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Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Three new indole alkaloids (1-3), named grandilodines A-C, and five known ones were obtained from the Malayan Kopsia grandifolia. The structures were established using NMR and MS analyses and, in the case of 1 and 2, were confirmed by X-ray diffraction analyses. Alkaloids 1, 3, and lapidilectine B (8) were found to reverse multidrug resistance in vincristine-resistant KB cells.

A total of 25 alkaloids were isolated from the leaf and stem-bark extracts of Alstonia spatulata, of which five are new alkaloids of the strychnan type (alstolucines A-E, 1-5) and the other, a new alkaloid of the secoangustilobine A type (alstolobine A, 6). The structures of these alkaloids were established using NMR and MS analysis and, in the case of alstolucine B (2), also confirmed by X-ray diffraction analysis. A reinvestigation of the stereochemical assignment of scholaricine (13) by NMR and X-ray analyses indicated that the configuration at C-20 required revision. Alkaloids 1, 2, 6, 7, 9, 10, and 13 reversed multidrug resistance in vincristine-resistant KB cells.

Bombolitin is the most abundant component of bumblebee venom and shares structural and biological properties with melittin, a component of honeybee venom. Here, we describe the molecular cloning and antimicrobial activity of bombolitin isolated from the venom of the bumblebee Bombus ignitus. The B. ignitus bombolitin gene consists of 2 exons encoding 56-amino acid residues. The bombolitin isolated from B. ignitus venom is a 1.99 kDa mature peptide with 18-amino acid residues, and it is created by the cleavage of the probombolitin domain between Ala38 and Leu39. B. ignitus bombolitin exhibits venom gland-specific expression. We also investigated the antimicrobial properties of B. ignitus bombolitin against bacteria and fungi. The B. ignitus bombolitin showed high antibacterial activity against two Gram-positive and two Gram-negative bacteria. In addition, the B. ignitus bombolitin displayed antifungal activity against the plant pathogenic fungi Fulvia fulva and Alternaria radicina., ((c) 2010 Elsevier Inc. All rights reserved.)

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated the regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that the loss of expression of BiPrx1 or BiTPx1 is compensated by the up-regulation of expression of the other peroxidase in response to H2O2 overload., (2009 Elsevier Inc. All rights reserved.)

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx or GPx4; EC 1.11.1.12) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln(82) and Trp(134). Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H(2)O(2) using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H(2)O(2) exposure, external temperature shock, and starvation. Under H(2)O(2) overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H(2)O(2).

Proteolytic enzymes are involved in insect molting and metamorphosis, and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compare the expression profiles of B. mori cathepsins B (BmCatB) and D (BmCatD) during normal development and after RNA interference (RNAi)-mediated inhibition. BmCatB is induced by 20-OH-ecdysone, and is expressed in the fat body of B. mori during molting and the larval-pupal and pupal-adult transformations, where its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation, and BmCatB RNAi treatment resulted in an arrest of the larval-pupal transformation. RNAi-mediated BmCatB knockdown sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, when BmCatD was inhibited via RNAi, the expression of BmCatB was upregulated. Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis, and that BmCatB and BmCatD contribute to B. mori metamorphosis.

Phospholipase A(2) (PLA(2)) is one of the main components of bee venom. Here, we identify a venom PLA(2) from the bumblebee, Bombus ignitus. Bumblebee venom PLA(2) (Bi-PLA(2)) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA(2) gene. Bi-PLA(2) is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA(2) (136 amino acids) possesses features consistent with other bee PLA(2)s, including ten conserved cysteine residues, as well as a highly conserved Ca(2+)-binding site and active site. Phylogenetic analysis of bee PLA(2)s separated the bumblebee and honeybee PLA(2) proteins into two groups. The mature Bi-PLA(2) purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA(2). Immunofluorescence staining of Bi-PLA(2)-treated insect Sf9 cells revealed that Bi-PLA(2) binds at the cell membrane and induces apoptotic cell death.

