Mass spectrometry imaging–based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme

Aminotransferases (ATs) catalyze pyridoxal 5′-phosphate–dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging–based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI–mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime–mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.     

THE ROLE OF LIGHT IN FRUCTIFICATION OF THE BASIDIOMYCETE CYATHUS STERCOREUS

Fructification in cultures of Cyathus stercoreus (Schw.) de Toni is a process in which photochemical reactions are involved. The amount of light energy required for fruiting to take place is a constant. This photoinductive constant is approximately 17200 foot-candle-hours considering optimum temperature (25 C) and light-saturation effect with reference to light intensity (240 ft-c). It is hypothesized that photoinduction becomes operative when a hypothetical “photoreceptive precursor” develops in the mycelium. The development of such a precursor is believed to occur when conditions unfavorable for good vegetative growth (e.g. shortage of food supply) develop in the culture. Internal metabolic pathways then shift to favor the production of the photoreceptive precursor. A linear function is derived which characterizes the biological photoinduction of fruit-body formation.     

Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D

Pig-to-human xenotransplantation has been proposed as a means to alleviate the shortage of human organs for transplantation, but cellular rejection remains a hurdle for successful xenograft survival. NK cells have been implicated in xenograft rejection and are tightly regulated by activating and inhibitory receptors recognizing ligands on potential target cells. The aim of the present study was to analyze the role of activating NK receptors including NKp30, NKp44, NKp46, and NKG2D in human xenogeneic NK cytotoxicity against porcine endothelial cells (pEC). (51)Cr release and Ab blocking assays were performed using freshly isolated, IL-2-activated polyclonal NK cell populations as well as a panel of NK clones. Freshly isolated NK cells are NKp44 negative and lysed pEC exclusively in an NKG2D-dependent fashion. In contrast, the lysis of pEC mediated by activated human NK cells depended on both NKp44 and NKG2D, since a complete protection of pEC was achieved only by simultaneous blocking of these activating NK receptors. Using a panel of NK clones, a highly significant correlation between anti-pig NK cytotoxicity and NKp44 expression levels was revealed. Other triggering receptors such as NKp30 and NKp46 were not involved in xenogeneic NK cytotoxicity. Finally, Ab-dependent cell-mediated cytotoxicity of pEC mediated by human NK cells in the presence of xenoreactive Ab was not affected by blocking of activating NK receptors. In conclusion, strategies aimed to inhibit interactions between NKp44 and NKG2D on human NK cells and so far unknown ligands on pEC may prevent direct NK responses against xenografts but not xenogeneic Ab-dependent cell-mediated cytotoxicity.     

Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age,Geography, and Lifestyle

1. Download high-res image (282KB)
2. Download full-size image

     

CC chemokine ligand 1 promotes recruitment of eosinophils but not Th2 cells during the development of allergic airways disease

One of the characteristic features of allergic asthma is recruitment of large numbers of inflammatory cells including eosinophils and Th2 lymphocytes to the lung. This influx of inflammatory cells is thought to be a controlled and coordinated process mediated by chemokines and their receptors. It is thought that distinct, differential expression of chemokine receptors allows selective migration of T cell subtypes in response to the chemokines that bind these receptors. Th2 cells preferentially express CCR8 and migrate selectively to its ligand, CC chemokine ligand (CCL)1. We studied the role of the CCR8 ligand, CCL1, in the specific recruitment of Th2 cells and eosinophils to the lung in a murine model of allergic airway disease. We have demonstrated for the first time that CCL1 is up-regulated in the lung following allergen challenge. Moreover, a neutralizing Ab to CCL1 reduced eosinophil migration to the lung, but had no effect on recruitment of Th2 cells following allergen challenge. In addition, there was no change in airway hyperresponsiveness or levels of Th2 cytokines. In a Th2 cell transfer system of pulmonary inflammation, anti-CCL1 also failed to affect recruitment of Th2 cells to the lung following allergen challenge. Significantly, intratracheal instillation of rCCL1 increased recruitment of eosinophils but not Th2 cells to the lung in allergen-sensitized and -challenged mice. In summary, our results indicate that CCL1 is important for the pulmonary recruitment of eosinophils, rather than allergen-specific Th2 cells, following allergen challenge.     

Emission of herbivore-induced volatiles in absence of a herbivore--response of Zea mays to green leaf volatiles and terpenoids

Green leaf volatiles (GLV), a series of saturated and monounsaturated six-carbon aldehydes, alcohols, and esters are emitted by plants upon mechanical damage. Evidence is increasing that intact plants respond to GLV by activating their own defense mechanisms, thus suggesting that they function in plant-plant communication. The present paper demonstrates that exposure of maize plants to naturally occurring GLV, including (Z)-3-, (E)-2- and saturated derivatives, induce the emission of volatile blends typically associated with herbivory. Position or configuration of a double bond, but not the functional group of the GLV influenced the strength of the emissions. (Z)-3-Configured compounds elicited stronger responses than (E)-2- and saturated derivatives. The response to (Z)-3-hexen-1-ol increased linearly with the dose between 200 and 1000 nmol per plant. Not only the naturally occurring (E)-2-hexenal, but also (E)-2-pentenal and (E)-2-heptenal induced maize plants, although to a lesser extent. Externally applied terpenoids [(3E)-4,8-dimethyl-1,3,7-nonatriene, beta-caryophyllene, and (E)-beta-farnesene] did not significantly increase the total amount of inducible volatiles in maize. Of three tested maize cultivars Delprim and Pactol responded much stronger than Attribut. Recovery experiments in the presence and absence of maize plants demonstrated that large proportions of externally applied GLV were assimilated by the plants, whereas (3E)-4,8-dimethyl-1,3,7-nonatriene was recovered in much higher amounts. The results furthermore suggested that plants converted a part of the assimilated leaf aldehydes and alcohols to the respective acetates. We propose that GLV not only can alert neighboring plants, but may facilitate intra-plant information transfer and can help mediate the systemic defense response in a plant.