amino acid (δ13C and δ15N), in muscle tissue, protein synthesis did not appear to occur substantially during starvation, whereas in liver, increases in δ13C and δ15N indicate that protein turnover occurred, probably for metabolic routing to energy-yielding processes. As a result, isotopic values of δ15N in muscle tissue do not change, whereas CSIA net change occurred in the liver tissue. During the study period of 35 days, muscle protein was largely conserved, being neither replenished from amino acid pools in the plasma and liver nor catabolized. PMID:28095488

  11. Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis

    NASA Astrophysics Data System (ADS)

    Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D.

    2016-03-01

    Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body. Electronic supplementary information (ESI) available. See

  12. Response of anaerobic carbon cycling to water table manipulation in an Alaskan rich fen

    USGS Publications Warehouse

    Kane, E.S.; Chivers, M.R.; Turetsky, M.R.; Treat, C.C.; Petersen, D.G.; Waldrop, M.; Harden, J.W.; McGuire, A.D.

    2013-01-01

    To test the effects of altered hydrology on organic soil decomposition, we investigated CO2 and CH4 production potential of rich-fen peat (mean surface pH = 6.3) collected from a field water table manipulation experiment including control, raised and lowered water table treatments. Mean anaerobic CO2 production potential at 10 cm depth (14.1 ± 0.9 μmol C g−1 d−1) was as high as aerobic CO2 production potential (10.6 ± 1.5 μmol C g−1 d−1), while CH4 production was low (mean of 7.8 ± 1.5 nmol C g−1 d−1). Denitrification enzyme activity indicated a very high denitrification potential (197 ± 23 μg N g−1 d−1), but net NO-3 reduction suggested this was a relatively minor pathway for anaerobic CO2 production. Abundances of denitrifier genes (nirK and nosZ) did not change across water table treatments. SO2-4 reduction also did not appear to be an important pathway for anaerobic CO2 production. The net accumulation of acetate and formate as decomposition end products in the raised water table treatment suggested that fermentation was a significant pathway for carbon mineralization, even in the presence of NO-3. Dissolved organic carbon (DOC) concentrations were the strongest predictors of potential anaerobic and aerobic CO2 production. Across all water table treatments, the CO2:CH4 ratio increased with initial DOC leachate concentrations. While the field water table treatment did not have a significant effect on mean CO2 or CH4 production potential, the CO2:CH4 ratio was highest in shallow peat incubations from the drained treatment. These data suggest that with continued drying or with a more variable water table, anaerobic CO2 production may be favored over CH4 production in this rich fen. Future research examining the potential for dissolved organic substances to facilitate anaerobic respiration, or alternative redox processes that limit the effectiveness of organic acids as substrates in anaerobic metabolism, would help explain additional

  13. Contrasting wetland CH4 emission responses to simulated glacial atmospheric CO2 in temperate bogs and fens.

    PubMed

    Boardman, Carl P; Gauci, Vincent; Watson, Jonathan S; Blake, Stephen; Beerling, David J

    2011-12-01

    Wetlands were the largest source of atmospheric methane (CH(4) ) during the Last Glacial Maximum (LGM), but the sensitivity of this source to exceptionally low atmospheric CO(2) concentration ([CO(2) ]) at the time has not been examined experimentally. We tested the hypothesis that LGM atmospheric [CO(2) ] reduced CH(4) emissions as a consequence of decreased photosynthate allocation to the rhizosphere. We exposed minerotrophic fen and ombrotrophic bog peatland mesocosms to simulated LGM (c. 200 ppm) or ambient (c. 400 ppm) [CO(2) ] over 21 months (n = 8 per treatment) and measured gaseous CH(4) flux, pore water dissolved CH(4) and volatile fatty acid (VFA; an indicator of plant carbon supply to the rhizosphere) concentrations. Cumulative CH(4) flux from fen mesocosms was suppressed by 29% (P < 0.05) and rhizosphere pore water [CH(4) ] by c. 50% (P < 0.01) in the LGM [CO(2) ], variables that remained unaffected in bog mesocosms. VFA analysis indicated that changes in plant root exudates were not the driving mechanism behind these results. Our data suggest that the LGM [CO(2) ] suppression of wetland CH(4) emissions is contingent on trophic status. The heterogeneous response may be attributable to differences in species assemblage that influence the dominant CH(4) production pathway, rhizosphere supplemented photosynthesis and CH(4) oxidation.

  14. Comparative Proteomic Study of Fatty Acid-treated Myoblasts Reveals Role of Cox-2 in Palmitate-induced Insulin Resistance.

    PubMed

    Chen, Xiulan; Xu, Shimeng; Wei, Shasha; Deng, Yaqin; Li, Yiran; Yang, Fuquan; Liu, Pingsheng

    2016-02-22

    Accumulated studies demonstrate that saturated fatty acids (FAs) such as palmitic acid (PA) inhibit insulin signaling in skeletal muscle cells and monounsaturated fatty acids such as oleic acid (OA) reverse the effect of PA on insulin signaling. The detailed molecular mechanism of these opposite effects remains elusive. Here we provide a comparative proteomic study of skeletal myoblast cell line C2C12 that were untreated or treated with PA, and PA plus OA. A total of 3437 proteins were quantified using SILAC in this study and 29 proteins fall into the pattern that OA reverses PA effect. Expression of some these proteins were verified using qRT-PCR and Western blot. The most significant change was cyclooxygenase-2 (Cox-2). In addition to whole cell comparative proteomic study, we also compared lipid droplet (LD)-associated proteins and identified that Cox-2 was one of three major altered proteins under the FA treatment. This finding was then confirmed using immunofluorescence. Finally, Cox-2 selective inhibitor, celecoxib protected cells from PA-reduced insulin signaling Akt phosphorylation. Together, these results not only provide a dataset of protein expression change in FA treatment but also suggest that Cox-2 and lipid droplets (LDs) are potential players in PA- and OA-mediated cellular processes.

  15. Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry

    PubMed Central

    Greaves, Jennifer; Munro, Kevin R.; Davidson, Stuart C.; Riviere, Matthieu; Wojno, Justyna; Smith, Terry K.; Tomkinson, Nicholas C. O.; Chamberlain, Luke H.

    2017-01-01

    S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity. PMID:28167757

  16. Proteomic-based stable isotope probing reveals taxonomically Distinct Patterns in Amino Acid Assimilation by Coastal Marine Bacterioplankton

    SciTech Connect

    Bryson, Samuel; Li, Zhou; Pett-Ridge, Jennifer; Robert L. Hettich; Mayali, Xavier; Pan, Chongle; Mueller, Ryan S.

    2016-04-26

    Heterotrophic marine bacterioplankton are a critical component of the carbon cycle, processing nearly a quarter of annual global primary production, yet defining how substrate utilization preferences and resource partitioning structure these microbial communities remains a challenge. In this study, we utilized proteomics-based stable isotope probing (proteomic SIP) to characterize the assimilation of amino acids by coastal marine bacterioplankton populations. We incubated microcosms of seawater collected from Newport, OR and Monterey Bay, CA with 1 M 13C-amino acids for 15 and 32 hours. Subsequent analysis of 13C incorporation into protein biomass quantified the frequency and extent of isotope enrichment for identified proteins. Using these metrics we tested whether amino acid assimilation patterns were different for specific bacterioplankton populations. Proteins associated with Rhodobacterales and Alteromonadales tended to have a significantly high number of tandem mass spectra from 13C-enriched peptides, while Flavobacteriales and SAR11 proteins generally had significantly low numbers of 13C-enriched spectra. Rhodobacterales proteins associated with amino acid transport and metabolism had an increased frequency of 13C-enriched spectra at time-point 2, while Alteromonadales ribosomal proteins were 13C- enriched across time-points. Overall, proteomic SIP facilitated quantitative comparisons of dissolved free amino acids assimilation by specific taxa, both between sympatric populations and between protein functional groups within discrete populations, allowing an unprecedented examination of population-level metabolic responses to resource acquisition in complex microbial communities.

  17. Understanding Prairie Fen Hydrology - a Hierarchical Multi-Scale Groundwater Modeling Approach

    NASA Astrophysics Data System (ADS)

    Sampath, P.; Liao, H.; Abbas, H.; Ma, L.; Li, S.

    2012-12-01

    Prairie fens provide critical habitat to more than 50 rare species and significantly contribute to the biodiversity of the upper Great Lakes region. The sustainability of these globally unique ecosystems, however, requires that they be fed by a steady supply of pristine, calcareous groundwater. Understanding the hydrology that supports the existence of such fens is essential in preserving these valuable habitats. This research uses process-based multi-scale groundwater modeling for this purpose. Two fen-sites, MacCready Fen and Ives Road Fen, in Southern Michigan were systematically studied. A hierarchy of nested steady-state models was built for each fen-site to capture the system's dynamics at spatial scales ranging from the regional groundwater-shed to the local fens. The models utilize high-resolution Digital Elevation Models (DEM), National Hydrologic Datasets (NHD), a recently-assembled water-well database, and results from a state-wide groundwater mapping project to represent the complex hydro-geological and stress framework. The modeling system simulates both shallow glacial and deep bedrock aquifers as well as the interaction between surface water and groundwater. Aquifer heterogeneities were explicitly simulated with multi-scale transition probability geo-statistics. A two-way hydraulic head feedback mechanism was set up between the nested models, such that the parent models provided boundary conditions to the child models, and in turn the child models provided local information to the parent models. A hierarchical mass budget analysis was performed to estimate the seepage fluxes at the surface water/groundwater interfaces and to assess the relative importance of the processes at multiple scales that contribute water to the fens. The models were calibrated using observed base-flows at stream gauging stations and/or static water levels at wells. Three-dimensional particle tracking was used to predict the sources of water to the fens. We observed from the

  18. Greenhouse gas emissions from fen soils used for forage production in northern Germany

    NASA Astrophysics Data System (ADS)

    Poyda, Arne; Reinsch, Thorsten; Kluß, Christof; Loges, Ralf; Taube, Friedhelm

    2016-09-01

    A large share of peatlands in northwestern Germany is drained for agricultural purposes, thereby emitting high amounts of greenhouse gases (GHGs). In order to quantify the climatic impact of fen soils in dairy farming systems of northern Germany, GHG exchange and forage yield were determined on four experimental sites which differed in terms of management and drainage intensity: (a) rewetted and unutilized grassland (UG), (b) intensive and wet grassland (GW), (c) intensive and moist grassland (GM) and (d) arable forage cropping (AR). Net ecosystem exchange (NEE) of CO2 and fluxes of CH4 and N2O were measured using closed manual chambers. CH4 fluxes were significantly affected by groundwater level (GWL) and soil temperature, whereas N2O fluxes showed a significant relation to the amount of nitrate in top soil. Annual balances of all three gases, as well as the global warming potential (GWP), were significantly correlated to mean annual GWL. A 2-year mean GWP, combined from CO2-C eq. of NEE, CH4 and N2O emissions, as well as C input (slurry) and C output (harvest), was 3.8, 11.7, 17.7 and 17.3 Mg CO2-C eq. ha-1 a-1 for sites UG, GW, GM and AR, respectively (standard error (SE) 2.8, 1.2, 1.8, 2.6). Yield-related emissions for the three agricultural sites were 201, 248 and 269 kg CO2-C eq. (GJ net energy lactation; NEL)-1 for sites GW, GM and AR, respectively (SE 17, 9, 19). The carbon footprint of agricultural commodities grown on fen soils depended on long-term drainage intensity rather than type of management, but management and climate strongly influenced interannual on-site variability. However, arable forage production revealed a high uncertainty of yield and therefore was an unsuitable land use option. Lowest yield-related GHG emissions were achieved by a three-cut system of productive grassland swards in combination with a high GWL (long-term mean ≤ 20 cm below the surface).

  19. Effects of Perfluorooctanoic Acid on Metabolic Profiles in Brain and Liver of Mouse Revealed by a High-throughput Targeted Metabolomics Approach

    NASA Astrophysics Data System (ADS)

    Yu, Nanyang; Wei, Si; Li, Meiying; Yang, Jingping; Li, Kan; Jin, Ling; Xie, Yuwei; Giesy, John P.; Zhang, Xiaowei; Yu, Hongxia

    2016-04-01

    Perfluorooctanoic acid (PFOA), a perfluoroalkyl acid, can result in hepatotoxicity and neurobehavioral effects in animals. The metabolome, which serves as a connection among transcriptome, proteome and toxic effects, provides pathway-based insights into effects of PFOA. Since understanding of changes in the metabolic profile during hepatotoxicity and neurotoxicity were still incomplete, a high-throughput targeted metabolomics approach (278 metabolites) was used to investigate effects of exposure to PFOA for 28 d on brain and liver of male Balb/c mice. Results of multivariate statistical analysis indicated that PFOA caused alterations in metabolic pathways in exposed individuals. Pathway analysis suggested that PFOA affected metabolism of amino acids, lipids, carbohydrates and energetics. Ten and 18 metabolites were identified as potential unique biomarkers of exposure to PFOA in brain and liver, respectively. In brain, PFOA affected concentrations of neurotransmitters, including serotonin, dopamine, norepinephrine, and glutamate in brain, which provides novel insights into mechanisms of PFOA-induced neurobehavioral effects. In liver, profiles of lipids revealed involvement of β-oxidation and biosynthesis of saturated and unsaturated fatty acids in PFOA-induced hepatotoxicity, while alterations in metabolism of arachidonic acid suggesting potential of PFOA to cause inflammation response in liver. These results provide insight into the mechanism and biomarkers for PFOA-induced effects.

  20. Effects of Perfluorooctanoic Acid on Metabolic Profiles in Brain and Liver of Mouse Revealed by a High-throughput Targeted Metabolomics Approach

    PubMed Central

    Yu, Nanyang; Wei, Si; Li, Meiying; Yang, Jingping; Li, Kan; Jin, Ling; Xie, Yuwei; Giesy, John P.; Zhang, Xiaowei; Yu, Hongxia

    2016-01-01

    Perfluorooctanoic acid (PFOA), a perfluoroalkyl acid, can result in hepatotoxicity and neurobehavioral effects in animals. The metabolome, which serves as a connection among transcriptome, proteome and toxic effects, provides pathway-based insights into effects of PFOA. Since understanding of changes in the metabolic profile during hepatotoxicity and neurotoxicity were still incomplete, a high-throughput targeted metabolomics approach (278 metabolites) was used to investigate effects of exposure to PFOA for 28 d on brain and liver of male Balb/c mice. Results of multivariate statistical analysis indicated that PFOA caused alterations in metabolic pathways in exposed individuals. Pathway analysis suggested that PFOA affected metabolism of amino acids, lipids, carbohydrates and energetics. Ten and 18 metabolites were identified as potential unique biomarkers of exposure to PFOA in brain and liver, respectively. In brain, PFOA affected concentrations of neurotransmitters, including serotonin, dopamine, norepinephrine, and glutamate in brain, which provides novel insights into mechanisms of PFOA-induced neurobehavioral effects. In liver, profiles of lipids revealed involvement of β-oxidation and biosynthesis of saturated and unsaturated fatty acids in PFOA-induced hepatotoxicity, while alterations in metabolism of arachidonic acid suggesting potential of PFOA to cause inflammation response in liver. These results provide insight into the mechanism and biomarkers for PFOA-induced effects. PMID:27032815

  1. BOREAS TF-11 SSA-Fen Soil Surface CO2 Flux Data

    NASA Technical Reports Server (NTRS)

    Arkebauer, Timothy J.; Hall, Forrest G. (Editor); Knapp, David E. (Editor)

    2000-01-01

    The BOREAS TF-11 team gathered a variety of data to complement its tower flux measurements collected at the SSA-Fen site. These data are soil surface CO 2 flux data at the SSA-Fen site from 27- May-1994 to 23-Sep-1994 and from 13-May-1995 to 03-Oct-1995. A portable gas exchange system was used to make these measurements. The data are stored in tabular ASCII files.

  2. Differences in potential for amino acid change after mutation reveals distinct strategies for kappa and lambda light-chain variation.

    PubMed

    Hershberg, Uri; Shlomchik, Mark J

    2006-10-24

    B cells generate varied yet functional clones under high rates of mutation of their V genes. It has been proposed that as a result of the opposing demands of diversification and preservation of integrity, the V genes of heavy and light chains have evolved to overexpress codons prone to amino acid change in their complementarity determining regions (CDR) compared with the framework (FW) regions. We have analyzed the germ-line V genes of heavy and light chains (both kappa and lambda), comparing codons of CDR and FW of the germ-line V regions both to each other and to control regions. We found that in both germ-line heavy chains and lambda chains, CDR codons are prone to replacement mutations, whereas in the FW, the opposite is true. Furthermore, the difference between CDR and FW in heavy chains and lambda chains is based on codons that are prone to nonconservative changes of amino acid. In contrast, in germ-line kappa chains, the codons in both CDR and FW are more prone to replacement mutations. We also demonstrated that negative selection during immune responses is more sensitive to nonconservative amino acid substitutions than overall amino acid change, demonstrating the applicability of our analysis to real-time process of selection in the immune system. The differences in germ-line kappa and lambda light chains' potential reaction to mutation suggests that via these two differently evolved light-chain types, the B cell repertoire encompasses two different strategies to balance diversity and stability in an immune response.

  3. Scale-location specific relations between soil nutrients and topographic factors in the Fen River Basin, Chinese Loess Plateau

    NASA Astrophysics Data System (ADS)

    Zhu, Hongfen; Bi, Rutian; Duan, Yonghong; Xu, Zhanjun

    2016-09-01

    Understanding scale- and location-specific variations of soil nutrients in cultivated land is a crucial consideration for managing agriculture and natural resources effectively. In the present study, wavelet coherency was used to reveal the scale-location specific correlations between soil nutrients, including soil organic matter (SOM), total nitrogen (TN), available phosphorus (AP), and available potassium (AK), as well as topographic factors (elevation, slope, aspect, and wetness index) in the cultivated land of the Fen River Basin in Shanxi Province, China. The results showed that SOM, TN, AP, and AK were significantly inter-correlated, and that the scales at which soil nutrients were correlated differed in different landscapes, and were generally smaller in topographically rougher terrain. All soil nutrients but TN were significantly influenced by the wetness index at relatively large scales (32-72 km) and AK was significantly affected by the aspect at large scales at partial locations, showing localized features. The results of this study imply that the wetness index should be taken into account during farming practices to improve the soil nutrients of cultivated land in the Fen River Basin at large scales.

  4. High throughput lipidomic profiling of schizophrenia and bipolar disorder brain tissue reveals alterations of free fatty acids, phosphatidylcholines, and ceramides.

    PubMed

    Schwarz, Emanuel; Prabakaran, Sudhakaran; Whitfield, Phil; Major, Hilary; Leweke, F M; Koethe, Dagmar; McKenna, Peter; Bahn, Sabine

    2008-10-01

    A mass spectrometry based high throughput approach was employed to profile white and gray matter lipid levels in the prefrontal cortex (Brodmann area 9) of 45 subjects including 15 schizophrenia and 15 bipolar disorder patients as well as 15 controls samples. We found statistically significant alterations in levels of free fatty acids and phosphatidylcholine in gray and white matter of both schizophrenia and bipolar disorder samples compared to controls. Also, ceramides were identified to be significantly increased in white matter of both neuropsychiatric disorders as compared to control levels. The patient cohort investigated in this study includes a number of drug naive as well as untreated patients, allowing the assessment of drug effects on lipid levels. Our findings indicate that while gray matter phosphatidylcholine levels were influenced by antipsychotic medication, this was not the case for phosphatidylcholine levels in white matter. Changes in free fatty acids or ceramides in either white or gray matter also did not appear to be influenced by antipsychotic treatment. To assess lipid profiles in the living patient, we also profiled lipids of 40 red blood cell samples, including 7 samples from drug naive first onset patients. We found significant alterations in the concentrations of free fatty acids as well as ceramide. Overall, our findings suggest that lipid abnormalities may be a disease intrinsic feature of both schizophrenia and bipolar disorder reflected by significant changes in the central nervous system as well as peripheral tissues.

  5. Volatile profiling reveals intracellular metabolic changes in Aspergillus parasiticus: veA regulates branched chain amino acid and ethanol metabolism

    PubMed Central

    2010-01-01

    Background Filamentous fungi in the genus Aspergillus produce a variety of natural products, including aflatoxin, the most potent naturally occurring carcinogen known. Aflatoxin biosynthesis, one of the most highly characterized secondary metabolic pathways, offers a model system to study secondary metabolism in eukaryotes. To control or customize biosynthesis of natural products we must understand how secondary metabolism integrates into the overall cellular metabolic network. By applying a metabolomics approach we analyzed volatile compounds synthesized by Aspergillus parasiticus in an attempt to define the association of secondary metabolism with other metabolic and cellular processes. Results Volatile compounds were examined using solid phase microextraction - gas chromatography/mass spectrometry. In the wild type strain Aspergillus parasiticus SU-1, the largest group of volatiles included compounds derived from catabolism of branched chain amino acids (leucine, isoleucine, and valine); we also identified alcohols, esters, aldehydes, and lipid-derived volatiles. The number and quantity of the volatiles produced depended on media composition, time of incubation, and light-dark status. A block in aflatoxin biosynthesis or disruption of the global regulator veA affected the volatile profile. In addition to its multiple functions in secondary metabolism and development, VeA negatively regulated catabolism of branched chain amino acids and synthesis of ethanol at the transcriptional level thus playing a role in controlling carbon flow within the cell. Finally, we demonstrated that volatiles generated by a veA disruption mutant are part of the complex regulatory machinery that mediates the effects of VeA on asexual conidiation and sclerotia formation. Conclusions 1) Volatile profiling provides a rapid, effective, and powerful approach to identify changes in intracellular metabolic networks in filamentous fungi. 2) VeA coordinates the biosynthesis of secondary

  6. Fens as Whole-Ecosystem Gauges of Groundwater Recharge Under Climate Change

    NASA Astrophysics Data System (ADS)

    Drexler, J. Z.; Knifong, D. L.; Tuil, J.; Flint, L. E.; Flint, A. L.

    2011-12-01

    Over the past century, mean air temperature has increased approximately 1 C in California. Since the 1950s, there has been an earlier onset of snowmelt and reduced snowpack (measured as snow water equivalent) in California as well as in much of the western United States. Because the snowpack is the main source of groundwater recharge in the mountainous west, reduced snowpack could result in decreased groundwater recharge through time. This could have important ramifications because groundwater recharge maintains groundwater springs, soil moisture, river baseflows and cool water temperatures. Reductions in groundwater recharge could not only impact water availability for human populations, but could also threaten long-term viability of ecosystems reliant on groundwater flows. Groundwater-fed peatlands called fens are potentially ideal ecosystems for studying changes in groundwater recharge, because they are groundwater discharge sites that rely upon corresponding recharge sites for their sustenance. When the amount of groundwater flow to a fen is reduced, the elevation of the water table decreases leading to desiccation, compaction and increased microbial oxidation of the organic soil. In cases where groundwater flow is reduced over an extended period of time, conversion of fen into wet or mesic meadow or even pine forest can occur resulting in contraction of fen boundaries. The purpose of this study is to evaluate the usefulness of fens as whole-ecosystem gauges of groundwater recharge. We studied 7 fens distributed throughout the Sierra Nevada by tracking their areas over 70+ years with aerial photography. All photos were geo-registered using the 2005 National Agricultural Inventory Program orthophotography as the base. Images were projected to UTM zone 10, NAD 83 using ArcGIS 10.0. Fen vegetation was identified in the aerial photos predominantly by (1) dark brownish-green coloring (or various shades of gray and black in black and white imagery) and (2) mottling

  7. Surface properties and intracellular speciation revealed an original adaptive mechanism to arsenic in the acid mine drainage bio-indicator Euglena mutabilis.

    PubMed

    Halter, David; Casiot, Corinne; Heipieper, Hermann J; Plewniak, Frédéric; Marchal, Marie; Simon, Stéphane; Arsène-Ploetze, Florence; Bertin, Philippe N

    2012-02-01

    Euglena mutabilis is a protist ubiquitously found in extreme environments such as acid mine drainages which are often rich in arsenic. The response of E. mutabilis to this metalloid was compared to that of Euglena gracilis, a protist not found in such environments. Membrane fatty acid composition, cell surface properties, arsenic accumulation kinetics, and intracellular arsenic speciation were determined. The results revealed a modification in fatty acid composition leading to an increased membrane fluidity in both Euglena species under sublethal arsenic concentrations exposure. This increased membrane fluidity correlated to an induced gliding motility observed in E. mutabilis in the presence of this metalloid but did not affect the flagellar dependent motility of E. gracilis. Moreover, when compared to E. gracilis, E. mutabilis showed highly hydrophobic cell surface properties and a higher tolerance to water-soluble arsenical compounds but not to hydrophobic ones. Finally, E. mutabilis showed a lower accumulation of total arsenic in the intracellular compartment and an absence of arsenic methylated species in contrast to E. gracilis. Taken together, our results revealed the existence of a specific arsenical response of E. mutabilis that may play a role in its hypertolerance to this toxic metalloid.

  8. Metabolic characteristics of dominant microbes and key rare species from an acidic hot spring in Taiwan revealed by metagenomics

    SciTech Connect

    Lin, Kuei -Han; Liao, Ben -Yang; Chang, Hao -Wei; Huang, Shiao -Wei; Chang, Ting -Yan; Yang, Cheng -Yu; Wang, Yu -Bin; Lin, Yu-Teh Kirk; Wu, Yu -Wei; Tang, Sen -Lin; Yu, Hon -Tsen

    2015-12-03

    Microbial diversity and community structures in acidic hot springs have been characterized by 16S rRNA gene-based diversity surveys. However, our understanding regarding the interactions among microbes, or between microbes and environmental factors, remains limited. In the present study, a metagenomic approach, followed by bioinformatics analyses, were used to predict interactions within the microbial ecosystem in Shi-Huang-Ping (SHP), an acidic hot spring in northern Taiwan. Characterizing environmental parameters and potential metabolic pathways highlighted the importance of carbon assimilatory pathways. Four distinct carbon assimilatory pathways were identified in five dominant genera of bacteria. Of those dominant carbon fixers, Hydrogenobaculum bacteria outcompeted other carbon assimilators and dominated the SHP, presumably due to their ability to metabolize hydrogen and to withstand an anaerobic environment with fluctuating temperatures. Furthermore, most dominant microbes were capable of metabolizing inorganic sulfur-related compounds (abundant in SHP). However, Acidithiobacillus ferrooxidans was the only species among key rare microbes with the capability to fix nitrogen, suggesting a key role in nitrogen cycling. In addition to potential metabolic interactions, based on the 16S rRNAs gene sequence of Nanoarchaeum-related and its potential host Ignicoccus-related archaea, as well as sequences of viruses and CRISPR arrays, we inferred that there were complex microbe-microbe interactions. In conclusion, our study provided evidence that there were numerous microbe-microbe and microbe-environment interactions within the microbial community in an acidic hot spring. We proposed that Hydrogenobaculum bacteria were the dominant microbial genus, as they were able to metabolize hydrogen, assimilate carbon and live in an anaerobic environment with fluctuating temperatures.

  9. Metabolic characteristics of dominant microbes and key rare species from an acidic hot spring in Taiwan revealed by metagenomics

    DOE PAGES

    Lin, Kuei -Han; Liao, Ben -Yang; Chang, Hao -Wei; ...

    2015-12-03

    Microbial diversity and community structures in acidic hot springs have been characterized by 16S rRNA gene-based diversity surveys. However, our understanding regarding the interactions among microbes, or between microbes and environmental factors, remains limited. In the present study, a metagenomic approach, followed by bioinformatics analyses, were used to predict interactions within the microbial ecosystem in Shi-Huang-Ping (SHP), an acidic hot spring in northern Taiwan. Characterizing environmental parameters and potential metabolic pathways highlighted the importance of carbon assimilatory pathways. Four distinct carbon assimilatory pathways were identified in five dominant genera of bacteria. Of those dominant carbon fixers, Hydrogenobaculum bacteria outcompeted othermore » carbon assimilators and dominated the SHP, presumably due to their ability to metabolize hydrogen and to withstand an anaerobic environment with fluctuating temperatures. Furthermore, most dominant microbes were capable of metabolizing inorganic sulfur-related compounds (abundant in SHP). However, Acidithiobacillus ferrooxidans was the only species among key rare microbes with the capability to fix nitrogen, suggesting a key role in nitrogen cycling. In addition to potential metabolic interactions, based on the 16S rRNAs gene sequence of Nanoarchaeum-related and its potential host Ignicoccus-related archaea, as well as sequences of viruses and CRISPR arrays, we inferred that there were complex microbe-microbe interactions. In conclusion, our study provided evidence that there were numerous microbe-microbe and microbe-environment interactions within the microbial community in an acidic hot spring. We proposed that Hydrogenobaculum bacteria were the dominant microbial genus, as they were able to metabolize hydrogen, assimilate carbon and live in an anaerobic environment with fluctuating temperatures.« less

  10. The Fat from Frozen Mammals Reveals Sources of Essential Fatty Acids Suitable for Palaeolithic and Neolithic Humans

    PubMed Central

    Guil-Guerrero, José L.; Tikhonov, Alexei; Rodríguez-García, Ignacio; Protopopov, Albert; Grigoriev, Semyon; Ramos-Bueno, Rebeca P.

    2014-01-01

    The elucidation of the sources of n-3 fatty acids available for the humans in the Upper Palaeolithic and Neolithic is highly relevant in order to ascertain the availability of such nutrients in that time frame as well as to draw useful conclusions about healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen mammals found in the permafrost of Siberia (Russia). A total of 6 specimens were included in this study: 2 mammoths, i.e. baby female calf called “Lyuba” and a juvenile female called “Yuka”, both specimens approximately from the same time, i.e. Karginian Interstadial (41,000 and 34,000 years BP); two adult horses from the middle Holocene (4,600 and 4,400 years BP); and two bison very close to the Early Holocene (8,200 and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC-flame ionization detector (GLC-FID). As demonstrated in this work, the fat of single-stomached mammals often consumed by Palaeolithic/Neolithic hunters contained suitable amounts of n-3 and n-6 fatty acids, possibly in quantities sufficient to meet the today's recommended daily intake for good health. Moreover, the results also suggest that mammoths and horses at that time were hibernators. PMID:24416235

  11. The fat from frozen mammals reveals sources of essential fatty acids suitable for Palaeolithic and Neolithic humans.

    PubMed

    Guil-Guerrero, José L; Tikhonov, Alexei; Rodríguez-García, Ignacio; Protopopov, Albert; Grigoriev, Semyon; Ramos-Bueno, Rebeca P

    2014-01-01

    The elucidation of the sources of n-3 fatty acids available for the humans in the Upper Palaeolithic and Neolithic is highly relevant in order to ascertain the availability of such nutrients in that time frame as well as to draw useful conclusions about healthy dietary habits for present-day humans. To this end, we have analysed fat from several frozen mammals found in the permafrost of Siberia (Russia). A total of 6 specimens were included in this study: 2 mammoths, i.e. baby female calf called "Lyuba" and a juvenile female called "Yuka", both specimens approximately from the same time, i.e. Karginian Interstadial (41,000 and 34,000 years BP); two adult horses from the middle Holocene (4,600 and 4,400 years BP); and two bison very close to the Early Holocene (8,200 and 9,300 years BP). All samples were analysed by gas-liquid chromatography-mass spectrometry (GLC-MS) and GLC-flame ionization detector (GLC-FID). As demonstrated in this work, the fat of single-stomached mammals often consumed by Palaeolithic/Neolithic hunters contained suitable amounts of n-3 and n-6 fatty acids, possibly in quantities sufficient to meet the today's recommended daily intake for good health. Moreover, the results also suggest that mammoths and horses at that time were hibernators.

  12. Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis.

    PubMed

    Valbuena, Gabriel N; Rizzardini, Milena; Cimini, Sara; Siskos, Alexandros P; Bendotti, Caterina; Cantoni, Lavinia; Keun, Hector C

    2016-05-01

    Defects in energy metabolism are potential pathogenic mechanisms in amyotrophic lateral sclerosis (ALS), a rapidly fatal disease with no cure. The mechanisms through which this occurs remain elusive and their understanding may prove therapeutically useful. We used metabolomics and stable isotope tracers to examine metabolic changes in a well-characterized cell model of familial ALS, the motor neuronal NSC-34 line stably expressing human wild-type Cu/Zn superoxide dismutase (wtSOD1) or mutant G93A (G93ASOD1). Our findings indicate that wt and G93ASOD1 expression both enhanced glucose metabolism under serum deprivation. However, in wtSOD1 cells, this phenotype increased supply of amino acids for protein and glutathione synthesis, while in G93ASOD1 cells it was associated with death, aerobic glycolysis, and a broad dysregulation of amino acid homeostasis. Aerobic glycolysis was mainly due to induction of pyruvate dehydrogenase kinase 1. Our study thus provides novel insight into the role of deranged energy metabolism as a cause of poor adaptation to stress and a promoter of neural cell damage in the presence of mutant SOD1. Furthermore, the metabolic alterations we report may help explain why mitochondrial dysfunction and impairment of the endoplasmic reticulum stress response are frequently seen in ALS.

  13. New insights into the diets of harbor seals in the Salish Sea revealed by quantitative fatty acid signature analysis

    USGS Publications Warehouse

    Bromaghin, Jeffrey F.; Lance, Monique M.; Elliott, Elizabeth W.; Jeffries, Steven J.; Acevedo-Gutiérrez, Alejandro; Kennish, John M.

    2012-01-01

    Harbor seals (Phoca vitulina) are an abundant predator along the west coast of North America, and there is considerable interest in their diet composition, especially in regard to predation on valued fish stocks. Available information on harbor seal diets, primarily derived from scat analysis, suggests that adult salmon (Oncorhynchus spp.), Pacific Herring (Clupea pallasii), and gadids predominate. Because diet assessments based on scat analysis may be biased, we investigated diet composition through quantitative analysis of fatty acid signatures. Blubber samples from 49 harbor seals captured in western North America from haul-outs within the area of the San Juan Islands and southern Strait of Georgia in the Salish Sea were analyzed for fatty acid composition, along with 269 fish and squid specimens representing 27 potential prey classes. Diet estimates varied spatially, demographically, and among individual harbor seals. Findings confirmed the prevalence of previously identified prey species in harbor seal diets, but other species also contributed significantly. In particular, Black (Sebastes melanops) and Yellowtail (S. flavidus) Rockfish were estimated to compose up to 50% of some individual seal diets. Specialization and high predation rates on Black and Yellowtail Rockfish by a subset of harbor seals may play a role in the population dynamics of these regional rockfish stocks that is greater than previously realized.

  14. 5-aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast.

    PubMed

    Naeem, Muhammad S; Warusawitharana, Hasitha; Liu, Hongbo; Liu, Dan; Ahmad, Rashid; Waraich, Ejaz Ahmad; Xu, Ling; Zhou, Weijun

    2012-08-01

    5-Aminolevulinic acid (ALA) is an important plant growth regulator which is derived from 5-carbon aliphatic amino acid. The present study investigates the interaction of increasing NaCl-salinity and ALA on plant growth, leaf pigment composition, leaf and root Na(+)/K(+) ratio and chloroplast ultrastructure in mesophyll cells of oilseed rape (Brassica napus) leaves. The plants were treated hydroponically with three different salinity levels (0, 100, 200 mM) and foliar application of ALA (30 mg l(-1)) simultaneously. Ten days after treatment, higher NaCl-salinity significantly reduced the plant biomass and height. However, ALA application restored the plant biomass and plant height under saline conditions. A concentration-dependent increase in Na(+) uptake was observed in the aerial parts of B. napus plants. On the other hand, ALA reduced Na(+) uptake, leading to a significant decrease in Na(+)/K(+) ratio. Accumulation of Na(+) augmented the oxidative stress, which was evident by electron microscopic images, highlighting several changes in cell shape and size, chloroplast swelling, increased number of plastogloubli, reduced starch granules and dilations of the thylakoids. Foliar application of ALA improved the energy supply and investment in mechanisms (higher chlorophyll and carotenoid contents, enhanced photosynthetic efficiency), reduced the oxidative stress as evident by the regular shaped chloroplasts with more intact thylakoids. On the basis of these results we can suggest that ALA is a promising plant growth regulator which can improve plant survival under salinity.

