Stable Isotope Labeling Experiments Research Articles

CDS2 expression regulates de novo phosphatidic acid synthesis.

CDS enzymes (CDS1 and 2 in mammals) convert phosphatidic acid (PA) to CDP-DG, an essential intermediate in the de novo synthesis of PI. Genetic deletion of CDS2 in primary mouse macrophages resulted in only modest changes in the steady-state levels of major phospholipid species, including PI, but substantial increases in several species of PA, CDP-DG, DG and TG. Stable isotope labelling experiments employing both 13C6- and 13C6D7-glucose revealed loss of CDS2 resulted in a minimal reduction in the rate of de novo PI synthesis but a substantial increase in the rate of de novo PA synthesis from G3P, derived from DHAP via glycolysis. This increased synthesis of PA provides a potential explanation for normal basal PI synthesis in the face of reduced CDS capacity (via increased provision of substrate to CDS1) and increased synthesis of DG and TG (via increased provision of substrate to LIPINs). However, under conditions of sustained GPCR-stimulation of PLC, CDS2-deficient macrophages were unable to maintain enhanced rates of PI synthesis via the 'PI cycle', leading to a substantial loss of PI. CDS2-deficient macrophages also exhibited significant defects in calcium homeostasis which were unrelated to the activation of PLC and thus probably an indirect effect of increased basal PA. These experiments reveal that an important homeostatic response in mammalian cells to a reduction in CDS capacity is increased de novo synthesis of PA, likely related to maintaining normal levels of PI, and provides a new interpretation of previous work describing pleiotropic effects of CDS2 deletion on lipid metabolism/signalling.

Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways

Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.

Lipogenesis in Nasonia vitripennis: Influence of sugar chemistry, preferential production of triacylglycerides, and comparison of fatty acid biosynthetic capacity with Drosophilamelanogaster

Sugar consumption increases the fecundity and longevity in many species of parasitic wasps (parasitoids) but whether these insects use sugars to synthesize significant amounts of fatty acids and storage fat de novo (lipogenesis) is discussed controversially. It has long been assumed that parasitic wasps lost this ability during evolution, mainly because in several species wasps with ad libitum access to sugar did not increase teneral lipid levels. Recent studies demonstrated that many species are nonetheless capable of synthesizing fatty acids de novo from glucose. It is unclear, however, whether also other sugars are used for fatty acid biosynthesis and whether an increase of sugar concentration to levels occurring in natural sugar sources translates into higher fatty acid production. Furthermore, it has been suggested that fatty acid production in parasitoids is negligible compared to species increasing teneral fat reserves such as Drosophila melanogaster. Here we show by stable isotope labeling experiments that females of Nasonia vitripennis convert D-glucose, D-fructose, sucrose, and α,α-trehalose, major sugars consumed by adult parasitoids in nature, equally well to palmitic, stearic, oleic, and linoleic acid. Lipogenesis from D-galactose occurs as well albeit to a lesser extent. Sugar concentration is crucial for lipogenic activity, and almost 80% of de novo synthesized fatty acids were incorporated into storage fat (triacylglycerides). Comparison of fatty acid biosynthesis within a 48-h feeding period with D. melanogaster revealed that N. vitripennis produced approximately half as many fatty acids per body mass unit. Both species fed equal amounts of the glucose offered. We conclude that lipogenesis is far from negligible in N. vitripennis and plays an important role for the energy balance when teneral lipid reserves deplete.

Esterification of Cyclic N6-Threonylcarbamoyladenosine During RNA Sample Preparation.

