Chemodiversity of Nonacosan‐10‐ol and n‐Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pan?i?i. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C₁₈ to C₃₃. The most abundant n‐alkanes were C₂₃, C₂₇, and C₂₅ (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C₁₈? C₃₁) than the trees of the variety pan?i?i (C₁₈? C₃₃). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.     

ELPylated anti‐human TNF therapeutic single‐domain antibodies for prevention of lethal septic shock

Tumour necrosis factor (TNF) is a major pro‐inflammatory cytokine involved in multiple inflammatory diseases. The detrimental activity of TNF can be blocked by various antagonists, and commercial therapeutics based upon this principle have been approved for treatment of diseases including rheumatoid arthritis, Crohn’s disease and psoriasis. In a search for new, improved anti‐inflammatory therapeutics we have designed a single‐domain monoclonal antibody (V_HH), which recognizes TNF. The antibody component (TNF‐V_HH) is based upon an anti‐human TNF Camelidae heavy‐chain monoclonal antibody, which was linked to an elastin‐like polypeptide (ELP). We demonstrate that ELP fusion to the TNF‐V_HH enhances accumulation of the fusion protein during biomanufacturing in transgenic tobacco plants. With this study, we show for the first time that this plant‐derived anti‐human TNF‐V_HH antibody was biologically active in vivo. Therefore, therapeutic application of TNF‐V_HH‐ELP fusion protein was tested in humanized TNF mice and was shown to be effective in preventing death caused by septic shock. The in vivo persistence of the ELPylated antibody was ～24 fold longer than that of non‐ELPylated TNF‐V_HH.     

In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Discovery of alkenones with variable methylene‐interrupted double bonds: implications for the biosynthetic pathway

Alkenones (C₃₇–C₄₀) are highly specific biomarkers produced by certain haptophyte algae in ocean and lacustrine environments and have been widely used for paleoclimate studies. Unusual shorter‐chain alkenones (SCA; e.g., C₃₅ and C₃₆) have been found in environmental and culture samples, but the origin and structure of these compounds are much less understood. The marine alkenone producer, Emiliania huxleyi CCMP2758 strain, was reported with abundant C_35:2Me (?^{12, 19}) alkenones when cultured at 15°C (Prahl et al. 2006). Here we show, when this strain is cultured at 4°C–10°C, that CCMP2758 produces abundant C_35:3Me, C_36:3Me, and small amounts of C_36:3Et alkenones with unusual double‐bond positions of ?^{7, 12, 19}. We determine the double‐bond positions of the C_35:3Me and C_36:3Me alkenones by GC‐MS analysis of the dimethyl disulfide and cyclobutylamine derivatives, and we provide the first temperature calibrations based on the unsaturation ratios of the C₃₅ and C₃₆ alkenones. Previous studies have found C_35:2Me (?^{14, 19}) and C_36:2Et (?^{14, 19}) alkenones with three‐methylene interruption in the Black Sea sediments, but this is the first reported instance of alkenones with a mixed three‐ and five‐methylene interruption configuration in the double‐bond positions. The discovery of these alkenones allows us to propose a novel biosynthetic scheme, termed the SCA biosynthesis pathway, that simultaneously rationalizes the formation of both the C_35:3Me (?^{7, 12, 19}) alkenone in our culture and the ?^{14, 19} Black Sea type alkenones without invoking new desaturases for the unusual double‐bond positions.     

ω‐Turn: A novel β‐turn mimic in globular proteins stabilized by main‐chain to side‐chain CH···O interaction

Mimicry of structural motifs is a common feature in proteins. The 10‐membered hydrogen‐bonded ring involving the main‐chain C?O in a β‐turn can be formed using a side‐chain carbonyl group leading to Asx‐turn. We show that the N? H component of hydrogen bond can be replaced by a C^γ‐H group in the side chain, culminating in a nonconventional C? H···O interaction. Because of its shape this β‐turn mimic is designated as ω‐turn, which is found to occur ～three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C? H···O interaction occurring between the terminal residues, constraining the torsion angles ?_{i + 1}, ψ_{i + 1}, ?_{i + 2} and χ′_{1(i + 2)} (using the interacting C^γ atom). Based on these angles there are two types of ω‐turns, each of which can be further divided into two groups. C^β‐branched side‐chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal‐binding sites. N‐linked glycosylation occurs at the consensus pattern Asn‐Xaa‐Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω‐turn, which may be the recognition site for protein modification. Location between two β‐strands is the most common occurrence in protein tertiary structure, and being generally exposed ω‐turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. Proteins 2015; 83:203–214. © 2014 Wiley Periodicals, Inc.     