  15. Fatty acid and DNA analyses of Permian bacteria isolated from ancient salt crystals reveal differences with their modern relatives.

    PubMed

    Vreeland, Russell H; Rosenzweig, William D; Lowenstein, Tim; Satterfield, Cindy; Ventosa, Antonio

    2006-02-01

    The isolation of living microorganisms from primary 250-million-year-old (MYA) salt crystals has been questioned by several researchers. The most intense discussion has arisen from questions about the texture and age of the crystals used, the ability of organisms to survive 250 million years when exposed to environmental factors such as radiation and the close similarity between 16S rRNA sequences in the Permian and modern microbes. The data in this manuscript are not meant to provide support for the antiquity of the isolated bacterial strains. Rather, the data presents several comparisons between the Permian microbes and other isolates to which they appear related. The analyses include whole cell fatty acid profiling, DNA-DNA hybridizations, ribotyping, and random amplified polymorphic DNA amplification (RAPD). These data show that the Permian strains, studied here, differ significantly from their more modern relatives. These differences are accumulating in both phenotypic and molecular areas of the cells. At the fatty acid level the differences are approaching but have not reached separate species status. At the molecular level the variation appears to be distributed across the genome and within the gene regions flanking the highly conserved 16S rRNA itself. The data show that these bacteria are not identical and help to rule out questions of contamination by putatively modern strains.

  16. Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production.

    PubMed

    Zheng, Xiong; Su, Yinglong; Li, Xiang; Xiao, Naidong; Wang, Dongbo; Chen, Yinguang

    2013-05-07

    Short-chain fatty acids (SCFAs) have been regarded as the excellent carbon source of wastewater biological nutrient removal, and sludge alkaline (pH 10) fermentation has been reported to achieve highly efficient SCFAs production. In this study, the underlying mechanisms for the improved SCFAs production at pH 10 were investigated by using 454 pyrosequencing and fluorescent in situ hybridization (FISH) to analyze the microbial community structures in sludge fermentation reactors. It was found that sludge fermentation at pH 10 increased the abundances of Pseudomonas sp. and Alcaligenes sp., which were able to excrete extracellular proteases and depolymerases, and thus enhanced the hydrolysis of insoluble sludge protein and polyhydroxyalkanoates (PHA). Meanwhile, the abundance of acid-producing bacteria (such as Clostridium sp.) in the reactor of pH 10 was also higher than that of uncontrolled pH, which benefited the acidification of soluble organic substrates. Further study indicated that sludge fermentation at pH 10 significantly decreased the number of methanogenic archaea, resulting in lower SCFAs consumption and lower methane production. Therefore, anaerobic sludge fermentation under alkaline conditions increased the abundances of bacteria involved in sludge hydrolysis and acidification, and decreased the abundance of methanogenic archaea, which favored the competition of bacteria over methanogens and resulted in the efficient production of SCFAs.

  17. Holocene Climate Change and Ecohydrological Responses Preserved in Subalpine Fens in Colorado

    NASA Astrophysics Data System (ADS)

    Sullivan, D. G.; Gilmore, K. P.

    2012-12-01

    Stratigraphy and peat humification analysis of subalpine fens from a variety of sites in Colorado indicate that these peatlands preserve a unique Holocene record of climate change and ecohydrological feedback. Much of the research investigating the record of environmental change in peat archives focuses on high latitude or tropical bogs. Relatively little research has focused on midlatitude minerotrophic fens. However, a large number of fens occupy favorable sites in mountain environments. The conditions that favor peat development in the mountain west include the presence of springs fed by snowpack melting that extends into mid or late summer. This project has yielded a sensitive paleoenvironmental record extending through the Holocene. We were interested in determining how increases in summer temperatures early in the Holocene influenced precipitation, particularly summer monsoons, and thus, groundwater. A further question concerned the extent to which the fens preserve evidence of lower magnitude changes, including those associated with the Medieval Climate Anomaly (MCA) and the Little Ice Age (LIA). Due to the relatively marginal conditions that allow fens to persist in the region, fens are very sensitive to fluctuations in climate and consequent hydrological responses. Our results indicate that warmer conditions earlier in the Holocene led to earlier snowmelt, particularly at lower elevations. We have found that fens located near the lower margins of the subalpine zone (<3100 m elevation) ceased to accumulate peat and reverted to alluvial depositional environments during periods of warming earlier in the Holocene, and then returned to peat accumulation when cooling occurred. These results are similar to results of other researchers working in the Sierra Nevada, California. Colorado fens at higher elevation persisted as peatlands throughout the Holocene, but warmer periods produced changes in the rates of peat accumulation and the degree of humification during

  18. Investigation of a 6-MSA Synthase Gene Cluster in Aspergillus aculeatus Reveals 6-MSA-derived Aculinic Acid, Aculins A-B and Epi-Aculin A.

    PubMed

    Petersen, Lene M; Holm, Dorte K; Gotfredsen, Charlotte H; Mortensen, Uffe H; Larsen, Thomas O

    2015-10-12

    Aspergillus aculeatus, a filamentous fungus belonging to the Aspergillus clade Nigri, is an industrial workhorse in enzyme production. Recently we reported a number of secondary metabolites from this fungus; however, its genetic potential for the production of secondary metabolites is vast. In this study we identified a 6-methylsalicylic acid (6-MSA) synthase from A. aculeatus, and verified its functionality by episomal expression in A. aculeatus and heterologous expression in A. nidulans. Feeding studies with fully (13) C-labeled 6-MSA revealed that 6-MSA is incorporated into aculinic acid, which further incorporates into three compounds that we name aculins A and B, and epi-aculin A, described here for the first time. Based on NMR data and bioinformatic studies we propose the structures of the compounds as well as a biosynthetic pathway leading to formation of aculins from 6-MSA.

  19. Response of extraintestinal pathogenic Escherichia coli to human serum reveals a protective role for Rcs-regulated exopolysaccharide colanic acid.

    PubMed

    Miajlovic, Helen; Cooke, Niamh M; Moran, Gary P; Rogers, Thomas R F; Smith, Stephen G

    2014-01-01

    Extraintestinal Escherichia coli (ExPEC) organisms are the leading cause of Gram-negative bacterial bloodstream infections. These bacteria adapt to survival in the bloodstream through expression of factors involved in scavenging of nutrients and resisting the killing activity of serum. In this study, the transcriptional response of a prototypic ExPEC strain (CFT073) to human serum was investigated. Resistance of CFT073 to the bactericidal properties of serum involved increased expression of envelope stress regulators, including CpxR, σE, and RcsB. Many of the upregulated genes induced by active serum were regulated by the Rcs two-component system. This system is triggered by envelope stress such as changes to cell wall integrity. RcsB-mediated serum resistance was conferred through induction of the exopolysaccharide colanic acid. Production of this exopolysaccharide may be protective while cell wall damage caused by serum components is repaired.

  20. Yeast and bacterial diversity along a transect in an acidic, As-Fe rich environment revealed by cultural approaches.

    PubMed

    Delavat, François; Lett, Marie-Claire; Lièvremont, Didier

    2013-10-01

    Acid mine drainages (AMDs) are often thought to harbour low biodiversity, yet little is known about the diversity distribution along the drainages. Using culture-dependent approaches, the microbial diversity from the Carnoulès AMD sediment was investigated for the first time along a transect showing progressive environmental stringency decrease. In total, 20 bacterial genera were detected, highlighting a higher bacterial diversity than previously thought. Moreover, this approach led to the discovery of 16 yeast species, demonstrating for the first time the presence of this important phylogenetic group in this AMD. All in all, the location of the microbes along the transect helps to better understand their distribution in a pollution gradient.

  1. Folding study of Venus reveals a strong ion dependence of its yellow fluorescence under mildly acidic conditions.

    PubMed

    Hsu, Shang-Te Danny; Blaser, Georg; Behrens, Caroline; Cabrita, Lisa D; Dobson, Christopher M; Jackson, Sophie E

    2010-02-12

    Venus is a yellow fluorescent protein that has been developed for its fast chromophore maturation rate and bright yellow fluorescence that is relatively insensitive to changes in pH and ion concentrations. Here, we present a detailed study of the stability and folding of Venus in the pH range from 6.0 to 8.0 using chemical denaturants and a variety of spectroscopic probes. By following hydrogen-deuterium exchange of (15)N-labeled Venus using NMR spectroscopy over 13 months, residue-specific free energies of unfolding of some highly protected amide groups have been determined. Exchange rates of less than one per year are observed for some amide groups. A super-stable core is identified for Venus and compared with that previously reported for green fluorescent protein. These results are discussed in terms of the stability and folding of fluorescent proteins. Under mildly acidic conditions, we show that Venus undergoes a drastic decrease in yellow fluorescence at relatively low concentrations of guanidinium chloride. A detailed study of this effect establishes that it is due to pH-dependent, nonspecific interactions of ions with the protein. In contrast to previous studies on enhanced green fluorescence protein variant S65T/T203Y, which showed a specific halide ion-binding site, NMR chemical shift mapping shows no evidence for specific ion binding. Instead, chemical shift perturbations are observed for many residues primarily located in both lids of the beta-barrel structure, which suggests that small scale structural rearrangements occur on increasing ionic strength under mildly acidic conditions and that these are propagated to the chromophore resulting in fluorescence quenching.

  2. Dissociative Ionization Mechanism and Appearance Energies in Adipic Acid Revealed by Imaging Photoelectron Photoion Coincidence, Selective Deuteration, and Calculations.

    PubMed

    Heringa, Maarten F; Slowik, Jay G; Prévôt, André S H; Baltensperger, Urs; Hemberger, Patrick; Bodi, Andras

    2016-05-26

    Adipic acid, a model compound for oxygenated organic aerosol, has been studied at the VUV beamline of the Swiss Light Source. Internal energy selected cations were prepared by threshold photoionization using vacuum ultraviolet synchrotron radiation and imaging photoelectron photoion coincidence spectroscopy (iPEPICO). The threshold photoelectron spectrum yields a vertical ionization energy (IE) of 10.5 eV, significantly above the calculated adiabatic IE of 8.6 eV. The cationic minimum is accessible after vertical ionization by H-transfer from one of the γ-carbons to a carbonyl oxygen and is sufficiently energetic to decay by water loss at the ionization onset. The slope of the breakdown curves, quantum chemical calculations, and selective deuteration of the carboxylic hydrogens establish the dissociative photoionization mechanism. After ionization, one γ-methylene hydrogen and the two carboxylic hydrogens are randomized prior to H2O loss. On the basis of the deuteration degree in the H2O + CO-loss product at higher energies, a direct water-loss channel without complete randomization also exists. The breakdown diagram and center of gravity of the H2O + CO-loss peak were modeled to obtain 0 K appearance energies of 10.77, 10.32, and 11.53 eV for H2O + CO loss, CH2COOH loss, and H2O + CH2COOH loss from adipic acid. These agree well with the CBS-QB3 calculated values of 10.68, 10.45, and 11.57 eV, respectively, which shows that threshold photoionization can yield energetics data as long as the dissociation is statistical, even when the parent ion cannot be observed. The results can be used as a starting point for a deeper understanding of the ionization and low-energy fragmentation of organic aerosol components.

  3. Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation

    PubMed Central

    Bhati, Kaushal Kumar; Alok, Anshu; Kumar, Anil; Kaur, Jagdeep; Tiwari, Siddharth; Pandey, Ajay Kumar

    2016-01-01

    Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34–22% reduction of the phytic acid content in the mature grains (T4 seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals. PMID:27342224

  4. Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation.

    PubMed

    Bhati, Kaushal Kumar; Alok, Anshu; Kumar, Anil; Kaur, Jagdeep; Tiwari, Siddharth; Pandey, Ajay Kumar

    2016-07-01

    Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34-22% reduction of the phytic acid content in the mature grains (T4 seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals.

  5. The FEN1 L209P mutation interferes with long-patch base excision repair and induces cellular transformation

    PubMed Central

    Sun, H; He, L; Wu, H; Pan, F; Wu, X; Zhao, J; Hu, Z; Sekhar, C; Li, H; Zheng, L; Chen, H; Shen, B H; Guo, Z

    2017-01-01

    Flap endonuclease-1 (FEN1) is a multifunctional, structure-specific nuclease that has a critical role in maintaining human genome stability. FEN1 mutations have been detected in human cancer specimens and have been suggested to cause genomic instability and cancer predisposition. However, the exact relationship between FEN1 deficiency and cancer susceptibility remains unclear. In the current work, we report a novel colorectal cancer-associated FEN1 mutation, L209P. This mutant protein lacks the FEN, exonuclease (EXO) and gap endonuclease (GEN) activities of FEN1 but retains DNA-binding affinity. The L209P FEN1 variant interferes with the function of the wild-type FEN1 enzyme in a dominant-negative manner and impairs long-patch base excision repair in vitro and in vivo. Expression of L209P FEN1 sensitizes cells to DNA damage, resulting in endogenous genomic instability and cellular transformation, as well as tumor growth in a mouse xenograft model. These data indicate that human cancer-associated genetic alterations in the FEN1 gene can contribute substantially to cancer development. PMID:27270424

  6. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    PubMed Central

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  7. Protein Conformation Ensembles Monitored by HDX Reveal a Structural Rationale for Abscisic Acid Signaling Protein Affinities and Activities

    PubMed Central

    West, Graham M.; Pascal, Bruce D.; Ng, Ley-Moy; Soon, Fen-Fen; Melcher, Karsten; Xu, H. Eric; Chalmers, Michael J.; Griffin, Patrick R.

    2012-01-01

    Summary Plants regulate growth and respond to environmental stress through abscisic acid (ABA) regulated pathways, and as such these pathways are of primary interest for biological and agricultural research. The ABA response is first perceived by the PYR/PYL/RCAR class of START protein receptors. These ABA activated receptors disrupt phosphatase inhibition of Snf1-related kinases (SnRKs) enabling kinase signaling. Here, insights into the structural mechanism of proteins in the ABA signaling pathway (the ABA receptor PYL2, HAB1 phosphatase, and two kinases, SnRK2.3 and 2.6) are discerned through hydrogen/deuterium exchange (HDX) mass spectrometry. HDX on the phosphatase in the presence of binding partners provides evidence for receptor-specific conformations involving the Trp385 ‘lock’ that is necessary for signaling. Furthermore, kinase activity is linked to a more stable closed conformation. These solution-based studies complement the static crystal structures and provide a more detailed understanding of the ABA signaling pathway. PMID:23290725

  8. C-terminomics Screen for Natural Substrates of Cytosolic Carboxypeptidase 1 Reveals Processing of Acidic Protein C termini*

    PubMed Central

    Tanco, Sebastian; Tort, Olivia; Demol, Hans; Aviles, Francesc Xavier; Gevaert, Kris; Van Damme, Petra; Lorenzo, Julia

    2015-01-01

    Cytosolic carboxypeptidases (CCPs) constitute a new subfamily of M14 metallocarboxypeptidases associated to axonal regeneration and neuronal degeneration, among others. CCPs are deglutamylating enzymes, able to catalyze the shortening of polyglutamate side-chains and the gene-encoded C termini of tubulin, telokin, and myosin light chain kinase. The functions of these enzymes are not entirely understood, in part because of the lack of information about C-terminal protein processing in the cell and its functional implications. By means of C-terminal COFRADIC, a positional proteomics approach, we searched for cellular substrates targets of CCP1, the most relevant member of this family. We here identified seven new putative CCP1 protein substrates, including ribosomal proteins, translation factors, and high mobility group proteins. Furthermore, we showed for the first time that CCP1 processes both glutamates as well as C-terminal aspartates. The implication of these C termini in molecular interactions furthermore suggests that CCP1-mediated shortening of acidic protein tails might regulate protein–protein and protein–DNA interactions. PMID:25381060

  9. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    NASA Astrophysics Data System (ADS)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

  10. Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome

    PubMed Central

    Sarma, Kavitha; Levasseur, Pierre; Aristarkhov, Alexander; Lee, Jeannie T.

    2010-01-01

    A large fraction of the mammalian genome is transcribed into long noncoding RNAs. The RNAs remain largely uncharacterized as the field awaits new technologies to aid functional analysis. Here, we describe a unique use of locked nucleic acids (LNAs) for studying nuclear long noncoding RNA, an RNA subclass that has been less amenable to traditional knockdown techniques. We target LNAs at Xist RNA and show displacement from the X chromosome with fast kinetics. Xist transcript stability is not affected. By targeting different Xist regions, we identify a localization domain and show that polycomb repressive complex 2 (PRC2) is displaced together with Xist. Thus, PRC2 depends on RNA for both initial targeting to and stable association with chromatin. H3K27-trimethyl marks and gene silencing remain stable. Time-course analysis of RNA relocalization suggests that Xist and PRC2 bind to different regions of the X at the same time but do not reach saturating levels immediately. Thus, LNAs provide a tool for studying an emerging class of regulatory RNA and offer a window of opportunity to target epigenetic modifications with possible therapeutic applications. PMID:21135235

  11. Proteomic analysis of mouse testis reveals perfluorooctanoic acid-induced reproductive dysfunction via direct disturbance of testicular steroidogenic machinery.

    PubMed

    Zhang, Hongxia; Lu, Yin; Luo, Bin; Yan, Shengmin; Guo, Xuejiang; Dai, Jiayin

    2014-07-03

    Perfluorooctanoic acid (PFOA) is a ubiquitous environmental pollutant suspected of being an endocrine disruptor; however, mechanisms of male reproductive disorders induced by PFOA are poorly understood. In this study, male mice were exposed to 0, 0.31, 1.25, 5, and 20 mg PFOA/kg/day by oral gavage for 28 days. PFOA significantly damaged the seminiferous tubules and reduced testosterone and progesterone levels in the testis in a dose-dependent manner. Furthermore, PFOA exposure reduced sperm quality. We identified 93 differentially expressed proteins between the control and the 5 mg/kg/d PFOA treated mice using a quantitative proteomic approach. Among them, insulin like-factor 3 (INSL3) and cytochrome P450 cholesterol side-chain cleavage enzyme (CYP11A1) as Leydig-cell-specific markers were significantly decreased. We examined in detail the expression patterns of CYP11A1 and associated genes involved in steroidogenesis in the mouse testis. PFOA inhibited the mRNA and protein levels of CYP11A1 and the mRNA levels of 17β-hydroxysteroid dehydrogenase (17β-HSD) in a dose-dependent manner. Moreover, in vitro study showed the reduction in progesterone levels was accompanied by decreased expression of CYP11A1 in cAMP-stimulated mLTC-1 cells. Our findings indicate that PFOA exposure can impair male reproductive function, possibly by disturbing testosterone levels, and CPY11A1 may be a major steroidogenic enzyme targeted by PFOA.

  12. Genome-wide siRNA screen reveals amino acid starvation-induced autophagy requires SCOC and WAC

    PubMed Central

    McKnight, Nicole C; Jefferies, Harold B J; Alemu, Endalkachew A; Saunders, Rebecca E; Howell, Michael; Johansen, Terje; Tooze, Sharon A

    2012-01-01

    Autophagy is a catabolic process by which cytoplasmic components are sequestered and transported by autophagosomes to lysosomes for degradation, enabling recycling of these components and providing cells with amino acids during starvation. It is a highly regulated process and its deregulation contributes to multiple diseases. Despite its importance in cell homeostasis, autophagy is not fully understood. To find new proteins that modulate starvation-induced autophagy, we performed a genome-wide siRNA screen in a stable human cell line expressing GFP–LC3, the marker-protein for autophagosomes. Using stringent validation criteria, our screen identified nine novel autophagy regulators. Among the hits required for autophagosome formation are SCOC (short coiled-coil protein), a Golgi protein, which interacts with fasciculation and elongation protein zeta 1 (FEZ1), an ULK1-binding protein. SCOC forms a starvation-sensitive trimeric complex with UVRAG (UV radiation resistance associated gene) and FEZ1 and may regulate ULK1 and Beclin 1 complex activities. A second candidate WAC is required for starvation-induced autophagy but also acts as a potential negative regulator of the ubiquitin-proteasome system. The identification of these novel regulatory proteins with diverse functions in autophagy contributes towards a fuller understanding of autophagosome formation. PMID:22354037

  13. Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef

    PubMed Central

    Matheson, Nicholas J.; Sumner, Jonathan; Wals, Kim; Rapiteanu, Radu; Weekes, Michael P.; Vigan, Raphael; Weinelt, Julia; Schindler, Michael; Antrobus, Robin; Costa, Ana S.H.; Frezza, Christian; Clish, Clary B.; Neil, Stuart J.D.; Lehner, Paul J.

    2015-01-01

    Summary Critical cell surface immunoreceptors downregulated during HIV infection have previously been identified using non-systematic, candidate approaches. To gain a comprehensive, unbiased overview of how HIV infection remodels the T cell surface, we took a distinct, systems-level, quantitative proteomic approach. >100 plasma membrane proteins, many without characterized immune functions, were downregulated during HIV infection. Host factors targeted by the viral accessory proteins Vpu or Nef included the amino acid transporter SNAT1 and the serine carriers SERINC3/5. We focused on SNAT1, a β-TrCP-dependent Vpu substrate. SNAT1 antagonism was acquired by Vpu variants from the lineage of SIVcpz/HIV-1 viruses responsible for pandemic AIDS. We found marked SNAT1 induction in activated primary human CD4+ T cells, and used Consumption and Release (CoRe) metabolomics to identify alanine as an endogenous SNAT1 substrate required for T cell mitogenesis. Downregulation of SNAT1 therefore defines a unique paradigm of HIV interference with immunometabolism. PMID:26439863

  14. Group A streptococcus cell-associated pathogenic proteins as revealed by growth in hyaluronic acid-enriched media.

    PubMed

    Zhang, Meng; McDonald, Fiona M; Sturrock, Shane S; Charnock, Simon J; Humphery-Smith, Ian; Black, Gary W

    2007-05-01

    Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.

  15. Transcriptome profiling and physiological studies reveal a major role for aromatic amino acids in mercury stress tolerance in rice seedlings.

    PubMed

    Chen, Yun-An; Chi, Wen-Chang; Trinh, Ngoc Nam; Huang, Li-Yao; Chen, Ying-Chih; Cheng, Kai-Teng; Huang, Tsai-Lien; Lin, Chung-Yi; Huang, Hao-Jen

    2014-01-01

    Mercury (Hg) is a serious environmental pollution threat to the planet. The accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. To gain more insight into the cellular response to Hg, we performed a large-scale analysis of the rice transcriptome during Hg stress. Genes induced with short-term exposure represented functional categories of cell-wall formation, chemical detoxification, secondary metabolism, signal transduction and abiotic stress response. Moreover, Hg stress upregulated several genes involved in aromatic amino acids (Phe and Trp) and increased the level of free Phe and Trp content. Exogenous application of Phe and Trp to rice roots enhanced tolerance to Hg and effectively reduced Hg-induced production of reactive oxygen species. Hg induced calcium accumulation and activated mitogen-activated protein kinase. Further characterization of the Hg-responsive genes we identified may be helpful for better understanding the mechanisms of Hg in plants.

  16. Ecological niche of Neanderthals from Spy Cave revealed by nitrogen isotopes of individual amino acids in collagen.

    PubMed

    Naito, Yuichi I; Chikaraishi, Yoshito; Drucker, Dorothée G; Ohkouchi, Naohiko; Semal, Patrick; Wißing, Christoph; Bocherens, Hervé

    2016-04-01

    This study provides a refined view on the diet and ecological niche of Neanderthals. The traditional view is that Neanderthals obtained most of their dietary protein from terrestrial animals, especially from large herbivores that roamed the open landscapes. Evidence based on the conventional carbon and nitrogen isotopic composition of bulk collagen has supported this view, although recent findings based on plant remains in the tooth calculus, microwear analyses, and small game and marine animal remains from archaeological sites have raised some questions regarding this assumption. However, the lack of a protein source other than meat in the Neanderthal diet may be due to methodological difficulties in defining the isotopic composition of plants. Based on the nitrogen isotopic composition of glutamic acid and phenylalanine in collagen for Neanderthals from Spy Cave (Belgium), we show that i) there was an inter-individual dietary heterogeneity even within one archaeological site that has not been evident in bulk collagen isotopic compositions, ii) they occupied an ecological niche different from those of hyenas, and iii) they could rely on plants for up to ∼20% of their protein source. These results are consistent with the evidence found of plant consumption by the Spy Neanderthals, suggesting a broader subsistence strategy than previously considered.

  17. BOREAS TF-10 NSA-Fen Tower Flux and Meteorological Data

    NASA Technical Reports Server (NTRS)

    McCaughey, J. Harry; Hall, Forrest G. (Editor); Huemmrich, Karl (Editor); Jelinski, Dennis E.

    2000-01-01

    The BOREAS TF-10 team collected tower flux and meteorological data at two sites, a fen and a young jack pine forest, near Thompson, Manitoba, Canada, as part of BOREAS. A preliminary data set was assembled in August 1993 while field testing the instrument packages, and at both sites data were collected from 15-Aug to 31-Aug. The main experimental period was in 1994, when continuous data were collected from 08-Apr to 23-Sep at the fen site. A very limited experiment was run in the spring/ summer of 1995, when the fen site tower was operated from 08-Apr to 14-Jun in support of a hydrology experiment in an adjoining feeder basin. Upon examination of the 1994 data set, it became clear that the behavior of the heat, water, and carbon dioxide fluxes throughout the whole growing season was an important scientific question, and that the 1994 data record was not sufficiently long to capture the character of the seasonal behavior of the fluxes. Thus, the fen site was operated in 1996 in order to collect data from spring melt to autumn freeze-up. Data were collected from 29-Apr to 05-Nov at the fen site. All variables are presented as 30-minute averages. The data are stored in tabular ASCII files.

  18. Diversity of trypsins in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae), revealed by nucleic acid sequences and enzyme purification.

    PubMed

    Díaz-Mendoza, M; Ortego, F; García de Lacoba, M; Magaña, C; de la Poza, M; Farinós, G P; Castañera, P; Hernández-Crespo, P

    2005-09-01

    The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the

  19. Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945.

    PubMed

    Mitsunaga, Hitoshi; Meissner, Lena; Palmen, Thomas; Bamba, Takeshi; Büchs, Jochen; Fukusaki, Eiichiro

    2016-04-01

    Poly(γ-glutamic acid) (PGA) is a polymer composed of L- and/or D-glutamic acids that is produced by Bacillus sp. Because the polymer has various features as water soluble, edible, non-toxic and so on, it has attracted attention as a candidate for many applications such as foods, cosmetics and so on. However, although it is well known that the intracellular metabolism of Bacillus sp. is mainly regulated by catabolite control, the effect of the catabolite control on the PGA producing Bacillus sp. is largely unknown. This study is the first report of metabolome analysis on the PGA producing Bacillus sp. that reveals the effect of carbon catabolite control on the metabolism of PGA producing Bacillus licheniformis ATCC 9945. Results showed that the cells cultivated in glycerol-containing medium showed higher PGA production than the cells in glucose-containing medium. Furthermore, metabolome analysis revealed that the activators of CcpA and CodY, global regulatory proteins of the intracellular metabolism, accumulated in the cells cultivated in glycerol-containing and glucose-containing medium, respectively, with CodY apparently inhibiting PGA production. Moreover, the cells seemed to produce glutamate from citrate and ammonium using glutamine synthetase/glutamate synthase. Pulsed addition of di-ammonium hydrogen citrate, as suggested by the metabolome result, was able to achieve the highest value so far for PGA production in B. licheniformis.

  20. Transcriptional profiling of Zea mays roots reveals roles for jasmonic acid and terpenoids in resistance against Phytophthora cinnamomi.

    PubMed

    Allardyce, Jane Alisa; Rookes, James Edward; Hussain, Hashmath Inayath; Cahill, David Miles

    2013-06-01

    Phytophthora cinnamomi is a soil-borne plant pathogen that has caused widespread damage to vulnerable native ecosystems and agriculture systems across the world and shows no sign of abating. Management of the pathogen in the natural environment is difficult and the options are limited. In order to discover more about how resistant plants are able to defend themselves against this generalist pathogen, a microarray study of plant gene expression following root inoculation with P. cinnamomi was undertaken. Zea mays was used as a resistant model plant, and microarray analysis was conducted using the Affymetrix GeneChip Maize Genome Array on root samples collected at 6- and 24-h post-inoculation. Over 300 genes were differentially expressed in inoculated roots compared with controls across the two time points. Following Gene Ontology enrichment analysis and REVIGO visualisation of the up-regulated genes, many were implicated in plant defence responses to biotic stress. Genes that were up-regulated included those involved in phytoalexin biosynthesis and jasmonic acid/ethylene biosynthesis and other defence-related genes including those encoding glutathione S-transferases and serine-protease inhibitors. Of particular interest was the identification of the two most highly up-regulated genes, terpene synthase11 (Tps11) and kaurene synthase2 (An2), which are both involved in production of terpenoid phytoalexins. This is the first study that has investigated gene expression at a global level in roots in response to P. cinnamomi in a model plant species and provides valuable insights into the mechanisms involved in defence.

  1. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

    PubMed

    Mann, Paul A; Müller, Anna; Wolff, Kerstin A; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J; Pinho, Mariana G; Schneider, Tanja; Roemer, Terry

    2016-05-01

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

  2. Aspartic Acid Racemization and Collagen Degradation Markers Reveal an Accumulation of Damage in Tendon Collagen That Is Enhanced with Aging*

    PubMed Central

    Thorpe, Chavaunne T.; Streeter, Ian; Pinchbeck, Gina L.; Goodship, Allen E.; Clegg, Peter D.; Birch, Helen L.

    2010-01-01

    Little is known about the rate at which protein turnover occurs in living tendon and whether the rate differs between tendons with different physiological roles. In this study, we have quantified the racemization of aspartic acid to calculate the age of the collagenous and non-collagenous components of the high strain injury-prone superficial digital flexor tendon (SDFT) and low strain rarely injured common digital extensor tendon (CDET) in a group of horses with a wide age range. In addition, the turnover of collagen was assessed indirectly by measuring the levels of collagen degradation markers (collagenase-generated neoepitope and cross-linked telopeptide of type I collagen). The fractional increase in d-Asp was similar (p = 0.7) in the SDFT (5.87 × 10−4/year) and CDET (5.82 × 10−4/year) tissue, and d/l-Asp ratios showed a good correlation with pentosidine levels. We calculated a mean (±S.E.) collagen half-life of 197.53 (±18.23) years for the SDFT, which increased significantly with horse age (p = 0.03) and was significantly (p < 0.001) higher than that for the CDET (34.03 (±3.39) years). Using similar calculations, the half-life of non-collagenous protein was 2.18 (±0.41) years in the SDFT and was significantly (p = 0.04) lower than the value of 3.51 (±0.51) years for the CDET. Collagen degradation markers were higher in the CDET and suggested an accumulation of partially degraded collagen within the matrix with aging in the SDFT. We propose that increased susceptibility to injury in older individuals results from an inability to remove partially degraded collagen from the matrix leading to reduced mechanical competence. PMID:20308077

  3. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action

    PubMed Central

    Wang, Pengcheng; Xue, Liang; Batelli, Giorgia; Lee, Shinyoung; Hou, Yueh-Ju; Van Oosten, Michael J.; Zhang, Huiming; Tao, W. Andy; Zhu, Jian-Kang

    2013-01-01

    Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. PMID:23776212

  4. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

    PubMed Central

    Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E.; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R.; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J.; Pinho, Mariana G.; Schneider, Tanja; Roemer, Terry

    2016-01-01

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation. PMID:27144276

  5. Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin.

    PubMed

    Lu, Bangmin; Zhang, Bin; Qi, Wei; Zhu, Yanan; Zhao, Yan; Zhou, Nan; Sun, Rong; Bao, Jinku; Wu, Chuanfang

    2014-11-01

    Clematis montana lectin (CML), a novel mannose-binding lectin purified from C. montana Buch.-Ham stem (Ranunculaceae), has been proved to have hemagglutinating activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline resistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCl, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan (Trp) microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine (Arg) residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML conformation. Furthermore, the Trp, Arg, and sulfhydryl-modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form substrate-accessible conformation and suitable environment for carbohydrate binding.

  6. The glycerophospho-metabolome and its influence on amino acid homeostasis revealed by brain metabolomics of GDE1(-/-) mice

    PubMed Central

    Kopp, Florian; Komatsu, Toru; Nomura, Daniel K.; Trauger, Sunia A.; Thomas, Jason R.; Siuzdak, Gary; Simon, Gabriel M.; Cravatt, Benjamin F.

    2010-01-01

    GDE1 is a mammalian glycerophosphodiesterase (GDE) implicated by in vitro studies in the regulation of glycerophopho-inositol (GroPIns) and possibly other glycerophospho (GroP) metabolites. Here, we show using untargeted metabolomics that GroPIns is profoundly (> 20-fold) elevated in brain tissue from GDE1(-/-) mice. Furthermore, two additional GroP-metabolites not previously identified in eukaryotic cells, glycerophospho-serine (GroPSer) and glycerophospho-glycerate (GroPGate), were also highly elevated in GDE1(-/-) brains. Enzyme assays with synthetic GroP-metabolites confirmed that GroPSer and GroPGate are direct substrates of GDE1. Interestingly, our metabolomic profiles also revealed that serine (both L-and D-) levels were significantly reduced in brains of GDE1 (-/-) mice. These findings designate GroPSer as a previously unappreciated reservoir for free serine in the nervous system and suggest that GDE1, through recycling serine from GroPSer, may impact D-serine-dependent neural signaling processes in vivo. PMID:20797612

  7. Systematic analysis of potato acid invertase genes reveals that a cold-responsive member, StvacINV1, regulates cold-induced sweetening of tubers.

    PubMed

    Liu, Xun; Zhang, Chi; Ou, Yongbin; Lin, Yuan; Song, Botao; Xie, Conghua; Liu, Jun; Li, Xiu-Qing

    2011-08-01

    Acid invertase is believed to play a regulatory role during plant developmental processes and to respond to environmental stimuli. The expression profiles of the entire acid invertase family are not yet available for potato. By searching existing databases, it was determined that there are at least six acid invertase genes in potato, including four cell-wall invertase genes and two vacuolar invertase genes. They were subjected to comparative expression profiling in various organs of potato plants and in stored tubers to exploit their potential functions. The results revealed that each gene exhibited a unique expression pattern, which differed in transcript abundance or showed organ-specific features, pointing to the possible involvement of individual genes in plant development. The vacuolar invertase gene StvacINV1 had the highest expression level among three genes detected in the potato tubers. Further storage experiments showed that StvacINV1 was strongly induced by low temperatures, which is consistent with glucose accumulation in cold-stored tubers. Suppression of StvacINV1 by the antisense transformation in potato confirmed that lower StvacINV1 transcript abundance in transgenic tubers is related to lower reducing sugar content and lighter chip color in comparison with the wild type. The evidence strongly suggests that StvacINV1 is a gene involved in regulation of cold-induced sweetening of potato tubers. This provides an avenue for studying the mechanism involved in the regulation of the cold-induced sweetening trait and for agronomic enhancement.

  8. Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism

    PubMed Central

    Rohde, Antje; Morreel, Kris; Ralph, John; Goeminne, Geert; Hostyn, Vanessa; De Rycke, Riet; Kushnir, Sergej; Van Doorsselaere, Jan; Joseleau, Jean-Paul; Vuylsteke, Marnik; Van Driessche, Gonzalez; Van Beeumen, Jozef; Messens, Eric; Boerjan, Wout

    2004-01-01

    The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids. PMID:15377757

  9. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis.