The continuous deciphering of crucial biological roles of RNA modifications and their involvement in various pathological conditions, together with their key roles in the use of RNA-based therapeutics, has reignited interest in studying the occurrence and identity of non-canonical ribonucleoside structures during the past years. Discovery and structural elucidation of new modified structures is usually achieved by combination of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) at the nucleoside level and stable isotope labeling experiments. This approach, however, has its pitfalls as demonstrated in the course of the present study: we structurally elucidated a new nucleoside structure that showed significant similarities to the family of (c)t6A modifications and was initially considered a genuine modification, but subsequently turned out to be an in vitro formed glycerol ester of t6A. This artifact is generated from ct6A during RNA hydrolysis upon addition of enzymes stored in glycerol containing buffers in a mildly alkaline milieu, and was moreover shown to undergo an intramolecular transesterification reaction. Our results demand for extra caution, not only in the discovery of new RNA modifications, but also with regard to the quantification of known modified structures, in particular chemically labile modifications, such as ct6A, that might suffer from exposure to putatively harmless reagents during the diverse steps of sample preparation.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Coupled carbon and nitrogen cycling regulates the cnidarian–algal symbiosis

Efficient nutrient recycling underpins the ecological success of cnidarian-algal symbioses in oligotrophic waters. In these symbioses, nitrogen limitation restricts the growth of algal endosymbionts in hospite and stimulates their release of photosynthates to the cnidarian host. However, the mechanisms controlling nitrogen availability and their role in symbiosis regulation remain poorly understood. Here, we studied the metabolic regulation of symbiotic nitrogen cycling in the sea anemone Aiptasia by experimentally altering labile carbon availability in a series of experiments. Combining 13C and 15N stable isotope labeling experiments with physiological analyses and NanoSIMS imaging, we show that the competition for environmental ammonium between the host and its algal symbionts is regulated by labile carbon availability. Light regimes optimal for algal photosynthesis increase carbon availability in the holobiont and stimulate nitrogen assimilation in the host metabolism. Consequently, algal symbiont densities are lowest under optimal environmental conditions and increase toward the lower and upper light tolerance limits of the symbiosis. This metabolic regulation promotes efficient carbon recycling in a stable symbiosis across a wide range of environmental conditions. Yet, the dependence on resource competition may favor parasitic interactions, explaining the instability of the cnidarian-algal symbiosis as environmental conditions in the Anthropocene shift towards its tolerance limits.

A large-scale LC-MS dataset of murine liver proteome from time course of heavy water metabolic labeling

Metabolic stable isotope labeling with heavy water followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein turnover studies. Several algorithms and tools have been developed to determine the turnover rates of peptides and proteins from time-course stable isotope labeling experiments. The availability of benchmark mass spectrometry data is crucial to compare and validate the effectiveness of newly developed techniques and algorithms. In this work, we report a heavy water-labeled LC-MS dataset from the murine liver for protein turnover rate analysis. The dataset contains eighteen mass spectral data with their corresponding database search results from nine different labeling durations and quantification outputs from d2ome+ software. The dataset also contains eight mass spectral data from two-dimensional fractionation experiments on unlabeled samples.

Discovery and Biosynthetic Origin of Quinolizidomycins A and B, Two Quinolizidine Alkaloids from Streptomyces sp. KIB-1714.

Quinolizidomycins A (1) and B (2), two unprecedented quinolizidine alkaloids featuring a tricyclic 6/6/5 ring system, were isolated from Streptomyces sp. KIB-1714. Their structures were assigned by detailed spectroscopic data analyses and X-ray diffraction. Stable isotope labeling experiments suggested that compounds 1 and 2 are derived from lysine, ribose 5-phosphate, and acetate units, which indicates an unprecedented manner of assembly of the quinolizidine (1-azabicyclo[4.4.0]decane) scaffold in quinolizidomycin biosynthesis. Quinolizidomycin A (1) was active in an acetylcholinesterase inhibitory assay.

13CO2-labelling and Sampling in Algae for Flux Analysis of Photosynthetic and Central Carbon Metabolism.

