A partial-complementary adapter for an improved and simplified ligation-mediated suppression PCR technique

Ligation-mediated suppression PCR (LMS-PCR) is a powerful tool for walking in unknown genomic DNA regions from known adjacent sequences. This approach has made it feasible to obtain promoter sequences and to enable researchers to identify full-length gene sequences or isoforms of multigene families. However, the advantages of LMS-PCR can be obviated by the presence of incomplete base modifications on the suppression adapters. We propose here that a 'partial-complementary adapter' is a more reliable suppression adapter, demanding only 5'-end phosphorylation. We also describe a simplified procedure for the easier preparation of PCR templates with very small quantities of DNA and a fast and direct characterization of the suppression-PCR products. A set of practical guidelines is proposed for pre-checking the efficiency of the adapter modification using two model systems: bacteriophage lambda (lambda) and Arabidopsis.     

在无有机碳源和缺氧条件下自养菌和异养菌脱氨氮作用

在无有机碳源和缺氧条件下,应用^15N示踪考察4株具有氨氧化作用的菌株在膜反应器中的氨氧化产气情况。当溶氧浓度（DO）〈0．5mg／L的缺氧条件下,自养菌Nitrosomonas sp．单株培养,能将6．3％的氨氮转化为氮气,^15N2产生量占氨氮消耗量的21．86％。自养菌株与异养菌株混合培养,可将30．86％的氨氮转化为氮气,^15N2产生量占氨氮消耗茸的80．38％。企无有机碳的条件下,自养菌和异养菌的协同代时作用,可大大提高缺氧氩氧化产氮气的效率。     

Isotopomer analysis of myocardial substrate metabolism: a systems biology approach

The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network.     

Two-step regio- and stereoselective syntheses of [19F]- and [18F]-2-deoxy-2-(R)-fluoro-beta-D-allose

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.     

Mycorrhizal‐mediated nitrogen acquisition in switchgrass under elevated temperatures and N enrichment

Arbuscular mycorrhizal fungi (AMF) can perform key roles in ecosystem functioning through improving host nutrient acquisition. Nitrogen (N) is an essential nutrient for plant growth, however, anthropogenic N loading (e.g. crop fertilization and deposition from combustion sources) is increasing so that N now threatens ecosystem sustainability around the world by causing terrestrial and aquatic eutrophication and acidification. It is important to better understand the capacity of AMF to directly uptake N from soils and transfer it to host plants because this process may increase N recycling and retention within ecosystems. In addition to understanding the role of AMF in the N cycle in the present day it is important to understand how AMF function may change as global change proceeds. Currently the net effects of N enrichment and elevated temperature predicted with global change on AMF are unknown. In this study, we examined the effects of N enrichment by simulated N‐deposition loading, elevated temperatures expected by future global changes and their interactions on growth and AMF‐mediated N acquisition of switchgrass (Panicum virgatum var. Alamo), an important species for biofuel production. Switchgrass plants were grown in microcosm units that divided mycorrhizal roots from AMF hyphae and organic residues enriched with ¹⁵N by compartments separated by an air gap to reduce N diffusion. While AMF did not enhance switchgrass biomass, mycorrhizas significantly increased ¹⁵N in shoots and total shoot N. Neither N enrichment nor elevated temperatures influenced this mycorrhizal‐mediated N uptake and transfer. Results from this study can aid in developing sustainable bioethanol and switchgrass production practices that are less reliant on synthetic fertilizers and more dependent on internal N recycling from AMF.     

Light enhanced amino acid uptake by dominant bacterioplankton groups in surface waters of the Atlantic Ocean

(35)S-Methionine and (3)H-leucine bioassay tracer experiments were conducted on two meridional transatlantic cruises to assess whether dominant planktonic microorganisms use visible sunlight to enhance uptake of these organic molecules at ambient concentrations. The two numerically dominant groups of oceanic bacterioplankton were Prochlorococcus cyanobacteria and bacteria with low nucleic acid (LNA) content, comprising 60% SAR11-related cells. The results of flow cytometric sorting of labelled bacterioplankton cells showed that when incubated in the light, Prochlorococcus and LNA bacteria increased their uptake of amino acids on average by 50% and 23%, respectively, compared with those incubated in the dark. Amino acid uptake of Synechococcus cyanobacteria was also enhanced by visible light, but bacteria with high nucleic acid content showed no light stimulation. Additionally, differential uptake of the two amino acids by the Prochlorococcus and LNA cells was observed. The populations of these two types of cells on average completely accounted for the determined 22% light enhancement of amino acid uptake by the total bacterioplankton community, suggesting a plausible way of harnessing light energy for selectively transporting scarce nutrients that could explain the numerical dominance of these groups in situ.     