    PubMed

    Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

    2016-12-01

    Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS.

  10. Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis

    PubMed Central

    Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

    2016-01-01

    Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS. PMID:27874013

  11. Long-term behavior of groundwater chemistry in a periodically rewetted fen area covered with macrophytes.

    PubMed

    Maassen, Sebastian; Balla, Dagmar; Dannowski, Ralf

    2015-03-01

    We studied the long-term changes in groundwater composition in the context of peat restoration at a degraded water-table managed peatland site typical for many agriculturally used fen areas in the northern hemisphere. At the study site, peatland rewetting with groundwater control and pumped canal water was carried out in two periods: from 1997 to 2002 and from 2011 to 2013. The site was not managed between 2002 and 2011, which led to an unstable groundwater table that had declined in part. The aim of this study was to investigate the consequences of rewetting and desiccation on groundwater chemistry. We pursued a multivariate approach using nonlinear principal component analysis (Isomap) to identify the prevailing processes that control the groundwater quality in this system. Sixteen years after peatland restoration, the groundwater quality had significantly improved. Principal component analysis revealed that hydrological processes had a major impact on groundwater quality, i.e. fluctuations between upwelling of local, salt-influenced groundwater and downwelling of surface and rainwater (first principal component) as well as upwelling of regional groundwater from deeper layers (second principal component) which originated from the catchment. In particular, the upwelling of regional deep groundwater had a strong positive impact on the groundwater quality of upper layers at the Biesenbrow site. Another major impact on groundwater quality was nutrient withdrawal by macrophytes and incorporation into organic matter. In the upper groundwater layer, peat mineralization processes resulted in substantially increased SO4 concentrations. We concluded that potential matter release after rewetting is buffered by hydrological barriers, and seems to be marginal with little impact on adjacent environments in the long term. The ecosystem is sustainably stabilized, and therefore has no negative impact on groundwater quality during periods of water shortage. Due to the strong

  12. Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid*

    PubMed Central

    Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

    2016-01-01

    Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51–64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic. PMID:26499837

  13. Proteomic Analysis Revealed the Important Role of Vimentin in Human Cervical Carcinoma HeLa Cells Treated With Gambogic Acid.

    PubMed

    Yue, Qingxi; Feng, Lixing; Cao, Biyin; Liu, Miao; Zhang, Dongmei; Wu, Wanying; Jiang, Baohong; Yang, Min; Liu, Xuan; Guo, Dean

    2016-01-01

    Gambogic acid (GA) is an anticancer agent in phase IIb clinical trial in China. In HeLa cells, GA inhibited cell proliferation, induced cell cycle arrest at G2/M phase and apoptosis, as showed by results of MTT assay and flow cytometric analysis. Possible target-related proteins of GA were searched using comparative proteomic analysis (2-DE) and nine proteins at early (3 h) stage together with nine proteins at late (24 h) stage were found. Vimentin was the only target-related protein found at both early and late stage. Results of both 2-DE analysis and Western blotting assay suggested cleavage of vimentin induced by GA. MS/MS analysis of cleaved vimentin peptides indicated possible cleavage sites of vimentin at or near ser51 and glu425. Results of targeted proteomic analysis showed that GA induced change in phosphorylation state of the vimentin head domain (aa51-64). Caspase inhibitors could not abrogate GA-induced cleavage of vimentin. Over-expression of vimentin ameliorated cytotoxicity of GA in HeLa cells. The GA-activated signal transduction, from p38 MAPK, heat shock protein 27 (HSP27), vimentin, dysfunction of cytoskeleton, to cell death, was predicted and then confirmed. Results of animal study showed that GA treatment inhibited tumor growth in HeLa tumor-bearing mice and cleavage of vimentin could be observed in tumor xenografts of GA-treated animals. Results of immunohistochemical staining also showed down-regulated vimentin level in tumor xenografts of GA-treated animals. Furthermore, compared with cytotoxicity of GA in HeLa cells, cytotoxicity of GA in MCF-7 cells with low level of vimentin was weaker whereas cytotoxicity of GA in MG-63 cells with high level of vimentin was stronger. These results indicated the important role of vimentin in the cytotoxicity of GA. The effects of GA on vimentin and other epithelial-to-mesenchymal transition (EMT) markers provided suggestion for better usage of GA in clinic.

  14. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

    SciTech Connect

    Cornish, Adam J.; Ginovska, Bojana; Thelen, Adam; da Silva, Julio C. S.; Soares, Thereza A.; Raugei, Simone; Dupuis, Michel; Shaw, Wendy J.; Hegg, Eric L.

    2016-06-07

    The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a modeled water molecule. Recently, a computational analysis of this pathway revealed that the solvent-exposed residue of the pathway (Glu282) could form hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implicating that these residues could function in regulating proton transfer. Substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in a 2.5-fold enhancement in H2 production activity, suggesting that Arg286 serves an important role in controlling the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate approximately 5-fold, implying that it either acts as a member of the pathway or influences Glu282 to enable proton transfer. Interestingly, QM/MM and molecular dynamics calculations indicate that Ser320 does not play an electronic or structural role. QM calculations also estimate that including Ser320 in the pathway does not significantly change the barrier to proton movement, providing further support for its role as a member of the proton pathway. While further studies are needed to quantify the role of Ser320, collectively, these data provide evidence that the enzyme scaffold plays a significant role in modulating the activity of the enzyme, demonstrating that the rate of intraprotein proton transfer can be accelerated, particularly in a non-biological context. This work was supported by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science, DE-FC02-07ER64494). In addition, support from the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences (WJS, BGP, SR) is gratefully acknowledged. Computational resources were provided at W. R. Wiley Environmental Molecular Science Laboratory (EMSL), a national scientific user facility sponsored by the Department of Energy’s Office of

  15. X-ray structure of Pur-alpha reveals a Whirly-like fold and an unusual nucleic-acid binding surface.

    PubMed

    Graebsch, Almut; Roche, Stéphane; Niessing, Dierk

    2009-11-03

    The PUR protein family is a distinct and highly conserved class that is characterized by its sequence-specific RNA- and DNA-binding. Its best-studied family member, Pur-alpha, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-alpha-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-alpha is mediated by its central core region, for which no structural information is available. We determined the x-ray structure of residues 40 to 185 from Drosophila melanogaster Pur-alpha, which constitutes a major part of the core region. We found that this region contains two almost identical structural motifs, termed "PUR repeats," which interact with each other to form a PUR domain. DNA- and RNA-binding studies confirmed that PUR domains are indeed functional nucleic-acid binding domains. Database analysis show that PUR domains share a fold with the Whirly class of nucleic-acid binding proteins. Structural analysis combined with mutational studies suggest that a PUR domain binds nucleic acids through two independent surface regions involving concave beta-sheets. Structure-based sequence alignment revealed that the core region harbors a third PUR repeat at its C terminus. Subsequent characterization by small-angle x-ray scattering (SAXS) and size-exclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-alpha. Surface envelopes calculated from SAXS data show that the Pur-alpha dimer consisting of repeats I to III is arranged in a Z-like shape. This unexpected domain organization of the entire core domain of Pur-alpha has direct implications for ssDNA/ssRNA and dsDNA binding.

  16. High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues*

    PubMed Central

    Szabó, András; Héja, Dávid; Szakács, Dávid; Zboray, Katalin; Kékesi, Katalin A.; Radisky, Evette S.; Sahin-Tóth, Miklós; Pál, Gábor

    2011-01-01

    Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2′ (Leu or Asp), and P4′ (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2′ (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4′ (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (KD 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a KD of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys51, Arg56, and Arg80) on the surface of human CTRC that interact with the P4′ acidic residue of the inhibitor. The acidic preference of CTRC at P4′ is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation. PMID:21515688

  17. NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implications for oligomer formation by 3D domain swapping.

    PubMed

    López-Alonso, Jorge Pedro; Bruix, Marta; Font, Josep; Ribó, Marc; Vilanova, Maria; Jiménez, María Angeles; Santoro, Jorge; González, Carlos; Laurents, Douglas V

    2010-02-10

    Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal alpha-helix, C-terminal beta-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the beta-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, I(N), with non-native X-Pro peptide bonds. I(N) is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation.

  18. Characterization of VuMATE1 Expression in Response to Iron Nutrition and Aluminum Stress Reveals Adaptation of Rice Bean (Vigna umbellata) to Acid Soils through Cis Regulation

    PubMed Central

    Liu, Meiya; Xu, Jiameng; Lou, Heqiang; Fan, Wei; Yang, Jianli; Zheng, Shaojian

    2016-01-01

    Rice bean (Vigna umbellata) VuMATE1 appears to be constitutively expressed at vascular system but root apex, and Al stress extends its expression to root apex. Whether VuMATE1 participates in both Al tolerance and Fe nutrition, and how VuMATE1 expression is regulated is of great interest. In this study, the role of VuMATE1 in Fe nutrition was characterized through in planta complementation assays. The transcriptional regulation of VuMATE1 was investigated through promoter analysis and promoter-GUS reporter assays. The results showed that the expression of VuMATE1 was regulated by Al stress but not Fe status. Complementation of frd3-1 with VuMATE1 under VuMATE1 promoter could not restore phenotype, but restored with 35SCaMV promoter. Immunostaining of VuMATE1 revealed abnormal localization of VuMATE1 in vasculature. In planta GUS reporter assay identified Al-responsive cis-acting elements resided between -1228 and -574 bp. Promoter analysis revealed several cis-acting elements, but transcription is not simply regulated by one of these elements. We demonstrated that cis regulation of VuMATE1 expression is involved in Al tolerance mechanism, while not involved in Fe nutrition. These results reveal the evolution of VuMATE1 expression for better adaptation of rice bean to acid soils where Al stress imposed but Fe deficiency pressure released. PMID:27148333

  19. Characterization of VuMATE1 Expression in Response to Iron Nutrition and Aluminum Stress Reveals Adaptation of Rice Bean (Vigna umbellata) to Acid Soils through Cis Regulation.

    PubMed

    Liu, Meiya; Xu, Jiameng; Lou, Heqiang; Fan, Wei; Yang, Jianli; Zheng, Shaojian

    2016-01-01

    Rice bean (Vigna umbellata) VuMATE1 appears to be constitutively expressed at vascular system but root apex, and Al stress extends its expression to root apex. Whether VuMATE1 participates in both Al tolerance and Fe nutrition, and how VuMATE1 expression is regulated is of great interest. In this study, the role of VuMATE1 in Fe nutrition was characterized through in planta complementation assays. The transcriptional regulation of VuMATE1 was investigated through promoter analysis and promoter-GUS reporter assays. The results showed that the expression of VuMATE1 was regulated by Al stress but not Fe status. Complementation of frd3-1 with VuMATE1 under VuMATE1 promoter could not restore phenotype, but restored with 35SCaMV promoter. Immunostaining of VuMATE1 revealed abnormal localization of VuMATE1 in vasculature. In planta GUS reporter assay identified Al-responsive cis-acting elements resided between -1228 and -574 bp. Promoter analysis revealed several cis-acting elements, but transcription is not simply regulated by one of these elements. We demonstrated that cis regulation of VuMATE1 expression is involved in Al tolerance mechanism, while not involved in Fe nutrition. These results reveal the evolution of VuMATE1 expression for better adaptation of rice bean to acid soils where Al stress imposed but Fe deficiency pressure released.

  20. Soil Iron Content as a Predictor of Carbon and Nutrient Mobilization in Rewetted Fens

    PubMed Central

    Emsens, Willem-Jan; Aggenbach, Camiel J. S.; Schoutens, Ken; Smolders, Alfons J. P.; Zak, Dominik; van Diggelen, Rudy

    2016-01-01

    Rewetted, previously drained fens often remain sources rather than sinks for carbon and nutrients. To date, it is poorly understood which soil characteristics stimulate carbon and nutrient mobilization upon rewetting. Here, we assess the hypothesis that a large pool of iron in the soil negatively affects fen restoration success, as flooding-induced iron reduction (Fe3+ to Fe2+) causes a disproportionate breakdown of organic matter that is coupled with a release of inorganic compounds. We collected intact soil cores in two iron-poor and two iron-rich drained fens, half of which were subjected to a rewetting treatment while the other half was kept drained. Prolonged drainage led to the mobilization of nitrate (NO3-, > 1 mmol L-1) in all cores, regardless of soil iron content. In the rewetted iron-rich cores, a sharp increase in pore water iron (Fe) concentrations correlated with concentrations of inorganic carbon (TIC, > 13 mmol L-1) and dissolved organic carbon (DOC, > 16 mmol L-1). Additionally, ammonium (NH4+) accumulated up to phytotoxic concentrations of 1 mmol L-1 in the pore water of the rewetted iron-rich cores. Disproportionate mobilization of Fe, TIC, DOC and NH4+ was absent in the rewetted iron-poor cores, indicating a strong interaction between waterlogging and iron-mediated breakdown of organic matter. Concentrations of dissolved phosphorus (P) rose slightly in all cores upon rewetting, but remained low throughout the experiment. Our results suggest that large pools of iron in the top soil of drained fens can hamper the restoration of the fen’s sink-service for ammonium and carbon upon rewetting. We argue that negative effects of iron should be most apparent in fens with fluctuating water levels, as temporary oxygenation allows frequent regeneration of Fe3+. We conclude that rewetting of iron-poor fens may be more feasible for restoration. PMID:27050837

  1. Key players of methane dynamics in alpine fens: interaction of vascular plants and microbial communities

    NASA Astrophysics Data System (ADS)

    Cheema, S.; Zeyer, J. A.; Henneberger, R.

    2014-12-01

    Natural wetlands are important emitters of the potent greenhouse gas methane (CH4), contributing an estimated 26 - 42% to the global emissions. In these habitats CH4 is generated by methanogenic archaea mediating the terminal steps of organic matter degradation under anoxic conditions. The produced CH4 is partly oxidized by methanotrophic bacteria in oxic zones, thereby mitigating CH4 release. Various factors can influence CH4 emissions from wetlands, including the presence of vascular plants, as their aerenchyma can serve as conduits for CH4 release to the atmosphere. In the present study, we investigated the CH4 dynamics in two Swiss alpine fens (1900 - 2300 m a.s.l), and sampling locations within these fens were characterized by distinct dominant vascular plants, namely Carex spp. and Eriophorum spp.. Analyses of the microbial communities present in the fen soils were complemented by in situ measurements of CH4 emissions and analyses of physico-chemical pore water properties. Methane emissions and pore water concentrations varied depending on fen and dominating plant species, with generally higher CH4 emissions observed from the Carex dominated locations. Active methanotrophic and methanogenic microorganisms (transcripts of specific marker genes) were detected at different depths, independent of O2 and CH4 pore water concentrations. The expected separation of oxic methanotrophic and anoxic methanogenic zones was not observed. Yet, composition analyses of the different communities showed a clear clustering according to fen and dominating plant species. Within each location, variation of composition with depth was only observed for the methanogenic communities. Detailed profiling of CH4 emissions with respect to changes in light and temperature is currently being carried out. Our results represent a comprehensive in situ study on factors affecting CH4 emissions from alpine fens, highlighting the influence of vascular plants on the microbial communities involved.

  2. Fate and Transport of Road Salt During Snowmelt Through a Calcareous Fen: Kampoosa Bog, Stockbridge, Massachusetts

    NASA Astrophysics Data System (ADS)

    Rhodes, A. L.; Guswa, A. J.; Pufall, A.

    2007-12-01

    Kampoosa Bog is the largest and most ecologically diverse calcareous lake-basin fen in Massachusetts. Situated within a 4.7 km2 drainage basin, the open fen (approx. 20 acres) consists of a floating mat of sedges (incl. Carex aquatilis and Cladium mariscoides) that overlie peat and lake clay deposits. Mineral weathering of marble bedrock within the drainage basin supplies highly alkaline ground and surface waters to the fen basin. The natural chemistry has been greatly altered by road salt runoff from the Massaschusetts Turnpike, and in question is whether disturbance from the Turnpike and a gas pipline has facilitated aggressive growth by the invasive species Phragmites australis. Considered to be one of the most significant rare species habitats in the state, Massachusetts has designated Kampoosa Bog an Area of Critical Environmental Concern, and a committee representing several local, regional, and state agencies, organizations, and citizens manages the wetland. The purpose of this study is to characterize the hydrologic and chemical response of the wetland during snowmelt events to understand the fate and movement of road salt (NaCl). Concentrations of Na and Cl in the fen groundwater are greatest close to the Turnpike. Concentrations decrease with distance downstream but are still greatly elevated relative to sites upstream of the Turnpike. During snowmelt events, the fen's outlet shows a sharp rise in Na and Cl concentrations at the onset of melting that is soon diluted by the added meltwater. The Na and Cl flux, however, is greatest at peak discharge, suggesting that high-flow events are significant periods of export of dissolved salts from the fen. Pure dissolution of rock salt produces an equal molar ratio between Na and Cl, and sodium and chloride imbalances in stream and ground waters suggest that ~20% of the Na is stored on cation exchange sites within the peat. The largest imbalances between Na and Cl occur deeper within the peat, where the peat is

  3. Performance of a constructed fen peatland: Reclamation of oil sands landscapes

    NASA Astrophysics Data System (ADS)

    Price, J. S.

    2013-12-01

    Wetlands cover >60% of the landscape in the Athabasca oils sands development region. These are predominantly fen peatlands, that rely to varying extent on base-rich surface- and/or ground-water derived from adjacent mineral lands. Large areas of peatland are removed by surface stripping to access the bitumen. Reclamation of post-mined landscapes to peatland has not previously been attempted. Groundwater modeling was used to guide design choices on the optimal geometry, placement (layering) and hydraulic properties of locally available materials; these include processed tailings sand, as well peat and forest soils from newly developed lease areas. The constructed system comprises a ~3 ha fen with 2 m of peat and an adjacent upland specifically designed to supply the requisite water to maintain suitable hydrological conditions. The upland is primarily tailings sand covered by a forest soil. The constructed fen/upland system was set within a ~30 ha basin that was reclaimed using standard techniques and materials, and supplements water inputs to the designed system. Both fen and upland were placed at water contents well below equilibrium levels, so there was concern about the wetland function during the years following construction. However, in its first year following construction (2013), a higher than normal snowpack flooded the system, but by surface inflow rather than seepage from the upland. Ground-ice in the fen limited the general infiltration and percolation of meltwater into the peat, although fortuitously a localized recharge pipe developed through thermal erosion and substantially recharged the fen. Water was redistributed beneath the fen through a highly permeable sublayer (petroleum coke) designed for this purpose, and created artesian pressures. Backflow into the upland also resulted, although much of the upland remains well below design water contents. Modification of the upland soils by plowing, and development of local recharge basins, is expected to

  4. Influence of NO and (NO)2 adsorption on the properties of Fe-N4 porphyrin-like graphene sheets.

    PubMed

    Ashori, Elham; Nazari, Fariba; Illas, Francesc

    2017-01-25

    Detection of NO in biological systems and removing or reducing NO for environment protection is paramount. Herein, we investigate the influence of NO and (NO)2 adsorption on the properties of Fe-N4 porphyrin-like graphene (G-Fe-N4) sheets using periodic DFT calculations with the dispersion correction. The results show that NO can be converted into N2O through adsorbed (NO)2 with a total energy barrier of 0.92 eV. The adsorption of N2O and of two NO on O/G-Fe-N4 sheets can proceed through (N2)gas + (O2)ads and (N2O)gas + (O2)ads, respectively. Both paths have a rate-determining step with a high (∼1.80 eV) energy barrier. Nevertheless, the formation of (O2)ads on the G-Fe-N4 can be regarded as an oxygen reduction reaction (ORR) precursor. Detailed analyses of the electronic properties of the various systems involved in this reaction reveal the increased spin filter characteristics for some structures. Hence, the obtained spin filter parameters of the NO@G-Fe-N4 and (NO)2@G-Fe-N4 structures are 72.53% and 47.96%, respectively. Also, it is found that the adsorption of NO gas molecules induces different energy antiresonant dips not found in G-Fe-N4, which are induced by quasi-bound states related to the adsorbate and Fe-N4 defect.

  5. Diversity in the structures and ligand-binding sites of nematode fatty acid and retinol-binding proteins revealed by Na-FAR-1 from Necator americanus

    PubMed Central

    Rey-Burusco, M. Florencia; Ibáñez-Shimabukuro, Marina; Gabrielsen, Mads; Franchini, Gisela R.; Roe, Andrew J.; Griffiths, Kate; Zhan, Bin; Cooper, Alan; Kennedy, Malcolm W.; Córsico, Betina; Smith, Brian O.

    2015-01-01

    Fatty acid and retinol-binding proteins (FARs) comprise a family of unusual α-helix rich lipid-binding proteins found exclusively in nematodes. They are secreted into host tissues by parasites of plants, animals and humans. The structure of a FAR protein from the free-living nematode Caenorhabditis elegans is available, but this protein [C. elegans FAR-7 (Ce-FAR-7)] is from a subfamily of FARs that does not appear to be important at the host/parasite interface. We have therefore examined [Necator americanus FAR-1 (Na-FAR-1)] from the blood-feeding intestinal parasite of humans, N. americanus. The 3D structure of Na-FAR-1 in its ligand-free and ligand-bound forms, determined by NMR (nuclear magnetic resonance) spectroscopy and X-ray crystallography respectively, reveals an α-helical fold similar to Ce-FAR-7, but Na-FAR-1 possesses a larger and more complex internal ligand-binding cavity and an additional C-terminal α-helix. Titration of apo-Na-FAR-1 with oleic acid, analysed by NMR chemical shift perturbation, reveals that at least four distinct protein–ligand complexes can be formed. Na-FAR-1 and possibly other FARs may have a wider repertoire for hydrophobic ligand binding, as confirmed in the present study by our finding that a range of neutral and polar lipids co-purify with the bacterially expressed recombinant protein. Finally, we show by immunohistochemistry that Na-FAR-1 is present in adult worms with a tissue distribution indicative of possible roles in nutrient acquisition by the parasite and in reproduction in the male. PMID:26318523

  6. Solute movement through unsaturated fen peat: Lab and greenhouse experiments for transport study of contaminants from Athabasca oil sands tailing pond water

    NASA Astrophysics Data System (ADS)

    Price, J. S.; Rezanezhad, F.; Graf, M.; Rochefort, L.

    2009-12-01

    In the Athabasca oil sands region, wetlands specially peatland dominate the landscape. Processing oil sands produces large volumes of wet material called oil sands tailing water. Discharge of organic liquid contaminants such as Naphthenic Acids (NA) and Sodium (Na) from tailing waters have a toxic effect on plants in this region. One of the greatest barriers to peatland creation will be the elevated amount of toxins (naphthenic acid, metals and salinity) present in the post-mined landscapes. Variability in solute transport properties in the unsaturated zone is of growing concern due to environmental hazards and there are no many scientific challenges in the field of organic liquid contaminants transport through the unsaturated peat soils. The attenuation, degradation and transport of NA and Na in peat are essentially unknown. The ionizable nature of NA and Na along with the complex structure of peat soils poses challenges to characterizing the transport properties of NA and Na in the filed and laboratory. In this experimental research project, we examine the plant responses in 64 greenhouse tubs filled with peat and process-water; and study the transport and attenuation processes of NA and Na through peat in a series of laboratory column experiments. We developed an analytical method for evaluating the transport and adsorption characteristics of NA and Na to derive a clear understanding of the transport, sorption mechanisms and desorption behaviour of NA and Na with temporal evolution of the solute concentration distribution from groundwater to fen plants. The goal of this research project is to investigate how oil sands process-affected waters will affect peatland vegetation, specifically fen vegetation. In particular, we would like to know how contaminants present in oil sand process affected water will be transported through peat and how typical fen vegetation will react to a realistic contamination scenario in a controlled macrocosm environment? Research that

  7. Sea anemone peptide with uncommon β-hairpin structure inhibits acid-sensing ion channel 3 (ASIC3) and reveals analgesic activity.

    PubMed

    Osmakov, Dmitry I; Kozlov, Sergey A; Andreev, Yaroslav A; Koshelev, Sergey G; Sanamyan, Nadezhda P; Sanamyan, Karen E; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Mineev, Konstantin S; Arseniev, Alexander S; Grishin, Eugene V

    2013-08-09

    Three novel peptides were isolated from the venom of the sea anemone Urticina grebelnyi. All of them are 29 amino acid peptides cross-linked by two disulfide bridges, with a primary structure similar to other sea anemone peptides belonging to structural group 9a. The structure of the gene encoding the shared precursor protein of the identified peptides was determined. One peptide, π-AnmTX Ugr 9a-1 (short name Ugr 9-1), produced a reversible inhibition effect on both the transient and the sustained current of human ASIC3 channels expressed in Xenopus laevis oocytes. It completely blocked the transient component (IC50 10 ± 0.6 μM) and partially (48 ± 2%) inhibited the amplitude of the sustained component (IC50 1.44 ± 0.19 μM). Using in vivo tests in mice, Ugr 9-1 significantly reversed inflammatory and acid-induced pain. The other two novel peptides, AnmTX Ugr 9a-2 (Ugr 9-2) and AnmTX Ugr 9a-3 (Ugr 9-3), did not inhibit the ASIC3 current. NMR spectroscopy revealed that Ugr 9-1 has an uncommon spatial structure, stabilized by two S-S bridges, with three classical β-turns and twisted β-hairpin without interstrand disulfide bonds. This is a novel peptide spatial structure that we propose to name boundless β-hairpin.

  8. Structure-Function Analyses of a Caffeic Acid O-Methyltransferase from Perennial Ryegrass Reveal the Molecular Basis for Substrate Preference[W][OA

    PubMed Central

    Louie, Gordon V.; Bowman, Marianne E.; Tu, Yi; Mouradov, Aidyn; Spangenberg, German; Noel, Joseph P.

    2010-01-01

    Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-l-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT’s catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde. PMID:21177481

  9. Disruption of OPR7 and OPR8 Reveals the Versatile Functions of Jasmonic Acid in Maize Development and Defense[W

    PubMed Central

    Yan, Yuanxin; Christensen, Shawn; Isakeit, Tom; Engelberth, Jürgen; Meeley, Robert; Hayward, Allison; Emery, R.J. Neil; Kolomiets, Michael V.

    2012-01-01

    Here, multiple functions of jasmonic acid (JA) in maize (Zea mays) are revealed by comprehensive analyses of JA-deficient mutants of the two oxo-phytodienoate reductase genes, OPR7 and OPR8. Single mutants produce wild-type levels of JA in most tissues, but the double mutant opr7 opr8 has dramatically reduced JA in all organs tested. opr7 opr8 displayed strong developmental defects, including formation of a feminized tassel, initiation of female reproductive buds at each node, and extreme elongation of ear shanks; these defects were rescued by exogenous JA. These data provide evidence that JA is required for male sex determination and suppression of female reproductive organ biogenesis. Moreover, opr7 opr8 exhibited delayed leaf senescence accompanied by reduced ethylene and abscisic acid levels and lack of anthocyanin pigmentation of brace roots. Remarkably, opr7 opr8 is nonviable in nonsterile soil and under field conditions due to extreme susceptibility to a root-rotting oomycete (Pythium spp), demonstrating that these genes are necessary for maize survival in nature. Supporting the importance of JA in insect defense, opr7 opr8 is susceptible to beet armyworm. Overall, this study provides strong genetic evidence for the global roles of JA in maize development and immunity to pathogens and insects. PMID:22523204

  10. The Crystal Structure of Rv0813c from Mycobacterium tuberculosis Reveals a New Family of Fatty Acid-Binding Protein-Like Proteins in Bacteria▿

    PubMed Central

    Shepard, William; Haouz, Ahmed; Graña, Martin; Buschiazzo, Alejandro; Betton, Jean-Michel; Cole, Stewart T.; Alzari, Pedro M.

    2007-01-01

    The gene Rv0813c from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is conserved within the order Actinomycetales but absent elsewhere. The crystal structure of Rv0813c reveals a new family of proteins that resemble the fatty acid-binding proteins (FABPs) found in eukaryotes. Rv0813c adopts the 10-stranded β-barrel fold typical of FABPs but lacks the double-helix insert that covers the entry to the binding site in the eukaryotic proteins. The barrel encloses a deep cavity, at the bottom of which a small cyclic ligand was found to bind to the hydroxyl group of Tyr192. This residue is part of a conserved Arg-X-Tyr motif much like the triad that binds the carboxylate group of fatty acids in FABPs. Most of the residues forming the internal surface of the cavity are conserved in homologous protein sequences found in CG-rich prokaryotes, strongly suggesting that Rv0813c is a member of a new family of bacterial FABP-like proteins that may have roles in the recognition, transport, and/or storage of small molecules in the bacterial cytosol. PMID:17172346

  11. Cloning of wrinkle-free, a previously uncharacterized mouse mutation, reveals crucial roles for fatty acid transport protein 4 in skin and hair development.

    PubMed

    Moulson, Casey L; Martin, Daniel R; Lugus, Jesse J; Schaffer, Jean E; Lind, Anne C; Miner, Jeffrey H

    2003-04-29

    Wrinkle-free (wrfr) is a previously uncharacterized, spontaneous, autosomal recessive mouse mutation resulting in very tight, thick skin. wrfr mutant mice exhibit severe breathing difficulties secondary to their tight skin and die shortly after birth. This phenotype is strikingly similar to a very rare human genetic disorder, restrictive dermopathy. wrfr mutant mice display a defective skin barrier, which is normally imparted by the cornified envelope, a composite of protein and lipid that prevents loss of water from within and entry of potentially harmful substances from without. In addition, hair growth from grafted wrfr skin is impaired. Positional cloning of the wrfr mutation revealed a retrotransposon insertion into a coding exon of Slc27a4, the gene encoding fatty acid transport protein (FATP)4. FATP4 is the primary intestinal FATP and is thought to play a major role in dietary fatty acid uptake; it therefore is viewed as a target to prevent or reverse obesity. However, its function in vivo had not been determined. Our results demonstrate an unexpected yet critical role for FATP4 in skin and hair development and suggest Slc27a4 to be a candidate gene for restrictive dermopathy.

  12. Metabolic signatures of extreme longevity in northern Italian centenarians reveal a complex remodeling of lipids, amino acids, and gut microbiota metabolism.

    PubMed

    Collino, Sebastiano; Montoliu, Ivan; Martin, François-Pierre J; Scherer, Max; Mari, Daniela; Salvioli, Stefano; Bucci, Laura; Ostan, Rita; Monti, Daniela; Biagi, Elena; Brigidi, Patrizia; Franceschi, Claudio; Rezzi, Serge

    2013-01-01

    The aging phenotype in humans has been thoroughly studied but a detailed metabolic profiling capable of shading light on the underpinning biological processes of longevity is still missing. Here using a combined metabonomics approach compromising holistic (1)H-NMR profiling and targeted MS approaches, we report for the first time the metabolic phenotype of longevity in a well characterized human aging cohort compromising mostly female centenarians, elderly, and young individuals. With increasing age, targeted MS profiling of blood serum displayed a marked decrease in tryptophan concentration, while an unique alteration of specific glycerophospholipids and sphingolipids are seen in the longevity phenotype. We hypothesized that the overall lipidome changes specific to longevity putatively reflect centenarians' unique capacity to adapt/respond to the accumulating oxidative and chronic inflammatory conditions characteristic of their extreme aging phenotype. Our data in centenarians support promotion of cellular detoxification mechanisms through specific modulation of the arachidonic acid metabolic cascade as we underpinned increased concentration of 8,9-EpETrE, suggesting enhanced cytochrome P450 (CYP) enzyme activity. Such effective mechanism might result in the activation of an anti-oxidative response, as displayed by decreased circulating levels of 9-HODE and 9-oxoODE, markers of lipid peroxidation and oxidative products of linoleic acid. Lastly, we also revealed that the longevity process deeply affects the structure and composition of the human gut microbiota as shown by the increased extrection of phenylacetylglutamine (PAG) and p-cresol sulfate (PCS) in urine of centenarians. Together, our novel approach in this representative Italian longevity cohort support the hypothesis that a complex remodeling of lipid, amino acid metabolism, and of gut microbiota functionality are key regulatory processes marking exceptional longevity in humans.

  13. Molecular cloning and functional characterization of Xenopus tropicalis frog transient receptor potential vanilloid 1 reveal its functional evolution for heat, acid, and capsaicin sensitivities in terrestrial vertebrates.

    PubMed

    Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2012-01-20

    The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca(2+)](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca(2+)](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.

  14. Direct Comparison of Mice Null for Liver or Intestinal Fatty Acid-binding Proteins Reveals Highly Divergent Phenotypic Responses to High Fat Feeding*

    PubMed Central

    Gajda, Angela M.; Zhou, Yin Xiu; Agellon, Luis B.; Fried, Susan K.; Kodukula, Sarala; Fortson, Walter; Patel, Khamoshi; Storch, Judith

    2013-01-01

    The enterocyte expresses two fatty acid-binding proteins (FABP), intestinal FABP (IFABP; FABP2) and liver FABP (LFABP; FABP1). LFABP is also expressed in liver. Despite ligand transport and binding differences, it has remained uncertain whether these intestinally coexpressed proteins, which both bind long chain fatty acids (FA), are functionally distinct. Here, we directly compared IFABP−/− and LFABP−/− mice fed high fat diets containing long chain saturated or unsaturated fatty acids, reasoning that providing an abundance of dietary lipid would reveal unique functional properties. The results showed that mucosal lipid metabolism was indeed differentially modified, with significant decreases in FA incorporation into triacylglycerol (TG) relative to phospholipid (PL) in IFABP−/− mice, whereas LFABP−/− mice had reduced monoacylglycerol incorporation in TG relative to PL, as well as reduced FA oxidation. Interestingly, striking differences were found in whole body energy homeostasis; LFABP−/− mice fed high fat diets became obese relative to WT, whereas IFABP−/− mice displayed an opposite, lean phenotype. Fuel utilization followed adiposity, with LFABP−/− mice preferentially utilizing lipids, and IFABP−/− mice preferentially metabolizing carbohydrate for energy production. Changes in body weight and fat may arise, in part, from altered food intake; mucosal levels of the endocannabinoids 2-arachidonoylglycerol and arachidonoylethanolamine were elevated in LFABP−/−, perhaps contributing to increased energy intake. This direct comparison provides evidence that LFABP and IFABP have distinct roles in intestinal lipid metabolism; differential intracellular functions in intestine and in liver, for LFABP−/− mice, result in divergent downstream effects at the systemic level. PMID:23990461

  15. Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates*

    PubMed Central

    Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2012-01-01

    The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca2+]i). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca2+]i increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function. PMID:22130664

  16. Tissue-Specific Inactivation of Type 2 Deiodinase Reveals Multilevel Control of Fatty Acid Oxidation by Thyroid Hormone in the Mouse

    PubMed Central

    Fonseca, Tatiana L.; Werneck-De-Castro, Joao Pedro; Castillo, Melany; Bocco, Barbara M.L.C.; Fernandes, Gustavo W.; McAninch, Elizabeth A.; Ignacio, Daniele L.; Moises, Caio C.S.; Ferreira, Alexandre; Gereben, Balázs

    2014-01-01

    Type 2 deiodinase (D2) converts the prohormone thyroxine (T4) to the metabolically active molecule 3,5,3′-triiodothyronine (T3), but its global inactivation unexpectedly lowers the respiratory exchange rate (respiratory quotient [RQ]) and decreases food intake. Here we used FloxD2 mice to generate systemically euthyroid fat-specific (FAT), astrocyte-specific (ASTRO), or skeletal-muscle-specific (SKM) D2 knockout (D2KO) mice that were monitored continuously. The ASTRO-D2KO mice also exhibited lower diurnal RQ and greater contribution of fatty acid oxidation to energy expenditure, but no differences in food intake were observed. In contrast, the FAT-D2KO mouse exhibited sustained (24 h) increase in RQ values, increased food intake, tolerance to glucose, and sensitivity to insulin, all supporting greater contribution of carbohydrate oxidation to energy expenditure. Furthermore, FAT-D2KO animals that were kept on a high-fat diet for 8 weeks gained more body weight and fat, indicating impaired brown adipose tissue (BAT) thermogenesis and/or inability to oxidize the fat excess. Acclimatization of FAT-D2KO mice at thermoneutrality dissipated both features of this phenotype. Muscle D2 does not seem to play a significant metabolic role given that SKM-D2KO animals exhibited no phenotype. The present findings are unique in that they were obtained in systemically euthyroid animals, revealing that brain D2 plays a dominant albeit indirect role in fatty acid oxidation via its sympathetic control of BAT activity. D2-generated T3 in BAT accelerates fatty acid oxidation and protects against diet-induced obesity. PMID:24487027

  17. Comparative transcriptome analysis reveals different molecular mechanisms of Bacillus coagulans 2-6 response to sodium lactate and calcium lactate during lactic acid production.