A novel endogenous beta-1,4-endoglucanase (EG) gene belonging to the glycosyl hydrolase family 9 (GHF 9) that is distributed throughout the digestive tract of the cricket Teleogryllus emma was cloned and characterized. This gene, named TeEG-I, consists of eight exons encoding 453 amino acid residues and exists as a single copy in the T. emma genome. TeEG-I possesses all the features, including signature motifs and catalytic domains, of GHF 9 members, sharing high levels of identity with the termite, Mastotermes darwiniensis (64% protein sequence identity), and the cockroach, Panesthia cribrata (62%), GHF 9 cellulases. Recombinant TeEG-I, which is expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells, showed an optimal pH and temperature of pH 5.0 and 40 degrees C. The K(m) and V(max) values for digestion of carboxymethyl cellulose were 5.4 mg/ml and 3118.4 U/mg, respectively. Northern and Western blot analyses revealed that TeEG-I is present throughout the digestive tract, which correlated with the TeEG-I distribution and cellulase activity in the digestive tract as assayed by immunofluorescence staining and enzyme activity assay, respectively. These results indicate that TeEG-I is distributed throughout the entire digestive tract of T. emma, suggesting a functional role of endogenous TeEG-I in a sequential cellulose digestion process throughout the T. emma digestion tract.

Four antibacterial peptide genes (apidaecin, hymenoptaecin, abaecin, and defensin) were cloned from the bumblebee Bombus ignitus, and cDNAs and their genomic structures were sequenced and characterized. Comparative analysis revealed that the four antibacterial peptides of B. ignitus had similar characteristics to other bee antibacterial peptides identified to date. The transcriptional expression profiles of the four antibacterial peptide genes in the fat body of B. ignitus workers revealed that all four antibacterial peptide genes were acutely induced in a similar manner by PBS injection or LPS stimulation, indicating that antibacterial peptides from various classes are simultaneously expressed in a single insect upon infection or injury.

Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the gene itself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface as a GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by alpha3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing alpha3beta4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.

In insects transferrin is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone regulated protein. Here, a novel functional role for insect transferrin as an antioxidant protein is demonstrated. Stressors, such as heat shock, fungal challenge, and H(2)O(2) exposure, cause upregulation of the white-spotted flower chafer Protaetia brevitarsis (Coleoptera: Scarabaeidae) transferrin (PbTf) mRNA in the fat body and increases PbTf protein levels in the hemolymph. RNA interference (RNAi) treated PbTf reduction causes increased iron and H(2)O(2) levels in the hemolymph and results in induction of apoptotic cell death in the fat body during exposure to stress. The observed effects of PbTf RNAi suggest that PbTf inhibits stress-induced apoptosis by diminishing the Fenton reaction via the binding of iron, thus supporting an antioxidant role for PbTf in stress responses.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.

A thioredoxin (Trx) gene was cloned from the silkworm, Bombyx mori. The B. mori Trx (BmTrx) cDNA contains an open reading frame of 318 bp encoding 106 amino acid residues with a conserved active site (CGPC). Northern blot analysis revealed the presence of BmTrx transcripts in all tissues examined. The cDNA encoding BmTrx was expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells. The recombinant BmTrx proved to be biologically active, using an insulin reduction assay, and was also able to activate thioredoxin peroxidase from B. mori. When H2O2 or paraquat was injected into the body cavity of B. mori larvae, BmTrx mRNA expression was upregulated in the fat body tissue. In addition, the expression levels of BmTrx mRNA in the fat body were greatly increased when B. mori larvae were exposed to low or high temperatures, or injected with microorganisms. These results suggest that BmTrx possibly protects against oxidative stress caused by extreme temperatures and microbial infection as well as by intracellularly generated reactive oxygen species during metabolism.

Background: Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis., Results: Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage., Conclusion: Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.

