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Capacity of enzymes of the euphorbiacea Aleurites montana involved in CO2-fixation, compared to plants having C3-, C4- and Crassulacean acid metabolism.

Capacities of phosphoenolpyruvate carboxylase (PEP-Co), ribulose bisphosphate carboxylase (Rubisco), NADP+ malic enzyme (ME) and of malate dehydrogenase (MDH) were measured in the Euphorbiacea Aleurites montana, grown under 700 ppm CO2 for four weeks prior to enzyme extraction. For comparison Bryophyllum daigremontiana (CAM). Saccharum officinarum (C4) and Capsicum frutescens (C3) were treated in the same way. PEP-Co capacity of Aleurites was in the range of 12-, that of Capsicum approx. 26 nmol x min(-1) x mg protein(-1), without significant influence of the light period or CO2-treatment. In contrast, the activity of the enzyme from Saccharum was, depending on the duration of light, 160- respectively 96 times higher than that of the tung-oil tree. In Bryophyllum a rather low activity in the morning was increased during the day to approx. 230 nmol x min(-1) x mg protein(-1) in plants grown in the greenhouse and to approx. 115 nmol x min(-1) x mg protein(-1) in those from the growth chamber. Malate was hardly detectable in extracts of Aleurites, whereas it was high in Bryophyllum, depending on the light period. The ratio of average PEP-Co to Rub-Co capacity was high for the CAM-plant (20:1), somewhat lower for sugar cane (10:1), but almost at equality for Aleurites (0.9:1) and chilli (0.8:1). For the NADP+ malic enzyme, low capacity (20 to 28 nmol x min(-1) x mg protein(-1)) was found for Aleurites and for Capsicum, whereas it was 10 to 17 times higher in Saccharum. In Bryophyllum, the activity was up to 80 nmol x min(-1) x mg protein, dependent on light period. MDH capacity was extremely high in all plants investigated. Highest rates (10-20 micromol x min(-1) x mg protein(-1)), were obtained for Bryophyllum, followed by sugar cane and Capsicum with 5-8 micromol x min(-1) x mg protein(-1). Again, the lowest capacity was found in extracts of Aleurites with approx. 1.3 to 1.6 micromol x min(-1) x m protein(-1). Thus, in Aleurites montana no indication for C4- or Crassulacean acid metabolism was obtained. Therefore, the earlier observed very efficient uptake of CO2 cannot be explained by a high expression of the PEP-Co protein, known to occur in CAM- and C4-plants.

Capsicum↗

Two outbreaks of acute Tung Nut (Aleurites fordii) poisoning.

OBJECTIVE: Aleurites fordii, widely distributed in the Southeastern US, Taiwan, mainland China, Japan and India, is commonly known as Tung Nut, Tung Oil Tree or Chinawood Oil Tree. The seeds are the most toxic part. CASE REPORTS: We report two outbreaks of Aleurites fordii poisoning, occurring on November 27, 1992 and November 29, 1994. Thirty-five elementary school students and 29 senior high school students misidentified Aleurites fordii seeds as chestnuts and ingested variable amounts. METHODS: We conducted a survey by questionnaire to supplement the hospital record information. Simple descriptive statistics and Chi-square (Fisher's exact) tests were calculated. RESULTS: The three most common symptoms of the patients were vomiting, abdominal pain and diarrhea. The more serious clinical presentations occurred in younger victims. Our information suggests that food attenuates intestinal irritation perhaps by delaying absorption of the toxic principle. With symptomatic treatment, all of the symptoms and signs subsided within one to two days. CONCLUSIONS: Aleurites fordii can be cultivated and is easily accessible to the community and schools. Public education about the toxicity of Tung Nut seeds in areas of ready availability may reduce the chance of misidentification and subsequent poisoning.

Adolescent↗

Hypolipidaemic activity of methanol extract of Aleurites moluccana.

The lipid-lowering action of the leaves of the Aleurites moluccana methanol extract was studied in Triton W-1339 and high-fat-diet fed rats. The serum lipids (total cholesterol, LDL- and HDL-cholesterol and triglycerides) and body weight were found to be lowered by A. moluccana (300 mg/kg, b.w.) in rats with Triton-induced hypercholesterolaemia and on a hyperlipaemic diet. The results suggest that the lipid lowering action of this natural product is mediated through inhibition of hepatic cholesterol biosynthesis and reduction of lipid absorption in the intestine.

Aleurites↗

Accumulation of hydrolyzable tannins by Aleurites fordii callus culture.