    PubMed

    Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

    2015-01-01

    Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that 'ATP-binding cassette transporters' were significantly affected by calcium lactate stress, and 'amino sugar and nucleotide sugar metabolism' was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of 'glycolysis/gluconeogenesis' genes but positive effect on the expression of 'citrate cycle (TCA cycle)' genes. However, calcium lactate stress had positive influence on the expression of 'glycolysis/gluconeogenesis' genes and had minor influence on 'citrate cycle (TCA cycle)' genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans.

  18. Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

    PubMed Central

    Exell, Jack C.; Thompson, Mark J.; Finger, L. David; Shaw, Steven J.; Debreczeni, Judit; Ward, Thomas A.; McWhirter, Claire; Siöberg, Catrine L. B.; Martinez Molina, Daniel; Mark Abbott, W.; Jones, Clifford D.; Nissink, J. Willem M.; Durant, Stephen T.; Grasby, Jane A.

    2016-01-01

    The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed. PMID:27526030

  19. FEN1 participates in repair of the 5'-phosphotyrosyl terminus of DNA single-strand breaks.

    PubMed

    Kametani, Yukiko; Takahata, Chiaki; Narita, Takashi; Tanaka, Kiyoji; Iwai, Shigenori; Kuraoka, Isao

    2016-01-01

    Etoposide is a widely used anticancer drug and a DNA topoisomerase II (Top2) inhibitor. Etoposide produces Top2-attached single-strand breaks (Top2-SSB complex) and double-strand breaks (Top2-DSB complex) that are thought to induce cell death in tumor cells. The Top2-SSB complex is more abundant than the Top2-DSB complex. Human tyrosyl-DNA phosphodiesterase 2 (TDP2) is required for efficient repair of Top2-DSB complexes. However, the identities of the proteins involved in the repair of Top2-SSB complexes are unknown, although yeast genetic data indicate that 5' to 3' structure-specific DNA endonuclease activity is required for alternative repair of Top2 DNA damage. In this study, we purified a flap endonuclease 1 (FEN1) and xeroderma pigmentosum group G protein (XPG) in the 5' to 3' structure-specific DNA endonuclease family and synthesized single-strand break DNA substrates containing a 5'-phoshotyrosyl bond, mimicking the Top2-SSB complex. We found that FEN1 and XPG did not remove the 5'-phoshotyrosyl bond-containing DSB substrates but removed the 5'-phoshotyrosyl bond-containing SSB substrates. Under DNA repair conditions, FEN1 efficiently repaired the 5'-phoshotyrosyl bond-containing SSB substrates in the presence of DNA ligase and DNA polymerase. Therefore, FEN1 may play an important role in the repair of Top2-SSB complexes in etoposide-treated cells.

  20. Promotion of CO oxidation on the Fe/N X clusters embedded graphene

    NASA Astrophysics Data System (ADS)

    Zhao, Ming Yu; Zhao, Ru Meng; Niu, Meng Meng; Li, Wei; Ma, Ya Qiang; Li, Yi; Tang, Ya Nan; Dai, Xian Qi

    2017-03-01

    CO catalytic oxidation on the two-dimensional (2D) Fe/N X clusters embedded graphene (G) (X = 4, 3, 2, 1) with D 4h, C 2v, C 2h, and C S symmetries is investigated using the first-principle method. Fe/N2-G system with C 2h symmetry facilitates the O2 adsorption. The strong interaction between the Fe atom and O2 in Fe/N2-G system can be contributed from the high energy of Fe's d_z^2 orbit. The CO oxidation reaction on Fe/N2-G system has a small energy barrier (0.43 eV) by the Langmuir-Hinshelwood (LH) mechanism (CO + O2 → OOCO → CO2 + Oads), which would be useful in evaluating the reactivity of Fe catalyst and serving as a good candidate for efficient non-noble metal catalyst. The results provide valuable guidance on selecting catalysts of low cost and superior activity to fabricate graphene-based materials.

  1. BOREAS TGB-1/TGB-3 CH4 Chamber Flux Data over the NSA Fen

    NASA Technical Reports Server (NTRS)

    Bubier, Jill L.; Moore, Tim R.; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor)

    2000-01-01

    The BOREAS TGB-3 team collected methane (CH4) chamber flux measurements at the NSA fen site during May-September 1994 and June-October 1996. Gas samples were extracted approximately every 7 days from chambers and analyzed at the NSA lab facility. The data are provided in tabular ASCII files.

  2. Changing Land Use: The Fens of England. A Case Study in Land Reclamation [And] Student Work Book.

    ERIC Educational Resources Information Center

    Laws, Kevin

    A social studies unit and student workbook explore changes in land use that have occurred in the Fenlands of England since the time it was first inhabited. Fens are lowlying land which is partially or completely covered with water. The English Fens are located on the eastern side of the British Isles and cover a total area of about 2,000 square…

  3. Impact of urban development on the chemical composition of ground water in a fen-wetland complex

    USGS Publications Warehouse

    Panno, S.V.; Nuzzo, V.A.; Cartwright, K.; Hensel, B.R.; Krapac, I.G.

    1999-01-01

    A 15-month-long hydrogeologic investigation of a fen-wetland complex in northeastern Illinois, USA indicated the encroachment of ground-water-borne anthropogenic contaminants into two of three high quality fens. Ground-water flow directions and chemical evidence indicated that plumes of ground water with anomalously large concentrations of Na+ and Cl- originated from a private septic system and from rock salt spread on an adjacent road. The contamination, in turn, had an adverse effect on fen vegetation; within the plumes, diverse vegetation was replaced by the more salt-tolerant narrow-leaf cattail (Typha angustifolia). Ground water of the third fen contained large concentrations of SO42- as high as 516 mg/L. The SO42- anomaly was observed on a transient and/or seasonal basis in the fen ground water and in an adjacent marsh and pond. Isotopically light ??34S values in these waters indicated that the addition of SO42- resulted from the oxidation of pyrite within underlying peat and/or pyritic gravel. However, the large SO42- concentrations had no discernible effect on fen vegetation. The results of this investigation indicate how easily construction of houses with private septic systems and deicing agents from roadway maintenance can contaminate fen ground water with relatively large concentrations of Na+ and Cl-, resulting in a significant loss of biodiversity in fens.

  4. The manipulation of auxin in the abscission zone cells of Arabidopsis flowers reveals that indoleacetic acid signaling is a prerequisite for organ shedding.

    PubMed

    Basu, Manojit M; González-Carranza, Zinnia H; Azam-Ali, Sayed; Tang, Shouya; Shahid, Ahmad Ali; Roberts, Jeremy A

    2013-05-01

    A number of novel strategies were employed to examine the role of indoleacetic acid (IAA) in regulating floral organ abscission in Arabidopsis (Arabidopsis thaliana). Analysis of auxin influx facilitator expression in β-glucuronidase reporter plants revealed that AUXIN RESISTANT1, LIKE AUX1, and LAX3 were specifically up-regulated at the site of floral organ shedding. Flowers from mutants where individual family members were down-regulated exhibited a reduction in the force necessary to bring about petal separation; however, the effect was not additive in double or quadruple mutants. Using the promoter of a polygalacturonase (At2g41850), active primarily in cells undergoing separation, to drive expression of the bacterial genes iaaL and iaaM, we have shown that it is possible to manipulate auxin activity specifically within the floral organ abscission zone (AZ). Analysis of petal breakstrength reveals that if IAA AZ levels are reduced, shedding takes place prematurely, while if they are enhanced, organ loss is delayed. The At2g41850 promoter was also used to transactivate the gain-of-function AXR3-1 gene in order to disrupt auxin signaling specifically within the floral organ AZ cells. Flowers from transactivated lines failed to shed their sepals, petals, and anthers during pod expansion and maturity, and these organs frequently remained attached to the plant even after silique desiccation and dehiscence had taken place. These observations support a key role for IAA in the regulation of abscission in planta and reveal, to our knowledge for the first time, a requirement for a functional IAA signaling pathway in AZ cells for organ shedding to take place.

  5. The Manipulation of Auxin in the Abscission Zone Cells of Arabidopsis Flowers Reveals That Indoleacetic Acid Signaling Is a Prerequisite for Organ Shedding1[C][W][OA

    PubMed Central

    Basu, Manojit M.; González-Carranza, Zinnia H.; Azam-Ali, Sayed; Tang, Shouya; Shahid, Ahmad Ali; Roberts, Jeremy A.

    2013-01-01

    A number of novel strategies were employed to examine the role of indoleacetic acid (IAA) in regulating floral organ abscission in Arabidopsis (Arabidopsis thaliana). Analysis of auxin influx facilitator expression in β-glucuronidase reporter plants revealed that AUXIN RESISTANT1, LIKE AUX1, and LAX3 were specifically up-regulated at the site of floral organ shedding. Flowers from mutants where individual family members were down-regulated exhibited a reduction in the force necessary to bring about petal separation; however, the effect was not additive in double or quadruple mutants. Using the promoter of a polygalacturonase (At2g41850), active primarily in cells undergoing separation, to drive expression of the bacterial genes iaaL and iaaM, we have shown that it is possible to manipulate auxin activity specifically within the floral organ abscission zone (AZ). Analysis of petal breakstrength reveals that if IAA AZ levels are reduced, shedding takes place prematurely, while if they are enhanced, organ loss is delayed. The At2g41850 promoter was also used to transactivate the gain-of-function AXR3-1 gene in order to disrupt auxin signaling specifically within the floral organ AZ cells. Flowers from transactivated lines failed to shed their sepals, petals, and anthers during pod expansion and maturity, and these organs frequently remained attached to the plant even after silique desiccation and dehiscence had taken place. These observations support a key role for IAA in the regulation of abscission in planta and reveal, to our knowledge for the first time, a requirement for a functional IAA signaling pathway in AZ cells for organ shedding to take place. PMID:23509178

  6. Phosphorus mobilization in rewetted fens: the effect of altered peat properties and implications for their restoration.

    PubMed

    Zak, Dominik; Wagner, Carola; Payer, Brian; Augustin, Jürgen; Gelbrecht, Jörg

    2010-07-01

    Rewetting of drained fens is necessary to stop further soil degradation and to reestablish important ecological functions. However, substantial changes of peat characteristics in the upper soil layers, due to drainage and land use, could counteract their recovery as nutrient-poor systems for an unknown period. We assessed the importance of altered peat properties, such as the degree of peat decomposition and the amount of redox-sensitive phosphorus (P) compounds, for P mobilization in different degraded fens. An experimental design involving 63 intact peat cores from fens with varying drainage and land-use histories was developed to quantify the mobilization of P, as well as that of iron (Fe), ammonium, carbon dioxide, and methane, all indicators of organic-matter decomposition and/or P-releasing processes. We found that net P release rates in peat cores with highly decomposed peat (range: 0.1-52.3 mg P x m(-2) x d(-1)) were significantly correlated to the amount of P bound to redox-sensitive compounds and the molar Fe:P as well as Al:P ratios of peat. We conclude that the following general rules apply for P mobilization in rewetted fens: (1) elevated levels of P release rates and P concentrations in pore water up to three orders of magnitude larger than under natural reference conditions can only be expected for rewetted fens whose surface soil layers consist of highly decomposed peat; (2) peat characteristics, such as the amount of P bound to redox-sensitive Fe(III) compounds (positive correlation) and molar ratios of Fe:P or Al:P (negative correlations), explain the high range of P release rates; and (3) a critical P export to adjacent lakes or rivers can only be expected if molar Fe:P ratios of highly decomposed peat are less than 10.

  7. Sphagnum establishment in alkaline fens: Importance of weather and water chemistry.

    PubMed

    Vicherová, Eliška; Hájek, Michal; Šmilauer, Petr; Hájek, Tomáš

    2017-02-15

    Sphagnum expansion to alkaline fens has accelerated during the last decades in Europe, leading to changes in diversity, habitat distributions and carbon storage. The causes are still not clearly understood and involve an interplay between climate change, hydrology, nutrient supply and Sphagnum physiology. We conducted a 4-year field experiment in eight fens in Central European highlands and assessed survival and establishment of individual apical shoot fragments of S. flexuosum, S. warnstorfii and S. squarrosum transplanted along the microtopographical gradient. In a laboratory experiment, we tested combined effects of desiccation and high calcium bicarbonate concentration on Sphagnum survival. We found that in unflooded positions, living shoots of Sphagnum and brown mosses lowered [Ca(2+)] and pH in their capillary water, in contrast to dead fragments; yet without differences between species. Survival and expansion of Sphagnum fragments, which did not die of acute calcium toxicity during first weeks/months, was negatively affected by dry weather and alkaline water chemistry, reflecting Sphagnum intolerance to desiccation and to combined high [Ca(2+)] and pH. Shoot fragments expanded to patches only when precipitation was high. Interestingly, non-toxic concentration of calcium bicarbonate reduced desiccation damage in Sphagnum, probably through protection of membranes or other cell components. This mechanism would facilitate Sphagnum survival in elevated, frequently desiccated microhabitats of calcareous fens such as brown-moss hummocks. However, since water-retaining capacity of few Sphagnum shoots is insufficient to change water chemistry in its surroundings, surface acidification may occur only once the environment (e.g. sufficient humidity) enabled expansion to larger mats. Then, the retained rainwater together with hardly decomposable Sphagnum litter would separate mire surface from groundwater, speeding up successional shift towards poor fens. Sphagnum

  8. XAS Characterization of a Nitridoiron(IV) Complex With a Very Short Fe-N Bond

    SciTech Connect

    Rohde, J.-U.; Betley, T.A.; Jackson, T.A.; Saouma, C.T.; Peters, J.C.; Que, L.; Jr.

    2009-06-04

    X-ray absorption spectroscopy has been used to characterize the novel nitridoiron(IV) units in two [PhBP{sup R}{sub 3}]Fe(N) complexes (R = iPr and CyCH{sub 2}) and obtain direct spectroscopic evidence for a very short Fe-N distance. The distance of 1.51--1.55 {angstrom} reflects the presence of an Fe{triple_bond}N triple bond in accord with the observed Fe{triple_bond}N vibration observed for one of these species (v{sub FeN}=1034 cm{sup -1}). This highly covalent bonding interaction results in the appearance of an unusually intense pre-edge peak, whose estimated area of 100(20) units is much larger than those of the related tetrahedral complexes with Fe{sup I}-N{sub 2}-Fe{sup I}, Fe{sup II}-NPh{sub 2}, and Fe{sup III}{triple_bond}NAd motifs, and those of recently described six-coordinate Fe{sup V}{triple_bond}N and Fe{sup VI}{triple_bond}N complexes. The observation that the Fe{sup IV}-N distances of two [PhBP{sup R}{sub 3}]Fe(N) complexes are shorter than the Fe{sup IV}-O bond lengths of oxoiron(IV) complexes may be rationalized on the basis of the greater ? basicity of the nitrido ligand than the oxo ligand and a lower metal coordination number for the Fe(N) complex.

  9. XAS Characterization of a Nitridoiron(IV) Complex with a Very Short Fe-N Bond

    SciTech Connect

    Rohde,J.; Betley, T.; Jackson, T.; Saouma, C.; Peters, J.; Que, Jr., L.

    2007-01-01

    X-ray absorption spectroscopy has been used to characterize the novel nitridoiron(IV) units in two [PhBP{sup R}{sub 3}]Fe(N) complexes (R = iPr and CyCH{sub 2}) and obtain direct spectroscopic evidence for a very short Fe-N distance. The distance of 1.51-1.55 {angstrom} reflects the presence of an Fe{triple_bond}N triple bond in accord with the observed Fe{triple_bond}N vibration observed for one of these species ({nu}{sub FeN} = 1034 cm{sup -1}). This highly covalent bonding interaction results in the appearance of an unusually intense pre-edge peak, whose estimated area of 100(20) units is much larger than those of the related tetrahedral complexes with Fe{sup I}-N{sub 2}-Fe{sup I}, Fe{sup II}-NPh{sub 2}, and Fe{sup III}NAd motifs, and those of recently described six-coordinate Fe{sup V}{triple_bond}N and Fe{sup VI}{triple_bond}N complexes. The observation that the Fe{sup IV}-N distances of two [PhBP{sup R}{sub 3}]Fe(N) complexes are shorter than the Fe{sup IV}-O bond lengths of oxoiron(IV) complexes may be rationalized on the basis of the greater {pi} basicity of the nitrido ligand than the oxo ligand and a lower metal coordination number for the Fe(N) complex.

  10. Seasonal and inter-annual variation in ecosystem scale methane emission from a boreal fen

    NASA Astrophysics Data System (ADS)

    Rinne, Janne; Li, Xuefei; Raivonen, Maarit; Peltola, Olli; Sallantaus, Tapani; Haapanala, Sami; Smolander, Sampo; Alekseychik, Pavel; Aurela, Mika; Korrensalo, Aino; Mammarella, Ivan; Tuittila, Eeva-Stiina; Vesala, Timo

    2016-04-01

    Northern wetlands are one of the major sources of atmospheric methane. We have measured ecosystem scale methane emissions from a boreal fen continuously since 2005. The site is an oligotrophic fen in boreal vegetation zone situated in Siikaneva wetland complex in Southern Finland. The mean annual temperature in the area is 3.3°C and total annual precipitation 710 mm. We have conducted the methane emission measurements by the eddy covariance method. Additionally we have measured fluxes of carbon dioxide, water vapor, and sensible heat together with a suite of other environmental parameters. We have analyzed this data alongside with a model run with University of Helsinki methane model. The measured fluxes show generally highest methane emission in late summers coinciding with the highest temperatures in saturated peat zone. During winters the fluxes show small but detectable emission despite the snow and ice cover on the fen. More than 90% of the annual methane emission occurs in snow-free period. The methane emission and peat temperature are connected in exponential manner in seasonal scales, but methane emission does not show the expected behavior with water table. The lack of water table position dependence also contrasts with the spatial variation across microtopography. There is no systematic variation in sub-diurnal time scale. The general seasonal cycle in methane emission is captured well with the methane model. We will show how well the model reproduces the temperature and water table position dependencies observed. The annual methane emission is typically around 10 gC m-2. This is a significant part of the total carbon exchange between the fen and the atmosphere and about twice the estimated carbon loss by leaching from the fen area. The inter-annual variability in the methane emission is modest. The June-September methane emissions from different years, comprising most of the annual emission, correlates positively with peat temperature, but not with

  11. Development of a high-throughput fluorescence polarization DNA cleavage assay for the identification of FEN1 inhibitors.

    PubMed

    McWhirter, Claire; Tonge, Michael; Plant, Helen; Hardern, Ian; Nissink, Willem; Durant, Stephen T

    2013-06-01

    Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5' overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z' factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

  12. Astrochemistry of transition metals? The selected cases of [FeN](+), [FeNH](+) and [(CO)2FeN](+): pathways toward CH3NH2 and HNCO.

    PubMed

    Fioroni, Marco

    2014-11-28

    Transition metals (TMs) are proposed to play a role in astrophysical environments in both gas and solid state astrochemistry by co-determining the homogeneous/heterogeneous chemistry represented by the gas-gas and gas-dust grain interactions. Their chemistry is a function of temperature, radiation field and chemical composition/coordination sphere and as a consequence, dependent on the astrophysical object in which TMs are localized. Here five main categories of TM compounds are proposed and classified as: (a) pure bulk and clusters; (b) TM naked ions; (c) TM oxides/minerals or inorganic compounds; (d) TM-L (L = ligand) with L = (σ and/or π)-donor/acceptor species like H/H2, N/N2, CO, and H2O and (e) TM-organoligands such as Cp, PAH, and R1=˙=˙=R2. Each of the classes is correlated to their possible localization within astrophysical objects. Because of this variety coupled with their ability to modulate reactivity and regio/enantioselectivity by ligand sphere composition, TM compounds can introduce a fine organic synthesis in astrochemistry. For the selection of small TM parental compounds to be analyzed as first examples, by constraining the TMs and the second element/molecule on the basis of their cosmic abundance and mutual reactivity, Fe atoms coupled with N and CO are studied by developing the chemistry of [FeN](+), [FeNH](+) and [(CO)2FeN](+). These molecules, due to their ability to perform C-C and C-H bond activation, are able to open the pathway toward nitrogenation/amination and carbonylation of organic substrates. By considering the simplest organic substrate CH4, the parental reaction schemes (gas phase, T = 30 K): (I) [FeN](+) + CH4 + H → [Fe](+) + H3C-NH2; (II) [FeNH](+) + CH4 → [Fe](+) + H3C-NH2 and (III) [(CO)2FeN](+) + H → [FeCO](+) + HNCO are analyzed by theoretical methods (B2PLYP double hybrid functional/TZVPPP basis set). All reactions are thermodynamically favored and first step transition states can follow a minimal energy path by

  13. Growth characteristics, nutrient allocation and photosynthesis ofCarex species from floating fens.

    PubMed

    Konings, H; Koot, E; Wolf, A T

    1989-03-01

    The purpose of this study was to investigate various growth parameters, dry matter and nitrogen, phosphorus and potassium allocation and photosynthesis ofCarex acutiformis, C. rostrata andC. diandra growing in fens with, in this order, decreasing nutrient availability and decreasing aboveground productivity. Plants were grown from cuttings at optimum nutrient conditions in a growth chamber. Growth analysis at sequential harvests revealed that the species had no inherently different relative growth rates which could explain their different productivity, but that their LAR (LWR and SLA) decreased in the orderC. acutiformis, C. rostrata, C. diandra and their NAR increased in this order. All growth parameters decreased during plant growth even under the controlled conditions of the experiment.C. acutiformis allocated relatively much dry matter to the leaves,C. rostrata to the rhizomes andC. diandra to the roots. This may, in part, explain the higher aboveground biomass production ofC. acutiformis in the field. Nitrogen, but not phosphorus and potassium, allocation patterns were different for the three species.C. diandra, the species from the nitrogen-poorest site, had the highest leaf N content of the three species and also a higher chlorophyll content. Related to this, this species had the highest photosynthetic activity of whole plants both when collected from the field and when grown in the growth chamber. The nitrogen productivity was similar for the three species and the photosynthetic nitrogen use efficiency, determined forC. acutiformis andC. diandra, was similar for these two species.C. diandra had the most finely branched root system, i.e., the highest specific root length of the three species and its root surface area to leaf surface area ratio was also the highest. All three species showed higher nitrate reductase activity in the leaves than in the roots when grown on nutrient solution. The growth ofC. diandra at a relatively nutrient-poor site and a rather

  14. Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum.

    PubMed

    Uribe, Juber Herrera; Collado-Romero, Melania; Zaldívar-López, Sara; Arce, Cristina; Bautista, Rocío; Carvajal, Ana; Cirera, Susanna; Claros, M Gonzalo; Garrido, Juan J

    2016-01-07

    Infected pork meat is an important source of non-typhoidal human salmonellosis. Understanding of molecular mechanisms involved in disease pathogenesis is important for the development of therapeutic and preventive strategies. Thus, hereby we study the transcriptional profiles along the porcine intestine during infection with Salmonella Typhimurium, as well as post-transcriptional gene modulation by microRNAs (miRNA). Sixteen piglets were orally challenged with S. Typhimurium. Samples from jejunum, ileum and colon, collected 1, 2 and 6 days post infection (dpi) were hybridized to mRNA and miRNA expression microarrays and analyzed. Jejunum showed a reduced transcriptional response indicating mild inflammation only at 2 dpi. In ileum inflammatory genes were overexpressed (e.g., IL-1B, IL-6, IL-8, IL1RAP, TNFα), indicating a strong immune response at all times of infection. Infection also down-regulated genes of the FXR pathway (e.g., NR1H4, FABP6, APOA1, SLC10A2), indicating disruption of the bile acid absorption in ileum. This result was confirmed by decreased high-density lipoprotein cholesterol in serum of infected pigs. Ileal inflammatory gene expression changes peaked at 2 dpi and tended to resolve at 6 dpi. Furthermore, miRNA analysis of ileum at 2 dpi revealed 62 miRNAs potentially regulating target genes involved in this inflammatory process (e.g., miR-374 and miR-451). In colon, genes involved in epithelial adherence, proliferation and cellular reorganization were down-regulated at 2 and 6 dpi. In summary, here we show the transcriptional changes occurring at the intestine at different time points of the infection, which are mainly related to inflammation and disruption of the bile acid metabolism.

  15. Cross sections of the 56Fe(n ,α ) 53Cr and 54Fe(n ,α ) 51Cr reactions in the MeV region

    NASA Astrophysics Data System (ADS)

    Wang, Zhimin; Fan, Xiao; Zhang, Luyu; Bai, Huaiyong; Chen, Jinxiang; Zhang, Guohui; Gledenov, Yu. M.; Sedysheva, M. V.; Krupa, L.; Khuukhenkhuu, G.

    2015-10-01

    Cross sections of the 56Fe(n ,α ) 53Cr and 54Fe(n ,α )51Cr reactions were measured at En=5.5 and 6.5 MeV and En=4.0 ,4.5 ,5.5 ,and 6.5 MeV , respectively, using a double-section gridded ionization chamber as the α -particle detector. Natural iron and enriched 56Fe and 54Fe foil samples were prepared. A deuterium gas target was used to produce monoenergetic neutrons through the 2H(d ,n )3He reaction. Two rounds of experiments were performed at the 4.5-MV Van de Graaff Accelerator of Peking University. The foreground and background were measured in separate runs. The neutron flux was monitored by a B F3 long counter, and the cross sections of the 238U(n ,f ) reaction were used as the standard. Present results are compared with those of the talys-1.6 code calculations, existing measurements, and evaluations.

  16. Intra-specific diet shift in manila clams (Ruditapes philippinarum) as revealed by carbon and nitrogen stable isotopes and fatty acid biomarker

    NASA Astrophysics Data System (ADS)

    Suh, Y.; Shin, K.

    2011-12-01

    Manila clams sampled in Seonjae Island, Korea with shell lengths (SL) below 19.76 mm in average showed a significantly depleted carbon and nitrogen isotope values (P<0.05) by 0.80~1.41 %. This size related variation can be caused by either altered carbon and nutrient source or by affected isotopic incorporation rates and discrimination factors. In order to examine size-related diet shift in manila clams, R. philippinarum with different sizes that were constantly fed on known mixed microalgae for several months were sampled from Incheon Fisheries Hacheries Research Institute (IFRI). These manila clams have shown a high intra-species variation in growth rate with a maximum difference of more or less 2.30 cm. The smallest size groups (3.68±0.17 mm and 6.88±0.21 mm) obtained their nutrition from both P. tricornutum and aggregated organic matter that consists of dead or decomposed microalgae or other detritus. Bigger size groups (10.92±0.34 mm and 14.81±0.25 mm) obtained most of their energy from P.tricorutum and also from other phytoplankton unlike the biggest size group (21.15±1.02 mm) that feeds mainly on fresh microalgae of all diets fed. This variation in diet reveals that smaller clams mostly inhale dead or decomposed microalgae that sinks on the bottom while the bigger clams uptake more fresh ones that are still alive. This variation in feeding behavior could have been caused by morphological constraints such as limited siphon length. The results suggest that manila clams greater than and below 19.76 mm in average have different feeding behavior and P. tricornutum and I. galbana were the two most preferred diets for manila clams cultured in IFHRI. The result of fatty acid composition of manila clams in relation to size or growth rate suggests that fast growing clams would have rapid metabolism of fatty acids not required by the animals and an accumulation of the essential fatty acids (PUFA). In addition, their higher energy requirement and more active state

  17. Promoter analyses and transcriptional profiling of eggplant polyphenol oxidase 1 gene (SmePPO1) reveal differential response to exogenous methyl jasmonate and salicylic acid.

    PubMed

    Shetty, Santoshkumar M; Chandrashekar, Arun; Venkatesh, Yeldur P

    2012-05-01

    The transcriptional regulation of multigenic eggplant (Solanum melongena) polyphenol oxidase genes (SmePPO) is orchestrated by their corresponding promoters which mediate developmentally regulated expression in response to myriad biotic and abiotic factors. However, information on structural features of SmePPO promoters and modulation of their expression by plant defense signals are lacking. In the present study, SmePPOPROMOTERs were cloned by genome walking, and their transcription start sites (TSS) were determined by RLM-RACE. Extensive sequence analyses revealed the presence of evolutionarily conserved and over-represented putative cis-acting elements involved in light-regulated transcription, biosynthetic pathways (phenylpropanoid/flavonoid), hormone signaling (abscisic acid, gibberellic acid, jasmonate and salicylate), elicitor and stress responses (cold/dehydration responses), sugar metabolism and plant defense signaling (W-BOX/WRKY) that are common to SmePPOPROMOTER1 and 2. The TSS for SmePPO genes are located 9-15bp upstream of ATG with variable lengths of 5' untranslated regions. Transcriptional profiling of SmePPOs in eggplant seedlings has indicated differential response to methyl jasmonate (MeJA) or salicylic acid (SA) treatment. In planta, while MeJA elicited expression of all the six SmePPOs, SA was only able to induce the expression of SmePPO4-6. Interestingly, in dual treatment, SA considerably repressed the MeJA-induced expression of SmePPOs. Functional dissection of SmePPOPROMOTER1 by deletion analyses using Agrobacterium-mediated transient expression in tobacco leaves has shown that MeJA enhances the SmePPOPROMOTER1-β-glucuronidase (GUS) expression in vivo, while SA does not. Histochemical and quantitative GUS assays have also indicated the negative effect of SA on MeJA-induced expression of SmePPOPROMOTER1. By combining in silico analyses, transcriptional profiling and expression of SmePPOPROMOTER1-GUS fusions, the role of SA on the modulation

  18. Novel Insights Into The Mode of Inhibition of Class A SHV-1 Beta-Lactamases Revealed by Boronic Acid Transition State Inhibitors

    SciTech Connect

    W Ke; J Sampson; C Ori; F Prati; S Drawz; C Bethel; R Bonomo; F van den Akker

    2011-12-31

    Boronic acid transition state inhibitors (BATSIs) are potent class A and C {beta}-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 {beta}-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 {beta}-lactamase with micromolar affinity that is considerably weaker than their inhibition of other {beta}-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other {beta}-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

  19. Geologic and geomorphic controls on the occurrence of fens in the Oregon Cascades and implications for vulnerability and conservation

    USGS Publications Warehouse

    Aldous, A.; Gannett, Marshall W.; Keith, Mackenzie K.; O'Connor, James E.

    2015-01-01

    Montane fens are biologically diverse peat-forming wetlands that develop at points of groundwater discharge. To protect these ecosystems, it is critical to understand their locations on the landscape and the hydrogeologic systems that support them. The upper Deschutes Basin has a groundwater flow system that supports baseflow in many rivers, but little is known about the wetland types and groundwater dependence of the thousands of wetlands within the watershed. In 292 randomly selected wetlands, we quantified landscape metrics thought useful for discriminating montane fens from non-peat-forming wetlands. We inspected these wetlands and classified 67 of them as fens. Of the landscape metrics, only geology reliably differentiated fens from other types of wetlands. Nearly all fens develop in low-permeability glacial till found at approximately 1400–1800 m in elevation, and are concentrated in areas mantled by pumice deposits that originated primarily from the eruption of Mt. Mazama approximately 7700 years BP. Stratigraphic and hydrologic factors indicate the fens are supplied by perched aquifers in glacial till, instead of the deeper regional aquifer system. Their hydrogeologic setting makes the fens highly vulnerable to expected changes to recharge associated with climate change, but not to groundwater pumping from the regional aquifer.

  20. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

    PubMed Central

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J.; Chang, Ta-Chau

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm−1) and lipid (~2845 cm−1) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans. PMID:27535493

  1. Suberoylanilide Hydroxamic Acid Treatment Reveals Crosstalks among Proteome, Ubiquitylome and Acetylome in Non-Small Cell Lung Cancer A549 Cell Line

    PubMed Central

    Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

    2015-01-01

    Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level. PMID:25825284

  2. Impaired Acid Catalysis by Mutation of a Protein Loop Hinge Residue in a YopH Mutant Revealed by Crystal Structures

    SciTech Connect

    Brandao, T.; Robinson, H; Johnson, S; Hengge, A

    2009-01-01

    Catalysis by the Yersinia protein-tyrosine phosphatase YopH is significantly impaired by the mutation of the conserved Trp354 residue to Phe. Though not a catalytic residue, this Trp is a hinge residue in a conserved flexible loop (the WPD-loop) that must close during catalysis. To learn why this seemingly conservative mutation reduces catalysis by 2 orders of magnitude, we have solved high-resolution crystal structures for the W354F YopH in the absence and in the presence of tungstate and vanadate. Oxyanion binding to the P-loop in W354F is analogous to that observed in the native enzyme. However, the WPD-loop in the presence of oxyanions assumes a half-closed conformation, in contrast to the fully closed state observed in structures of the native enzyme. This observation provides an explanation for the impaired general acid catalysis observed in kinetic experiments with Trp mutants. A 1.4 Angstroms structure of the W354F mutant obtained in the presence of vanadate reveals an unusual divanadate species with a cyclic [VO]2 core, which has precedent in small molecules but has not been previously reported in a protein crystal structure.

  3. Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes.

    PubMed

    McCammon, Mark T; Epstein, Charles B; Przybyla-Zawislak, Beata; McAlister-Henn, Lee; Butow, Ronald A

    2003-03-01

    To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in alpha-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth-enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in alpha-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.

  4. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

    NASA Astrophysics Data System (ADS)

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J.; Chang, Ta-Chau

    2016-08-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm‑1) and lipid (~2845 cm‑1) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans.

  5. Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line.

    PubMed

    Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

    2015-03-31

    Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level.

  6. Combined Transcriptomics and Chemical-Genetics Reveal Molecular Mode of Action of Valproic acid, an Anticancer Molecule using Budding Yeast Model

    PubMed Central

    Golla, Upendarrao; Joseph, Deepthi; Tomar, Raghuvir Singh

    2016-01-01

    Valproic acid (VA) is a pharmacologically important histone deacetylase inhibitor that recently garnered attention as an anticancer agent. Since the molecular mechanisms behind the multiple effects of VA are unclear, this study was aimed to unravel the comprehensive cellular processes affected by VA and its molecular targets in vivo using budding yeast as a model organism. Interestingly, genome-wide transcriptome analysis of cells treated with VA showed differential regulation of 30% of the genome. Functional enrichment analysis of VA transcriptome evidenced alteration of various cellular processes including cell cycle, cell wall biogenesis, DNA repair, ion homeostasis, metabolism, stress response, transport and ribosomal biogenesis, etc. Moreover, our genetic screening analysis revealed VA molecular targets belonging to oxidative and osmotic stress, DNA repair, cell wall integrity, and iron homeostasis. Further, our results demonstrated the activation of mitogen-activated protein kinases (MAPKs) Hog1 (p38) and Slt2 (p44/42) upon VA treatment. Our results also exhibited that VA acts through alteration of mitochondrial, ER architecture and functions. Especially, VA effects were neutralized in cells lacking lipid particles. Altogether, our results deciphered the novel molecular insights and mechanistic links to strengthen our knowledge on diverse cellular effects of VA along with its probable therapeutic targets and detoxification approaches. PMID:27734932

  7. Quantitative proteomic analysis to decipher the differential apoptotic response of bortezomib-treated APL cells before and after retinoic acid differentiation reveals involvement of protein toxicity mechanisms.