Introduction: We conducted a study to assess the impact of an asthma education programme (AEP) on knowledge of asthma and medication, compliance to treatment and inhaler technique, emergency department visits and hospital re-admissions., Methods: Patients hospitalised for asthma exacerbation were administered a questionnaire to test their baseline knowledge and beliefs on asthma, its medications and their compliance to treatment. Their inhaler technique was assessed. They then underwent an AEP consisting of two individualised education sessions. Re-testing was performed after three months. Per protocol approach and McNemar's test was used to analyse the statistical significance of the change in the pre- and post-AEP test scores. Hospital administrative data were used to determine the number of ED visits and hospital admissions pre- and post-AEP., Results: Among the 67 patients who completed the two-phase AEP, there was significant improvement in some knowledge aspects (ability to identify rescue medication [p-value is 0.031], that different stimuli can trigger asthma symptoms [p-value is 0.016], that a peak flow meter is used for monitoring asthma [p-value is 0.004], that asthma symptoms are caused by airway swelling/narrowing [p-value is less than 0.001], that steroid inhaler are to be used daily as preventive therapy [p-value is less than 0.001], in self-reported inhaler compliance (number of puffs per administration [p-value is less than 0.001] and per day [p-value is less than 0.001]), and in inhaler technique [p-value is 0.001]. There was also significant reduction in emergency department attendances (p-value is less than 0.001) and hospital admissions (p-value is less than 0.001) among all 97 subjects over a one-year period., Conclusion: This study demonstrated the effectiveness of an AEP in patients hospitalised for asthma exacerbation.

Some unidentified minor compounds have been observed in the residue from short-path distillation of transesterified palm oil that are not detected in the original palm oil. A method combining short-path distillation to enrich the unknowns with fractionation using solid-phase extraction is described. The fractionated components were identified using GC coupled with MS. The transesterified palm oil was found to contain methyl esters of up to C32 carbon atoms. In the very long chain FAME with carbon numbers > or = 20, both even and odd carbon numbers accounted for 0.26 wt%, with C24 and C26 being the major ones present in the residue after short-path distillation of transesterified palm oil.

The gene structure, expression and enzyme activity of a serine protease from the firefly, Pyrocoelia rufa (PrSP) were examined. The PrSP gene spans 1474 bp and consists of two introns and three exons coding for 257 amino acid residues. Southern blot analysis of genomic DNA suggested the presence of PrSP gene as a single copy. Western blot analysis and enzyme activity assay exhibited midgut-specific expression, suggesting that the midgut is the prime site where large quantities of PrSP are synthesized for degrading the absorbed protein from the diet. The cDNA encoding PrSP was expressed as a 31 kDa polypeptide in the baculovirus-infected insect Sf9 cells and the recombinant PrSP showed activity in the protease enzyme assay using gelatin as a substrate.

This paper discusses a rapid GC-FID technique for the simultaneous quantitative analysis of FFA, MAG, DAG, TAG, sterols, and squalene in vegetable oils, with special reference to palm oil. The FFA content determined had a lower SE compared with a conventional titrimetric method. Squalene and individual sterols, consisting of beta-sitosterol, stigmasterol, campesterol, and cholesterol, were accurately quantified without any losses. This was achieved through elimination of tedious conventional sample pretreatments, such as saponification and preparative TLC. With this technique, the separation of individual MAG, consisting of 16:0, 18:0, and 18:1 FA, and the DAG species, consisting of the 1,2(2,3)- and 1,3-positions, was sufficient to enable their quantification. This technique enabled the TAG to be determined according to their carbon numbers in the range of C44 to C56. Comparisons were made with conventional methods, and the results were in good agreement with those reported in the literature.

The application of supercritical fluid chromatography (SFC) coupled with a UV variable-wavelength detector to isolate the minor components (carotenes, vitamin E, sterols, and squalene) in crude palm oil (CPO) and the residual oil from palm-pressed fiber is reported. SFC is a good technique for the isolation and analysis of these compounds from the sources mentioned. The carotenes, vitamin E, sterols, and squalene were isolated in less than 20 min. The individual vitamin E isomers present in palm oil were also isolated into their respective components, alpha-tocopherol, alpha-tocotrienol, gamma-tocopherol, gamma-tocotrienol, and delta-tocotrienol. Calibration of all the minor components of palm as well as the individual components of palm vitamin E was carried out and was found to be comparable to those analyzed by other established analytical methods.