Variability of n‐Alkanes and Nonacosan‐10‐ol in Natural Populations of Picea omorika

This is the first report of population variability of the contents of n‐alkanes and nonacosan‐10‐ol in the needle epicuticular waxes of Serbian spruce (Picea omorika). The hexane extracts of needle samples originated from three natural populations in Serbia (Vranjak, Zmajeva?ki potok, and Mile?evka Canyon) were investigated by GC and GC/MS analyses. The amount of nonacosan‐10‐ol varied individually from 50.05 to 74.42% (65.74% in average), but the differences between the three investigated populations were not statistically confirmed. The results exhibited variability of the composition of n‐alkanes in the epicuticular waxes with their size ranging from C₁₈ to C₃₅. The most abundant n‐alkanes were C₂₉, C₃₁, and C₂₇ (35.22, 13.77, and 12.28% in average, resp.). The carbon preference index of all the n‐alkanes (CPI_total) of the P. omorika populations (average of populations I–III) ranged from 3.3 to 11.5 (mean of 5.9), while the average chain length (ACL) ranged from 26.6 to 29.2. The principal component and cluster analyses of the contents of nine n‐alkanes showed the greatest difference for the population growing in the Mile?evka Canyon. The obtained results were compared with previous literature data given for other Picea species, and this comparison was briefly discussed.     

Antioxidation and DNA‐Binding Properties of Binuclear Lanthanide(III) Complexes with a Schiff Base Ligand Derived from 8‐Hydroxyquinoline‐7‐carboxaldehyde and Benzoylhydrazine

8‐Hydroxyquinoline‐7‐carboxaldehyde (8‐HQ‐7‐CA), Schiff‐base ligand 8‐hydroxyquinoline‐7‐carboxaldehyde benzoylhydrazone, and binuclear complexes [LnL(NO₃)(H₂O)₂]₂ were prepared from the ligand and equivalent molar amounts of Ln(NO₃)?6 H₂O (Ln=La³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Dy³⁺, Ho³⁺, Er³⁺, Yb³⁺, resp.). Ligand acts as dibasic tetradentates, binding to Ln^III through the phenolate O‐atom, N‐atom of quinolinato unit, and C?N and ? O? C?N? groups of the benzoylhydrazine side chain. Dimerization of this monomeric unit occurs through the phenolate O‐atoms leading to a central four‐membered (LnO)₂ ring. Ligand and all of the Ln^III complexes can strongly bind to CT‐DNA through intercalation with the binding constants at 10⁵–10⁶ M ^?1. Moreover, ligand and all of the Ln^III complexes have strong abilities of scavenging effects for hydroxyl (HO^.) radicals. Both the antioxidation and DNA‐binding properties of Ln^III complexes are much better than that of ligand.     

Anti‐Androgenic Activity of Fatty Acids

In this study, we show that 5α‐reductase derived from rat fresh liver was inhibited by certain aliphatic free fatty acids. The influences of chain length, unsaturation, oxidation, and esterification on the potency to inhibit 5α‐reductase activity were studied. Among the fatty acids we tested, inhibitory saturated fatty acids had C₁₂–C₁₆ chains, and the presence of a C?C bond enhanced the inhibitory activity. Esterification and hydroxy compounds were totally inactive. Finally, we tested the prostate cancer cell proliferation effect of free fatty acids. In keeping with the results of the 5α‐reductase assay, saturated fatty acids with a C₁₂ chain (lauric acid) and unsaturated fatty acids (oleic acid and α‐linolenic acid) showed a proliferation inhibitory effect on lymph‐node carcinoma of the prostate (LNCaP) cells. At the same time, the testosterone‐induced prostate‐specific antigen (PSA) mRNA expression was down‐regulated. These results suggested that fatty acids with 5α‐reductase inhibitory activity block the conversion of testosterone to 5α‐dihydrotestosterone (DHT) and then inhibit the proliferation of prostate cancer cells.     