    PubMed

    Uttenweiler-Joseph, Sandrine; Bouyssié, David; Calligaris, David; Lutz, Pierre G; Monsarrat, Bernard; Burlet-Schiltz, Odile

    2013-01-01

    The ubiquitin-proteasome system allows the targeted degradation of proteins and plays a critical role in the regulation of many cellular processes. Proteasome inhibition is a recent antitumor therapeutic strategy and bortezomib was the first proteasome inhibitor approved for clinical use. In this study, we used the NB4 cell line to investigate the effects of bortezomib toward acute promyelocytic leukemia cells before and after retinoic acid-induced differentiation. We showed that apoptosis level after bortezomib treatment is higher in NB4 cells than in differentiated NB4 cells. To compare early protein variations upon bortezomib treatment in both NB4 cell populations, we performed a quantitative proteomic analysis based on iTRAQ peptide labeling followed by data analysis with in-house developed scripts. This strategy revealed the regulation of 14 proteins principally involved in protein stress response and apoptosis in NB4 cells after proteasome inhibition. Altogether, our results suggest that the differential level of apoptosis induced by bortezomib treatment in both NB4 cell populations could result from distinct protein toxicity level.

  8. Microarray Analysis Reveals Higher Gestational Folic Acid Alters Expression of Genes in the Cerebellum of Mice Offspring—A Pilot Study

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Chadman, Kathryn K.; Brown, W. Ted; Junaid, Mohammed A.

    2015-01-01

    Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA) in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development. PMID:25629700

  9. CH4 emissions from two floodplain fens of differing nutrient status

    NASA Astrophysics Data System (ADS)

    Stanley, Kieran; Heppell, Catherine; Belyea, Lisa; Baird, Andrew

    2014-05-01

    Floodplain fens emit large amounts of CH4 in comparison with ombrotrophic bogs. Little is known about the effect of fluvial nitrogen (N) and phosphorus (P) on CH4 dynamics in fens, although N and P affect carbon (C) dynamics indirectly in other environments by controlling plant growth and root exudate release, as well as by altering microbial biomass and decomposition rates. This study aimed to compare CH4 emissions from two floodplain fen sites which differ in nutrient status, Sutton Fen (52°45'N 001°30'E) and Strumpshaw Fen (52°36'N 001°27'E), in the Norfolk Broadland of England. Sutton and Strumpshaw Fen are under conservation management and both sites have water levels that vary within a few decimetres above and below the surface. The sites are dominated by reed (Phragmites australis). Areas within the fens where the reed was cut in 2009 were chosen for this study. Average plant height and mean aboveground biomass were significantly greater at Strumpshaw (107.2 ± 7.8 cm and 1578 ± 169 g m-2, respectively) than Sutton (56.5 ± 5.1 cm and 435 ± 42 g m-2) as were mean foliar N and P contents (21.8 ± 1.5 g kg-1 and 2.0 ± 0.2 g kg-1 at Strumpshaw, versus 16.3 ± 1.5 g kg-1 and 1.1 ± 0.1 g kg-1 at Sutton). Foliar NPK ratios showed Strumpshaw to be N limited, whereas Sutton was both N and P limited, depending on microsite. Surface peat N and P contents were also greater at Strumpshaw (28.3 ± 0.35 g kg-1 and 0.78 ± 0.02 g kg-1, respectively) than Sutton (18.32 ± 0.87 g kg-1 and 0.43 ± 0.1 g kg-1). These results indicate clear differences in nutrient status between the two sites despite their geographical proximity and other similarities. CH4 emissions were monitored monthly between 19th June 2012 and 2nd September 2013 using tall static chambers and glass funnel-traps, the latter for ebullition. Steady fluxes did not follow a clear seasonal pattern; however, emission was greatest in the summer months. Strumpshaw had a greater range in efflux (0.25 to 134

  10. Interacting partners of FEN1 and its role in the development of anticancer therapeutics.

    PubMed

    Kathera, Chandrasekhar; Zhang, Jing; Janardhan, Avilala; Sun, Hongfang; Ali, Wajid; Zhou, Xiaolong; He, Lingfeng; Guo, Zhigang

    2017-02-07

    Protein-protein interaction (PPI) plays a key role in cellular communication, Protein-protein interaction connected with each other with hubs and nods involved in signaling pathways. These interactions used to develop network based biomarkers for early diagnosis of cancer. FEN1(Flap endonuclease 1) is a central component in cellular metabolism, over expression and decrease of FEN1 levels may cause cancer, these regulation changes of Flap endonuclease 1reported in many cancer cells, to consider this data may needs to develop a network based biomarker. The current review focused on types of PPI, based on nature, detection methods and its role in cancer. Interacting partners of Flap endonuclease 1 role in DNA replication repair and development of anticancer therapeutics based on Protein-protein interaction data.

  11. BOREAS TF-11 SSA-Fen 1996 Water Surface Film Capping Data

    NASA Technical Reports Server (NTRS)

    Billesbach, David P.; Hall, Forrest G. (Editor); Knapp, David E. (Editor)

    2000-01-01

    The BOREAS TF-11 team gathered a variety of data to complement its tower flux measurements collected at the SSA-Fen site. The data described in this document were made by the TF-11 team at the SSA-Fen site to quantify the effect that the films observed to form on open water surfaces had on the transfer of carbon dioxide and methane from the water to the air. Measurements of fluxes of carbon dioxide and methane were made in 1994 and in 1996 using the chamber flux method. A gas chromatograph and a LI-COR LI-6200 were used to measure concentrations and to calculate the fluxes. The data are stored in tabular ASCII files.

  12. BOREAS TF-11 CO2 and CH4 Flux Data from the SSA-Fen

    NASA Technical Reports Server (NTRS)

    Valentine, David W.; Hall, Forrest G. (Editor); Conrad, Sara (Editor)

    2000-01-01

    The BOREAS TF-11 team collected several data sets in its efforts to fully describe the flux and site characteristics at the SSA-Fen site. This data set contains fluxes of methane and carbon dioxide at the SSA-Fen site measured using static chambers. The measurements were conducted as part of a 2 x 2 factorial experiment in which we added carbon (300 g/sq m as wheat straw) and nitrogen (6 g/sq m as urea) to four replicate locations in the vicinity of the TF-11 tower. In addition to siting and treatment variables, it reports air temperature and water table height relative to the average peat surface during each measurement. The data set covers the period from the first week of June 1994 through the second week of September 1994. The data are stored in tabular ASCII files.

  13. BOREAS TF-11 SSA-Fen 1995 Leaf Area Index Data

    NASA Technical Reports Server (NTRS)

    Arkebauer, Timothy J.; Hall, Forrest G. (Editor); Knapp, David E. (Editor)

    2000-01-01

    The BOREAS TF-11 team gathered a variety of data to complement its tower flux measurements collected at the SSA-Fen site. These data are LAI measurements made by the TF-11 team throughout the 1995 growing season. The data include the LAI of plants that fall into six categories: total, Carex spp., Betula pumila, Menyanthes trifoliata, Salix spp., and other vascular plants. The data are stored in tabular ASCII files.

  14. Field Observations and Numerical Modeling of the Thermal Effects of Groundwater Flow Through a Subarctic Fen

    NASA Astrophysics Data System (ADS)

    Sjöberg, Y.; Coon, E.; Sannel, B.; Pannetier, R.; Harp, D. R.; Frampton, A.; Painter, S. L.; Lyon, S. W.

    2015-12-01

    Field observations and numerical modeling of ground temperatures are the main tools for understanding current and projecting future permafrost changes in the rapidly warming Arctic. Traditionally, most studies have focused on vertical fluxes of heat through the ground. Groundwater can transport heat in both lateral and vertical directions but its influence on ground temperatures at local scales in permafrost environments is not well understood. In this study field observations from a subarctic fen located within the sporadic permafrost zone are combined with numerical simulations for investigating coupled water and thermal fluxes. Ground temperature profiles and groundwater levels were observed in boreholes at the Tavvavuoma study site in northern Sweden. Based on these observations, one- and two-dimensional simulations down to 2 m depth across a gradient of permafrost conditions both within and surrounding the fen, were set up. To quantify the influence of groundwater flows on the ground temperature, two-dimensional scenarios representing the fen under various groundwater fluxes were developed. The observations suggest that lateral groundwater flows significantly affect ground temperatures. This is corroborated by modeling results that show seasonal ground ice melts 1 month earlier when a lateral groundwater flux is present. Further, although the thermal regime may be dominated by vertically conducted heat fluxes during most of the year, isolated high groundwater flow events can be potentially important for ground temperatures. Sporadic permafrost environments contain substantial portions of unfrozen ground, often with active groundwater flow paths such as fens. Knowledge of this heat transport mechanism is therefore important for understanding permafrost dynamics in these environments.

  15. Methane dynamics in a montane fen: Factors controlling production, accumulation and emissions

    NASA Astrophysics Data System (ADS)

    Mwakanyamale, K. E.; Yeung, H.; Strack, M.

    2014-12-01

    Characterization of methane dynamics in peatlands is essential to improve understanding of peatlands contribution to carbon balance and interaction with climate. Of the two peatland types, natural fens are known to be a larger contributor of methane emissions to the atmosphere than natural bogs. This study uses geophysical methods integrated with in-situ direct measurements and chamber fluxes to improve understanding of temporal and spatial variation in methane production, accumulation and emissions from natural montane fen in Alberta Canada. Meteorological data and peat cores (~150 cm) were collected to study factors affecting methane production, accumulation and emissions from the Sibbald Research Wetland, a montane fen in the Rocky Mountains in southern Alberta. Our results show a direct correlation between methane accumulation and degree of peat humification, substrate quality and porosity. Changes in temperature, pressure and water table position were shown to relate to ebullition events, with the highest number of ebullition events occurring from late August to early November. The geophysical results indicate a small spatial variation in free phase biogenic gas accumulation within the studied area. Diffusive methane fluxes were correlated to plant productivity on both daily and seasonal time scales with patterns varying between plots dominated by Juncus sp. and Carex spp. These results highlight the interacting ecological and physical controls on peatland methane dynamics.

  16. The role of peat double porosity on groundwater-surface water interaction in a drained fen

    NASA Astrophysics Data System (ADS)

    Rossi, P. M.; Ala-aho, P.; Ronkanen, A.-K.

    2012-04-01

    Groundwater discharge from an esker aquifer to a fen was studied to understand relevant hydrological processes for surface-groundwater interaction in an esker-peatland hillslope. Piezometric levels of the peat layer and esker sand layer were continuously monitored and compared to climate data. Groundwater exfiltration points were spatially mapped and related to peat depth. The study showed a clear interaction between fen surface water and sand esker groundwater although the hydraulic conductivity of peat was low and the peat depth thick. In the artesian aquifer beneath the fen, the piezometric head showed a clear response to precipitation and evapotranspiration. Wetting of the peat is rapidly seen as pressure increase in the confined aquifer. This provides new information on hydraulics of wetlands found in aquifer discharge areas. Groundwater exfiltrated through thick peat layers in vertical preferential point discharges demonstrating how double porosity provides flow through the peat layer with low hydraulic conductivity in the peat matrix. The "pipe flow" channels were found in the deep peat area in the transition zones from deep peat to shallow peat. Diffuse seepage through the ditch bottom was found where ditches cut into the sand layer. Increased discharge through the ditch bed reduced the flow resistance at the aquifer boundary which can lower the groundwater level in the esker. The results show new processes in the groundwater discharge zone that are useful for groundwater modeling and policy development for future groundwater protection.

  17. Groundwater-surface water interaction between an esker aquifer and a drained fen

    NASA Astrophysics Data System (ADS)

    Rossi, Pekka M.; Ala-aho, Pertti; Ronkanen, Anna-Kaisa; Kløve, Bjørn

    2012-04-01

    SummaryGroundwater discharge from an esker aquifer to a fen was studied to understand relevant hydrological processes for groundwater-surface water interaction in an esker-peatland hillslope. Piezometric levels of the peat layer and esker sand layer were continuously monitored and compared to climate data. Groundwater exfiltration points were spatially mapped and related to peat depth. The study showed a clear interaction between fen surface water and sand esker groundwater although the hydraulic conductivity of peat was low and the peat depth thick. In the artesian aquifer beneath the fen, the piezometric head showed a clear response to precipitation and evapotranspiration. Wetting of the peat is rapidly seen as pressure increase in the confined aquifer. This provides new information on hydraulics of wetlands found in aquifer discharge areas. Groundwater exfiltrated through thick peat layers in vertical preferential point discharges demonstrating how double porosity provides flow through the peat layer with low hydraulic conductivity in the peat matrix. The "pipe flow" channels were found in the deep peat area in the transition zones from deep peat to shallow peat. Diffuse seepage through the ditch bottom was found where ditches cut into the sand layer. Increased discharge through the ditch bed reduced the flow resistance at the aquifer boundary which can lower the groundwater level in the esker. The results show new processes in the groundwater discharge zone that are useful for groundwater modelling and policy development for future groundwater protection.

  18. Evaluating cumulative effects of disturbance on the hydrologic function of bogs, fens, and mires

    SciTech Connect

    Siegel, D.I.

    1988-01-01

    Few quantitative studies have been done on the hydrology of fens, bogs, and mires and, consequently, any predictions of the cumulative impacts of disturbances on their hydrologic functions is extremely difficult. Bogs and fens are, in a sense, hydrobiologic systems, and any evaluation of cumulative impacts on them will have to consider the complicated interactions, barely understood, among the wetland hydrology, water chemistry, and biota, and place the effect of individual wetland impacts within the context of the cumulative impacts contributed to the watershed from other geomorphic areas and land uses. It is difficult to evaluate the potential cumulative impacts on wetland hydrology because geologic settings of wetlands are often complex and the methods used to measure wetland stream flow, ground-water flow, and evapotranspiration are inexact. Their very scale makes it difficult to quantify the hydrologic function accurately. The paper reviews current understanding of the hydrologic function of bogs, fens, and mires at different scales and in different physiographic settings, and presents hypotheses on potential cumulative impacts on the hydrologic function that might occur with multiple disturbances.

  19. Metabolomics analysis reveals elevation of 3-indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: Investigation of nicotinic acid receptor agonists

    SciTech Connect

    Zgoda-Pols, Joanna R.; Chowdhury, Swapan; Wirth, Mark; Milburn, Michael V.; Alexander, Danny C.; Alton, Kevin B.

    2011-08-15

    An investigative renal toxicity study using metabolomics was conducted with a potent nicotinic acid receptor (NAR) agonist, SCH 900424. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were used to identify small molecule biomarkers of acute kidney injury (AKI) that could aid in a better mechanistic understanding of SCH 900424-induced AKI in mice. The metabolomics study revealed 3-indoxyl sulfate (3IS) as a more sensitive marker of SCH 900424-induced renal toxicity than creatinine or urea. An LC-MS assay for quantitative determination of 3IS in mouse matrices was also developed. Following treatment with SCH 900424, 3IS levels were markedly increased in murine plasma and brain, thereby potentially contributing to renal- and central nervous system (CNS)-related rapid onset of toxicities. Furthermore, significant decrease in urinary excretion of 3IS in those animals due to compromised renal function may be associated with the elevation of 3IS in plasma and brain. These data suggest that 3IS has a potential to be a marker of renal and CNS toxicities during chemically-induced AKI in mice. In addition, based on the metabolomic analysis other statistically significant plasma markers including p-cresol-sulfate and tryptophan catabolites (kynurenate, kynurenine, 3-indole-lactate) might be of toxicological importance but have not been studied in detail. This comprehensive approach that includes untargeted metabolomic and targeted bioanalytical sample analyses could be used to investigate toxicity of other compounds that pose preclinical or clinical development challenges in a pharmaceutical discovery and development. - Research Highlights: > Nicotinic acid receptor agonist, SCH 900424, caused acute kidney injury in mice. > MS-based metabolomics was conducted to identify potential small molecule markers of renal toxicity. > 3-indoxyl-sulfate was found to be as a more sensitive marker of renal toxicity than creatinine

  20. Wastewater contaminant transport and treatment in a nutrient limited ribbed fen

    NASA Astrophysics Data System (ADS)

    McCarter, C. P. R.; Price, J. S.; Branfireun, B. A.

    2015-12-01

    To minimize the discharge of wastewater contaminants from remote northern communities and mining operations, fen peatlands in sub-arctic regions are used for tertiary wastewater treatment to detain, transform, and remove these contaminants. However, there is a limited understanding of contaminant transport and treatment in fen peatlands, particularly in sub-arctic Canada. To better characterize wastewater contaminant transport and treatment in these systems, approximately 44 m3 day-1 of simulated wastewater, concentrated custom-blend fertilizer (NO3-, PO33-, and SO42-) and Cl- diluted with water, was pumped into a small 0.5 ha sub-arctic ribbed fen continuously for 47 days (July 15th -August 31st 2014). Contaminant concentration of 3 similar ribbed fens varied between 0.0-3.0 mg L-1 over the study period (May - September 2014). An exponential increase in transmissivity (2.4 to 16.8 m2 day-1) as the water table rose (~0.16 m) increased the average linear groundwater velocity (0.5 to 3.4 m day-1) and resulted in rapid SO42- (0.8 m day-1) and Cl- (1.9 m day-1) transport. Notwithstanding the rapid transport of Cl-, diffusion into inactive pores still retarded Cl- transport by a factor of 1.8. Contrary to the rapid transport of SO42- and Cl-, the other contaminants were rapidly removed from the pore water (likely through biological uptake or adsorption) and minimal transport was observed (0.29 and 0.04 m day-1 for PO33- and NO3-, respectively). Northern ribbed fens have a large capacity to detain certain wastewater contaminants (e.g., NO3- and PO33-), yet allow rapid transport of others (e.g., SO42- and Cl-). Thus, these peatlands have the potential to significantly decrease wastewater contamination in northern aquatic environment by both biogeochemical and physical processes but careful management of the hydrology is required to prevent the release of mobile contaminants.

  1. Dynamics of peat accumulation and marl flat formation in a calcareous fen, midwestern United States

    USGS Publications Warehouse

    Miner, J.J.; Ketterling, D.B.

    2003-01-01

    The age and sequence of peat accumulation were investigated at a calcareous fen in northeastern Illinois, USA. The purpose of this study was to identify the processes that form and sustain marl flats, which are areas of marl or tufa substrate within the fen that contain numerous rare plant species. Geomorphic, stratigraphic, and radiocarbon evidence was used to establish the processes and chronology of peat accumulation and erosion adjacent to each marl flat. The age of the base of the peat deposit varies greatly throughout the fen, ranging from 14,679 calibrated years before present (cal. years BP) to nearly modern, indicating that colonization of the sand and gravel substrate by peat occurred throughout the period from the Late Pleistocene to present. Adjacent to one marl flat, trends in basal peat age and thickness show that peat accumulation has progressed laterally inward from both sides, suggesting that the marl flat has been infilling with peat progressively by accumulation at the margins since at least 5,370 cal. years BP or longer. A second marl flat in the fen is surrounded by older, thick peat of differing ages on either edge and is bounded by fresh scarps, indicating that the marl flat currently is expanding laterally by erosion into the preexisting peat blanket. These two examples suggest a continuously repeating process, where erosion of the accumulated peat blanket forms a marl flat, which is later covered by peat accumulation. Trends in basal peat age elsewhere in the fen suggest that other marl flats may have existed in the past that have been completely infilled with peat. This study suggests that marl flat formation is a natural process that has been occurring for millennia, continuously creating habitat for the rare plant species that occupy marl flats. There is no evidence that the marl flats at this site are indicative of anthropogenic disturbance, so that management options for these areas are limited to maintaining the quality and quantity

  2. Adsorption and self-assembly of octyl hydroxamic acid at a fluorite surface as revealed by sum-frequency vibrational spectroscopy.

    PubMed

    Wang, Xuming; Liu, Jin; Miller, Jan D

    2008-09-15

    In the study described here, the surface structure of a self-assembly octyl hydroxamic acid at a calcium fluoride (CaF(2)) surface is evaluated using sum-frequency vibrational spectroscopy (SFVS). Of particular significance are the results that show octyl hydroxamic acid adsorbs at the fluorite surface from octanol solution and has more ordering and molecular conformation than the octyl hydroxamic acid adsorbed from solution. At the fluorite/0.1 M octyl hydroxamic acid octanol solution interface a bilayer-like structure consisting of an octyl hydroxamic acid layer in contact with fluorite and a tilted alcohol layer was observed by SFVS. The alcohol molecules are oriented with respect to the hydroxamic acid monolayer with the OH groups directed towards the bulk alcohol phase and the terminal CH(3) group oriented to face the alkyl chains of the hydroxamic acid monolayer.

  3. Mutagenesis of hetR reveals amino acids necessary for HetR function in the heterocystous cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Risser, Douglas D; Callahan, Sean M

    2007-03-01

    HetR is the master regulator of heterocyst differentiation in the filamentous cyanobacterium Anabaena sp. strain PCC 7120. Genetic selection was used to identify 33 amino acid substitutions in HetR that reduced the proportion of cells undergoing heterocyst differentiation to less than 2%. Conservative substitutions in the wild-type HetR protein revealed three mutations that dramatically reduced the amount of heterocyst differentiation when the mutant allele was present in place of the wild-type allele on a replicating plasmid in a mutant lacking hetR on the chromosome. An H69Y substitution resulted in heterocyst formation among less than 0.1% of cells, and D17E and G36A substitutions resulted in a Het- phenotype, compared to heterocyst formation among approximately 25% of cells with the wild-type hetR under the same conditions. The D17E substitution prevented DNA binding activity exhibited by wild-type HetR in mobility shift assays, whereas G36A and H69Y substitutions had no affect on DNA binding. D17E, G36A, and H69Y substitutions also resulted in higher levels of the corresponding HetR protein than of the wild-type protein when each was expressed from an inducible promoter in a hetR deletion strain, suggesting an effect on HetR protein turnover. Surprisingly, C48A and S152A substitutions, which were previously reported to result in a Het- phenotype, were found to have no effect on heterocyst differentiation or patterning when the corresponding mutations were introduced into an otherwise wild-type genetic background in Anabaena sp. strain PCC 7120. The clustering of mutations that satisfied the positive selection near the amino terminus suggests an important role for this part of the protein in HetR function.

  4. High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach*

    PubMed Central

    Schlessinger, Avner; Wittwer, Matthias B.; Dahlin, Amber; Khuri, Natalia; Bonomi, Massimiliano; Fan, Hao; Giacomini, Kathleen M.; Sali, Andrej

    2012-01-01

    The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABAA). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families. PMID:22932902

  5. Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex

    PubMed Central

    Liu, Songbai; Lu, Guojun; Ali, Shafat; Liu, Wenpeng; Zheng, Li; Dai, Huifang; Li, Hongzhi; Xu, Hong; Hua, Yuejin; Zhou, Yajing; Ortega, Janice; Li, Guo-Min; Kunkel, Thomas A; Shen, Binghui

    2015-01-01

    During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5′ ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism. PMID:25921062

  6. Identification of a Lipoteichoic Acid Glycosyltransferase Enzyme Reveals that GW-Domain-Containing Proteins Can Be Retained in the Cell Wall of Listeria monocytogenes in the Absence of Lipoteichoic Acid or Its Modifications

    PubMed Central

    Percy, Matthew G.; Karinou, Eleni; Webb, Alexander J.

    2016-01-01

    ABSTRACT Listeria monocytogenes is a foodborne Gram-positive bacterial pathogen, and many of its virulence factors are either secreted proteins or proteins covalently or noncovalently attached to the cell wall. Previous work has indicated that noncovalently attached proteins with GW (glycine-tryptophan) domains are retained in the cell wall by binding to the cell wall polymer lipoteichoic acid (LTA). LTA is a glycerol phosphate polymer, which is modified in L. monocytogenes with galactose and d-alanine residues. We identified Lmo0933 as the cytoplasmic glycosyltransferase required for the LTA glycosylation process and renamed the protein GtlA, for glycosyltransferase LTA A. Using L. monocytogenes mutants lacking galactose or d-alanine modifications or the complete LTA polymer, we show that GW domain proteins are retained within the cell wall, indicating that other cell wall polymers are involved in the retention of GW domain proteins. Further experiments revealed peptidoglycan as the binding receptor as a purified GW domain fusion protein can bind to L. monocytogenes cells lacking wall teichoic acid (WTA) as well as purified peptidoglycan derived from a wild-type or WTA-negative strain. With this, we not only identify the first enzyme involved in the LTA glycosylation process, but we also provide new insight into the binding mechanism of noncovalently attached cell wall proteins. IMPORTANCE Over the past 20 years, a large number of bacterial genome sequences have become available. Computational approaches are used for the genome annotation and identification of genes and encoded proteins. However, the function of many proteins is still unknown and often cannot be predicted bioinformatically. Here, we show that the previously uncharacterized Listeria monocytogenes gene lmo0933 likely codes for a glycosyltransferase required for the decoration of the cell wall polymer lipoteichoic acid (LTA) with galactose residues. Using L. monocytogenes mutants lacking LTA

  7. Could Poor Fens BE More Sensitive than Bogs to Elevated N Deposition in the Oil Sands Region of Northern Alberta?

    NASA Astrophysics Data System (ADS)

    Wieder, R. K.; Vile, M. A.; Scott, K. D.

    2015-12-01

    Bogs and fens cover 29% of the 140,000 km2 Oil Sands Administrative Area (OSAA) in northern Alberta, a region characterized by quite low background N deposition (1-2 kg/ha/yr). However, development of the oil sands resource has led to increasing emission of nitrogen oxides, which are then returned to regional ecosystems as elevated atmospheric N deposition. Given the nutrient deficient nature of bogs and poor fens, elevated N deposition from oil sands development could potentially affect peatland ecosystem structure and function. To evaluate the ecosystem-level effects of N deposition, since 2011, we have experimentally applied N to a bog and a poor fen near Mariana Lakes, Alberta, located far enough from the OSAA to be unaffected by oil sands emissions. Treatments include simulated rainfall equivalent to N deposition of 0, 5, 10, 15, 20, and 25 kg/ha/yr, plus control plots receiving no added water (3 replicate plots per site per N treatment). Concentrations of NH4+-N, NO3- N, and DON at the top of the peatland water table did not increase with increasing N deposition, averaging 0.61, 0.09, and 1.07 mg/L, respectively, in the bog, and 0.53, 0.10, and 0.81 mg/L, respectively, in the poor fen. Ericaceous shrub abundance increased with increasing N deposition in both the bog and the poor fen, although plot-scale greenness (hand-held spectral measurement of the Normalized Difference Red Edge (NDRE) index) increased with N deposition in the poor fen, but not in the bog. Segmented regression indicated that in the poor fen, at N deposition above 14-16 kg/ha/yr, total microbial, bacterial, and fungal biomass in the top 5 cm of peat increased with N deposition, with no effect at lower N deposition. No effect of N deposition on microbial, bacterial, or fungal biomass was observed at 5-10 cm in the poor fen, or at either 0-5 or 5-10 cm in the bog. In the poor fen, microbial, bacterial, and fungal biomass increased with NDRE, but the effect was not significant in the bog

  8. Integrated Systems Biology Analysis of Transcriptomes Reveals Candidate Genes for Acidity Control in Developing Fruits of Sweet Orange (Citrus sinensis L. Osbeck)

    PubMed Central

    Huang, Dingquan; Zhao, Yihong; Cao, Minghao; Qiao, Liang; Zheng, Zhi-Liang

    2016-01-01

    Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). Fruits of these varieties at 45 days post anthesis (DPA), which corresponds to Stage I (cell division), had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion). Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis). Our network analysis shows that the acid-correlated genes belong to three distinct network modules. Several of these candidate fruit acidity genes encode regulatory proteins involved in transport (such as AHA10), degradation (such as APD2) and transcription (such as AIL6) and act as hubs in the citrate accumulation gene networks. Taken together, our integrated systems biology analysis has provided new insights into the fruit citrate accumulation gene network and led to the identification of candidate genes likely associated with the fruit acidity control. PMID:27092171

  9. Integrated Systems Biology Analysis of Transcriptomes Reveals Candidate Genes for Acidity Control in Developing Fruits of Sweet Orange (Citrus sinensis L. Osbeck).

    PubMed

    Huang, Dingquan; Zhao, Yihong; Cao, Minghao; Qiao, Liang; Zheng, Zhi-Liang

    2016-01-01

    Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). Fruits of these varieties at 45 days post anthesis (DPA), which corresponds to Stage I (cell division), had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion). Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis). Our network analysis shows that the acid-correlated genes belong to three distinct network modules. Several of these candidate fruit acidity genes encode regulatory proteins involved in transport (such as AHA10), degradation (such as APD2) and transcription (such as AIL6) and act as hubs in the citrate accumulation gene networks. Taken together, our integrated systems biology analysis has provided new insights into the fruit citrate accumulation gene network and led to the identification of candidate genes likely associated with the fruit acidity control.

  10. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  11. Characterization of heterotrophic nitrifying bacteria with respiratory ammonification and denitrification activity--description of Paenibacillus uliginis sp. nov., an inhabitant of fen peat soil and Paenibacillus purispatii sp. nov., isolated from a spacecraft assembly clean room.

    PubMed

    Behrendt, Undine; Schumann, Peter; Stieglmeier, Michaela; Pukall, Rüdiger; Augustin, Jürgen; Spröer, Cathrin; Schwendner, Petra; Moissl-Eichinger, Christine; Ulrich, Andreas

    2010-10-01

    In the course of studying the influence of N-fertilization on N(2) and N(2)O flux rates in relation to soil bacterial community composition of a long-term fertilization experiment in fen peat grassland, a strain group was isolated that was related to a strain isolated from a spacecraft assembly clean room during diversity studies of microorganisms, which withstood cleaning and bioburden reduction strategies. Both the fen soil isolates and the clean room strain revealed versatile physiological capacities in N-transformation processes by performing heterotrophic nitrification, respiratory ammonification and denitrification activity. Phylogenetic analysis based on 16S rRNA gene sequences demonstrated that the investigated isolates belonged to the genus Paenibacillus. Sequence similarities lower than 97% in comparison to established species indicated a separate species position. Except for the peptidoglycan type (A4alpha L-Lys-D-Asp), chemotaxonomic features of the isolates matched the genus description, but differences in several physiological characteristics separated them from related species and supported their novel species status. Despite a high 16S rRNA gene sequence similarity between the clean room isolate ES_MS17(T) and the representative fen soil isolate N3/975(T), DNA-DNA hybridization studies revealed genetic differences at the species level. These differences were substantiated by MALDI-TOF MS analysis, ribotyping and several distinct physiological characteristics. On the basis of these results, it was concluded that the fen soil isolates and the clean room isolate ES_MS17(T) represented two novel species for which the names Paenibacillus uliginis sp. nov. (type strain N3/975(T)=DSM 21861(T)=LMG 24790(T)) and Paenibacillus purispatii sp. nov. (type strain ES_MS17(T)=DSM 22991(T)=CIP 110057(T)) are proposed.

  12. CO2 and CH4 exchange from ditch networks in two floodplain fens.

    NASA Astrophysics Data System (ADS)

    Stanley, Kieran; Heppell, Catherine; Belyea, Lisa; Baird, Andrew

    2014-05-01

    Historically, many peatlands were drained using ditches. These ditches are now often used to control water levels in sites under conservation management, though they can be a significant atmospheric source of both CO2 and CH4. Studies have already been undertaken on ditches in ombrotrophic peatlands (bogs), but little work has been done on C exchanges in drainage ditches in floodplain fens. This study quantified C exchange in drainage ditches at two floodplain fens, Sutton Fen (52°45'N 001°30'E) and Strumpshaw Fen (52°36'N 001°27'E), in The Norfolk Broadlands of England. C exchange was quantified between 19th June 2012 and 2nd September 2013 using floating static chambers. Ecosystem respiration (Reco), net ecosystem exchange (NEE) and CH4 fluxes were measured and a seasonal pattern was observed in all three, with the highest fluxes in late summer. Both sites were sources of CO2 and CH4. The few previous studies of C fluxes from ditches in floodplain fens have suggested flux ranges of 69.6 to 199 mg CO2 m2 h-1 for both Reco and NEE, and 1.2 to 366 mg CH4 m2 h-1 (positive means a flux to the atmosphere). For Reco, NEE and CH4, our values were generally at the higher end of the reported values. Reco ranged from 3.66 to 420.18 mg CO2 m-2 h-1 and 28.35 to 257.07 mg CO2 m-2 h-1 for Sutton and Strumpshaw, respectively. The highest flux was observed in September 2013 at Strumpshaw (506.03 mg CO2 m-2 h-1). NEE ranged from -80.64 to 786.2 mg CO2 m-2 h-1 and -70.72 to 229.75 mg CO2 m-2 h-1 for Sutton and Strumpshaw, respectively. The majority of NEE fluxes were positive (a source), apart from April, June and July 2013, where a small uptake occurred. Sutton had significantly greater CH4 emissions than Strumpshaw, with values ranging from 0.46 to 733.72 and 0.05 to 89.11 mg CH4 m-2 h-1, respectively. This difference may have been related to ditch water SO42- concentrations, which were higher at Strumpshaw (23.11 ± 1.26 mg L-1) than at Sutton (17.78 ± 1.68 mg L-1

  13. Seasonal Trace Gas Dynamics on Minerotrophic Fen Peatlands in NE-Germany

    NASA Astrophysics Data System (ADS)

    Giebels, Michael; Beyer, Madlen; Augustin, Jürgen; Minke, Merten; Juszczak, Radoszlav; Serba, Tomasz

    2010-05-01

    In Germany more than 99 % of fens have lost their carbon and nutrient sink function due to heavy drainage and agricultural land use especially during the last decades and thus resulted in compression and heavy peat loss (CHARMAN 2002; JOOSTEN & CLARKE 2002; SUCCOW & JOOSTEN 2001; AUGUSTIN et al. 1996; KUNTZE 1993). Therefore fen peatlands play an important part (4-5 %) in the national anthropogenic trace gas budget. But only a small part of drained and agricultural used fens in NE Germany can be restored. Knowledge of the influence of land use to trace gas exchange is important for mitigation of the climate impact of the anthropogenic peatland use. We study carbon exchanges of several fen peatland use areas between soil and atmosphere at different sites in NE-Germany. Our research covers peatlands of supposed strongly climate forcing land use (cornfield and intensive pasture) and of probably less forcing, alternative types (meadow and extensive pasture) as well as rewetted (formerly drained) areas and near-natural sites like a low-degraded fen and a wetted alder woodland. We measured trace gas fluxes with manual and automatic chambers in periodic routines since spring 2007. The used chamber technique bases on DROESLER (2005). In total we now do research at 22 sites situated in 5 different locations covering agricultural, varying states of rewetted and near-natural treatments. We present results of at least 2 years of measurements and show significant differences in their annual trace gas balances depending on the genesis of the observed sites and the seasonal dynamics. Crosswise comparison of different site treatments combined with the seasonal environmental observations give good hints for the identification of main flux driving parameters. That is that a reduced intensity in land use as a supposed mitigating treatment did not show the expected effect, though a normal meadow treatment surprisingly resulted in the lowest balances in both years. For implementing a

  14. Seasonal Carbon Dynamics on Selected Fen Peatland Sites in NE-Germany

    NASA Astrophysics Data System (ADS)

    Giebels, Michael; Beyer, Madlen; Augustin, Jürgen; Minke, Merten; Juszczak, Radoszlav; Serba, Tomasz

    2010-05-01

    In Germany more than 99 % of fens have lost their carbon and nutrient sink function due to heavy drainage and agricultural land use especially during the last decades and thus resulted in compression and heavy peat loss (CHARMAN 2002; JOOSTEN & CLARKE 2002; SUCCOW & JOOSTEN 2001; AUGUSTIN et al. 1996; KUNTZE 1993). Therefore fen peatlands play an important part (4-5 %) in the national anthropogenic trace gas budget. But only a small part of drained and agricultural used fens in NE Germany can be restored. Knowledge of the influence of land use to trace gas exchange is important for mitigation of the climate impact of the anthropogenic peatland use. We study carbon exchanges of several fen peatland use areas between soil and atmosphere at different sites in NE-Germany. Our research covers peatlands of supposed strongly climate forcing land use (cornfield and intensive pasture) and of probably less forcing, alternative types (meadow and extensive pasture) as well as rewetted (formerly drained) areas and near-natural sites like a low-degraded fen and a wetted alder woodland. We measured trace gas fluxes with manual and automatic chambers in periodic routines since spring 2007. The used chamber technique bases on DROESLER (2005). In total we now do research at 22 sites situated in 5 different locations covering agricultural, varying states of rewetted and near-natural treatments. We present results of at least 2 years of measurements and show significant differences in their annual carbon balances depending on the genesis of the observed sites and the seasonal dynamics. Crosswise comparison of different site treatments combined with the seasonal environmental observations give good hints for the identification of main flux driving parameters. That is that a reduced intensity in land use as a supposed mitigating treatment did not show the expected effect, though a normal meadow treatment surprisingly resulted in the lowest CO2 balances in both years. For implementing a

  15. A season of heat, water vapor, total hydrocarbon, and ozone fluxes at a subarctic fen

    NASA Technical Reports Server (NTRS)

    Moore, Kathleen E.; Fitzjarrald, David R.; Wofsy, Steven C.; Daube, Bruce C.; Munger, J. William; Bakwin, Peter S.; Crill, Patrick

    1994-01-01

    High-latitude environments are thought to play several critical roles in the global balance of radiatively active trace gases. Adequate documentation of the source and sink strengths for trace gases requires long time series of detailed measurements, including heat and moisture budgets. A fen near Schefferville, Quebec, was instrumented during the summer of 1990 for the measurement of the surface energy, radiation, and moisture balances as well as for eddy correlation estimates of ozone and methane flux. Despite the limited fetch at this site, analysis of the tower flux 'footprint' indicates that at least 80% of the flux observed originates from sources within the fen. Sensible heat fluxes averaged 25% of the daytime net radiation at the site, while the latent heat flux, determined from the energy balance, was 63%; the Bowen ratio varied from 0.2 to 0.8 from day to day, without a seasonal trend to the variation. The competing effects of rooted macrophyte development (with concomitant effects on roughness and transpiration) and the normal shift in synoptic pattern around day 200 to warm, dry conditions results in a lack of net seasonal effect on the energy partitioning. Over the period from days 170 to 230, the evaporation (167 mm) was double the rainfall, while the decline in water level was 107 mm, leaving a net runoff of 0.44 mm/d. The total hydrocarbon flux was 75-120 mg m(exp -2)/d, following a diurnal pattern similar to heat or moisture flux, while the daytime ozone flux was about -1.11 x 10(exp 11) molecules cm(exp -2)/s. A period near the end of the experiment, during week 30, produced the strongest total hydrocarbon flux, associated with warmer deep (1 m) soil temperatures, lower fen water levels, and the late summer shift in wind direction at that time. An early summer 'flush' of total hydrocarbon was not observed.