Previous reports showed that vitamin E in palm oil consists of various isomers of tocopherols and tocotrienols [alpha-tocopherol (alpha-T), alpha-tocotrienol, gamma-tocopherol, gamma-tocotrienol, and delta-tocotrienol), and this is normally analyzed using silica column HPLC with fluorescence detection. In this study, an HPLC-fluorescence method using a C30 silica stationary phase was developed to separate and analyze the vitamin E isomers present in palm oil. In addition, an alpha-tocomonoenol (alpha-T1) isomer was quantified and characterized by MS and NMR. (alpha-T1 constitutes about 3-4% (40+/-5 ppm) of vitamin E in crude palm oil (CPO) and is found in the phytonutrient concentrate (350+/-10 ppm) from palm oil, whereas its concentration in palm fiber oil (PFO) is about 11% (430+/-6 ppm). The relative content of each individual vitamin E isomer before and after interesterification/transesterification of CPO to CPO methyl esters, followed by vacuum distillation of CPO methyl esters to yield the residue, remained the same except for alpha-T and gamma-T3. Whereas alpha-T constitutes about 36% of the total vitamin E in CPO, it is present at a level of 10% in the phytonutrient concentrate. On the other hand, the composition of gamma-T3 increases from 31% in CPO to 60% in the phytonutrient concentrate. Vitamin is present at 1160+/-43 ppm, and its concentrations in PFO and the phytonutrient concentrate are 4,040+/-41 and 13,780+/-65 ppm, respectively. The separation and quantification of alpha-T1 in palm oil will lead to more in-depth knowledge of the occurrence of vitamin E in palm oil.

Two new venalstonine derivatives, viz., venacarpines A and B, and one new dioxokopsan derivative, kopsorinine, in addition to the kopsifolines A-F, and 11 other known alkaloids, were isolated from a Malayan Kopsia species. The structures of the new alkaloids were determined using NMR and MS analysis.

The concentration of vitamin E isomers, namely, alpha-tocopherol (alpha-T), alpha-tocotrienol, gamma-tocotrienol, and delta-tocotrienol in palm mesocarp at 4, 8, 12, 16, and 20 wk after anthesis (WAA) were quantified using HPLC coupled with fluorescence detection. alpha-T was detected throughout the palm fruits' maturation process, whereas unsaturated tocotrienols were found only in ripe palm fruits. These developmental results indicate that tocotrienols are synthesized between 16 and 20 WAA.

Six new indole alkaloids, viz., (3S)-3-cyanocoronaridine (2), (3S)-3-cyanoisovoacangine (3), conolobine A (5), conolobine B (6), conolidine (7), and (3R/3S)-3-ethoxyvoacangine (8), in addition to 36 known ones, were obtained from the stem-bark extract of the Malayan Tabernaemontana divaricata. The structures were determined by NMR and MS analysis. The CN-substituted alkaloids showed appreciable cytotoxicity towards the KB human oral epidermoid carcinoma cell-line.

Ten new indole alkaloids, alstomaline (1), 10,11-dimethoxynareline (2), alstohentine (3), alstomicine (4), 16-hydroxyalstonisine (5), 16-hydroxyalstonal (6), 16-hydroxy-N(4)-demethylalstophyllal oxindole (7), alstophyllal (8), 6-oxoalstophylline (9), and 6-oxoalstophyllal (10), in addition to 21 other known ones, were obtained from the leaf extract of the Malayan Alstonia macrophylla. The structures were determined using NMR and MS analysis.

Six new alkaloids, viz., alstolactone, affinisine oxindole, lagumicine, N(4)-demethylalstonerine, N(4)-demethylalstonerinal, and 10-methoxycathafoline N(4)-oxide, in addition to 36 other known alkaloids, were obtained from the leaf extract of Alstonia angustifolia var. latifolia. The structures of the new alkaloids were determined using NMR and MS analysis.

