Tracing mangrove carbon in suspended matter and aquatic fauna of the Gautami–Godavari Delta,Bay of Bengal (India)

Stable carbon isotopic composition and C/N ratio were used to trace the input of carbon associated with mangrove litter into the estuary of the Godavari–Gautami delta system and Kakinada bay (Andhra Pradesh, India). Suspended organic matter in the mangrove channels was more depleted in ¹³C (average ¹³C = –24.5) than in Kakinada bay which showed ¹³C values for suspended matter (average ¹³C = –22.7) closer to those expected for marine phytoplankton. Suspended organic matter from mangrove channels was enriched in nitrogen (average C/N atom ratio 12.7) and ¹³C (average ¹³C = –24.5) relative to mangrove leaf litter, which had a C/N ratio of 75 and a ¹³C value of –28. Lowest C/N ratios for suspended matter were observed during southwest monsoon when rainfall was highest. Although in general, mangrove litter fall was also lower during this period, no clear correlation was observed between litter fall and C/N ratio of suspended matter. In general, the composition of suspended matter pointed towards phytoplankton as a major component. Isotopic composition of zooplankton suggested selective feeding on ¹³C-enriched, marine phytoplankton in open Kakinada bay and on ¹³C-depleted organic matter, such as estuarine phytoplankton and mangrove litter, in the mangrove channels. From the ¹³C signature, it appeared that mangrove carbon was present to some extent in zooplankton and macrofauna from the mangrove mudflats and channels, but the signal rapidly decreased in Kakinada bay. Nitrogen isotopic composition of zooplankton and macrofauna indicated a progressive enrichment of ¹⁵N away from the mangrove forest towards the northern part of Kakinada bay, in approach of Kakinada city. This is thought to reflect input of anthropogenic nitrogen enriched in ¹⁵N and subsequent uptake of this enriched nitrogen into the aquatic food chain.     

Influences of different nitrogen sources on nitrogen- and water-use efficiency, and carbon isotope discrimination, in C3 Triticum aestivum L. and C4 Zea mays L. plants

The impacts of various nitrogen sources, i.e. NO⁻ ₃, NH₄ ⁺ or NH₄NO₃ in combination with gaseous NH₃, on nitrogen-, carbon- and water-use efficiency and ¹³C discrimination (δ¹³C) by plants of the C₃ species Triticum aestivum L. (wheat) and the C₄ species Zea mays L. (maize) were studied. Triticum aestivum and Z. mays were hydroponically grown with 2 mol · m⁻³ of N supplied as NO⁻ ₃, NH₄ ⁺ or NH₄NO₃ for 21 and 18 d, respectively, and thereafter exposed to gaseous NH₃ at 320 μg · m⁻³ or to ambient air for 7 d. In T. aestivum and Z. mays over a 7-d growth period, nitrogen-use efficiency (NUE) values were influenced by N-sources in the decreasing order NH₄NO₃-N > NO⁻ ₃-N > NH₄ ⁺-N and NO⁻ ₃-N > NH₄NO₃-N > NH₄ ⁺-N, respectively. Fumigation with NH₃ decreased the NUE values of plants grown with any of the N-forms. During 28- and 7-d growth periods, N-sources affected water-use efficiency (WUE) values in the decreasing order of NH₄ ⁺-N > NO⁻ ₃-N≈NH₄NO₃-N in non-fumigated T. aestivum, while fumigation with NH₃ increased the WUE of NO⁻ ₃-grown plants. There were insignificant effects of N-sources on WUE values of Z. mays over 25- and 7-d growth periods. Furthermore, δ¹³C values in plant tissues (leaves, stubble and roots) were higher (less negative) in NH₄ ⁺-grown plants of T. aestivum and Z. mays than in those supplied with NH₄NO₃ or NO⁻ ₃. Regardless of the N-form supplied to the roots of the plant species, exposure to NH₃ caused more-positive δ¹³C values in the plant tissues. These results indicate that the variations in N-source were associated with small but significant variations in δ¹³C values in plants of T. aestivum and Z. mays. These differences in δ¹³C values are in the direction expected from differences in WUE values over long or short growth periods and with differences in the extent of non-Rubisco (ribulose-1,5-bisphosphate carboxylase-oxygenase, EC 4.1.1.39) carboxylate contribution to net C acquisition, as a function of N-source. Received: 12 September 1997 / Accepted: 13 January 1998     