  16. Mosses influence phosphorus cycling in rich fens by driving redox conditions in shallow soils.

    PubMed

    Crowley, Katherine F; Bedford, Barbara L

    2011-09-01

    Mosses play an integral role in the hydrologic regimes of ecosystems where they cover the soil surface, and thus affect biogeochemical cycling of elements influenced by soil oxidation-reduction (redox) reactions, including the plant growth-limiting nutrients, nitrogen and phosphorus (P). In rich fens where P often limits plant growth, we hypothesized that feedbacks between mosses and redox conditions would determine P availability to shallow-rooted forb species that constitute much of these wetlands' unusually high plant species diversity. In a moss removal experiment in three fens, forb tissue P and microbial P were greater while anion exchange membrane (AEM) resin P was lower where mosses occurred than where they were removed, suggesting both higher availability and greater demand for P in moss-covered soils. Coupled physicochemical and biological mechanisms drove moss effects on P cycling, ultimately through effects on soil oxygenation or reduction: higher redox potential underlying mosses corresponded to greater microbial activity, phosphatase enzyme activity, and colonization by arbuscular mycorrhizal fungi (AMF), all of which can promote greater P availability to plants. These more oxidized soils stimulated: (1) greater microbial activity and root vigor; (2) correspondingly greater P demand via microbial uptake, forb uptake, and iron (Fe)-P reactions; and (3) greater P supply through soil and root phosphatase activity and AMF colonization. This work demonstrates that mosses improve vascular plant P acquisition by alleviating stresses caused by reducing conditions that would otherwise prevail in shallow underlying soils, thus providing a mechanism by which mosses facilitate plant species diversity in rich fens.

  17. Evaluation on the Efficiency of Subsurface Drainage in Chiu-Fen Landslide at Northern Taiwan

    NASA Astrophysics Data System (ADS)

    Ying, L. Y.; Lin, D. G.

    2015-12-01

    For administrative district, the Chiu-Fen landslide is situated at northern Taiwan and comes within the jurisdiction of Ruei-Fang district, New Taipei City Government. Chiu-Fen village is a famous spot for sightseeing and tourism in Southeast Asia. In the last decade, for economic purpose, a vast area of slope land in Chiu-Fen area was reclaimed into business and commercial districts. However, due to the complicated geological and hydrological conditions, improper reclamation, and lack of appropriate soil and water conservation facilities, large scale landslides are frequently triggered by typhoon rainfall and causes damages to the transportation and residential building in the community. As a consequence, the government initiated a comprehensive field investigations and remediation plans to stabilize the landslide from 1997 and the remediation works were concentrated on subsurface drainages, namely the application of drainage well (a vertical shaft with multi-level horizontal drainage boreholes). To investigate the efficiency of drainage wells on the landslide, the A1-profile in the landslide which covers the drainage wells W2 and W4 was selected for a series of rainfall seepage and slope stability analyses. In addition, a 48-hrs design rainfall with return period of 25, 50 and 100 years based on the local meteorological data bank was adopted for the analyses. The numerical results indicate the factor safety FS of the three potential sliding surfaces within A1-profile are constantly keeping greater than one (FS > 1.0) and without decreasing with the elapsed time during rainfall. This implies that the subsurface drainage works can drain off the infiltrated rainwater from a high intensity and long duration rainfall and preserve the slope stability of landslides from deterioration. Finally, the efficiency of the drainage wells can be evaluated quantitatively in terms of the time-dependent factor of safety and the pore water pressure distribution on several potential

  18. BOREAS TGB-3 Plant Species Composition Data over the NSA Fen

    NASA Technical Reports Server (NTRS)

    Bubier, Jill L.; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor)

    2000-01-01

    The BOReal Ecosystem-Atmosphere Study Trace Gas Biogeochemistry (BOREAS TGB-3) team collected several data sets that contributed to understanding the measured trace gas fluxes over sites in the Northern Study Area (NSA). This data set contains information about the composition of plant species that were within the collars used to measure Net Ecosystem Exchange of CO2 (NEE). The species composition was identified to understand the differences in NEE among the various plant communities in the NSA fen. The data were collected in July of 1994 and 1996. The data are contained in comma-delimited, ASCII files.

  19. BOREAS TGB-1/TGB-3 NEE Data over the NSA Fen

    NASA Technical Reports Server (NTRS)

    Bellisario, Lianne; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor); Moore, Tim R.

    2000-01-01

    The BOReal Ecosystem-Atmosphere Study Trace Gas Biogeochemistry (BOREAS TGB-1) and TGB-3 teams collected several data sets that contributed to understanding the measured trace gas fluxes over sites in the Northern Study Area (NSA). This data set contains Net Ecosystem Exchange of CO2 (NEE) measurements collected with chambers at the NSA fen in 1994 and 1996. Gas samples were extracted approximately every 7 days from chambers and analyzed at the NSA lab facility. The data are provided in tabular ASCII files.

  20. Structure and specificity of FEN-1 from Methanopyrus kandleri

    SciTech Connect

    Shah, Santosh; Dunten, Pete; Stiteler, Amanda; Park, Chad K.; Horton, Nancy C.

    2014-11-18

    DNA repair is fundamental to genome stability and is found in all three domains of life. However, many archaeal species, such as Methanopyrus kandleri, contain only a subset of the eukaryotic nucleotide excision repair (NER) homologues, and those present often contain significant differences compared to their eukaryotic homologues. To clarify the role of the NER XPG-like protein Mk0566 from M. kandleri, its biochemical activity and three dimensional structure were investigated. Ultimately, we found both to be more similar to human FEN-1 than human XPG, suggesting a biological role in replication and long-patch base excision repair rather than in NER.

  1. In Silico and Wet Lab Studies Reveal the Cholesterol Lowering Efficacy of Lauric Acid, a Medium Chain Fat of Coconut Oil.

    PubMed

    Lekshmi Sheela, Devi; Nazeem, Puthiyaveetil Abdulla; Narayanankutty, Arunaksharan; Manalil, Jeksy Jos; Raghavamenon, Achuthan C

    2016-12-01

    The coconut oil (CO) contains 91 % of saturated fatty acids in which 72 % are medium chain fatty acids (MCFAs) like lauric, capric and caprylic acids. In contrast to animal fat, coconut oil has no cholesterol. Despite this fact, CO is sidelined among other vegetable oils due to the health hazards attributed to the saturated fatty acids. Though various medicinal effects of CO have been reported including the hypolipidemic activity, people are still confused in the consumption of this natural oil. In silico analyses and wet lab experiments have been carried out to identify the hypolipidemic properties of MCFAs and phenolic acids in CO by using different protein targets involved in cholesterol synthesis. The molecular docking studies were carried out using CDOCKER protocol in Accelery's Discovery Studio, by taking different proteins like HMG- CoA reductase and cholesterol esterase as targets and the different phytocompounds in coconut as ligands. Molecular docking highlighted the potential of lauric acid in inhibiting the protein targets involved in hyperlipidemics. Further, validation of in silico results was carried out through in vivo studies. The activity of key enzymes HMG- CoA reductase and lipoprotein lipase were found reduced in animals fed with lauric acid and CO.

  2. Stable isotope resolved metabolomics revealed the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in Amaranthus palmeri biotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using stable isotope resolved metabolomics (SIRM), we characterized the role of anabolic (de novo synthesis) vs catabolic (protein catalysis) processes contributing to free amino acid pools in glyphosate susceptible (S) and resistant (R) Amaranthus palmeri biotypes. Following exposure to glyphosate ...

  3. Genetic analysis of two OsLpa1-like genes in Arabidopsis reveals that only one is required for wild-type seed phytic acid levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytic acid (inositol-1,2,3,4,5,6-hexakisphosphate or InsP6) is the primary storage form of phosphorus in plant seeds. The rice OsLpa1 encodes a novel protein required for wild-type levels of seed InsP6 and was identified from a low phytic acid (lpa) mutant exhibiting a 45-50% reduction in seed InsP...

  4. Comparison and evaluation on image fusion methods for GaoFen-1 imagery

    NASA Astrophysics Data System (ADS)

    Zhang, Ningyu; Zhao, Junqing; Zhang, Ling

    2016-10-01

    Currently, there are many research works focusing on the best fusion method suitable for satellite images of SPOT, QuickBird, Landsat and so on, but only a few of them discuss the application of GaoFen-1 satellite images. This paper proposes a novel idea by using four fusion methods, such as principal component analysis transform, Brovey transform, hue-saturation-value transform, and Gram-Schmidt transform, from the perspective of keeping the original image spectral information. The experimental results showed that the transformed images by the four fusion methods not only retain high spatial resolution on panchromatic band but also have the abundant spectral information. Through comparison and evaluation, the integration of Brovey transform is better, but the color fidelity is not the premium. The brightness and color distortion in hue saturation-value transformed image is the largest. Principal component analysis transform did a good job in color fidelity, but its clarity still need improvement. Gram-Schmidt transform works best in color fidelity, and the edge of the vegetation is the most obvious, the fused image sharpness is higher than that of principal component analysis. Brovey transform, is suitable for distinguishing the Gram-Schmidt transform, and the most appropriate for GaoFen-1 satellite image in vegetation and non-vegetation area. In brief, different fusion methods have different advantages in image quality and class extraction, and should be used according to the actual application information and image fusion algorithm.

  5. Effect of land cover data on nitrous oxide inventory in fen meadows.

    PubMed

    Nol, Linda; Verburg, Peter H; Heuvelink, Gerard B M; Molenaar, Karin

    2008-01-01

    Landscape representations based on land cover databases differ significantly from the real landscape. Using a land cover database with high uncertainty as input for emission inventory analyses can cause propagation of systematic and random errors. The objective of this study was to analyze how different land cover representations introduce systematic errors into the results of regional N2O emission inventories. Surface areas of grassland, ditches, and ditch banks were estimated for two polders in the Dutch fen meadow landscape using five land cover representations: four commonly used databases and a detailed field map, which most closely resembles the real landscape. These estimated surface areas were scaled up to the Dutch western fen meadow landscape. Based on the estimated surface areas agricultural N2O emissions were estimated using different inventory techniques. All four common databases overestimated the grassland area when compared to the field map. This caused a considerable overestimation of agricultural N2O emissions, ranging from 9% for more detailed databases to 11% for the coarsest database. The effect of poor land cover representation was larger for an inventory method based on a process model than for inventory methods based on simple emission factors. Although the effect of errors in land cover representations may be small compared to the effect of uncertainties in emission factors, these effects are systematic (i.e., cause bias) and do not cancel out by spatial upscaling. Moreover, bias in land cover representations can be quantified or reduced by careful selection of the land cover database.

  6. Atmospheric methane sources - Alaskan tundra bogs, an alpine fen, and a subarctic boreal marsh

    NASA Technical Reports Server (NTRS)

    Sebacher, D. I.; Harriss, R. C.; Grice, S. S.; Bartlett, K. B.; Sebacher, S. M.

    1986-01-01

    Methane (CH4) flux measurements from Alaska tundra bogs, an alpine fen, and a subarctic boreal marsh were obtained at field sites ranging from Prudhoe Bay on the coast of the Arctic Ocean to the Alaskan Range south of Fairbanks during August 1984. In the tundra, average CH4 emission rates varied from 4.9 mg CH4 per sq m per day (moist tundra) to 119 mg CH4 per sq m per day (waterlogged tundra). Fluxes averaged 40 mg CH4 per sq m per day from wet tussock meadows in the Brooks Range and 289 mg Ch4 per sq m per day from an alpine fen in the Alaskan Range. The boreal marsh had an average CH4 emission rate of 106 mg CH4 per sq m per day. Significant emissions were detected in tundra areas where peat temperatures were as low as 4 C, and permafrost was only 25 cm below the ground surface. Emission rates from the 17 sites sampled were found to be logarithmically related to water levels at the sites. Extrapolation of the data to an estimate of the total annual CH4 emission from all arctic and boreal wetlands suggests that these ecosystems are a major source of atmospheric CH4 and could account for up to 23 percent of global CH4 emissions from wetlands.

  7. The Role of Sedges in Methane Production and Emission From a Temperate Fen

    NASA Astrophysics Data System (ADS)

    Noyce, G. L.; Szarkowski, E. R.; Bubier, J. L.; Varner, R. K.

    2008-12-01

    Northern peatlands are the largest single natural source of atmospheric methane (CH4) and thus any changes in these fluxes could have global consequences. Positive correlations have been observed between sedge biomass and high CH4 emissions from peatlands. We examined the effect that Carex rostrata has on methane production and transport in Sallie's Fen, Barrington, NH. During the summer of 2008, CH4 fluxes, pore-water CH4 concentrations, and C. rostrata biomass, along with temperature, net ecosystem CO2 exchange (NEE), and water table depth, were measured from both clipped and unclipped plots. In clipped plots, all C. rostrata were clipped below the peat surface and then sealed with petroleum jelly and plastic bags. Unclipped plots were left undisturbed. We observed a strong positive correlation between C. rostrata biomass and CH4 fluxes. Initial findings suggest that sedges have a strong effect on gas transport. The average 18 cm (C. rostrata rooting depth) pore water CH4 concentrations in the clipped plots (6141 ppm CH4) were significantly higher (p<0.001) than in the unclipped plots (3912 ppm CH4). The past 20 years of data at Sallie's Fen also show that plots with more C. rostrata have higher mid-season CH4 fluxes, on average, than shrub-dominated plots. Depth to water table, however, may be a stronger control on CH4 fluxes than vegetation.

  8. Environmental factors controlling fluxes of dimethyl sulfide in a New Hampshire fen

    NASA Technical Reports Server (NTRS)

    Demello, William Zamboni; Hines, Mark E.

    1992-01-01

    The major environmental factors controlling fluxes of dimethyl sulfide (DMS) in a Sphagnum-dominated peatland were investigated in a poor fen in New Hampshire. DMS emissions from the surface of the peatland varied greatly over 24 hours and seasonally. Maximum DMS emissions occurred in summer with minima in the late fall. Temperature was the major environmental factor controlling these variabilities. There was also some evidence that the changes in water table height might have contributed to the seasonable variability in DMS emission. The influence of the water table was greater during periods of elevated temperature. DMS and MSH were the most abundant dissolved volatile sulfur compound (VSC) in the surface of the water table. Concentrations of dissolved VSC's varied with time and space throughout the fen. Dissolved MDS, MSH, and OCS in the surface of the water table were supersaturated with respect to their concentrations in the atmosphere suggesting that the peat surface was a source of VSC's in the peatland. VCS in peatlands seemed to be produced primarily by microbial processes in the anoxic surface layers of the peat rich in organic matter and inorganic sulfide. Sphagnum mosses were not a direct source of VSC's. However, they increased transport of DMS from the peat surface to the atmosphere.

  9. RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae.

    PubMed

    Dong, Yachen; Hu, Jingjin; Fan, Linlin; Chen, Qihe

    2017-02-17

    As a typical harmful inhibitor in cellulosic hydrolyzates, acetic acid not only hinders bioethanol production, but also induces cell death in Saccharomyces cerevisiae. Herein, we conducted both transcriptomic and metabolomic analyses to investigate the global responses under acetic acid stress at different stages. There were 295 up-regulated and 427 down-regulated genes identified at more than two time points during acetic acid treatment (150 mM, pH 3.0). These differentially expressed genes (DEGs) were mainly involved in intracellular homeostasis, central metabolic pathway, transcription regulation, protein folding and stabilization, ubiquitin-dependent protein catabolic process, vesicle-mediated transport, protein synthesis, MAPK signaling pathways, cell cycle, programmed cell death, etc. The interaction network of all identified DEGs was constructed to speculate the potential regulatory genes and dominant pathways in response to acetic acid. The transcriptional changes were confirmed by metabolic profiles and phenotypic analysis. Acetic acid resulted in severe acidification in both cytosol and mitochondria, which was different from the effect of extracellular pH. Additionally, the imbalance of intracellular acetylation was shown to aggravate cell death under this stress. Overall, this work provides a novel and comprehensive understanding of stress responses and programmed cell death induced by acetic acid in yeast.

  10. Real-Time Tracking of BODIPY-C12 Long-Chain Fatty Acid in Human Term Placenta Reveals Unique Lipid Dynamics in Cytotrophoblast Cells

    PubMed Central

    Louey, Samantha; Varlamov, Oleg; Thornburg, Kent

    2016-01-01

    While the human placenta must provide selected long-chain fatty acids to support the developing fetal brain, little is known about the mechanisms underlying the transport process. We tracked the movement of the fluorescently labeled long-chain fatty acid analogue, BODIPY-C12, across the cell layers of living explants of human term placenta. Although all layers took up the fatty acid, rapid esterification of long-chain fatty acids and incorporation into lipid droplets was exclusive to the inner layer cytotrophoblast cells rather than the expected outer syncytiotrophoblast layer. Cytotrophoblast is a progenitor cell layer previously relegated to a repair role. As isolated cytotrophoblasts differentiated into syncytialized cells in culture, they weakened their lipid processing capacity. Syncytializing cells suppress previously active genes that regulate fatty-acid uptake (SLC27A2/FATP2, FABP4, ACSL5) and lipid metabolism (GPAT3, LPCAT3). We speculate that cytotrophoblast performs a previously unrecognized role in regulating placental fatty acid uptake and metabolism. PMID:27124483

  11. RNA-Seq-based transcriptomic and metabolomic analysis reveal stress responses and programmed cell death induced by acetic acid in Saccharomyces cerevisiae

    PubMed Central

    Dong, Yachen; Hu, Jingjin; Fan, Linlin; Chen, Qihe

    2017-01-01

    As a typical harmful inhibitor in cellulosic hydrolyzates, acetic acid not only hinders bioethanol production, but also induces cell death in Saccharomyces cerevisiae. Herein, we conducted both transcriptomic and metabolomic analyses to investigate the global responses under acetic acid stress at different stages. There were 295 up-regulated and 427 down-regulated genes identified at more than two time points during acetic acid treatment (150 mM, pH 3.0). These differentially expressed genes (DEGs) were mainly involved in intracellular homeostasis, central metabolic pathway, transcription regulation, protein folding and stabilization, ubiquitin-dependent protein catabolic process, vesicle-mediated transport, protein synthesis, MAPK signaling pathways, cell cycle, programmed cell death, etc. The interaction network of all identified DEGs was constructed to speculate the potential regulatory genes and dominant pathways in response to acetic acid. The transcriptional changes were confirmed by metabolic profiles and phenotypic analysis. Acetic acid resulted in severe acidification in both cytosol and mitochondria, which was different from the effect of extracellular pH. Additionally, the imbalance of intracellular acetylation was shown to aggravate cell death under this stress. Overall, this work provides a novel and comprehensive understanding of stress responses and programmed cell death induced by acetic acid in yeast. PMID:28209995

  12. Activity of FEN1 endonuclease on nucleosome substrates is dependent upon DNA sequence but not flap orientation.

    PubMed

    Jagannathan, Indu; Pepenella, Sharon; Hayes, Jeffrey J

    2011-05-20

    We demonstrated previously that human FEN1 endonuclease, an enzyme involved in excising single-stranded DNA flaps that arise during Okazaki fragment processing and base excision repair, cleaves model flap substrates assembled into nucleosomes. Here we explore the effect of flap orientation with respect to the surface of the histone octamer on nucleosome structure and FEN1 activity in vitro. We find that orienting the flap substrate toward the histone octamer does not significantly alter the rotational orientation of two different nucleosome positioning sequences on the surface of the histone octamer but does cause minor perturbation of nucleosome structure. Surprisingly, flaps oriented toward the nucleosome surface are accessible to FEN1 cleavage in nucleosomes containing the Xenopus 5S positioning sequence. In contrast, neither flaps oriented toward nor away from the nucleosome surface are cleaved by the enzyme in nucleosomes containing the high-affinity 601 nucleosome positioning sequence. The data are consistent with a model in which sequence-dependent motility of DNA on the nucleosome is a major determinant of FEN1 activity. The implications of these findings for the activity of FEN1 in vivo are discussed.

  13. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    SciTech Connect

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  14. Altering the Mitochondrial Fatty Acid Synthesis (mtFASII) Pathway Modulates Cellular Metabolic States and Bioactive Lipid Profiles as Revealed by Metabolomic Profiling

    PubMed Central

    Clay, Hayley B.; Parl, Angelika K.; Mitchell, Sabrina L.; Singh, Larry; Bell, Lauren N.; Murdock, Deborah G.

    2016-01-01

    Despite the presence of a cytosolic fatty acid synthesis pathway, mitochondria have retained their own means of creating fatty acids via the mitochondrial fatty acid synthesis (mtFASII) pathway. The reason for its conservation has not yet been elucidated. Therefore, to better understand the role of mtFASII in the cell, we used thin layer chromatography to characterize the contribution of the mtFASII pathway to the fatty acid composition of selected mitochondrial lipids. Next, we performed metabolomic analysis on HeLa cells in which the mtFASII pathway was either hypofunctional (through knockdown of mitochondrial acyl carrier protein, ACP) or hyperfunctional (through overexpression of mitochondrial enoyl-CoA reductase, MECR). Our results indicate that the mtFASII pathway contributes little to the fatty acid composition of mitochondrial lipid species examined. Additionally, loss of mtFASII function results in changes in biochemical pathways suggesting alterations in glucose utilization and redox state. Interestingly, levels of bioactive lipids, including lysophospholipids and sphingolipids, directly correlate with mtFASII function, indicating that mtFASII may be involved in the regulation of bioactive lipid levels. Regulation of bioactive lipid levels by mtFASII implicates the pathway as a mediator of intracellular signaling. PMID:26963735

  15. Quantitative Profiling of Hydroxy Lipid Metabolites in Mouse Organs Reveals Distinct Lipidomic Profiles and Modifications Due to Elevated n-3 Fatty Acid Levels

    PubMed Central

    Chiu, Cheng-Ying; Smyl, Christopher; Dogan, Inci; Rothe, Michael; Weylandt, Karsten-H.

    2017-01-01

    Polyunsaturated fatty acids (PUFA) are precursors of bioactive metabolites and mediators. In this study, the profile of hydroxyeicosatetraenoic (HETE), hydroxyeicosapentaenoic (HEPE) and hydroxydocosahexaenoic (HDHA) acids derived from arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in colon, liver, lung, spleen, muscle, heart and kidney tissue of healthy wildtype mice were characterized, and compared to profiles in organs from transgenic fat-1 mice engineered to express the Caenorhabditis elegans fat-1 gene encoding an n-3 desaturase and thereby with endogenously elevated n-3 PUFA levels. PUFAs were measured using gas chromatography. The lipid metabolites were assayed using LC-MS/MS. AA and DHA were the prominent PUFAs in wildtype and fat-1 mice. EPA levels were low in both groups even though there was a significant increase in fat-1 organs with an up to 12-fold increase in fat-1 spleen and kidney. DHA levels increased by approximately 1.5-fold in fat-1 as compared to wildtype mice. While HETEs remained the same or decreased moderately and HDHAs increased 1- to 3-fold, HEPE formation in fat-1 tissues increased from 8- (muscle) to 44-fold (spleen). These findings indicate distinct profiles of monohydroxy lipid metabolites in different organs and strong utilization of EPA for HEPE formation, by which moderate EPA supplementation might trigger formation of biologically active EPA-derived resolvins. PMID:28165385

  16. N-H Bond Dissociation Enthalpies and Facile H Atom Transfers for Early Intermediates of Fe-N2 and Fe-CN Reductions.

    PubMed

    Rittle, Jonathan; Peters, Jonas C

    2017-03-01

    Fe-mediated biological nitrogen fixation is thought to proceed via either a sequence of proton and electron transfer steps, concerted H atom transfer steps, or some combination thereof. Regardless of the specifics and whether the intimate mechanism for N2-to-NH3 conversion involves a distal pathway, an alternating pathway, or some hybrid of these limiting scenarios, Fe-NxHy intermediates are implicated that feature reactive N-H bonds. Thermodynamic knowledge of the N-H bond strengths of such species is scant, and is especially difficult to obtain for the most reactive early stage candidate intermediates (e.g., Fe-N═NH, Fe═N-NH2, Fe-NH═NH). Such knowledge is essential to considering various mechanistic hypotheses for biological (and synthetic) nitrogen fixation and to the rational design of improved synthetic N2 fixation catalysts. We recently reported several reactive complexes derived from the direct protonation of Fe-N2 and Fe-CN species at the terminal N atom (e.g., Fe═N-NH2, Fe-C≡NH, Fe≡C-NH2). These same Fe-N2 and Fe-CN systems are functionally active for N2-to-NH3 and CN-to-CH4/NH3 conversion, respectively, when subjected to protons and electrons, and hence provide an excellent opportunity for obtaining meaningful N-H bond strength data. We report here a combined synthetic, structural, and spectroscopic/analytic study to estimate the N-H bond strengths of several species of interest. We assess the reactivity profiles of species featuring reactive N-H bonds and estimate their homolytic N-H bond enthalpies (BDEN-H) via redox and acidity titrations. Very low N-H bond dissociation enthalpies, ranging from 65 (Fe-C≡NH) to ≤37 kcal/mol (Fe-N═NH), are determined. The collective data presented herein provide insight into the facile reactivity profiles of early stage protonated Fe-N2 and Fe-CN species.

  17. Electronic structures and magnetic moments of Co{sub 3}FeN thin films grown by molecular beam epitaxy

    SciTech Connect

    Ito, Keita; Sanai, Tatsunori; Yasutomi, Yoko; Toko, Kaoru; Honda, Syuta; Suemasu, Takashi; Zhu, Siyuan; Kimura, Akio; Ueda, Shigenori; Takeda, Yukiharu; Saitoh, Yuji; Imai, Yoji

    2013-12-02

    We evaluated electronic structures and magnetic moments in Co{sub 3}FeN epitaxial films on SrTiO{sub 3}(001). The experimentally obtained hard x-ray photoemission spectra of the Co{sub 3}FeN film have a good agreement with those calculated. Site averaged spin magnetic moments deduced by x-ray magnetic circular dichroism were 1.52 μ{sub B} per Co atom and 2.08 μ{sub B} per Fe atom at 100 K. They are close to those of Co{sub 4}N and Fe{sub 4}N, respectively, implying that the Co and Fe atoms randomly occupy the corner and face-centered sites in the Co{sub 3}FeN unit cell.

  18. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

    PubMed

    Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  19. Overexpression of Human Fatty Acid Transport Protein 2/Very Long Chain Acyl-CoA Synthetase 1 (FATP2/Acsvl1) Reveals Distinct Patterns of Trafficking of Exogenous Fatty Acids

    PubMed Central

    Melton, Elaina M.; Cerny, Ronald L.; DiRusso, Concetta C.; Black, Paul N.

    2014-01-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4hr. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  20. FEN1 −69G>A and +4150G>T polymorphisms and breast cancer risk

    PubMed Central

    Rezaei, Maryam; Hashemi, Mohammad; Sanaei, Sara; Mashhadi, Mohammad Ali; Hashemi, Seyed Mehdi; Bahari, Gholamreza; Taheri, Mohsen

    2016-01-01

    Flap endonuclease 1 (FEN1), a DNA repair protein, is important in preventing carcinogenesis. Two functional germ line variants −69G>A (rs174538) and +4150G>T (rs4246215) in the FEN1 gene have been associated with risk of various types of cancer. The aim of the present study was to evaluate the possible impact of FEN1 polymorphisms on risk of breast cancer (BC) in a sample of Iranian subjects. The FEN1 −69G>A and +4150G>T polymorphisms were analyzed in a case-control study that included 266 BC patients and 225 healthy females. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to genotype the variants. The findings demonstrated that the FEN1 −69G>A and +4150G>T polymorphisms were not associated with BC risk in co-dominant, dominant and recessive inheritance models. The findings indicated that GG/GT, GA/GG and GA/TT genotypes significantly decreased the risk of BC when compared with −69GG/+4150GG. Furthermore, haplotype analysis indicated that −69G/+4150T as well as −69A/+4150G significantly decreased the risk of BC compared with −69G/+4150G. Thus, these findings demonstrated that haplotypes of FEN1 −69G>A and +4150G>T polymorphisms decreased the risk of BC in an Iranian population. Further studies with larger sample sizes and different ethnicities are required to validate the present findings. PMID:27699013

  1. The response of soil organic carbon of a rich fen peatland in interior Alaska to projected climate change.

    PubMed

    Fan, Zhaosheng; David McGuire, Anthony; Turetsky, Merritt R; Harden, Jennifer W; Michael Waddington, James; Kane, Evan S

    2013-02-01

    It is important to understand the fate of carbon in boreal peatland soils in response to climate change because a substantial change in release of this carbon as CO2 and CH4 could influence the climate system. The goal of this research was to synthesize the results of a field water table manipulation experiment conducted in a boreal rich fen into a process-based model to understand how soil organic carbon (SOC) of the rich fen might respond to projected climate change. This model, the peatland version of the dynamic organic soil Terrestrial Ecosystem Model (peatland DOS-TEM), was calibrated with data collected during 2005-2011 from the control treatment of a boreal rich fen in the Alaska Peatland Experiment (APEX). The performance of the model was validated with the experimental data measured from the raised and lowered water-table treatments of APEX during the same period. The model was then applied to simulate future SOC dynamics of the rich fen control site under various CO2 emission scenarios. The results across these emissions scenarios suggest that the rate of SOC sequestration in the rich fen will increase between year 2012 and 2061 because the effects of warming increase heterotrophic respiration less than they increase carbon inputs via production. However, after 2061, the rate of SOC sequestration will be weakened and, as a result, the rich fen will likely become a carbon source to the atmosphere between 2062 and 2099. During this period, the effects of projected warming increase respiration so that it is greater than carbon inputs via production. Although changes in precipitation alone had relatively little effect on the dynamics of SOC, changes in precipitation did interact with warming to influence SOC dynamics for some climate scenarios.

  2. The response of soil organic carbon of a rich fen peatland in interior Alaska to projected climate change

    USGS Publications Warehouse

    Fan, Zhaosheng; McGuire, Anthony David; Turetsky, Merritt R.; Harden, Jennifer W.; Waddington, James Michael; Kane, Evan S.

    2013-01-01

    It is important to understand the fate of carbon in boreal peatland soils in response to climate change because a substantial change in release of this carbon as CO2 and CH4 could influence the climate system. The goal of this research was to synthesize the results of a field water table manipulation experiment conducted in a boreal rich fen into a process-based model to understand how soil organic carbon (SOC) of the rich fen might respond to projected climate change. This model, the peatland version of the dynamic organic soil Terrestrial Ecosystem Model (peatland DOS-TEM), was calibrated with data collected during 2005–2011 from the control treatment of a boreal rich fen in the Alaska Peatland Experiment (APEX). The performance of the model was validated with the experimental data measured from the raised and lowered water-table treatments of APEX during the same period. The model was then applied to simulate future SOC dynamics of the rich fen control site under various CO2 emission scenarios. The results across these emissions scenarios suggest that the rate of SOC sequestration in the rich fen will increase between year 2012 and 2061 because the effects of warming increase heterotrophic respiration less than they increase carbon inputs via production. However, after 2061, the rate of SOC sequestration will be weakened and, as a result, the rich fen will likely become a carbon source to the atmosphere between 2062 and 2099. During this period, the effects of projected warming increase respiration so that it is greater than carbon inputs via production. Although changes in precipitation alone had relatively little effect on the dynamics of SOC, changes in precipitation did interact with warming to influence SOC dynamics for some climate scenarios.

  3. Novel properties of the wheat aluminum tolerance organic acid transporter (TaALMT1) revealed by electrophysiological characterization in Xenopus oocytes: Functional and structural implications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many plant species avoid the phytotoxic effects of aluminum (Al) by exuding organic acid anions which chelate Al3+ and prevent its entry into the root. Several novel genes that encode membrane transporters from the ALMT and MATE families have recently been cloned and implicated in mediating the orga...

  4. High-Quality Draft Genome Sequence of Kallotenue papyrolyticum JKG1T Reveals Broad Heterotrophic Capacity Focused on Carbohydrate and Amino Acid Metabolism.