Leaf wax n‐alkane patterns of six tropical montane tree species show species‐specific environmental response

It remains poorly understood how the composition of leaf wax n‐alkanes reflects the local environment. This knowledge gap inhibits the interpretation of plant responses to the environment at the community level and, by extension, inhibits the applicability of n‐alkane patterns as a proxy for past environments. Here, we studied the n‐alkane patterns of five Miconia species and one Guarea species, in the Ecuadorian Andes (653–3,507 m a.s.l.). We tested for species‐specific responses in the average chain length (ACL), the C₃₁/(C₃₁ + C₂₉) ratio (ratio), and individual odd n‐alkane chain lengths across an altitudinally driven environmental gradient (mean annual temperature, mean annual relative air humidity, and mean annual precipitation). We found significant correlations between the environmental gradients and species‐specific ACL and ratio, but with varying magnitude and direction. We found that the n‐alkane patterns are species‐specific at the individual chain length level, which could explain the high variance in metrics like ACL and ratio. Although we find species‐specific sensitivity and responses in leaf n‐alkanes, we also find a general decrease in “shorter” (<C₂₉) and an increase in “longer” (>C₃₁) chain lengths with the environmental gradients, most strongly with temperature, suggesting n‐alkanes are useful for reconstructing past environments.     

LEW3, encoding a putative α‐1,2‐mannosyltransferase (ALG11) in N‐linked glycoprotein,plays vital roles in cell‐wall biosynthesis and the abiotic stress response in Arabidopsis thaliana

N‐linked glycosylation is an essential protein modification that helps protein folding, trafficking and translocation in eukaryotic systems. The initial process for N‐linked glycosylation shares a common pathway with assembly of a dolichol‐linked core oligosaccharide. Here we characterize a new Arabidopsis thaliana mutant lew3 (leaf wilting 3), which has a defect in an α‐1,2‐mannosyltransferase, a homolog of ALG11 in yeast, that transfers mannose to the dolichol‐linked core oligosaccharide in the last two steps on the cytosolic face of the ER in N‐glycan precursor synthesis. LEW3 is localized to the ER membrane and expressed throughout the plant. Mutation of LEW3 caused low‐level accumulation of Man₃GlcNAc₂ and Man₄GlcNAc₂ glycans, structures that are seldom detected in wild‐type plants. In addition, the lew3 mutant has low levels of normal high‐mannose‐type glycans, but increased levels of complex‐type glycans. The lew3 mutant showed abnormal developmental phenotypes, reduced fertility, impaired cellulose synthesis, abnormal primary cell walls, and xylem collapse due to disturbance of the secondary cell walls. lew3 mutants were more sensitive to osmotic stress and abscisic acid (ABA) treatment. Protein N‐glycosylation was reduced and the unfolded protein response was more activated by osmotic stress and ABA treatment in the lew3 mutant than in the wild‐type. These results demonstrate that protein N‐glycosylation plays crucial roles in plant development and the response to abiotic stresses.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

2H‐Pyran‐2‐one and 2H‐Furan‐2‐one Derivatives from the Plant Endophytic Fungus Pestalotiopsis fici

Two new α‐pyrones (=2H‐pyran‐2‐ones), ficipyrones A and B ( 1 and 2 , resp.), and two new α‐furanones (=2H‐furan‐2‐ones), ficifuranones A and B ( 3 and 4 , resp.), together with three known metabolites, antibiotic F 0368 ( 5 ), hydroxyseiridin ( 6 ), and hydroxyisoseiridin ( 7 ), were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute configuration of 1 was deduced from the circular‐dichroism (CD) data. Compound 1 showed antifungal activity against the plant pathogen Gibberella zeae (CGMCC 3.2873) with an IC₅₀ value of 15.9 μM .     

Synthesis,characterization and luminescent properties of europium complexes with 2,4,6‐tris‐(2‐pyridyl)‐s‐triazine as highly efficient sensitizers

Using 2,4,6‐tris‐(2‐pyridyl)‐s‐triazine (TPTZ) as a neutral ligand, and p‐hydroxybenzoic acid, terephthalic acid and nitrate as anion ligands, five novel europium complexes have been synthesized. These complexes were characterized using elemental analysis, rare earth coordination titrations, UV/vis absorption spectroscopy and infrared spectroscopy. Luminescence spectra, luminescence lifetime and quantum efficiency were investigated and the mechanism discussed in depth. The results show that the complexes have excellent emission intensities, long emission lifetimes and high quantum efficiencies. The superior luminescent properties of the complexes may be because the triplet energy level of the ligands matches well with the lowest excitation state energy level of Eu³⁺. Moreover, changing the ratio of the ligands and metal ions leads to different luminescent properties. Among the complexes, Eu₂(TPTZ)₂(C₈H₄O₄)(NO₃)₄(C₂H₅OH)·H₂O shows the strongest luminescence intensity, longest emission lifetime and highest quantum efficiency. Copyright © 2015 John Wiley & Sons, Ltd.     