The flux in photosynthesis can be studied by performing 13CO2 pulse labelling and analysing the temporal labelling kinetics of metabolic intermediates using gas or liquid chromatography linked to mass spectrometry. Metabolic flux analysis (MFA) is the primary approach for analysing metabolic network function and quantifying intracellular metabolic fluxes. Different MFA approaches differ based on the metabolic state (steady vs. non-steady state) and the use of stable isotope tracers. The main methodology used to investigate metabolic systems is metabolite steady state associated with stable isotope labelling experiments. Specifically, in biological systems like photoautotrophic organisms, isotopic non-stationary 113C metabolic flux analysis at metabolic steady state with transient isotopic labelling (13C-INST-MFA) is required. The common requirement for metabolic steady state, alongside its very short half-timed reactions, complicates robust MFA of photosynthetic metabolism. While custom gas chambers design has addressed these challenges in various model plants, no similar tools were developed for liquid photosynthetic cultures (e.g., algae, cyanobacteria), where diffusion and equilibration of inorganic carbon species in the medium entails a new dimension of complexity. Recently, a novel tailor-made microfluidics labelling system has been introduced, supplying short 13CO2 pulses at steady state, and resolving fluxes across most photosynthetic metabolic pathways in algae. The system involves injecting algal cultures and medium containing pre-equilibrated inorganic 13C into a microfluidic mixer, followed by rapid metabolic quenching, enabling precise seconds-level label pulses. This was complemented by a 13CO2-bubbling-based open labelling system (photobioreactor), allowing long pulses (minutes-hours) required for investigating fluxes into central C metabolism and major products. This combined labelling procedure provides a comprehensive fluxome cover for most algal photosynthetic and central C metabolism pathways, thus allowing comparative flux analyses across algae and plants.

Conditional Fragment Ion Probabilities Improve Database Searching for Nonmonoisotopic Precursors.

Stochastic, intensity-based precursor isolation can result in isotopically enriched fragment ions. This problem is exacerbated for large peptides and stable isotope labeling experiments using deuterium or 15N. For stable isotope labeling experiments, incomplete and ubiquitous labeling strategies result in the isolation of peptide ions composed of many distinct structural isomers. Unfortunately, existing proteomics search algorithms do not account for this variability in isotopic incorporation, and thus often yield poor peptide and protein identification rates. We sought to resolve this shortcoming by deriving the expected isotopic distributions of each fragment ion and incorporating them into the theoretical mass spectra used for peptide-spectrum-matching. We adapted the Comet search platform to integrate a modified spectral prediction algorithm we term Conditional fragment Ion Distribution Search (CIDS). Comet-CIDS uses a traditional database searching strategy, but for each candidate peptide we compute the isotopic distribution of each fragment to better match the observed m/z distributions. Evaluating previously generated D2O and 15N labeled data sets, we found that Comet-CIDS identified more confident peptide spectral matches and higher protein sequence coverage compared to traditional theoretical spectra generation, with the magnitude of improvement largely determined by the amount of labeling in the sample.

Abstract 13709: Cardioprotective Mechanisms of FTO-Regulated m 6 A in Heart Failure

Introduction: Post-transcriptional modifications of mRNA play an important role in cardiac remodeling. We recently reported an aberrant increase in global N6-methyladenosine (m 6 A) in failing hearts associated with reduction in expression of m 6 A eraser, fat and obesity-associated protein (FTO). Overexpression of FTO increased m 6 A, and improved cardiomyocyte and cardiac function in both in vitro and in vivo models for myocardial infarction (MI). Here, we investigated the mRNA targets of FTO and mechanism of FTO-dependent cardiac protein expression in failing hearts. Hypothesis: We hypothesized that FTO mediates cardioprotection via demethylation of selective mRNA transcripts in the nucleus and cytoplasm of cardiomyocytes. Methods and Results: To identify subcellular localization of FTO-mRNA interaction, we examined FTO expression in human left ventricular cardiomyocytes under hypoxia in vitro and in mouse and human failing hearts in vivo . FTO was detected in both nucleus and cytoplasm of healthy cardiomyocytes and tissues, but not in hypoxic cardiomyocytes and MI mouse and human heart tissues. To reveal functional targets of FTO, we performed m 6 A RNA sequencing of nuclear and cytoplasmic RNA in cultured human cardiomyocytes. We identified distinct sets of RNA transcripts in nucleus and cytoplasm of cardiomyocytes involved in cardiac contraction, angiogenesis, cardiomyogenesis, hypermethylated by FTO. Methylated RNA immunoprecipitation experiments revealed that SERCA2a (involved in cardiomyocytes contraction) and FSTL1 (pro-angiogenic) mRNAs directly bind to FTO. Loss of FTO resulted in decreased nuclear export of SERCA2a and FSTL1 mRNAs. Our stable isotope labeling (SILAC) experiment confirmed that FTO regulates de-novo synthesis of SERCA2A and FSTL1 proteins. Conclusion: We conclude that FTO selectively targets the cardioprotective transcripts, regulates their nuclear export and protein expression. Our study will reveal FTO binding sites on cardiac transcripts and a novel mechanism of FTO-dependent protein expression in cardiomyocytes.