    PubMed

    Hedlund, Brian P; Murugapiran, Senthil K; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Stamatis, Dimitrios; Reddy, T B K; Ngan, Chew Yee; Daum, Chris; Duffy, Kecia; Shapiro, Nicole; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Williams, Amanda J; Cole, Jessica K; Dodsworth, Jeremy A; Woyke, Tanja

    2015-12-03

    The draft genome of Kallotenue papyrolyticum JKG1(T), a member of the order Kallotenuales, class Chloroflexia, consists of 4,475,263 bp in 4 contigs and encodes 4,010 predicted genes, 49 tRNA-encoding genes, and 3 rRNA operons. The genome is consistent with a heterotrophic lifestyle including catabolism of polysaccharides and amino acids.

  5. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-02

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

  6. NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

    PubMed

    D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

    2009-01-01

    The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

  7. Investigations on dendrimer space reveal solid and liquid tumor growth-inhibition by original phosphorus-based dendrimers and the corresponding monomers and dendrons with ethacrynic acid motifs.

    PubMed

    El Brahmi, Nabil; Mignani, Serge M; Caron, Joachim; El Kazzouli, Saïd; Bousmina, Mosto M; Caminade, Anne-Marie; Cresteil, Thierry; Majoral, Jean-Pierre

    2015-03-07

    The well-known reactive diuretic ethacrynic acid (EA, Edecrin), with low antiproliferative activities, was chemically modified and grafted onto phosphorus dendrimers and the corresponding simple branched phosphorus dendron-like derivatives affording novel nanodevices showing moderate to strong antiproliferative activities against liquid and solid tumor cell lines, respectively.

  8. Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport.

    PubMed

    Ribardo, Deborah A; Hendrixson, David R

    2011-11-01

    Campylobacter jejuni is a leading cause of diarrheal disease in humans and an intestinal commensal in poultry and other agriculturally important animals. These zoonotic infections result in significant amounts of C. jejuni present in the food supply to contribute to disease in humans. We previously found that a transposon insertion in Cjj81176_1038, encoding a homolog of the Escherichia coli LivJ periplasmic binding protein of the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system, reduced the commensal colonization capacity of C. jejuni 81-176 in chicks. Cjj81176_1038 is the first gene of a six-gene locus that encodes homologous components of the E. coli LIV system. By analyzing mutants with in-frame deletions of individual genes or pairs of genes, we found that this system constitutes a LIV transport system in C. jejuni responsible for a high level of leucine acquisition and, to a lesser extent, isoleucine and valine acquisition. Despite each LIV protein being required for branched-chain amino acid transport, only the LivJ and LivK periplasmic binding proteins were required for wild-type levels of commensal colonization of chicks. All LIV permease and ATPase components were dispensable for in vivo growth. These results suggest that the biological functions of LivJ and LivK for colonization are more complex than previously hypothesized and extend beyond a role for binding and acquiring branched-chain amino acids during commensalism. In contrast to other studies indicating a requirement and utilization of other specific amino acids for colonization, acquisition of branched-chain amino acids does not appear to be a determinant for C. jejuni during commensalism.

  9. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts.

  10. Integration of Genome-Wide SNP Data and Gene-Expression Profiles Reveals Six Novel Loci and Regulatory Mechanisms for Amino Acids and Acylcarnitines in Whole Blood

    PubMed Central

    Beutner, Frank; Holdt, Lesca M.; Gross, Arnd; Teren, Andrej; Tönjes, Anke; Becker, Susen; Krohn, Knut; Kovacs, Peter; Stumvoll, Michael; Teupser, Daniel; Thiery, Joachim; Ceglarek, Uta; Scholz, Markus

    2015-01-01

    Profiling amino acids and acylcarnitines in whole blood spots is a powerful tool in the laboratory diagnosis of several inborn errors of metabolism. Emerging data suggests that altered blood levels of amino acids and acylcarnitines are also associated with common metabolic diseases in adults. Thus, the identification of common genetic determinants for blood metabolites might shed light on pathways contributing to human physiology and common diseases. We applied a targeted mass-spectrometry-based method to analyze whole blood concentrations of 96 amino acids, acylcarnitines and pathway associated metabolite ratios in a Central European cohort of 2,107 adults and performed genome-wide association (GWA) to identify genetic modifiers of metabolite concentrations. We discovered and replicated six novel loci associated with blood levels of total acylcarnitine, arginine (both on chromosome 6; rs12210538, rs17657775), propionylcarnitine (chromosome 10; rs12779637), 2-hydroxyisovalerylcarnitine (chromosome 21; rs1571700), stearoylcarnitine (chromosome 1; rs3811444), and aspartic acid traits (chromosome 8; rs750472). Based on an integrative analysis of expression quantitative trait loci in blood mononuclear cells and correlations between gene expressions and metabolite levels, we provide evidence for putative causative genes: SLC22A16 for total acylcarnitines, ARG1 for arginine, HLCS for 2-hydroxyisovalerylcarnitine, JAM3 for stearoylcarnitine via a trans-effect at chromosome 1, and PPP1R16A for aspartic acid traits. Further, we report replication and provide additional functional evidence for ten loci that have previously been published for metabolites measured in plasma, serum or urine. In conclusion, our integrative analysis of SNP, gene-expression and metabolite data points to novel genetic factors that may be involved in the regulation of human metabolism. At several loci, we provide evidence for metabolite regulation via gene-expression and observed overlaps with GWAS

  11. Domain Mapping of Heat Shock Protein 70 Reveals That Glutamic Acid 446 and Arginine 447 Are Critical for Regulating Superoxide Dismutase 2 Function.

    PubMed

    Afolayan, Adeleye J; Alexander, Maxwell; Holme, Rebecca L; Michalkiewicz, Teresa; Rana, Ujala; Teng, Ru-Jeng; Zemanovic, Sara; Sahoo, Daisy; Pritchard, Kirkwood A; Konduri, Girija G

    2017-02-10

    Stress-inducible heat shock protein 70 (hsp70) interacts with superoxide dismutase 2 (SOD2) in the cytosol after synthesis to transfer the enzyme to the mitochondria for subsequent activation. However, the structural basis for this interaction remains to be defined. To map the SOD2-binding site in hsp70, mutants of hsp70 were made and tested for their ability to bind SOD2. These studies showed that SOD2 binds in the amino acid 393-537 region of the chaperone. To map the hsp70-binding site in SOD2, we used a series of pulldown assays and showed that hsp70 binds to the amino-terminal domain of SOD2. To better define the binding site, we used a series of decoy peptides derived from the primary amino acid sequence in the SOD2-binding site in hsp70. This study shows that SOD2 specifically binds to hsp70 at (445)GERAMT(450) Small peptides containing GERAMT inhibited the transfer of SOD2 to the mitochondria and decreased SOD2 activity in vitro and in vivo To determine the amino acid residues in hsp70 that are critical for SOD2 interactions, we substituted each amino acid residue for alanine or more conservative residues, glutamine or asparagine, in the GERAMT-binding site. Substitutions of E446A/Q and R447A/Q inhibited the ability of the GERAMT peptide to bind SOD2 and preserved SOD2 function more than other substitutions. Together, these findings indicate that the GERAMT sequence is critical for hsp70-mediated regulation of SOD2 and that Glu(446) and Arg(447) cooperate with other amino acid residues in the GERAMT-binding site for proper chaperone-dependent regulation of SOD2 antioxidant function.

  12. BOREAS TF-11 CO2 and CH4 Concentration Data from the SSA-Fen

    NASA Technical Reports Server (NTRS)

    Hall, Forrest G. (Editor); Conrad, Sara (Editor); Valentine, David W.

    2000-01-01

    The BOREAS TF-11 team collected several data sets in its efforts to fully describe the flux and site characteristics at the SSA-Fen site. This data set contains temperature, pH, and concentration profiles of methane and carbon dioxide within the surface 50 cm of peat. The measurements were conducted as part of a 2 x 2 factorial experiment in which we added carbon (300 g/sq m as wheat straw) and nitro-gen (6 g/sq m as urea) to four replicate locations in the vicinity of the TF-11 tower. The data set covers the period from the first week of June 1994 through the second week of September 1994. The data are stored in tabular ASCII files.

  13. BOREAS TGB-6 Soil Methane Oxidation and Production from NSA BP and Fen Sites

    NASA Technical Reports Server (NTRS)

    Deck, Bruce; Wahlen, Martin; Hall, Forrest G. (Editor); Conrad, Sara K. (Editor)

    2000-01-01

    The BOReal Ecosystem-Atmosphere Study Trace Gas Biogeochemistry (BOREAS TGB-6) team collected soil methane measurements at several sites in the Southern Study Area (SSA) and Northern Study Area (NSA). This data set contains soil methane consumption (bacterial CH4 oxidation) and associated C-13 fractionation effects in samples that were collected at various sites in 1994 and 1996 from enclosures (chambers). Methane C-13 data in soil gas samples from the NSA Young Jack Pine (YJP) and Old Jack Pine (OJP) sites for 1994 and 1996 are also given. Additional data on the isotopic composition of methane (carbon and hydrogen isotopes) produced in the NSA beaver ponds and fen bog in 1993 and 1994 are given as well. The data are stored in tabular ASCII files.

  14. Solute movement in drained fen peat: a field tracer study in a Somerset (UK) wetland

    NASA Astrophysics Data System (ADS)

    Baird, Andrew J.; Gaffney, Simon W.

    2000-10-01

    Little is known about solute transport in peats, despite the obvious importance of solute transport on eco-hydrological processes in both managed and natural peatlands. To address this lack of knowledge, we investigated solute transport processes in an agricultural fen peat using a conservative KBr tracer. The main aim of the study was to elucidate solute transport behaviour in general in this peat, with a more specific aim of investigating whether preferential or bypassing flow occurred. The tracer moved through the peat more rapidly than expected, and the pattern of movement showed clear evidence of plot-scale bypassing flow. The data also provide evidence that bypassing flow occurs in pores at smaller scales. The implications of this study for management of wetland pastures in the Somerset Moors in south-west England are discussed.

  15. Exchange coupling and noncollinear magnetic states in Ni/Fen/Ni(1 0 0) multilayers

    NASA Astrophysics Data System (ADS)

    Malonda-Boungou, B. R.; Stojić, N.; Binggeli, N.; M'Passi-Mabiala, B.

    2015-01-01

    The Ni interlayer exchange coupling (IEC) and the atomic-scale magnetic configurations in fcc Ni /Fen /Ni (1 0 0) multilayers, with ultrathin Fe spacers, are investigated using first-principles density-functional theory including the noncollinear spin formalism. The trends with changing Fe thickness (n) between 3 and 5 monolayers (MLs) are examined. For n = 3 and 4 MLs, we find the ground state to display antiferromagnetic IEC between the Ni films, while for the 5-ML Fe spacer, the IEC changes into ferromagnetic. Upon reversal of the magnetization alignment, from antiparallel to parallel, between the Ni films with 3- and 4-ML thick Fe spacer, we find noncollinear magnetic configurations in the Fe layer as the lowest-energy states, which are related to the magnetic instability towards noncollinear solutions in bulk γ -Fe.

  16. Fenómenos solares que afectan la atmósfera terrestre

    NASA Astrophysics Data System (ADS)

    Rovira, M.

    Se describen los principales fenómenos solares como fulguraciones, prominencias eruptivas, viento solar y eyecciones de masa coronal (CME) que tienen consecuencias en la atmósfera terrestre. En algunos casos el material es eyectado a velocidades superiores a los 900 km/seg y tarda menos de 48 horas en llegar a la Tierra. En general, estos CME no son peligrosos ya que el campo magnético terrestre actúa como protector siendo un aislante efectivo. Si el viento solar es muy intenso puede comprimir la magnetósfera y producir una tormenta geomagnética. En casos extremos, puede interferir la transmisión de potencia eléctrica, perturbar los satélites y producir auroras polares.

  17. The Wonderful World of Wetlands (WWW): Bogs, fens, marshes and swamps and their global environmental significance

    NASA Astrophysics Data System (ADS)

    Shotyk, W.

    2012-04-01

    Bogs, fens, marshes, and swamps are waterlogged ecosystems where organic soils form and peat accumulates. These are remarkably diverse ecosystems and represent an important component of the biodiversity found on Earth. Their geochemical function is dominated by their predominately anoxic condition which has some important consequences. Best known as reservoirs and reactors for a significant part of our surface freshwater resources, and impacting their chemical composition in remarkable ways, they also have a significant influence on the atmosphere, removing CO2 and adding CH4. The contemporary view during the past centuries was that these were wastelands in need of improving by drainage. Today, however, in some circles at least, the remaining wetlands are valued ecosystems, and the soils they contain archives of climate change, human history and atmospheric pollution.

  18. Thermal neutron capture cross section for 56Fe(n ,γ )

    NASA Astrophysics Data System (ADS)

    Firestone, R. B.; Belgya, T.; Krtička, M.; Bečvář, F.; Szentmikloṡi, L.; Tomandl, I.

    2017-01-01

    The 56Fe(n ,γ ) thermal neutron capture cross section and the 57Fe level scheme populated by this reaction have been investigated in this work. Singles γ -ray spectra were measured with an isotopically enriched 56Fe target using the guided cold neutron beam at the Budapest Reactor, and γ γ -coincidence data were measured with a natural Fe target at the LWR-15 research reactor in Řež, Czech Republic. A detailed level scheme consisting of 448 γ rays populating/depopulating 97 levels and the capture state in 57Fe has been constructed, and ≈99 % of the total transition intensity has been placed. The transition probability of the 352-keV γ ray was determined to be Pγ(352 ) =11.90 ±0.07 per 100 neutron captures. The 57Fe level scheme is substantially revised from earlier work and ≈33 previously assigned levels could not be confirmed while a comparable number of new levels were added. The 57Feγ -ray cross sections were internally calibrated with respect to 1H and 32Sγ -ray cross section standards using iron(III) acetylacetonate (C15H21FeO6) and iron pyrite (FeS2) targets. The thermal neutron cross section for production of the 352-keV γ -ray cross section was determined to be σγ(352 ) =0.2849 ±0.015 b. The total 56Fe(n ,γ ) thermal radiative neutron cross section is derived from the 352-keV γ -ray cross section and transition probability as σ0=2.394 ±0.019 b. A least-squares fit of the γ rays to the level scheme gives the 57Fe neutron separation energy Sn=7646.183 ±0.018 keV.

  19. Spatial characterization of hydrogeochemistry within a constructed fen, Greene County, Ohio

    SciTech Connect

    Hite, C.D.; Cheng, S.

    1996-05-01

    An artificial fen environment was constructed near the municipality of Beavercreek, Ohio. The constructed wetland environment utilizes local ground-water flow patterns to maintain a high degree of water saturation. Chemical reactions driven by the activity of plant roots and soil microorganisms affect the spatial distribution and magnitude of hydrogeochemical parameters, including alkalinity, pH, redox potential, and concentrations of Ca{sup +2}, Mg{sup +2}, total iron, NO{sub 3}{sup {minus}}, and SO{sub 4}{sup {minus}2}. The purpose of this study is to investigate the chemical interactions between wetland plants and ground water by means of characterizing the spatial variation in hydrogeochemical parameters. Nested piezometers within the artificial fen are used to monitor ground-water chemistry within, and immediately adjacent to, the rhizosphere of cultivated plant species. The major reactions at the site include oxidation of organic matter by Fe(OH){sub 3} and dissolution of carbonate minerals driven by high CO{sub 2} production in the root zone. The relationship between alkalinity and dissolved concentrations of Ca{sup +2} and Mg{sup +2} suggests that organic anions could also be major alkalinity contributors. Redox potential is buffered by the reduction of Fe(OH){sub 3} and is typically maintained in the range of 100--200 mV. This buffering effect prevents the formation of sulfide and methane commonly found in similar wetland environments. This finding suggests that methane production associated with rice cultivation can be prevented by the addition of Fe(OH){sub 3} to rice fields.

  20. Season-long measurement of carbon dioxide exchange in a boreal fen

    NASA Astrophysics Data System (ADS)

    Suyker, Andrew E.; Verma, Shashi B.; Arkebauer, Timothy J.

    1997-12-01

    Atmospheric CO2 exchange was measured in a boreal minerotrophic patterned fen in central Saskatchewan, Canada, using the eddy correlation technique. The study was conducted from mid-May to early October 1994, as part of the Boreal Ecosystem-Atmosphere Study (BOREAS). Herbaceous vegetation was dominated by buckbean (Menyanthes trifoliata) and various species of sedges (Carex and Eriphorum spp). Bog birch (Betula pumila) and willow species (Salve spp.) were dominant shrubs. Brown mosses were the predominant nonvascular vegetation. Canopy photosynthesis approached light saturation for PAR (photosynthetically active radiation) above 1000-1200 μmol m-2 s-1. High temperature (>20°C) and vapor pressure deficit (>1.5 kPa) decreased photosynthesis significantly. On cool days with low vapor pressure deficit, canopy photosynthesis tended to follow incident PAR. The diurnal pattern of canopy photosynthesis exhibited a midmorning maximum on days with high temperature and vapor pressure deficit. Canopy photosynthesis reached a peak of 0.59 mg CO2 m-2 s-1 (midday) in early July, corresponding to the period of maximum leaf area index. Another increase in photosynthesis occurred in late August as the canopy recovered from a brief rise in water table that inundated some of the leaf area. The daily net CO2 exchange showed significant day-to-day variability resulting from changes in environmental conditions. The integrated value of the net ecosystem-CO2 exchange during the measurement period (mid-May to early October) was about 88 g C m-2. Consistent with the high productivity and high water table, this fen exhibited magnitudes of CO2 exchange larger than other northern wetlands reported in the literature.

  1. Changes of Phosphatidylcholine and Fatty Acids in Germ Cells during Testicular Maturation in Three Developmental Male Morphotypes of Macrobrachium rosenbergii Revealed by Imaging Mass Spectrometry

    PubMed Central

    Siangcham, Tanapan; Chansela, Piyachat; Hayasaka, Takahiro; Masaki, Noritaka; Sroyraya, Morakot; Poljaroen, Jaruwan; Suwansa-ard, Saowaros; Engsusophon, Attakorn; Hanna, Peter J.; Sobhon, Prasert; Setou, Mitsutoshi

    2015-01-01

    Testis maturation, germ cell development and function of sperm, are related to lipid composition. Phosphatidylcholines (PCs) play a key role in the structure and function of testes. As well, increases of polyunsaturated fatty acids (PUFA) and highly unsaturated fatty acids (HUFA), especially arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are essential for male fertility. This study is the first report to show the composition and distribution of PCs and total fatty acids (FAs) in three groups of seminiferous tubules (STs) classified by cellular associations [i.e., A (STs with mostly early germ cells), B (STs with mostly spermatids), and C (STs with spermatozoa)], in three morphotypes of Macrobrachium rosenbergii, [i.e., small male (SM), orange claw male (OC), and blue claw male (BC)]. Thin layer chromatography exhibited levels of PCs reaching maxima in STs of group B. Imaging mass spectrometry showed remarkably high signals corresponding to PC (16:0/18:1), PC (18:0/18:2), PC (18:2/20:5), and PC (16:0/22:6) in STs of groups A and B. Moreover, most signals were detected in the early developing cells and the intertubular area, but not at the area containing spermatozoa. Finally, gas chromatography-mass spectrometry indicated that the major FAs present in the testes were composed of 14:0, 16:0, 17:0, 18:0, 16:1, 18:1, 18:2, 20:1, 20:2, 20:4, 20:5, and 22:6. The testes of OC contained the greatest amounts of these FAs while the testes of BC contained the least amounts of these FAs, and there was more EPA (20:5) in the testes of SM and OC than those in the BC. The increasing amounts of FAs in the SM and OC indicate that they are important for spermatogenesis and spermiogenesis. This knowledge will be useful in formulating diets containing PUFA and HUFA for prawn broodstocks in order to improve testis development, and lead to increased male fecundity. PMID:25781176

  2. Turn on, fade out - methane exchange in a coastal fen over a period of six years after rewetting

    NASA Astrophysics Data System (ADS)

    Jurasinski, Gerald; Glatzel, Stephan; Hahn, Juliane; Koch, Stefan; Koch, Marian; Koebsch, Franziska

    2016-04-01

    The rewetting of drained peatlands is widely regarded as an adequate measure for the mitigation of greenhouse gas emissions. Therefore, especially in NE Germany, many peatlands are being rewetted. Our knowledge about greenhouse gas exchange associated with rewetting is mainly based on short-term experiments or space-for-time substitutions. These approaches do not consider the transient character of ecosystem acclimatization to flooding by rewetting. Moreover data in this regard on coastal peatland ecosystems are sparse. Here, we present 7 years of data on CH4-exchange in a coastal fen after rewetting by flooding. On the site „Rodewiese", which is located within the NSG "Hütelmoor und Heiligensee" in the Northeast of Rostock, NE Germany, we have established a long term research observatory addressing atmospheric C-exchange. The site is part of the TERENO network. Since summer 2009 we determine CH4 fluxes with closed chambers distributed widely across the study site and CO2-exchange with eddy covariance as well as ancillary data on vegetation, hydrology, and biogeochemistry. This talk addresses the CH4-exchange over time whereas CO2-exchange data are presented by Koebsch et al. in the same session. Rewetting turned the site from a summer dry fen with mean annual water levels of around -0.08m into a shallow lake with water levels up to 0.60m. In the first year after flooding, we observed a substantial die-back of vegetation, especially in stands of Carex acutiformis. Flooding increased methane release rates to extremely high levels of up to 4.3 t ha-1 a-1 for sedge stands and 2.7 t ha-1 a-1 on average, which amounts to 75.6 t ha-1 a-1 in CO2-equivalents. Thereafter, the averaged annual CH4 emissions decreased asymptotically and where at an average of 0.5 t ha-1 a-1 (14 t ha-1 a-1 in CO2-equivalents) in 2015. Factoring in the NEE of the growing season (from Eddy measurements) suggests that the system may be slightly above neutral with respect to the greenhouse

  3. Investigations on dendrimer space reveal solid and liquid tumor growth-inhibition by original phosphorus-based dendrimers and the corresponding monomers and dendrons with ethacrynic acid motifs

    NASA Astrophysics Data System (ADS)

    El Brahmi, Nabil; Mignani, Serge M.; Caron, Joachim; El Kazzouli, Saïd; Bousmina, Mosto M.; Caminade, Anne-Marie; Cresteil, Thierry; Majoral, Jean-Pierre

    2015-02-01

    The well-known reactive diuretic ethacrynic acid (EA, Edecrin), with low antiproliferative activities, was chemically modified and grafted onto phosphorus dendrimers and the corresponding simple branched phosphorus dendron-like derivatives affording novel nanodevices showing moderate to strong antiproliferative activities against liquid and solid tumor cell lines, respectively.The well-known reactive diuretic ethacrynic acid (EA, Edecrin), with low antiproliferative activities, was chemically modified and grafted onto phosphorus dendrimers and the corresponding simple branched phosphorus dendron-like derivatives affording novel nanodevices showing moderate to strong antiproliferative activities against liquid and solid tumor cell lines, respectively. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05983b

  4. High-Quality Draft Genome Sequence of Kallotenue papyrolyticum JKG1T Reveals Broad Heterotrophic Capacity Focused on Carbohydrate and Amino Acid Metabolism

    PubMed Central

    Murugapiran, Senthil K.; Huntemann, Marcel; Clum, Alicia; Pillay, Manoj; Palaniappan, Krishnaveni; Varghese, Neha; Mikhailova, Natalia; Stamatis, Dimitrios; Reddy, T. B. K.; Ngan, Chew Yee; Daum, Chris; Duffy, Kecia; Shapiro, Nicole; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Williams, Amanda J.; Cole, Jessica K.; Dodsworth, Jeremy A.; Woyke, Tanja

    2015-01-01

    The draft genome of Kallotenue papyrolyticum JKG1T, a member of the order Kallotenuales, class Chloroflexia, consists of 4,475,263 bp in 4 contigs and encodes 4,010 predicted genes, 49 tRNA-encoding genes, and 3 rRNA operons. The genome is consistent with a heterotrophic lifestyle including catabolism of polysaccharides and amino acids. PMID:26634758

  5. Metabolomic analysis reveals key metabolites related to the rapid adaptation of Saccharomyce cerevisiae to multiple inhibitors of furfural, acetic acid, and phenol.

    PubMed

    Wang, Xin; Li, Bing-Zhi; Ding, Ming-Zhu; Zhang, Wei-Wen; Yuan, Ying-Jin

    2013-03-01

    During hydrolysis of lignocellulosic biomass, a broad range of inhibitors are generated, which interfere with yeast growth and bioethanol production. In order to improve the strain tolerance to multiple inhibitors--acetic acid, furfural, and phenol (three representative lignocellulose-derived inhibitors) and uncover the underlying tolerant mechanism, an adaptation experiment was performed in which the industrial Saccharomyces cerevisiae was cultivated repeatedly in a medium containing multiple inhibitors. The adaptation occurred quickly, accompanied with distinct increase in growth rate, glucose utilization rate, furfural metabolism rate, and ethanol yield, only after the first transfer. A similar rapid adaptation was also observed for the lab strains of BY4742 and BY4743. The metabolomic analysis was employed to investigate the responses of the industrial S. cereviaise to three inhibitors during the adaptation. The results showed that higher levels of 2-furoic acid, 2, 3-butanediol, intermediates in glycolytic pathway, and amino acids derived from glycolysis, were discovered in the adapted strains, suggesting that enhanced metabolic activity in these pathways may relate to resistance against inhibitors. Additionally, through single-gene knockouts, several genes related to alanine metabolism, GABA shunt, and glycerol metabolism were verified to be crucial for the resistance to multiple inhibitors. This study provides new insights into the tolerance mechanism against multiple inhibitors, and guides for the improvement of tolerant ethanologenic yeast strains for lignocellulose-bioethanol fermentation.

  6. Differential molecular responses of rice and wheat coleoptiles to anoxia reveal novel metabolic adaptations in amino acid metabolism for tissue tolerance.

    PubMed

    Shingaki-Wells, Rachel N; Huang, Shaobai; Taylor, Nicolas L; Carroll, Adam J; Zhou, Wenxu; Millar, A Harvey

    2011-08-01

    Rice (Oryza sativa) and wheat (Triticum aestivum) are the most important starch crops in world agriculture. While both germinate with an anatomically similar coleoptile, this tissue defines the early anoxia tolerance of rice and the anoxia intolerance of wheat seedlings. We combined protein and metabolite profiling analysis to compare the differences in response to anoxia between the rice and wheat coleoptiles. Rice coleoptiles responded to anoxia dramatically, not only at the level of protein synthesis but also at the level of altered metabolite pools, while the wheat response to anoxia was slight in comparison. We found significant increases in the abundance of proteins in rice coleoptiles related to protein translation and antioxidant defense and an accumulation of a set of enzymes involved in serine, glycine, and alanine biosynthesis from glyceraldehyde-3-phosphate or pyruvate, which correlates with an observed accumulation of these amino acids in anoxic rice. We show a positive effect on wheat root anoxia tolerance by exogenous addition of these amino acids, indicating that their synthesis could be linked to rice anoxia tolerance. The potential role of amino acid biosynthesis contributing to anoxia tolerance in cells is discussed.

  7. Metabolomics using GC-TOF-MS followed by subsequent GC-FID and HILIC-MS/MS analysis revealed significantly altered fatty acid and phospholipid species profiles in plasma of smokers.

    PubMed

    Müller, Daniel C; Degen, Christian; Scherer, Gerhard; Jahreis, Gerhard; Niessner, Reinhard; Scherer, Max

    2014-09-01

    Mass spectrometry is an ideal tool for investigations of the metabolome in human plasma. To investigate the impact of smoking on the human metabolome, we performed an untargeted metabolic fingerprinting using GC-TOF-MS with EDTA-plasma samples from 25 smokers and 25 non-smokers. The observed elevated levels in the monounsaturated fatty acids (MUFAs) in smokers were verified by a targeted analysis using GC-FID, which revealed also significantly alterations in saturated and polyunsaturated fatty acids in smokers (p<0.05, Mann-Whitney U test). Since the main fraction of fatty acids in plasma is esterified to phospholipids, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species composition in the plasma samples of the same subjects. The profiles of 39 PC and 40 PE species were analyzed with a newly developed and validated HILIC-ESI-MS/MS method. We were able to baseline separate the two lipid classes (PC from PE) by maintaining co-elution of individual lipid species of each class. The method shows a linear range from 0.5μM to 2000μM and an inter- and intraday coefficient of variation (CV)<20% across all analytes. Application of the validated method to the plasma samples of smokers and non-smokers, derived from a diet-controlled smoking study, revealed significantly elevated levels of PC and PE species containing MUFAs in smokers. In summary, we could demonstrate that there is a significantly altered total fatty acid profile, with increased MUFAs, in the plasma of smokers compared to non-smokers. Results obtained with the new HILIC-MS/MS method indicate that the altered fatty acid profile is also reflected in the PC and PE profile of smokers.

  8. Analysis of a pair of END+ and END− viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

    PubMed Central

    KOZASA, Takashi; ABE, Yuri; MITSUHASHI, Kazuya; TAMURA, Tomokazu; AOKI, Hiroshi; ISHIMARU, Masatoshi; NAKAMURA, Shigeyuki; OKAMATSU, Masatoshi; KIDA, Hiroshi; SAKODA, Yoshihiro

    2014-01-01

    The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein Npro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of Npro determine the difference in characteristics between END-phenomenon-positive (END+) and END-phenomenon-negative (END−) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END+ and END− viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END+ and END− viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of Npro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV. PMID:25648277

  9. Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

    PubMed

    Kozasa, Takashi; Abe, Yuri; Mitsuhashi, Kazuya; Tamura, Tomokazu; Aoki, Hiroshi; Ishimaru, Masatoshi; Nakamura, Shigeyuki; Okamatsu, Masatoshi; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-05-01

    The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.

  10. Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis.

    PubMed

    Neely, Benjamin A; Ferrante, Jason A; Chaves, J Mauro; Soper, Jennifer L; Almeida, Jonas S; Arthur, John M; Gulland, Frances M D; Janech, Michael G

    2014-01-01

    Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers.

  11. Expression pattern of NMDA receptors reveals antiepileptic potential of apigenin 8-C-glucoside and chlorogenic acid in pilocarpine induced epileptic mice.

    PubMed

    Aseervatham, G Smilin Bell; Suryakala, U; Doulethunisha; Sundaram, S; Bose, P Chandra; Sivasudha, T

    2016-08-01

    The present study was aimed to evaluate the effect of apigenin 8-C-glucoside (Vitexin) and chlorogenic acid on epileptic mice induced by pilocarpine and explored its possible mechanisms. Intraperitonial administration of pilocarpine (85mg/kg) induced seizure in mice was assessed by behavior observations, which is significantly (p>0.05) reduced by apigenin 8-C-glucoside (AP8CG) (10mg/kg) and chlorogenic acid (CA) (5mg/kg), similar to diazepam. Seizure was accompanied by an imbalance in the levels of Gamma-aminobutyric acid (GABA) and glutamate in the pilocarpine administered group. Moreover, convulsion along with reduced acetylcholinesterase, increased monoamine oxidase and oxidative stress was observed in epileptic mice brain. AP8CG and CA significantly restored back to normal levels even at lower doses. Further, increased lipid peroxidation and nitrite content was also significantly attenuated by AP8CG and CA. However, CA was found to be more effective when compared to AP8CG. In addition, the mRNA expression of N-methyl-d-aspartate receptor (NMDAR), mGluR1 and mGlu5 was significantly (P≤0.05) inhibited by AP8CG and CA in a lower dose. The mRNA expression of GRIK1 did not differ significantly in any of the group and showed a similar pattern of expression. Our result shows that AP8CG and CA selectively inhibit NMDAR, mGluR1 and mGlu5 expression. Modification in the provoked NMDAR calcium response coupled with neuronal death. Hence, these findings underline that the polyphenolics, AP8CG and CA have exerted antiepileptic and neuroprotective activity by suppressing glutamate receptors.

  12. Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis

    PubMed Central

    Neely, Benjamin A.; Ferrante, Jason A.; Chaves, J. Mauro; Soper, Jennifer L.; Almeida, Jonas S.; Arthur, John M.; Gulland, Frances M. D.; Janech, Michael G.

    2015-01-01

    Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers. PMID:25919366

  13. FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments.

    PubMed

    Ridley, S H; Ktistakis, N; Davidson, K; Anderson, K E; Manifava, M; Ellson, C D; Lipp, P; Bootman, M; Coadwell, J; Nazarian, A; Erdjument-Bromage, H; Tempst, P; Cooper, M A; Thuring, J W; Lim, Z Y; Holmes, A B; Stephens, L R; Hawkins, P T

    2001-11-01

    FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line

  14. Differences in acid tolerance between Bifidobacterium breve BB8 and its acid-resistant derivative B. breve BB8dpH, revealed by RNA-sequencing and physiological analysis.

    PubMed

    Yang, Xu; Hang, Xiaomin; Tan, Jing; Yang, Hong

    2015-06-01

    Bifidobacteria are common inhabitants of the human gastrointestinal tract, and their application has increased dramatically in recent years due to their health-promoting effects. The ability of bifidobacteria to tolerate acidic environments is particularly important for their function as probiotics because they encounter such environments in food products and during passage through the gastrointestinal tract. In this study, we generated a derivative, Bifidobacterium breve BB8dpH, which displayed a stable, acid-resistant phenotype. To investigate the possible reasons for the higher acid tolerance of B. breve BB8dpH, as compared with its parental strain B. breve BB8, a combined transcriptome and physiological approach was used to characterize differences between the two strains. An analysis of the transcriptome by RNA-sequencing indicated that the expression of 121 genes was increased by more than 2-fold, while the expression of 146 genes was reduced more than 2-fold, in B. breve BB8dpH. Validation of the RNA-sequencing data using real-time quantitative PCR analysis demonstrated that the RNA-sequencing results were highly reliable. The comparison analysis, based on differentially expressed genes, suggested that the acid tolerance of B. breve BB8dpH was enhanced by regulating the expression of genes involved in carbohydrate transport and metabolism, energy production, synthesis of cell envelope components (peptidoglycan and exopolysaccharide), synthesis and transport of glutamate and glutamine, and histidine synthesis. Furthermore, an analysis of physiological data showed that B. breve BB8dpH displayed higher production of exopolysaccharide and lower H(+)-ATPase activity than B. breve BB8. The results presented here will improve our understanding of acid tolerance in bifidobacteria, and they will lead to the development of new strategies to enhance the acid tolerance of bifidobacterial strains.

  15. Chlorobaculum tepidum Modulates Amino Acid Composition in Response to Energy Availability, as Revealed by a Systematic Exploration of the Energy Landscape of Phototrophic Sulfur Oxidation.

    PubMed

    Levy, Amalie T; Lee, Kelvin H; Hanson, Thomas E

    2016-11-01

    Microbial sulfur metabolism, particularly the formation and consumption of insoluble elemental sulfur (S(0)), is an important biogeochemical engine that has been harnessed for applications ranging from bioleaching and biomining to remediation of waste streams. Chlorobaculum tepidum, a low-light-adapted photoautolithotrophic sulfur-oxidizing bacterium, oxidizes multiple sulfur species and displays a preference for more reduced electron donors: sulfide > S(0) > thiosulfate. To understand this preference in the context of light energy availability, an "energy landscape" of phototrophic sulfur oxidation was constructed by varying electron donor identity, light flux, and culture duration. Biomass and cellular parameters of C. tepidum cultures grown across this landscape were analyzed. From these data, a correction factor for colorimetric protein assays was developed, enabling more accurate biomass measurements for C. tepidum, as well as other organisms. C. tepidum's bulk amino acid composition correlated with energy landscape parameters, including a tendency toward less energetically expensive amino acids under reduced light flux. This correlation, paired with an observation of increased cell size and storage carbon production under electron-rich growth conditions, suggests that C. tepidum has evolved to cope with changing energy availability by tuning its proteome for energetic efficiency and storing compounds for leaner times.