The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

Editing of Multidimensional NMR Spectra of Partially Deuterated Proteins. Measurement of Amide Deuterium Isotope Effects on the Chemical Shifts of Protein Backbone Nuclei

Novel multidimensional NMR pulse sequences are presented for determination of amide deuterium isotope effects on the 13C chemical shifts of the backbone in proteins. The sequences edit heteronuclear triple resonance spectra into two subspectra according to whether a deuterium or a proton is attached to 15N for the pertinent correlations. The new experiments are demonstrated using 13C, 15N-labeled RAP 17-112 (N-terminal domain of 2-macroglobulin receptor associated protein).     

Synthesis of the 15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Summary Synthesis of zervamicin IIB, specifically labeled at the α-position of glutamine-11 with¹⁵N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB specifically¹⁵N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the¹⁵N-label was clearly detected. The isotope enrichment (98 ±2%) was determined by FAB-mass spectrometry.     

Use of stored carbon reserves in growth of temperate tree roots and leaf buds: analyses using radiocarbon measurements and modeling

Characterizing the use of carbon (C) reserves in trees is important for understanding regional and global C cycles, stress responses, asynchrony between photosynthetic activity and growth demand, and isotopic exchanges in studies of tree physiology and ecosystem C cycling. Using an inadvertent, whole-ecosystem radiocarbon (¹⁴C) release in a temperate deciduous oak forest and numerical modeling, we estimated that the mean age of stored C used to grow both leaf buds and new roots is 0.7 years and about 55% of new-root growth annually comes from stored C. Therefore, the calculated mean age of C used to grow new-root tissue is ∼0.4 years. In short, new roots contain a lot of stored C but it is young in age. Additionally, the type of structure used to model stored C input is important. Model structures that did not include storage, or that assumed stored and new C mixed well (within root or shoot tissues) before being used for root growth, did not fit the data nearly as well as when a distinct storage pool was used. Consistent with these whole-ecosystem labeling results, the mean age of C in new-root tissues determined using 'bomb-¹⁴C' in three additional forest sites in North America and Europe (one deciduous, two coniferous) was less than 1–2 years. The effect of stored reserves on estimated ages of fine roots is unlikely to be large in most natural abundance isotope studies. However, models of root C dynamics should take stored reserves into account, particularly for pulse-labeling studies and fast-cycling roots (<1 years).     

TNFalph 和 IL-1bet 靶点抗炎药物筛选细胞模型的构建

目的:构建两个高表达人TNFα和IL-1β细胞系,建立抗炎药物筛选细胞模型。方法:运用PCR的方法从载体pCMVSport-TNFα和p CMVSport-IL1β上扩增目的基因,以亚克隆方法将目的基因分别插入真核表达载体pcDNA3.1和pFLAG-CMV中,用单酶切、PCR扩增和基因测序的方法鉴定重组效果,然后将重组成功的质粒转入HEK293细胞系内,挑选能够稳定表达并遗传的单克隆细胞株,用蛋白免疫印迹(Western blot)法分析其表达效果。结果:三种鉴定方法均显示重组质粒构建成功。Western blot结果显示,细胞株T3、T4均能较高表达炎症因子TNF-α;细胞株I2、I3、I5均能较高表达炎症因子IL-1β。结论:成功构建了TNFα和IL-1β靶标的药物筛选细胞模型,为筛选具有抗炎作用的中药提供了一个新平台。     