Anthropogenic N deposition and the fate of 15NO 3 − in a northern hardwood ecosystem

Human activity has substantially increased atmospheric NO ₃ ^– deposition in many regions of the Earth, which could lead to the N saturation of terrestrial ecosystems. Sugar maple (Acer saccharum Marsh.) dominated northern hardwood forests in the Upper Great Lakes region may be particularly sensitive to chronic NO ₃ ^– deposition, because relatively moderate experimental increases (three times ambient) have resulted in substantial N leaching over a relatively short duration (5–7 years). Although microbial immobilization is an initial sink (i.e., within 1–2 days) for anthropogenic NO ₃ ^– in this ecosystem, we have an incomplete understanding of the processes controlling the longer-term (i.e., after 1 year) retention and flow of anthropogenic N. Our objectives were to determine: (i) whether chronic NO ₃ ^– additions have altered the N content of major ecosystem pools, and (ii) the longer-term fate of ¹⁵NO ₃ ^– in plots receiving chronic NO ₃ ^– addition. We addressed these objectives using a field experiment in which three northern hardwood plots receive ambient atmospheric N deposition (ca. 0.9 g N m^–2 year^–1) and three plots which receive ambient plus experimental N deposition (3.0 g NO₃ ^–-N m^–2 year^–1). Chronic NO ₃ ^– deposition significantly increased the N concentration and content (g N/m²) of canopy leaves, which contained 72% more N than the control treatment. However, chronic NO ₃ ^– deposition did not significantly alter the biomass, N concentration or N content of any other ecosystem pool. The largest portion of ¹⁵N recovered after 1 year occurred in overstory leaves and branches (10%). In contrast, we recovered virtually none of the isotope in soil organic matter (SOM), indicating that SOM was not a sink for anthropogenic NO ₃ ^– over a 1 year duration. Our results indicate that anthropogenic NO ₃ ^– initially assimilated by the microbial community is released into soil solution where it is subsequently taken up by overstory trees and allocated to the canopy. Anthropogenic N appears to be incorporated into SOM only after it is returned to the forest floor and soil via leaf litter fall. Short- and long-term isotope tracing studies provided very different results and illustrate the need to understand the physiological processes controlling the flow of anthropogenic N in terrestrial ecosystems and the specific time steps over which they operate.     

The feeding behaviour of a deep-sea holothurian, Stichopus tremulus (Gunnerus) based on in situ observations and experiments using a Remotely Operated Vehicle

Using a Remotely Operated Vehicle (ROV) to deploy an in situ cage experiment incorporating fluorescent Luminophore particle tracers, the gut throughput time of the deposit feeding holothurian, Stichopus tremulus (Gunnerus) was determined as 23.73 h (S.D.±2.3). For a range of individuals examined at different depths (350-500 m) and locations, throughput times varied between 19 and 26 h irrespective of animal size or gut tract length. In situ video observations of feeding behaviour showed that this species uses fine oral papillae in a ‘sweeping’ motion to target particles on the seafloor. Following detection of a food source fine-branched digitate tentacles collect a large range of sediment fragments from the seabed. The main types of particles ingested include silica fragments (<20 >500 μm), pelagic foraminifera, benthic foraminifera, fine phytodetrital remains and occasional larger rock fragments (∼1 cm). Ingested sediment consisted mainly of very fine silica fragments (∼50 μm) accounting for over 50% of the total gut contents. Frame-by-frame video analysis revealed that the particle handling time (i.e. the time taken for a tentacle insertion and the subsequent collection of food) was found to be ∼54 s. Only 10 of the 20 feeding tentacles were simultaneously employed during feeding. Use of tentacles appeared to be in sequence, alternating between the reserve and active tentacles. Estimating the rate of movement over the seabed and the total effective capture area of each tentacle, the impact of this animal on the turnover and quality of surface sediment at this deepwater site is potentially substantial. The in situ experiments provided a significant improvement over previous methods used to investigate deep-sea deposit feeders and represent a useful concept for further in situ deep-sea research using an industrial ROV.     

A Liquid Chromatography–Mass Spectrometry Method to Measure Stable Isotopic Tracer Enrichments of Glycerol and Glucose in Human Serum

Stable isotopes are commonly used as tracers for the measurement of glycerol and glucose kinetics in metabolic studies. Traditionally, the analysis of these isotopes has been performed using gas chromatography-mass spectrometry, which requires that the analytes first be derivatized. The derivatization process adds considerable complexity to the method. Liquid chromatography-mass spectrometry (LCMS) can measure many metabolites directly with limited sample preparation. We present a novel analytical method for the measurement of [1,1,2,3,3-(2)H(5)]glycerol (d(5)-glycerol) and [6,6-(2)H(2)]glucose (d(2)-glucose) isotopic tracer enrichments in human serum in a single run by LCMS. After a simple extraction step, the sample is separated isocratically by HPLC, and the isotopes are measured using positive electrospray ionization with selected ion monitoring of the sodium-adduct ions. The method is linear over a wide range of d(2)-glucose and d(5)-glycerol enrichments. The within-day standard deviation of measurement of serum samples was 0.05 mole% excess (MPE) for d(2)-glucose and 0.25 MPE for d(5)-glycerol. The variation of tracer enrichment among days was about double that measured within 1 day.