A callus culture of Aleurites fordii Hemsley (Euphorbiaceae) producing five galloylglucoses and an ellagitannin, geraniin, was established. The production of pentagalloylglucose was remarkably enhanced under light irradiation compared with that in the dark. Cell growth and tannin production were also greatly affected by changing the concentrations and composition of nitrogen sources.

Aleurites↗

Isolation of flavonoids from Aleurites moluccana using chitosan modified with benzaldehyde (CH-Bz) as chromatographic support.

This paper describes the preparation, characterization and use of a derivative of chitosan as a chromatographic sorbent. Chitosan modified with benzenic ring (CH-Bz) was used to separate two flavonoids, swertisin and 2"-O-rhamnosylswertisin, from ethyl acetate fraction of Aleurites moluccana. The results showed that CH-Bz can be used as a sorbent for the separation of flavonoid compounds. The studies showed that CH-Bz in column chromatography produces goods results, separation of the flavonoid compounds.

Acetates↗

An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds.

In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.

Ascomycota↗

Epstein-Barr virus activation by tung oil, extracts of Aleurites fordii and its diterpene ester 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate.

During the screening of plant oils for their Epstein-Barr virus (EBV)-activating potency, we found that tung oil possesses an activity comparable to croton oil. Tung oil from various sources and the extracts from its parental plant Aleurites fordii (Chinese tung oil tree), when used in combination with n-butyrate, were shown to efficiently activate EBV persisting in human lymphoblastoid Raji cells (non-producer). The major diterpene ester in the plant extract, 12-O-hexadecanoyl-16-hydroxyphorbol-13-acetate (HHPA), also exerted a similar activity. In producer P3HR-1 cells, both tung oil and HHPA increased the yield of infectious EBV by approximately five-fold. Since tung oil is used for the manufacture of oil paints, varnishes, waterproof substance, anticorrosives and other products, the implication of using such an agent with EBV-activating potency in our daily life is assessed and discussed.

Cells, Cultured↗

A new phorbol diester from Aleurites moluccana.

A new phorbol diester, 13-O-myristyl-20-O-acetyl-12-deoxyphorbol (1), has been isolated from the benzene extract of the heartwood of Aleurites moluccana. In addition, hentriacontane, 6,7-dimethoxycoumarin, 5,6,7-trimethoxycoumarin and beta-sitostenone are being reported for the first time from this species.

Coumarins↗

Separation of C-glycoside flavonoids from Aleurites moluccana using chitin and full N-acetylated chitin.

This paper describes a comparative study using different chromatographic supports (fully N-acetylated chitin, chitin and silica gel) to separate the flavonoids swertisin and 2"-O-rhamnosylswertisin from Aleurites moluccana. The results show that the flavonoids have apparently been separated by the hydrogen bond between the stationary phase (chitin and chitin-100) and flavonoids under the conditions studied.

Acetylation↗

Human polymorphonuclear leukocyte activating factor isolated from Aleurites Fordii Hemsl. (Euphorbiaceae) seed.

A human polymorphonuclear leukocyte (PMN) activating factor (AFH-S) was isolated from a tropical plant seed, Aleurites Fordii Hemsl. (AFH) in PBS. It is found that the AFH-S stimulate human PMNs and induced superoxide generation and chemotaxis. Superoxide generation was affected by the extracellular calcium ion or pretreatment with H-7 (PK-C inhibitor), but not by mepacrine (PLA2 inhibitor) or pertussis toxin (islet-activating protein: IAP). Furthermore, a lag time exists dose-dependently. In addition the cytosolic calcium was not increased by the stimulation with AFH-S. Thus, the receptor for AFH-S was suggested to be independent from Ni-like protein, PI-response, or PLA2-activation, and stimulate PMNs through activation of PK-C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

Separation of flavonoids from Aleurites moluccana leaves using chitosan modified with heptaldehyde.

Heptaldehyde-modified chitosan (heptyl-chitosan, CH-Hp) was investigated as adsorbent for chromatograhic separation of the flavonoids from A. moluccana. The amount of 2"-O-rhamnosylswertisin isolated (30.0 mg) was approx. twice as high as swertisin (17.5 mg). The improved surface hydrophobicity effected by the heptyl groups promoted the separation of flavonoids. From the results obtained, CH-Hp seems to be more suitable for separation of glycosylated flavonoids than other flavonoids. Thus, modified chitosan described here can be used for hydrophobic interaction chromatography as sucessfully illustrated with flavonoids.

