Membrane topology and identification of key residues of EaDAcT, a plant MBOAT with unusual substrate specificity.[Pubmed: 28715115]

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl-sn-glycerol (acetyl-TAG). EaDAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases (MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that EaDAcT colocalizes to the same fractions as an endoplasmic reticulum (ER)-specific marker. By mapping the membrane topology of EaDAcT, we obtained an experimentally determined topology model for a plant MBOAT. The EaDAcT model contains four transmembrane domains (TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl-TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for EaDAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of EaDAcT, suggesting that multiple amino acids are important for substrate recognition.

Screening of inhibitors of glycogen synthase kinase-3β from traditional Chinese medicines using enzyme-immobilized magnetic beads combined with high-performance liquid chromatography.[Pubmed: 26610618]

Glycogen synthase kinase-3β (GSK-3β) was immobilized on magnetic beads (MBs) by affinity method for the first time. The enzyme-immobilized MBs were coupled with high-performance liquid chromatography-ultraviolet (HPLC-UV) technique to establish a cost-effective and reliable method for screening of inhibitors of GSK-3β. A peptide substrate of GSK-3β containing a tyrosine residue was employed since it can be sensitively detected by UV detector at 214nm. The substrate and its phosphorylated product were separated by baseline within 10min. The enzyme activity was determined by the quantification of peak area of the product. Parameters including enzyme immobilization, enzyme reaction and the performance of immobilized-enzyme were investigated. The immobilized enzyme can be reused for 10 times and remain stable for 4 days at 4°C. The inhibitory activities of extracts of 15 traditional Chinese medicines (TCMs) were screened. As a result, three of them including Euonymus fortunei, Amygdalus communis and Garcinia xanthochymus were found possessing high inhibitory activities (inhibition rate >90%). From G. xanthochymus, a new inhibitor of GSK-3β, fukugetin, was discovered with an IC50 value of 3.18±0.07μM. Enzyme kinetics and molecular docking experiments further revealed the inhibitory mechanism, indicating fukugetin was a non-ATP competitive inhibitor interacting with the phosphate recognizing substrate binding site of GSK-3β.

[Research on quality standards of Euonymus fortunei].[Pubmed: 26390641]

To establish quality standards of Euonymus fortunei, and supply scientific evidence for the quality control of Euonymus fortunei. Empirical and microscopic identification methods were adopted to observe morphological and histological characters. The contents of water, total ash, acid-insoluble ash and alcohol-soluble extractive were analysed according to the methods of Chinese Pharmaco- poeia (2010). Dulcitol and reference herbs were used to identify materia medica of Euonymus fortunei by TLC method. The total flavonol glycosides contents were analysed by HPLC method, using quercetin and kaempferol as reference substances. Quercetin and kaempferol were separated on a C18 column (4.6 mm x 250 mm, 5 μm) with methanol-0.1% formic acid(51:49) as the mobile phase and detected at 366 nm. The flavonoid aglycones content was then multiplied by a conversion coefficient, and the result was the total flavonol glycosides content. The macroscopical identification, microscopic features and TLC methods were proper. The average contents of water, total ash, acid-insoluble ash, alcohol-soluble extractive and total flavonol glycosides were 8.76%, 6.48%, 0.31%, 17.48% and 0.211% , respectively. The quality standards established on the basis of the research results were suitable for the quality evaluation of Euonymus fortunei.

Chemical constituents of Euonymus fortunei.[Pubmed: 25921486]

A new flavonol glycoside, kaempferol-3-O-β-D-(2-O-E-p-coumaroyl)-glucopyranosyl-7-O-α-l-rhamnopyranoside (1), along with eleven known compounds including five flavonol glycosides (2-6), one phenolic glycoside (7), two megastigmane glycosides (8 and 9), two triterpenoids (10 and 11) and one alditol (12), was isolated from the aerial parts of Euonymus fortunei. Their structures were determined on the basis of spectroscopic analysis and chemical evidence. Compounds 2-4, 7, 8, and 10-12 were evaluated their antimicrobial activities against Ureaplasma urealyticumin vitro, but all tested compounds have no useful activities against Ureaplasma urealyticum.