Mesophyll CO2 conductance and leakiness are not responsive to short‐ and long‐term soil water limitations in the C4 plant Sorghum bicolor

Breeding economically important C₄ crops for enhanced whole‐plant water‐use efficiency (WUE_plant) is needed for sustainable agriculture. WUE_plant is a complex trait and an efficient phenotyping method that reports on components of WUE_plant, such as intrinsic water‐use efficiency (WUE_i, the rate of leaf CO₂ assimilation relative to water loss via stomatal conductance), is needed. In C₄ plants, theoretical models suggest that leaf carbon isotope composition (δ¹³C), when the efficiency of the CO₂‐concentrating mechanism (leakiness, ?) remains constant, can be used to screen for WUE_i. The limited information about how ? responds to water limitations confines the application of δ¹³C for WUE_i screening of C₄ crops. The current research aimed to test the response of ? to short‐ or long‐term moderate water limitations, and the relationship of δ¹³C with WUE_i and WUE_plant, by addressing potential mesophyll CO₂ conductance (g_m) and biochemical limitations in the C₄ plant Sorghum bicolor. We demonstrate that g_m and ? are not responsive to short‐ or long‐term water limitations. Additionally, δ¹³C was not correlated with gas‐exchange estimates of WUE_i under short‐ and long‐term water limitations, but showed a significant negative relationship with WUE_plant. The observed association between the δ¹³C and WUE_plant suggests an intrinsic link of δ¹³C with WUE_i in this C₄ plant, and can potentially be used as a screening tool for WUE_plant in sorghum.     

Engineering ‘designer’ glycomodules for boosting recombinant protein secretion in tobacco hairy root culture and studying hydroxyproline‐O‐glycosylation process in plants

The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of ‘Ser‐Hyp‐Hyp‐Hyp‐Hyp’ motif or (SP4)₁₈ and an arabinogalactan protein module consisting of 32 tandem repeats of ‘Ser‐Hyp’ motif or (SP)₃₂. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)₁₈ and (SP)₃₂ modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)₁₈‐EGFP and (SP)₃₂‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)₁₈ module and AGP‐styled (SP)₃₂ module is proposed.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

Arabidopsis ketoacyl‐CoA synthase 16 (KCS16) forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β‐ketoacyl‐CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C₃₅ and C₃₇ alkanes derived from C₃₆ and C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss‐of‐function mutants were depleted of C₃₅ and C₃₇ alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C₃₆ and C₃₈ fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C₃₄ to C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra‐long compounds in adjacent pavement cells.     

Interactive effects of elevated CO2 and nitrogen deposition on fatty acid molecular and isotope composition of above‐ and belowground tree biomass and forest soil fractions

Atmospheric carbon dioxide (CO₂) and reactive nitrogen (N) concentrations have been increasing due to human activities and impact the global carbon (C) cycle by affecting plant photosynthesis and decomposition processes in soil. Large amounts of C are stored in plants and soils, but the mechanisms behind the stabilization of plant‐ and microbial‐derived organic matter (OM) in soils are still under debate and it is not clear how N deposition affects soil OM dynamics. Here, we studied the effects of 4 years of elevated (¹³C‐depleted) CO₂ and N deposition in forest ecosystems established in open‐top chambers on composition and turnover of fatty acids (FAs) in plants and soils. FAs served as biomarkers for plant‐ and microbial‐derived OM in soil density fractions. We analyzed above‐ and belowground plant biomass of beech and spruce trees as well as soil density fractions for the total organic C and FA molecular and isotope (δ¹³C) composition. FAs did not accumulate relative to total organic C in fine mineral fractions, showing that FAs are not effectively stabilized by association with soil minerals. The δ¹³C values of FAs in plant biomass increased under high N deposition. However, the N effect was only apparent under elevated CO₂ suggesting a N limitation of the system. In soil fractions, only isotope compositions of short‐chain FAs (C₁₆₊₁₈) were affected. Fractions of ‘new’ (experimental‐derived) FAs were calculated using isotope depletion in elevated CO₂ plots and decreased from free light to fine mineral fractions. ‘New’ FAs were higher in short‐chain compared to long‐chain FAs (C_20?30), indicating a faster turnover of short‐chain compared to long‐chain FAs. Increased N deposition did not significantly affect the quantity of ‘new’ FAs in soil fractions, but showed a tendency of increased amounts of ‘old’ (pre‐experimental) C suggesting that decomposition of ‘old’ C is retarded by high N inputs.     

Cloning,expression, and identification of anti‐carbofuran single chain Fv gene

Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly₄Ser)₃. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC₅₀ value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009