Effects of grass species and grass growth on atmospheric nitrogen deposition to a bog ecosystem surrounded by intensive agricultural land use

We applied a ¹⁵N dilution technique called “Integrated Total Nitrogen Input” (ITNI) to quantify annual atmospheric N input into a peatland surrounded by intensive agricultural practices over a 2-year period. Grass species and grass growth effects on atmospheric N deposition were investigated using Lolium multiflorum and Eriophorum vaginatum and different levels of added N resulting in increased biomass production. Plant biomass production was positively correlated with atmospheric N uptake (up to 102.7 mg N pot⁻¹) when using Lolium multiflorum. In contrast, atmospheric N deposition to Eriophorum vaginatum did not show a clear dependency to produced biomass and ranged from 81.9 to 138.2 mg N pot⁻¹. Both species revealed a relationship between atmospheric N input and total biomass N contents. Airborne N deposition varied from about 24 to 55 kg N ha⁻¹ yr⁻¹. Partitioning of airborne N within the monitor system differed such that most of the deposited N was found in roots of Eriophorum vaginatum while the highest share was allocated in aboveground biomass of Lolium multiflorum. Compared to other approaches determining atmospheric N deposition, ITNI showed highest airborne N input and an up to fivefold exceedance of the ecosystem-specific critical load of 5–10 kg N ha⁻¹ yr⁻¹.     

Technical Note: Calcium and carbon stable isotope ratios as paleodietary indicators

Calcium stable isotope ratios are hypothesized to vary as a function of trophic level. This premise raises the possibility of using calcium stable isotope ratios to study the dietary behaviors of fossil taxa and to test competing hypotheses on the adaptive origins of euprimates. To explore this concept, we measured the stable isotope composition of contemporary mammals in northern Borneo and northwestern Costa Rica, two communities with functional or phylogenetic relevance to primate origins. We found that bone collagen δ¹³C and δ¹⁵N values could differentiate trophic levels in each assemblage, a result that justifies the use of these systems to test the predicted inverse relationship between bioapatite δ¹³C and δ⁴⁴Ca values. As expected, taxonomic carnivores (felids) showed a combination of high δ¹³C and low δ⁴⁴Ca values; however, the δ⁴⁴Ca values of other faunivores were indistinguishable from those of primary consumers. We suggest that the trophic insensitivity of most bioapatite δ⁴⁴Ca values is attributable to the negligible calcium content of arthropod prey. Although the present results are inconclusive, the tandem analysis of δ⁴⁴Ca and δ¹³C values in fossils continues to hold promise for informing paleodietary studies and we highlight this potential by drawing attention to the stable isotope composition of the Early Eocene primate Cantius. Am J Phys Anthropol 154:633–643, 2014. © 2014 Wiley Periodicals, Inc.     

Insight into the reaction mechanism of the Escherichia coli cyclopropane fatty acid synthase: Isotope exchange and kinetic isotope effects

Cyclopropanation of unsaturated lipids is an intriguing enzymatic reaction and a potential therapeutic target in Mycobacterium tuberculosis. Cyclopropane fatty acid synthase from Escherichia coli is the only in vitro model available to date for mechanistic and inhibition studies. While the overall reaction mechanism of this enzymatic process is now well accepted, some mechanistic issues are still debated. Using homogeneous E. coli enzyme we have shown that, contrary to previous report based on in vivo experiments, there is no exchange of the cylopropane methylene protons with the solvent during catalysis, as probed by ultra high resolution mass spectrometry. Using [methyl-¹⁴C]-labeled and [methyl-³H₃]-S-adenosyl-l-methionine we have measured a significant intermolecular primary tritium kinetic isotope effect (^TV/K_app = 1.8 ± 0.1) consistent with a partially rate determining deprotonation step. We conclude that both chemical steps of this enzymatic cyclopropanation, the methyl addition onto the double bond and the deprotonation step, are rate determining, a common situation in efficient enzymes.