Comparison of the structure of floral nectaries in two Euonymus L. species (Celastraceae).[Pubmed: 25391262]

The inconspicuous Euonymus L. flowers are equipped with open receptacular floral nectaries forming a quadrilateral green disc around the base of the superior ovary. The morphology and anatomy of the nectaries in Euonymus fortunei (Turcz.) Hand.-Mazz. and Euonymus europaeus L. flowers were analysed under a bright-field light microscope as well as stereoscopic and scanning electron microscopes. Photosynthetic nectaries devoid of the vascular tissue were found in both species. Nectar was exuded through typical nectarostomata (E. fortunei) or nectarostomata and secretory cell cuticle (E. europaeus). The nectaries of the examined species differed in their width and height, number of layers and thickness of secretory parenchyma, and the height of epidermal cells. Moreover, there were differences in the location and abundance of nectarostomata and the content of starch and phenolic compounds.

Field attraction of the vine weevil Otiorhynchus sulcatus to kairomones.[Pubmed: 22420269]

Root weevils in the genus Otiorhynchus are cited as one of the most important pests in the major nursery and small fruit production areas throughout the United States, western Canada, and northern Europe. A major problem in combating weevil attack is monitoring and timing of control measures. Because of the night-activity of the adult weevils growers do not observe the emerging weevils in a timely manner and oviposition often starts before effective control measures are taken. Several vine weevil electroantennogram-active plant volatiles were identified from a preferred host plant, Euonymus fortunei. Main compounds evoking antennal responses on the weevils' antennae were (Z)-2-pentenol, (E)-2-hexenol, (Z)-3-hexenol, methyl benzoate, linalool, (E)-4,8-dimethyl-1,3,7-nonatriene, methyl eugenol, and (E, E)-alpha-farnesene. Several of these compounds were tested alone and in mixtures on attractiveness for the vine weevil Otiorhynchus sulcatus (F.) in field-grown strawberry in Oregon. O. sulcatus were attracted to (Z)-2-pentenol (approximately 3 x more than control) and a 1:1 ratio mixture of (Z)-2-pentenol and methyl eugenol (4.5 x more than control). This is the first report of field-active attractants for O. sulcatus which holds promise for the development of new monitoring strategies for growers in the near future.

[Optimal extraction of melampyrit from Euonymus fortunei by central composite design-response surface methodology].[Pubmed: 19764338]

To optimize the process of extracting melampyrit from Euonymus fortunei by central composite design-response surface methodology.

Variability among species in the apoplastic pH signalling response to drying soils.[Pubmed: 19726633]

After the imposition of soil drying treatments, an elevation of xylem sap pH is one of the earliest observable responses in many herbaceous model plant species. It is theorized that alkalization of sap results in a concurrent elevation in abscisic acid (ABA) concentration delivered to transpiring tissues by preventing Henderson-Hasselbalch-regulated partitioning between the apoplast and symplast. However, here it is demonstrated that the sap alkalzation response to soil drying is far from universal in higher plant species. Tests were conducted to determine how universal the pH response to drying soil was in a range of perennial species from a diverse range of plant families. The response was not found in the majority of the 22 species tested. Four species exhibited significant increases in pH, but the majority showed no significant change in xylem sap pH. There was no evolutionary relationship between the species that showed alkalization under drought stress. However, the species that alkalized sap also exhibited good control over internal water status and were the most isohydric species of those tested. None of the species exhibiting anisohydric responses alkalized xylem sap under drought stress. Regardless of alkalization response, plants still retain the ability to respond to changes in xylem sap pH when manipulated by alkaline buffer foliar sprays. This finding indicates that plants have conserved the ability to respond to changes in xylem pH and redistribute ABA, even if they do not currently utilize the mechanism when exposed to drought stress. It was found in Buddleja davidii, Euonymus fortunei, and Hydrangea serrata that the xylem sap pH response to water deficits mirrored the natural pH changes that occur as sap is transported to the leaves, indicating that plants need to be able to have naturally occurring alkalization processes in place for them to be up-regulated under drought stress.

[Study on microwave extracting process of polysaccharide from Euonymus fortunei].[Pubmed: 19504977]

To study the best extraction process of the polysaccharides from Euonymus fortuei with microwave.