Larval and adult activity of the oriental beetle Exomala orientalis (Waterhouse), a pest of turfgrass in Korea, was investigated at four golf clubs in Pusan, Korea, from 1995 to 1999. Adult emergence was first observed on the greens in late May with peak activity occurring 2 wk later. During the day, E. orientalis adults were most active between 1800 and 2200 hours. First instars were found mostly in early July, second instars mostly in late July, and third instars from August to April. The density of larvae in fixed plots decreased steadily from the time of egg laying to pupation: 667/m3 on 26 July, 267/m3 on 29 August, and 122/m3 on 2 October 1997. All the observed E. orientalis completed one generation per year. Adult females were observed feeding on flowers of a late-blooming variety of Japanese chestnut (Castanea crenata Sieb & Zucc). E. orientalis larval densities were higher in greens with Japanese chestnut nearby, and where magpie, Pica pica sericea (Gould), feeding was observed. More E. orientalis adults emerged from the right, left, and back of greens than from the front or middle. The intensity of emergence was inversely proportional to the amount of golfer traffic on various parts of the green. Counting emergence holes may be a way that golf course superintendents can predict which greens and tees are most likely to be damaged from E. orientalis larvae without destructive sampling.

The oxidative susceptibilities of low density lipoproteins (LDL) isolated from rabbits fed high-fat atherogenic diets containing coconut, palm, or soybean oil were investigated. New Zealand white rabbits were fed atherogenic semisynthetic diets containing 0.5% cholesterol and either (i) 13% coconut oil and 2% corn oil (CNO), (ii) 15% refined, bleached, and deodorized palm olein (RBDPO), (iii) 15% crude palm olein (CPO), (iv) 15% soybean oil (SO), or (v) 15% refined, bleached, and deodorized palm olein without cholesterol supplementation [RBDPO(wc)], for a period of twelve weeks. Total fatty acid compositions of the plasma and LDL were found to be modulated (but not too drastically) by the nature of the dietary fats. Cholesterol supplementation significantly increased the plasma level of vitamin E and effectively altered the plasma composition of long-chain fatty acids in favor of increasing oleic acid. Oxidative susceptibilities of LDL samples were determined by Cu2(+)-catalyzed oxidation which provide the lag times and lag-phase slopes. The plasma LDL from all palm oil diets [RBDPO, CPO, and RBDPO(wc)] were shown to be equally resistant to the oxidation, and the LDL from SO-fed rabbits were most susceptible, followed by the LDL from the CNO-fed rabbits. These results reflect a relationship between the oxidative susceptibility of LDL due to a combination of the levels of polyunsaturated fatty acids and vitamin E.

d-alpha-Tocopherol and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but different hydrocarbon "tails". alpha-Tocotrienol has been shown to be more potent in protecting against free radical-induced oxidative stress than alpha-tocopherol. Simple models of phospholipid membrane systems were used to investigate the mechanism of the antioxidant potency of alpha-tocotrienol in terms of its effects on membrane order and reorientation dynamics. Chemiluminescence and fluorescence measurements demonstrated that alpha-tocotrienol exhibits significantly greater peroxyl radical scavenging potency than alpha-tocopherol in phosphatidylcholine liposomes, whereas both antioxidants have identical activity in hexane. This suggests that the antioxidant potency of alpha-tocotrienol requires the membrane environment. When alpha-tocopherol and alpha-tocotrienol were examined for their effects on phospholipid molecular order using conventional ESR spin labeling with 5- and 16-position-labeled doxylstearic acid, although both vitamin E constituents disordered the gel phase and stabilized the liquid-crystalline phase, no differences were observed between the effects of the two compounds. A slightly greater increase (19% vs 15%) in ordering of the liquid-crystalline state due to alpha-tocopherol, however, was discerned in noninvasive 2H NMR experiments. The difference is most noticeable near C10-C13 positions of the phospholipid chain, possibly suggesting alpha-tocotrienol is located closer to the membrane surface. Saturation-transfer ESR, furthermore, revealed that on the time scale tau c = 10(-7)-10(-3) s the rates of rotation about the long molecular axis and of the wobbling motion of the axis are modified to differing extents by the two forms of the vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)