  16. Functional characterization of NAT/NCS2 proteins of Aspergillus brasiliensis reveals a genuine xanthine-uric acid transporter and an intrinsically misfolded polypeptide.

    PubMed

    Krypotou, Emilia; Scazzocchio, Claudio; Diallinas, George

    2015-02-01

    The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H(+)/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.

  17. δ-Conotoxins Synthesized Using an Acid-cleavable Solubility Tag Approach Reveal Key Structural Determinants for NaV Subtype Selectivity*

    PubMed Central

    Peigneur, Steve; Paolini-Bertrand, Marianne; Gaertner, Hubert; Biass, Daniel; Violette, Aude; Stöcklin, Reto; Favreau, Philippe; Tytgat, Jan; Hartley, Oliver

    2014-01-01

    Conotoxins are venom peptides from cone snails with multiple disulfide bridges that provide a rigid structural scaffold. Typically acting on ion channels implicated in neurotransmission, conotoxins are of interest both as tools for pharmacological studies and as potential new medicines. δ-Conotoxins act by inhibiting inactivation of voltage-gated sodium channels (Nav). Their pharmacology has not been extensively studied because their highly hydrophobic character makes them difficult targets for chemical synthesis. Here we adopted an acid-cleavable solubility tag strategy that facilitated synthesis, purification, and directed disulfide bridge formation. Using this approach we readily produced three native δ-conotoxins from Conus consors plus two rationally designed hybrid peptides. We observed striking differences in Nav subtype selectivity across this group of compounds, which differ in primary structure at only three positions: 12, 23, and 25. Our results provide new insights into the structure-activity relationships underlying the Nav subtype selectivity of δ-conotoxins. Use of the acid-cleavable solubility tag strategy should facilitate synthesis of other hydrophobic peptides with complex disulfide bridge patterns. PMID:25352593

  18. Protein engineering of the quaternary sulfiredoxin.peroxiredoxin enzyme.substrate complex reveals the molecular basis for cysteine sulfinic acid phosphorylation.

    PubMed

    Jönsson, Thomas J; Johnson, Lynnette C; Lowther, W Todd

    2009-11-27

    Oxidative stress can damage the active site cysteine of the antioxidant enzyme peroxiredoxin (Prx) to the sulfinic acid form, Prx-SO(2)(-). This modification leads to inactivation. Sulfiredoxin (Srx) utilizes a unique ATP-Mg(2+)-dependent mechanism to repair the Prx molecule. Using selective protein engineering that involves disulfide bond formation and site-directed mutagenesis, a mimic of the enzyme.substrate complex has been trapped. Here, we present the 2.1 A crystal structure of human Srx in complex with PrxI, ATP, and Mg(2+). The Cys(52) sulfinic acid moiety was substituted by mutating this residue to Asp, leading to a replacement of the sulfur atom with a carbon atom. Because the Srx reaction cannot occur, the structural changes in the Prx active site that lead to the attack on ATP may be visualized. The local unfolding of the helix containing C52D resulted in the packing of Phe(50) in PrxI within a hydrophobic pocket of Srx. Importantly, this structural rearrangement positioned one of the oxygen atoms of Asp(52) within 4.3 A of the gamma-phosphate of ATP bound to Srx. These observations support a mechanism where phosphorylation of Prx-SO(2)(-) is the first chemical step.

  19. New insights into the biogeochemistry of extremely acidic environments revealed by a combined cultivation-based and culture-independent study of two stratified pit lakes.

    PubMed

    Falagán, Carmen; Sánchez-España, Javier; Johnson, David Barrie

    2014-01-01

    The indigenous microbial communities of two extremely acidic, metal-rich stratified pit lakes, located in the Iberian Pyrite Belt (Spain), were identified, and their roles in mediating transformations of carbon, iron, and sulfur were confirmed. A combined cultivation-based and culture-independent approach was used to elucidate microbial communities at different depths and to examine the physiologies of isolates, which included representatives of at least one novel genus and several species of acidophilic Bacteria. Phosphate availability correlated with redox transformations of iron, and this (rather than solar radiation) dictated where primary production was concentrated. Carbon fixed and released as organic compounds by acidophilic phototrophs acted as electron donors for acidophilic heterotrophic prokaryotes, many of which catalyzed the dissimilatory reduction in ferric iron; the ferrous iron generated was re-oxidized by chemolithotrophic acidophiles. Bacteria that catalyze redox transformations of sulfur were also identified, although these Bacteria appeared to be less abundant than the iron oxidizers/reducers. Primary production and microbial numbers were greatest, and biogeochemical transformation of carbon, iron, and sulfur, most intense, within a zone of c. 8-10 m depth, close to the chemocline, in both pit lakes. Archaea detected in sediments included two Thaumarchaeota clones, indicating that members of this recently described phylum can inhabit extremely acidic environments.

  20. Transcriptional analysis reveals a high impact of conjugated linoleic acid on stearoyl-Coenzyme A desaturase 1 mRNA expression in mice gastrocnemius muscle.

    PubMed

    Parra, Pilar; Serra, Francisca; Palou, Andreu

    2012-10-01

    We examined the potential implication of skeletal muscle in the fat-lowering effect observed in mice treated with moderate doses of CLA. In experiment 1, mice fed with a standard-fat diet were orally treated with sunflower oil (control) and 3 or 10 mg CLA mixture/day for 37 days. In experiment 2, mice were fed with a high-fat diet for 65 days. For the first 30 days, they received the same doses as in experiment 1 and, from that time onwards, animals received double doses. Gene expression of key proteins involved in fatty acid transport, oxidation, regulation of lipid and carbohydrate utilization, composition of muscle fiber, and thermogenesis were determined and, in most of them, no major impact of CLA was seen. Therefore, enhancement of fatty acid oxidation in muscle did not seem to contribute to the antiobesity effect of CLA as seen in other studies with higher CLA doses. However, a strong induction of classically associated lipogenic genes such as Fasn (up to twofold) and, particularly, Scd1 (up to ninefold) was found. This activation could contribute to a protective role in muscle cells, since expression of ER stress markers was decreased and inversely correlated with the induction of Scd1.

  1. Comparisons of soil nitrogen mass balances for an ombrotrophic bog and a minerotrophic fen in northern Minnesota.

    PubMed

    Hill, Brian H; Jicha, Terri M; Lehto, LaRae L P; Elonen, Colleen M; Sebestyen, Stephen D; Kolka, Randall K

    2016-04-15

    We compared nitrogen (N) storage and flux in soils from an ombrotrophic bog with that of a minerotrophic fen to quantify the differences in N cycling between these two peatlands types in northern Minnesota (USA). Precipitation, atmospheric deposition, and bog and fen outflows were analyzed for nitrogen species. Upland and peatland soil samples were analyzed for N content, and for ambient (DN) and potential (DEA) denitrification rates. Annual atmospheric deposition was: 0.88-3.07kg NH4(+)ha(-1)y(-1); 1.37-1.42kg NO3(-)ha(-1)y(-1); 2.79-4.69kg TNha(-1)y(-1). Annual N outflows were: bog-0.01-0.04kg NH4(+)ha(-1)y(-1), NO3(-) 0.01-0.06kgha(-1)y(-1), and TN 0.11-0.69kgha(-1)y(-1); fen-NH4(+) 0.01-0.16kgha(-1)y(-1), NO3(-) 0.29-0.48kgha(-1)y(-1), and TN 1.14-1.61kgha(-1)y(-1). Soil N content depended on location within the bog or fen, and on soil depth. DN and DEA rates were low throughout the uplands and peatlands, and were correlated with atmospheric N deposition, soil N storage, and N outflow. DEA was significantly greater than DN indicating C or N limitation of the denitrification process. We highlight differences between the bog and fen, between the upland mineral soils and peat, and the importance of biogeochemical hotspots within the peatlands. We point out the importance of organic N storage, as a source of N for denitrification, and propose a plausible link between organic N storage, denitrification and N export from peatlands. Finally, we considered the interactions of microbial metabolism with nutrient availability and stoichiometry, and how N dynamics might be affected by climate change in peatland ecosystems.

  2. Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

    SciTech Connect

    Goblirsch, BR; Frias, JA; Wackett, LP; Wilmot, CM

    2012-05-22

    OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acylcoenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatized structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short beta-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117 beta) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly, Glu117

  3. Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids

    SciTech Connect

    Goblirsch, Brandon R.; Frias, Janice A.; Wackett, Lawrence P.; Wilmot, Carrie M.

    2012-10-25

    OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acyl-coenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatized structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short {beta}-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117{beta}) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly

  4. Hydrogen Isotopes of N-Alkanoic Acids from Lake Sediments Reveal Strong Amount-Effect Influence over the Past 5,000 Years, Adak Island, Alaska

    NASA Astrophysics Data System (ADS)

    Vaillencourt, D.; Kaufman, D. S.; D'Andrea, W. J.; Anderson, R. S.

    2012-12-01

    The Aleutian Low (AL) pressure system is a major feature of North Pacific climate, especially during the winter. The AL is associated with a precipitation dipole, which features increased precipitation and storminess over the western North Pacific region and decreased precipitation and storminess in the east when the AL is weak, and the opposite pattern when it is strong. Changes in the amount of storminess at any site might therefore be associated with changes in the strength of the AL, or in its position, or both. Reconstructing the strength and position of the AL requires proxy records from both sides of the dipole, yet Holocene paleoclimate records are largely limited to sites in the eastern North Pacific. Our study site, Andrew Lake on Adak Island, Alaska (51.93° N 176.63° W, 5 m a.s.l) is in the central Aleutian Islands, in the western North Pacific region of the AL center of action. Sediment cores recovered from Andrew Lake were radiometrically dated using ten 14C ages, the peak in 240Pu activity representing 1963 AD, and a 210Pb profile. The 456-cm-long sedimentary sequence represents nearly 7200 years. A total of 76 samples were analyzed for hydrogen isotopes (δD) of long-chain n-alkanoic acids (fatty acids), organic compounds produced primarily by terrestrial vegetation. Each sample spans an average of 140 years, with higher resolution during the instrumental period. A calibration-in-time analysis shows that δD of the C28 fatty acid and October-May storminess (number of days with >19 mm (0.75 in) of precipitation) are inversely correlated (r2 = 0.58, p < 0.02, n = 19) at multi-annual scale during the instrumental record. This result is consistent with the amount effect, whereby intense storms produce precipitation with lower δD values relative to weaker storms. This period of strongest correlation (October-May) also coincides with the period of increased AL activity, when >75% of large storms pass over Adak Island. During the past 5 ka, δD of fatty

  5. Mouse sperm patch-clamp recordings reveal single Cl- channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction.

    PubMed

    Espinosa, F; de la Vega-Beltrán, J L; López-González, I; Delgado, R; Labarca, P; Darszon, A

    1998-04-10

    Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (approximately 3 microm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch-clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50 = 11 microM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl- channels participate in the sperm acrosome reaction in mammals.

  6. Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity.

    PubMed

    Hsu, Min-Feng; Fu, Hsu-Yuan; Cai, Chun-Jie; Yi, Hsiu-Pin; Yang, Chii-Shen; Wang, Andrew H-J

    2015-12-04

    Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg(82) and Thr(201), linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg(82)-Thr(201) hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping.

  7. Structural and Functional Studies of a Newly Grouped Haloquadratum walsbyi Bacteriorhodopsin Reveal the Acid-resistant Light-driven Proton Pumping Activity*

    PubMed Central

    Hsu, Min-Feng; Fu, Hsu-Yuan; Cai, Chun-Jie; Yi, Hsiu-Pin; Yang, Chii-Shen; Wang, Andrew H.-J.

    2015-01-01

    Retinal bound light-driven proton pumps are widespread in eukaryotic and prokaryotic organisms. Among these pumps, bacteriorhodopsin (BR) proteins cooperate with ATP synthase to convert captured solar energy into a biologically consumable form, ATP. In an acidic environment or when pumped-out protons accumulate in the extracellular region, the maximum absorbance of BR proteins shifts markedly to the longer wavelengths. These conditions affect the light-driven proton pumping functional exertion as well. In this study, wild-type crystal structure of a BR with optical stability under wide pH range from a square halophilic archaeon, Haloquadratum walsbyi (HwBR), was solved in two crystal forms. One crystal form, refined to 1.85 Å resolution, contains a trimer in the asymmetric unit, whereas another contains an antiparallel dimer was refined at 2.58 Å. HwBR could not be classified into any existing subgroup of archaeal BR proteins based on the protein sequence phylogenetic tree, and it showed unique absorption spectral stability when exposed to low pH values. All structures showed a unique hydrogen-bonding network between Arg82 and Thr201, linking the BC and FG loops to shield the retinal-binding pocket in the interior from the extracellular environment. This result was supported by R82E mutation that attenuated the optical stability. The negatively charged cytoplasmic side and the Arg82–Thr201 hydrogen bond may play an important role in the proton translocation trend in HwBR under acidic conditions. Our findings have unveiled a strategy adopted by BR proteins to solidify their defenses against unfavorable environments and maintain their optical properties associated with proton pumping. PMID:26483542

  8. Proteome analysis of fatty liver in feed-deprived dairy cows reveals interaction of fuel sensing, calcium, fatty acid, and glycogen metabolism.

    PubMed

    Kuhla, Björn; Albrecht, Dirk; Kuhla, Siegfried; Metges, Cornelia C

    2009-04-10

    The liver of dairy cows is involved in signaling the current hepatic metabolic state to the brain via metabolites and nerval afferents to control and adjust feed intake. Feed deprivation may result in mobilization of body reserves favoring hepatic steatosis. While the overall metabolic changes are well characterized, specific regulatory mechanisms are not readily understood. To identify molecular events associated with metabolic adaptation and the control of energy homeostasis, liver specimens from six ad libitum-fed and six feed-deprived cows were analyzed for selected metabolites, for the activation of AMP kinase, and for regulatory/regulated proteins using two-dimensional gel electrophoresis and MALDI-TOF-MS. Feed deprivation increased total liver fat and the calcium content, as well as augmented AMPK phosphorylation, while it decreased the contents of protein, glucose, glycogen, and cholesterol when expressed as a percentage of dry matter. Among 34 differentially expressed proteins identified, we found downregulation of proteins associated with fatty acid oxidation, glycolysis, electron transfer, protein degradation, and antigen processing, as well as cytoskeletal rearrangement. Proteins upregulated after feed deprivation included enzymes of the urea cycle, fatty acid or cholesterol transport proteins, an inhibitor of glycolysis, and previously unknown changes in calcium signaling network. Direct correlation was found between expression of glycolytic enzymes and glucose/glycogen content, whereas inverse correlation exists between expression of beta-oxidative enzymes and total liver fat content. In conclusion, the regulatory response of identified proteins may help to explain development and consequences of hepatic lipidosis but also offers novel candidates potentially involved in signaling for maintaining energy homeostasis.

  9. RNA-seq reveals the critical role of OtpR in regulating Brucella melitensis metabolism and virulence under acidic stress.

    PubMed

    Liu, Wenxiao; Dong, Hao; Li, Jing; Ou, Qixing; Lv, Yujin; Wang, Xiaolei; Xiang, Zuoshuang; He, Yongqun; Wu, Qingmin

    2015-08-05

    The response regulator OtpR is critical for the growth, morphology and virulence of Brucella melitensis. Compared to its wild type strain 16 M, B. melitensis 16 MΔotpR mutant has decreased tolerance to acid stress. To analyze the genes regulated by OtpR under acid stress, we performed RNA-seq whole transcriptome analysis of 16 MΔotpR and 16 M. In total, 501 differentially expressed genes were identified, including 390 down-regulated and 111 up-regulated genes. Among these genes, 209 were associated with bacterial metabolism, including 54 genes involving carbohydrate metabolism, 13 genes associated with nitrogen metabolism, and seven genes associated with iron metabolism. The 16 MΔotpR also decreased capacity to utilize different carbon sources and to tolerate iron limitation in culture experiments. Notably, OtpR regulated many Brucella virulence factors essential for B. melitensis intracellular survival. For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR. Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR. Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis.

  10. RNA-seq reveals the critical role of OtpR in regulating Brucella melitensis metabolism and virulence under acidic stress

    PubMed Central

    Liu, Wenxiao; Dong, Hao; Li, Jing; Ou, Qixing; Lv, Yujin; Wang, Xiaolei; Xiang, Zuoshuang; He, Yongqun; Wu, Qingmin

    2015-01-01

    The response regulator OtpR is critical for the growth, morphology and virulence of Brucella melitensis. Compared to its wild type strain 16 M, B. melitensis 16 MΔotpR mutant has decreased tolerance to acid stress. To analyze the genes regulated by OtpR under acid stress, we performed RNA-seq whole transcriptome analysis of 16 MΔotpR and 16 M. In total, 501 differentially expressed genes were identified, including 390 down-regulated and 111 up-regulated genes. Among these genes, 209 were associated with bacterial metabolism, including 54 genes involving carbohydrate metabolism, 13 genes associated with nitrogen metabolism, and seven genes associated with iron metabolism. The 16 MΔotpR also decreased capacity to utilize different carbon sources and to tolerate iron limitation in culture experiments. Notably, OtpR regulated many Brucella virulence factors essential for B. melitensis intracellular survival. For instance, the virB operon encoding type IV secretion system was significantly down-regulated, and 36 known transcriptional regulators (e.g., vjbR and blxR) were differentially expressed in 16 MΔotpR. Selected RNA-seq results were experimentally confirmed by RT-PCR and RT-qPCR. Overall, these results deciphered differential phenomena associated with virulence, environmental stresses and cell morphology in 16 MΔotpR and 16 M, which provided important information for understanding the detailed OtpR-regulated interaction networks and Brucella pathogenesis. PMID:26242322

  11. Salicylic and jasmonic acid pathways are necessary for defence against Dickeya solani as revealed by a novel method for Blackleg disease screening of in vitro grown potato.

    PubMed

    Burra, D D; Mühlenbock, P; Andreasson, E

    2015-09-01

    Potato is major crop ensuring food security in Europe, and blackleg disease is increasingly causing losses in yield and during storage. Recently, one blackleg pathogen, Dickeya solani has been shown to be spreading in Northern Europe that causes aggressive disease development. Currently, identification of tolerant commercial potato varieties has been unsuccessful; this is confounded by the complicated etiology of the disease and a strong environmental influence on disease development. There is currently a lack of efficient testing systems. Here, we describe a system for quantification of blackleg symptoms on shoots of sterile in vitro potato plants, which saves time and space compared to greenhouse and existing field assays. We found no evidence for differences in infection between the described in vitro-based screening method and existing greenhouse assays. This system facilitates efficient screening of blackleg disease response of potato plants independent of other microorganisms and variable environmental conditions. We therefore used the in vitro screening method to increase understanding of plant mechanisms involved in blackleg disease development by analysing disease response of hormone- related (salicylic and jasmonic acid) transgenic potato plants. We show that both jasmonic (JA) and salicylic (SA) acid pathways regulate tolerance to blackleg disease in potato, a result unlike previous findings in Arabidopsis defence response to necrotrophic bacteria. We confirm this by showing induction of a SA marker, pathogenesis-related protein 1 (StPR1), and a JA marker, lipoxygenase (StLOX), in Dickeya solani infected in vitro potato plants. We also observed that tubers of transgenic potato plants were more susceptible to soft rot compared to wild type, suggesting a role for SA and JA pathways in general tolerance to Dickeya.

  12. Methane Exchange in a Coastal Fen in the First Year after Flooding - A Systems Shift

    PubMed Central

    Hahn, Juliane; Köhler, Stefan; Glatzel, Stephan; Jurasinski, Gerald

    2015-01-01

    Background Peatland restoration can have several objectives, for example re-establishing the natural habitat, supporting unique biodiversity attributes or re-initiating key biogeochemical processes, which can ultimately lead to a reduction in greenhouse gas (GHG) emissions. Every restoration measure, however, is itself a disturbance to the ecosystem. Methods Here, we examine an ecosystem shift in a coastal fen at the southern Baltic Sea which was rewetted by flooding. The analyses are based on one year of bi-weekly closed chamber measurements of methane fluxes gathered at spots located in different vegetation stands. During measurement campaigns, we recorded data on water levels, peat temperatures, and chemical properties of peat water. In addition we analyzed the first 20 cm of peat before and after flooding for dry bulk density (DBD), content of organic matter and total amounts of carbon (C), nitrogen (N), sulfur (S), and other nutrients. Results Rewetting turned the site from a summer dry fen into a shallow lake with water levels up to 0.60 m. We observed a substantial die-back of vegetation, especially in stands of sedges (Carex acutiformis Ehrh). Concentrations of total organic carbon and nitrogen in the peat water, as well as dry bulk density and concentrations of C, N and S in the peat increased. In the first year after rewetting, the average annual exchange of methane amounted to 0.26 ± 0.06 kg m-2. This is equivalent to a 190-times increase in methane compared to pre-flooding conditions. Highest methane fluxes occurred in sedge stands which suffered from the heaviest die-back. None of the recorded environmental variables showed consistent relationships with the amounts of methane exchanged. Conclusions Our results suggest that rewetting projects should be monitored not only with regard to vegetation development but also with respect to biogeochemical conditions. Further, high methane emissions that likely occur directly after rewetting by flooding should

  13. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics.

    PubMed

    Bell-Temin, Harris; Culver-Cochran, Ashley E; Chaput, Dale; Carlson, Christina M; Kuehl, Melanie; Burkhardt, Brant R; Bickford, Paula C; Liu, Bin; Stevens, Stanley M

    2015-12-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells.

  14. Novel Molecular Insights into Classical and Alternative Activation States of Microglia as Revealed by Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Proteomics*

    PubMed Central

    Bell-Temin, Harris; Culver-Cochran, Ashley E.; Chaput, Dale; Carlson, Christina M.; Kuehl, Melanie; Burkhardt, Brant R.; Bickford, Paula C.; Liu, Bin; Stevens, Stanley M.

    2015-01-01

    Microglia, the resident immune cells of the brain, have been shown to display a complex spectrum of roles that span from neurotrophic to neurotoxic depending on their activation status. Microglia can be classified into four stages of activation, M1, which most closely matches the classical (pro-inflammatory) activation stage, and the alternative activation stages M2a, M2b, and M2c. The alternative activation stages have not yet been comprehensively analyzed through unbiased, global-scale protein expression profiling. In this study, BV2 mouse immortalized microglial cells were stimulated with agonists specific for each of the four stages and total protein expression for 4644 protein groups was quantified using SILAC-based proteomic analysis. After validating induction of the various stages through a targeted cytokine assay and Western blotting of activation states, the data revealed novel insights into the similarities and differences between the various states. The data identify several protein groups whose expression in the anti-inflammatory, pro-healing activation states are altered presumably to curtail inflammatory activation through differential protein expression, in the M2a state including CD74, LYN, SQST1, TLR2, and CD14. The differential expression of these proteins promotes healing, limits phagocytosis, and limits activation of reactive nitrogen species through toll-like receptor cascades. The M2c state appears to center around the down-regulation of a key member in the formation of actin-rich phagosomes, SLP-76. In addition, the proteomic data identified a novel activation marker, DAB2, which is involved in clathrin-mediated endocytosis and is significantly different between M2a and either M1 or M2b states. Western blot analysis of mouse primary microglia stimulated with the various agonists of the classical and alternative activation states revealed a similar trend of DAB2 expression compared with BV2 cells. PMID:26424600

  15. Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel: Implication for simultaneous modulation in ASICs.

    PubMed

    Smith, Rachel N; Gonzales, Eric B

    2014-01-01

    Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a "molecular wedge" that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli.

  16. Fe3C nanoparticle decorated Fe/N doped graphene for efficient oxygen reduction reaction electrocatalysis

    NASA Astrophysics Data System (ADS)

    Niu, Yanli; Huang, Xiaoqin; Hu, Weihua

    2016-11-01

    Oxygen reduction reaction (ORR) electrocatalysts with high activity, low cost and good durability are crucial to promote the large-scale practical application of fuel cells. Particularly, iron carbide (Fe3C) supported on nitrogen-doped carbon has recently demonstrated compelling promise for ORR electrocatalysis. In this paper, we report the facile synthesis of mesoporous Fe/N-doped graphene with encapsulated Fe3C nanoparticles (Fe3C@Fe/N-graphene) and its superior ORR catalytic activity. This hybrid material was synthesized by the spontaneous oxidative polymerization of dopamine on graphene oxide (GO) sheets in the presence of iron ion, followed by thermal annealing in Argon (Ar) atmosphere. As-prepared material shows high ORR catalytic activity with overwhelming four-electron reduction pathway, long-term durability and high methanol tolerance in alkaline media. This work reports a facile method to synthesize promising ORR electrocatalysis with multiple components and hierarchical architecture, and may offer valuable insight into the underlying mechanism of Fe3C-boosted ORR activity of Fe/N doped carbon.

  17. Carbonaceous thin film coating with Fe-N4 site for enhancement of dioxovanadium ion reduction

    NASA Astrophysics Data System (ADS)

    Maruyama, Jun; Hasegawa, Takahiro; Iwasaki, Satoshi; Fukuhara, Tomoko; Orikasa, Yuki; Uchimoto, Yoshiharu

    2016-08-01

    It has been found that carbonaceous materials containing a transition metal coordinated by 4 nitrogens in the square-planar configuration (metal-N4 site) on the surface possessed a catalytic activity for various electrochemical reactions related to energy conversion and storage; i.e., oxygen reduction, hydrogen evolution, and quite recently, the electrode reactions in vanadium redox flow batteries (VRFB). The catalyst for the VRFB positive electrode discharge reaction, i.e., the dioxovanadium ion reduction, was formed by coating the surface of cup-stack carbon nanotubes with a carbonaceous thin film with the Fe-N4 site generated by the sublimation, deposition, and pyrolysis of iron phthalocyanine. In this study, the influence of the physical properties of the catalyst on the electrochemical reactions was investigated to optimize the coating. With an increase in the coating, the specific surface area increased, whereas the pore size decreased. The surface Fe concentration was increased in spite of the Fe aggregation inside the carbon matrix. The catalytic activity enhancement was achieved due to the increase in the specific surface area and the surface Fe concentration, but was lowered due to the decrease in the pore size, which was disadvantageous for the penetration of the electrolyte and the mass transfer.

  18. Strategies to mitigate diffuse phosphorus pollution during rewetting of fen peat soils.

    PubMed

    Meissner, R; Rupp, H; Seeger, J; Leinweber, P

    2010-01-01

    The cultivation of fen peat soils (Eutric Histosols) for agricultural purposes, started in Europe about 250 years ago, resulting in decreased soil fertility, increased oxidation of peat and corresponding greenhouse gas fluxes to the atmosphere, nutrient transfer to aquatic ecosystems and losses in total area of the former native wetlands. To prevent these negative environmental effects set-aside programs and rewetting measures were promoted in recent years. Literature results and practical experiences showed that large scale rewetting of intensively used agricultural Histosols may result in mobilisation of phosphorus (P), its transport to adjacent surface waters and an accelerated eutrophication. The paper summarises results from an international European Community sponsored research project and demonstrates how results obtained at different scales and from different scientific disciplines were compiled to derive a strategy to carry out rewetting measures. Based on this findings a simple decision support system (DSS) for a hydrologically sensitive area in the Droemling catchment in north-eastern Germany was developed and since 2005 practically used to prevent freshwater resources from non point P pollution.

  19. Seasonal dynamics of methane emissions from a subarctic fen in the Hudson Bay Lowlands

    NASA Astrophysics Data System (ADS)

    Hanis, K. L.; Tenuta, M.; Amiro, B. D.; Papakyriakou, T. N.

    2013-07-01

    Ecosystem-scale methane (CH4) flux (FCH4) over a subarctic fen at Churchill, Manitoba, Canada was measured to understand the magnitude of emissions during spring and fall shoulder seasons, and the growing season in relation to physical and biological conditions. FCH4 was measured using eddy covariance with a closed-path analyser in four years (2008-2011). Cumulative measured annual FCH4 (shoulder plus growing seasons) ranged from 3.0 to 9.6 g CH4 m-2 yr-1 among the four study years, with a mean of 6.5 to 7.1 g CH4 m-2 yr-1 depending upon gap-filling method. Soil temperatures to depths of 50 cm and air temperature were highly correlated with FCH4, with near-surface soil temperature at 5 cm most correlated across spring, fall, and the shoulder and growing seasons. The response of FCH4 to soil temperature at the 5 cm depth and air temperature was more than double in spring to that of fall. Emission episodes were generally not observed during spring thaw. Growing season emissions also depended upon soil and air temperatures but the water table also exerted influence, with FCH4 highest when water was 2-13 cm below and lowest when it was at or above the mean peat surface.

  20. Seasonal dynamics of methane emissions from a subarctic fen in the Hudson Bay Lowlands

    NASA Astrophysics Data System (ADS)

    Hanis, K. L.; Tenuta, M.; Amiro, B. D.; Papakyriakou, T. N.

    2013-03-01

    Ecosystem-scale methane (CH4) flux (FCH4) over a subarctic fen at Churchill, Manitoba, Canada was measured to understand the magnitude of emissions during spring and fall shoulder seasons, and the growing season in relation to physical and biological conditions. FCH4 was measured using eddy covariance with a closed-path analyzer in four years (2008-2011). Cumulative measured annual FCH4 (shoulder plus growing seasons) ranged from 3.0 to 9.6 g CH4 m-2 yr-1 among the four study years, with a mean of 6.5 to 7.1 g CH4 m-2 yr-1 depending upon gap-filling method. Soil temperatures to depths of 50 cm and air temperature were highly correlated with FCH4, with near surface soil temperature at 5 cm most correlated across spring, fall, and the whole season. The response of FCH4 to soil temperature at the 5 cm depth and air temperature was more than double in spring to that of fall. Emission episodes were generally not observed during spring thaw. Growing season emissions also depended upon soil and air temperatures but water table also exerted influence with FCH4 highest when water was 2-13 cm below and least when it was at or above the mean peat surface.

  1. Transcriptome Analysis Reveals the Mechanism Underlying the Production of a High Quantity of Chlorogenic Acid in Young Leaves of Lonicera macranthoides Hand.-Mazz

    PubMed Central

    Chen, Zexiong; Tang, Ning; You, Yuming; Lan, Jianbin; Liu, Yiqing; Li, Zhengguo

    2015-01-01

    Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides. PMID:26381882

  2. Differences between main-channel and off-channel food webs in the upper Mississippi River revealed by fatty acid profiles of consumers

    USGS Publications Warehouse

    Larson, James H.; Bartsch, Michelle; Gutreuter, Steve; Knights, Brent C.; Bartsch, Lynn; Richardson, William B.; Vallazza, Jonathan M.; Arts, Michael T.

    2015-01-01

    Large river systems are often thought to contain a mosaic of patches with different habitat characteristics driven by differences in flow and mixing environments. Off-channel habitats (e.g., backwater areas, secondary channels) can become semi-isolated from main-channel water inputs, leading to the development of distinct biogeochemical environments. Observations of adult bluegill (Lepomis macrochirus) in the main channel of the Mississippi River led to speculation that the main channel offered superior food resources relative to off-channel areas. One important aspect of food quality is the quantity and composition of polyunsaturated fatty acids (PUFA). We sampled consumers from main-channel and backwater habitats to determine whether they differed in PUFA content. Main-channel individuals for relatively immobile species (young-of-year bluegill, zebra mussels [Dreissena polymorpha], and plain pocketbook mussels [Lampsilis cardium]) had significantly greater PUFA content than off-channel individuals. No difference in PUFA was observed for the more mobile gizzard shad (Dorsoma cepedianum), which may move between main-channel and off-channel habitats even at early life-history stages. As off-channel habitats become isolated from main-channel waters, flow and water column nitrogen decrease, potentially improving conditions for nitrogen-fixing cyanobacteria and vascular plants that, in turn, have low PUFA content. We conclude that main-channel food webs of the upper Mississippi River provide higher quality food resources for some riverine consumers as compared to food webs in off-channel habitats.

  3. Proteomic analysis reveals differences in tolerance to acid rain in two broad-leaf tree species, Liquidambar formosana and Schima superba.

    PubMed

    Chen, Juan; Hu, Wen-Jun; Wang, Chao; Liu, Ting-Wu; Chalifour, Annie; Chen, Juan; Shen, Zhi-Jun; Liu, Xiang; Wang, Wen-Hua; Zheng, Hai-Lei

    2014-01-01

    Acid rain (AR) is a serious environmental issue inducing harmful impacts on plant growth and development. It has been reported that Liquidambar formosana, considered as an AR-sensitive tree species, was largely injured by AR, compared with Schima superba, an AR-tolerant tree species. To clarify the different responses of these two species to AR, a comparative proteomic analysis was conducted in this study. More than 1000 protein spots were reproducibly detected on two-dimensional electrophoresis gels. Among them, 74 protein spots from L. formosana gels and 34 protein spots from S. superba gels showed significant changes in their abundances under AR stress. In both L. formosana and S. superba, the majority proteins with more than 2 fold changes were involved in photosynthesis and energy production, followed by material metabolism, stress and defense, transcription, post-translational and modification, and signal transduction. In contrast with L. formosana, no hormone response-related protein was found in S. superba. Moreover, the changes of proteins involved in photosynthesis, starch synthesis, and translation were distinctly different between L. formosana and S. superba. Protein expression analysis of three proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, ascorbate peroxidase and glutathione-S-transferase) by Western blot was well correlated with the results of proteomics. In conclusion, our study provides new insights into AR stress responses in woody plants and clarifies the differences in strategies to cope with AR between L. formosana and S. superba.

  4. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

    NASA Technical Reports Server (NTRS)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  5. Real-time analysis of endogenous protoporphyrin IX fluorescence from δ-aminolevulinic acid and its derivatives reveals distinct time- and dose-dependent characteristics in vitro

    NASA Astrophysics Data System (ADS)

    Kiesslich, Tobias; Helander, Linda; Illig, Romana; Oberdanner, Christian; Wagner, Andrej; Lettner, Herbert; Jakab, Martin; Plaetzer, Kristjan

    2014-08-01

    Photodynamic therapy (PDT) and photodiagnosis based on the intracellular production of the photosensitizer protoporphyrin IX (PPIX) by administration of its metabolic precursor δ-aminolevulinic acid (ALA) achieved their breakthrough upon the clinical approval of MAL (ALA methyl ester) and HAL (ALA hexyl ester). For newly developed ALA derivatives or application in new tumor types, in vitro determination of PPIX formation involves multiparametric experiments covering variable pro-drug concentrations, medium composition, time points of analysis, and cell type(s). This study uses a fluorescence microplate reader with a built-in temperature and atmosphere control to investigate the high-resolution long-term kinetics (72 h) of cellular PPIX fueled by administration of either ALA, MAL, or HAL for each 10 different concentrations. For simultaneous proliferation correction, A431 cells were stably transfected with green fluorescent protein. The results indicate that the peak PPIX level is a function of both, incubation concentration and period: maximal PPIX is generated with 1 to 2-mM ALA/MAL or 0.125-mM HAL; also, the PPIX peak shifts to longer incubation periods with increasing pro-drug concentrations. The results underline the need for detailed temporal analysis of PPIX formation to optimize ALA (derivative)-based PDT or photodiagnosis and highlight the value of environment-controlled microplate readers for automated in vitro analysis.

  6. X-ray structure of acid-sensing ion channel 1-snake toxin complex reveals open state of a Na(+)-selective channel.

    PubMed

    Baconguis, Isabelle; Bohlen, Christopher J; Goehring, April; Julius, David; Gouaux, Eric

    2014-02-13

    Acid-sensing ion channels (ASICs) detect extracellular protons produced during inflammation or ischemic injury and belong to the superfamily of degenerin/epithelial sodium channels. Here, we determin