The Novel Monooxygenase Gene dipD in the dip Gene Cluster of Alcaligenes faecalis JQ135 Is Essential for the Initial Catabolism of Dipicolinic Acid.

Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.

Rapid response of the giant protist xenophyophores (Foraminifera, Rhizaria) to organic matter supply at abyssal depths revealed by an in situ dual stable isotope labeling experiment

The trophic ecology of xenophyophores (Foraminifera, Rhizaria) was examined during a 2-day in situ feeding experiment on the seafloor (water depths of 5370 m) off the east coast of Torishima Island, Japan. Both 13C-labeled glucose (as an example of labile dissolved organic matter) and 15N-labeled diatoms (as an example of labile particulate organic matter sinking from the ocean surface) were supplied to the specimens captured in in-situ incubation cores using the manned submersible Shinkai 6500. We found that xenophyophores took up both dissolved and particulate organic matter during the 2 days of incubation in situ. The higher concentrations of 15N-labeled diatoms in the xenophyophores than in the surrounding sediments suggest efficient collection and use of food resources by xenophyophores, and may support their rapid growth. It is likely that the agglutinated test of xenophyophores, which forms a contorted plate-like structure with upper open fan-like and funnel-like elements, also contributes to their ability to collect phytodetritus derived from the ocean surface.

New insight into soil carbon fixation rate: The intensive co-occurrence network of autotrophic bacteria increases the carbon fixation rate in depositional sites

Whilst it has been shown that different microbial populations can significantly affect ecosystem functions, little is known about the potential effect of soil erosion on autotrophic bacterial co-occurrence networks and if the networks can influence carbon fixation potential. Here, the random matrix theory (RMT)-based molecular ecological network (MEN) approach with a stable isotope labeling experiment were performed to discern the relationship between autotrophic bacterial networks with the carbon fixing ability and quantify the 13CO2 (carbon dioxide) fixation rate in different soil erosion regions (the Chinese Loess Plateau, the T groups, and the hilly red soil erosion region in Southern China, the H groups). The rates of carbon fixation (RS) were ranked in the following order: H group depositional sites (HD; 30.86 Mg C km−2 yr−1) > H group erosion sites (HE; 18.04 Mg C km−2 yr−1) > T group depositional sites (TD; 5.00 Mg C km−2 yr−1) > T group erosion sites (TE; 1.03 Mg C km−2 yr−1). The results of the random forest modeling and variation partitioning analysis indicated that the bacterial co-occurrence networks were significantly associated with the carbon fixation rate (P < 0.05), and can explain 85.9% variations of carbon fixation rate in different erosion and deposition sites. With the results of multiple stepwise linear regression analysis, pH and C: N were considered to be the key abiotic factors that influenced the carbon fixing rate, which resulted in the carbon fixation rate of H groups were higher than what in T groups. Further, the ZP-plot suggested that the Proteobacteria were the keystones connecting and promoting other species to perform the C-fixing function. The statistical evidence of co-occurrence networks, which are positively associated with the autotrophic bacterial carbon fixation rate, advances the understanding of microbial networks in the function of carbon fixation.

