Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Dendritic cell-derived IL-12p40 homodimer contributes to susceptibility in cutaneous leishmaniasis in BALB/c mice

Protection against Leishmania major in resistant C57BL/6 mice is mediated by Th1 cells, whereas susceptibility in BALB/c mice is the result of Th2 development. IL-12 release by L. major-infected dendritic cells (DC) is critically involved in differentiation of Th1 cells. Previously, we reported that strain differences in the production of DC-derived factors, e.g., IL-1alphabeta, are in part responsible for disparate disease outcome. In the present study, we analyzed the release of IL-12 from DC in more detail. Stimulated DC from C57BL/6 and BALB/c mice released comparable amounts of IL-12p40 and p70. In the absence of IL-4, BALB/c DC produced significantly more IL-12p40 than C57BL/6 DC. Detailed analyses by Western blot and ELISA revealed that one-tenth of IL-12p40 detected in DC supernatants was released as the IL-12 antagonist IL-12p40 homodimer (IL-12p80). BALB/c DC released approximately 2-fold more IL-12p80 than C57BL/6 DC both in vitro and in vivo. Local injection of IL-12p80 during the first 3 days after infection resulted in increased lesion volumes for several weeks in both L. major-infected BALB/c or C57BL/6 mice, in higher lesional parasite burdens, and decreased Th1-cytokine production. Finally, IL-12p40-transgenic C57BL/6 mice characterized by overexpression of p40 showed increased levels of serum IL-12p80 and enhanced disease susceptibility. Thus, in addition to IL-1alphabeta, strain-dependent differences in the release of other DC-derived factors such as IL-12p80 may influence genetically determined disease outcome.     

How Many Wolves (Canis lupus) Fit into Germany? The Role of Assumptions in Predictive Rule-Based Habitat Models for Habitat Generalists

Due to legislative protection, many species, including large carnivores, are currently recolonizing Europe. To address the impending human-wildlife conflicts in advance, predictive habitat models can be used to determine potentially suitable habitat and areas likely to be recolonized. As field data are often limited, quantitative rule based models or the extrapolation of results from other studies are often the techniques of choice. Using the wolf (Canis lupus) in Germany as a model for habitat generalists, we developed a habitat model based on the location and extent of twelve existing wolf home ranges in Eastern Germany, current knowledge on wolf biology, different habitat modeling techniques and various input data to analyze ten different input parameter sets and address the following questions: (1) How do a priori assumptions and different input data or habitat modeling techniques affect the abundance and distribution of potentially suitable wolf habitat and the number of wolf packs in Germany? (2) In a synthesis across input parameter sets, what areas are predicted to be most suitable? (3) Are existing wolf pack home ranges in Eastern Germany consistent with current knowledge on wolf biology and habitat relationships? Our results indicate that depending on which assumptions on habitat relationships are applied in the model and which modeling techniques are chosen, the amount of potentially suitable habitat estimated varies greatly. Depending on a priori assumptions, Germany could accommodate between 154 and 1769 wolf packs. The locations of the existing wolf pack home ranges in Eastern Germany indicate that wolves are able to adapt to areas densely populated by humans, but are limited to areas with low road densities. Our analysis suggests that predictive habitat maps in general, should be interpreted with caution and illustrates the risk for habitat modelers to concentrate on only one selection of habitat factors or modeling technique.     

Top-Down Lipidomics Reveals Ether Lipid Deficiency in Blood Plasma of Hypertensive Patients

Background

Dyslipoproteinemia, obesity and insulin resistance are integrative constituents of the metabolic syndrome and are major risk factors for hypertension. The objective of this study was to determine whether hypertension specifically affects the plasma lipidome independently and differently from the effects induced by obesity and insulin resistance.