Acetylation↗

The use of chitosan modified with glutaraldehyde and glyoxal as chromatographic support for the separation of flavonoids from Aleurites moluccana leaves.

This paper describes the preparation of chitosan modified with dialdehydes, glutaraldehyde (CH-Glu) and glyoxal (CH-Gly) and its application in the isolation of the flavonoids swertisin and 2"-O-rhamnosylswertisin from A. mollucana. The additional non-polar alkyl groups increase the hydrophobicity of the sorbent. The results show that the separation was mediated by hydrophobic interaction (CH-Glu), as well as hydrogen bonding, between phenolic OH or rhamnosil residues from the flavonoids, and the free amine groups (CH-Gly).

Aleurites↗

Further studies on a chemoattractant derived from Aleurites Fordii Hemsl. seed.

1. Activities stimulatory to PMN superoxide generation and chemotactic function were present in two separate gel chromatographic fractions of AFH-S and were designated AFH-S1 (500 kD) and AFH-S2 (38 kD). 2. Characterization studies of these activities revealed physiologic and pharmacologic characteristics dissimilar to those of other previously studied chemoattractants such as bacterial peptide FMLP.

Calcium↗

Diurnal and seasonal variations in leaf hydraulic conductance in evergreen and deciduous trees.

We studied changes in the hydraulic conductance of leaves (K(leaf)) between dawn and dusk during the growth period (July) and at midday at the beginning of autumn in four tree species. The main objectives of the study were to check the extent of diurnal and seasonal changes in K(leaf) and the relationships between K(leaf), irradiance and leaf gas exchange. Two evergreen (Aleurites moluccana and Persea americana) and two deciduous trees (Platanus orientalis and Quercus rubra) were studied. Leaf hydraulic conductance was measured every 2 h between 0700 and 1900 h in July and compared with values measured between 0900 and 1300 h in October. Other variables measured were photosynthetically active radiation (PAR), leaf conductance to water vapor (gL) and water potential (psiL). In July, K(leaf) varied by up to 75% in Pe. americana on a diurnal basis and by at least 44% in Q. rubra. The diurnal time course of K(leaf) showed a distinct increase between dawn and late morning (1100 h) and a subsequent decrease in the evening in A. moluccana and Pl. orientalis, whereas in the other two species, K(leaf) was highest just after dawn and lowest in the evening. In October, K(leaf) of all the species studied was lower than in July, with differences of 20 to 28% for A. moluccana and Pl. orientalis and of 66 to over 70% in Pe. americana and Q. rubra, respectively. Significant correlations were found between PAR and K(leaf) (in all species) as well as between gL and K(leaf) (in three out of four species). Leaf habit (evergreen or deciduous) did not influence absolute values of K(leaf) or its diurnal variation.

Aleurites↗

Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity.

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.

Aleurites↗

Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome b5 to either endoplasmic reticulum or mitochondria.

Tail-anchored membrane proteins are a class of proteins that are targeted posttranslationally to various organelles and integrated by a single segment of hydrophobic amino acids located near the C terminus. Although the localization of tail-anchored proteins in specific subcellular compartments in plant cells is essential for their biological function, the molecular targeting signals responsible for sorting these proteins are not well defined. Here, we describe the biogenesis of four closely related tung (Aleurites fordii) cytochrome b5 isoforms (Cb5-A, -B, -C, and -D), which are small tail-anchored proteins that play an essential role in many cellular processes, including lipid biosynthesis. Using a combination of in vivo and in vitro assays, we show that Cb5-A, -B, and -C are targeted exclusively to the endoplasmic reticulum (ER), whereas Cb5-D is targeted specifically to mitochondrial outer membranes. Comprehensive mutational analyses of ER and mitochondrial Cb5s revealed that their C termini, including transmembrane domains (TMD) and tail regions, contained several unique physicochemical and sequence-specific characteristics that defined organelle-specific targeting motifs. Mitochondrial targeting of Cb5 was mediated by a combination of hydrophilic amino acids along one face of the TMD, an enrichment of branched beta-carbon-containing residues in the medial portion of the TMD, and a dibasic -R-R/K/H-x motif in the C-terminal tail. By contrast, ER targeting of Cb5 depended primarily upon the overall length and hydrophobicity of the TMD, although an -R/H-x-Y/F- motif in the tail was also a targeting determinant. Collectively, the results presented provide significant insight into the early biogenetic events required for entry of tail-anchored proteins into either the ER or mitochondrial targeting pathways.

Aleurites↗