Mfapy: An open-source Python package for 13C-based metabolic flux analysis

13C-based metabolic flux analysis (13C-MFA) is an essential tool for estimating intracellular metabolic flux levels in metabolic engineering and biology. In 13C-MFA, a metabolic flux distribution that explains the observed isotope labeling data was computationally estimated using a non-linear optimization method. Herein, we report the development of mfapy, an open-source Python package developed for more flexibility and extensibility for 13C-MFA. mfapy compels users to write a customized Python code by describing each step in the data analysis procedures of the isotope labeling experiments. The flexibility and extensibility provided by mfapy can support trial-and-error performance in the routine estimation of metabolic flux distributions, experimental design by computer simulations of 13C-MFA experiments, and development of new data analysis techniques for stable isotope labeling experiments. mfapy is available to the public from the Github repository (https://github.com/fumiomatsuda/mfapy).

IsoSolve: An Integrative Framework to Improve Isotopic Coverage and Consolidate Isotopic Measurements by Mass Spectrometry and/or Nuclear Magnetic Resonance.

Stable-isotope labeling experiments are widely used to investigate the topology and functioning of metabolic networks. Label incorporation into metabolites can be quantified using a broad range of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy methods, but in general, no single approach can completely cover isotopic space, even for small metabolites. The number of quantifiable isotopic species could be increased and the coverage of isotopic space improved by integrating measurements obtained by different methods; however, this approach has remained largely unexplored because no framework able to deal with partial, heterogeneous isotopic measurements has yet been developed. Here, we present a generic computational framework based on symbolic calculus that can integrate any isotopic data set by connecting measurements to the chemical structure of the molecules. As a test case, we apply this framework to isotopic analyses of amino acids, which are ubiquitous to life, central to many biological questions, and can be analyzed by a broad range of MS and NMR methods. We demonstrate how this integrative framework helps to (i) clarify and improve the coverage of isotopic space, (ii) evaluate the complementarity and redundancy of different techniques, (iii) consolidate isotopic data sets, (iv) design experiments, and (v) guide future analytical developments. This framework, which can be applied to any labeled element, isotopic tracer, metabolite, and analytical platform, has been implemented in IsoSolve (available at https://github.com/MetaSys-LISBP/IsoSolve and https://pypi.org/project/IsoSolve), an open-source software that can be readily integrated into data analysis pipelines.

Isopentylamine is a novel defence compound induced by insect feeding in rice.

Plants produce a broad variety of defensive metabolites to protect themselves against herbivorous insects. Although polyamines have been implicated in various responses to abiotic and biotic stress, there have been no studies focused on amines in response to insect herbivory. By screening for bioactive amines, we identified isopentylamine as a novel type of herbivory-induced compound in rice leaves, which was derived from the amino acid leucine in stable isotope labelling experiments. Accumulation of isopentylamine increased during herbivory by the brown planthopper (Nilaparvata lugens, BPH) and the rice-feeding armyworm (Mythimna loreyi), as well as in response to treatment with the plant hormone, jasmonic acid. Likewise, isopentylamine accumulation was compromised in rice jasmonate biosynthesis mutants, hebiba and Osjar1. In bio-assays, BPH insects feeding on rice seedlings submerged in 50 mg/L isopentylamine solution had a higher mortality compared with BPH feeding on seedlings submerged in water. Notably, the rice leaves submerged in 50 mg/L solution showed the endogenous concentrations of isopentylamine similar to that induced by BPHs. These results suggest that isopentylamine functions as a new type of plant defence metabolite that is rapidly induced by herbivore attack and deters insect herbivores in rice.

Endogenous Carbon Monoxide Signaling Modulates Mitochondrial Function and Intracellular Glucose Utilization: Impact of the Heme Oxygenase Substrate Hemin.