Methodology/Principal Findings

We screened the plasma lipidome of 19 men with hypertension and 51 normotensive male controls by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 95 lipid species of 10 major lipid classes. Obesity resulted in generally higher lipid load in blood plasma, while the content of tri- and diacylglycerols increased dramatically. Insulin resistance, defined by HOMA-IR >3.5 and controlled for BMI, had little effect on the plasma lipidome. Importantly, we observed that in blood plasma of hypertensive individuals the overall content of ether lipids decreased. Ether phosphatidylcholines and ether phosphatidylethanolamines, that comprise arachidonic (20∶4) and docosapentaenoic (22∶5) fatty acid moieties, were specifically diminished. The content of free cholesterol also decreased, although conventional clinical lipid homeostasis indices remained unaffected.

Conclusions/Significance

Top-down shotgun lipidomics demonstrated that hypertension is accompanied by specific reduction of the content of ether lipids and free cholesterol that occurred independently of lipidomic alterations induced by obesity and insulin resistance. These results may form the basis for novel preventive and dietary strategies alleviating the severity of hypertension.     

Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca²⁺ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca²⁺ influx in hα3β4 AChRs with ～9-fold higher potency than that for PCP, (b) [³H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (K_d = 0.46 ± 0.06 μM), and ibogaine inhibits [³H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [³H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time.     

Localization and Functionality of the Inflammasome in Neutrophils

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1β and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1β release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1β, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1β and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.     

Local and landscape drivers of arthropod diversity and decomposition processes in oil palm leaf axils

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Parasitization of bats by bat flies (Streblidae) in fragmented habitats

Parasites represent a large fraction of the world's biodiversity. They control host population sizes and contribute to ecosystem functioning. However, surveys on species diversity rarely include parasitic species. Bats often present traits favoring parasite diversity, such as large home ranges, long life spans, and large colonies. The most conspicuous bat parasites are the highly host-specific, blood-sucking bat flies (Diptera: Streblidae, Nycteribiidae). Recent studies have found a direct effect of habitat alteration on the abundance of bat species. We expected, therefore, that changes in the host community in response to anthropogenic habitat modification will also result in changes in the associated parasite community. We captured bats in three different habitats in Central Panama between 2013 and 2015. We recorded information on prevalence and intensity of bat fly parasitization of the seven most commonly captured bat species. Prevalence and intensity were both significantly influenced by roost type, abundance, and host sex and age. We found that habitat variables and matrix type significantly influenced the prevalence and intensity of parasitization, while the direction of the responses was host species- and parasite species-specific. In general, roosting conditions and behavior of host bats appear to be fundamental in explaining changes in prevalence and intensity of parasitization between different habitat types, as bat flies are bound to the roost during their reproductive cycle. Habitat alterations affect next to the host community composition also the availability of possible roost structures as well as microclimatic conditions, which all three reflect in parasitization.     

Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134

2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate. The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction. Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funnelled into the 3-oxoadipate pathway by a second reductive step. There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.Abbreviations 2,4-D 2,4-dichlorophenoxyacetate     

In‐depth protein profiling of the postsynaptic density from mouse hippocampus using data‐independent acquisition proteomics

Located at neuronal terminals, the postsynaptic density (PSD) is a highly complex network of cytoskeletal scaffolding and signaling proteins responsible for the transduction and modulation of glutamatergic signaling between neurons. Using ion‐mobility enhanced data‐independent label‐free LC‐MS/MS, we established a reference proteome of crude synaptosomes, synaptic junctions, and PSD derived from mouse hippocampus including TOP3‐based absolute quantification values for identified proteins. The final dataset across all fractions comprised 49 491 peptides corresponding to 4558 protein groups. Of these, 2102 protein groups were identified in highly purified PSD in at least two biological replicates. Identified proteins play pivotal roles in neurological and synaptic processes providing a rich resource for studies on hippocampal PSD function as well as on the pathogenesis of neuropsychiatric disorders. All MS data have been deposited in the ProteomeXchange with identifier PXD000590 ( http://proteomecentral.proteomexchange.org/dataset/PXD000590 ).