Stress-inducible heme oxygenase-1 (HO-1) catalyzes the oxidative cleavage of heme yielding biliverdin, ferrous iron, and carbon monoxide (CO). Heme oxygenase activity has been attributed to antioxidant defense via the redox cycling system of biliverdin and bilirubin. There is increasing evidence that CO is a gaseous signaling molecule and plays a role in the regulation of energy metabolism. Inhibitory effects of CO on the respiratory chain are well established, but the implication of such a process on the cellular stress response is not well understood. By means of extracellular flux analyses and isotopic tracing, we studied the effects of CO, either released from the CO donor CORM-401 or endogenously produced by heme oxygenases, on the respiratory chain and glucose metabolism. CORM-401 was thereby used as a tool to mimic endogenous CO production by heme oxygenases. In the long term (>60 min), CORM-401-derived CO exposure inhibited mitochondrial respiration, which was compensated by increased glycolysis accompanied by a loss of the ATP production rate and an increase in proton leakage. This effect pattern was likewise observed after endogenous CO production by heme oxygenases. However, in the present setting, these effects were only observed when sufficient substrate for heme oxygenases (hemin) was provided. Modulation of the HO-1 protein level was less important. The long-term influence of CO on glucose metabolism via glycolysis was preceded by a short-term response (<30 min) of the cells to CO. Stable isotope-labeling experiments and metabolic flux analysis revealed a short-term shift of glucose consumption from glycolysis to the pentose phosphate pathway (PPP) along with an increase in reactive oxygen species (ROS) generation. Overall, we suggest that signaling by endogenous CO stimulates the rapid formation of reduction equivalents (NADPH) via the PPP, and plays an additional role in antioxidant defense, e.g., via feed-forward stimulation of the bilirubin/biliverdin redox cycling system.

Relevance of aboveground litter for soil organic matter formation – a soil profile perspective

Abstract. In contrast to mineral topsoils, in subsoils the origin and processes leading to the formation and stabilization of organic matter (OM) are still not well known. This study addresses the fate of litter-derived carbon (C) in whole soil profiles with regard to the conceptual cascade model, which proposes that OM formation in subsoils is linked to sorption–microbial processing–remobilization cycles during the downward migration of dissolved organic carbon (DOC). Our main objectives were to quantify the contribution of recent litter to subsoil C stocks via DOC translocation and to evaluate the stability of litter-derived OM in different functional OM fractions. A plot-scale stable isotope-labeling experiment was conducted in a temperate beech forest by replacing the natural litter layer with 13C enriched litter on an area of 20 m2 above a Dystric Cambisol. After 22 months of field exposure, the labeled litter was replaced again by natural litter and soil cores were drilled down to 180 cm soil depth. Water extraction and density fractionation were combined with stable isotope measurements in order to link the fluxes of recent litter-derived C to its allocation into different functional OM fractions. A second sampling was conducted 18 months later to further account for the stability of translocated young litter-derived C. Almost no litter-derived particulate OM (POM) entered the subsoil, suggesting root biomass as the major source of subsoil POM. The contribution of aboveground litter to the formation of mineral-associated OM (MAOM) in topsoils (0–10 cm) was 1.88±0.83 g C m−2 and decreased to 0.69±0.19 g C m−2 in the upper subsoil (10–50 cm) and 0.01±0.02 g C m−2 in the deep subsoil &gt;100 cm soil depth during the 22 months. This finding suggests a subordinate importance of recent litter layer inputs via DOC translocation to subsoil C stocks, and implies that most of the OM in the subsoil is of older age. Smaller losses of litter-derived C within MAOM of about 66 % compared to POM (77 %–89 %) over 18 months indicate that recent carbon can be stabilized by interaction with mineral surfaces; although the overall stabilization in the sandy study soils is limited. Our isotope-labeling approach supports the concept of OM undergoing a sequence of cycles of sorption, microbial processing, and desorption while migrating down a soil profile, which needs to be considered in models of soil OM formation and subsoil C cycling.

Antibacterial pyrrolidinyl and piperidinyl substituted 2,4-diacetylphloroglucinols from Pseudomonas protegens UP46.

In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG (1) and 6-(piperidin-2-yl)DAPG (2). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with minimal inhibitory concentration (MIC) 2 and 4 μg ml-1, respectively, for 1, and 2 μg ml-1 for both pathogens for 2. The MICs for 1 and 2, against all tested Gram-negative bacteria, were >32 μg ml-1. The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 μg ml-1, respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2, and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2, but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.