Insect growth inhibition by tocotrienols and hydroquinones from Roldana barba-johannis

The methanol extract from the aerial parts of Roldana barba-johannis (Asteraceae) afforded sargachromenol, sargahydroquinoic acid, and sargaquinoic acid. These natural products and their corresponding acetylated and methylated derivatives showed insecticidal and insect growth regulatory activities against the Fall Armyworm [Spodoptera frugiperda J.E. Smith, (Lepidoptera: Noctuidae)], an important insect pest of corn. The most active compounds were sargachromenol and its acetylated derivative; sargahydroquinoic acid and its acetylated derivative; and a mixture of sargachromenol, sargahydroquinoic acid, and sargaquinoic acid (6:3:1) and the acetylated form of this mixture. All these compounds and mixtures had significant inhibitory effects between 5.0 and 20.0 ppm in diets. Most compounds were insecticidal to larvae, with lethal doses between 20 and 35 ppm. In addition, these substances also demonstrated scavenging properties toward 2,2-diphenyl-1-picrylhydrazyl radical in TLC autographic and spectrophotometric assays. These compounds appear to have selective effects on the pre-emergence metabolism of the insect. The results from these compounds were fully comparable in activity to those known natural insect growth inhibitors such as gedunin and methanol extracts of Cedrela salvadorensis and Yucca periculosa. These substances may be useful as natural insecticidal agents.     

Peroxynitrite-scavenging constituents from the brown alga<Emphasis Type="Italic">Sargassum thunbergii</Emphasis>

Peroxynitrite formationin vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products for their treatment. The three components (1–3) isolated fromSargassum thunbergii as well as the organic solventsoluble fractions and the aqueous layer ofS. thunbergii were evaluated for their potential to scavenge authentic ONOO and ONOO derived from 3-morpholinosydnonimine (SIN-1). The antioxidant activity of the individual fractions was in the order of 85% aquaous (aq.) MeOH>n-BuOH>n-hexane> H₂O. The three known compounds sargahydroquinoic acid (1), sargaquinoic acid (2) and sargachromenol (3) showed peroxynitrite-scavenging activities comparable to those of L-ascorbic acid and penicillamine. These results showed a possible antioxidant activity in major constituents ofS. thunbergii.     

Selective vasodilatation effect of sargahydroquinoic acid, an active constituent of Sargassum micracanthum, on the basilar arteries of rabbits

Sargahydroquinoic acid (2), a major active constituent of Sargassum micracanthum collected from the coast of the East Sea in Korea, showed a selective vasodilatation effect on the basilar arteries of rabbits. Therefore, treatment with sargahydroquinoic acid may selectively accelerate cerebral blood flow through dilatation of the basilar artery without lowering systemic blood pressure.     

Final steps in juvenile hormone biosynthesis in the desert locust, Schistocerca gregaria

Two genes coding for enzymes previously reported to be involved in the final steps of juvenile hormone (JH) biosynthesis in different insect species, were characterised in the desert locust, Schistocerca gregaria. Juvenile hormone acid O-methyltransferase (JHAMT) was previously described to catalyse the conversion of farnesoic acid (FA) and JH acid to their methyl esters, methyl farnesoate (MF) and JH respectively. A second gene, CYP15A1 was reported to encode a cytochrome P450 enzyme responsible for the epoxidation of MF to JH. Additionally, a third gene, FAMeT (originally reported to encode a farnesoic acid methyltransferase) was included in this study. Using q-RT-PCR, all three genes (JHAMT, CYP15A1 and FAMeT) were found to be primarily expressed in the CA of the desert locust, the main biosynthetic tissue of JH. An RNA interference approach was used to verify the orthologous function of these genes in S. gregaria. Knockdown of the three genes in adult animals followed by the radiochemical assay (RCA) for JH biosynthesis and release showed that SgJHAMT and SgCYP15A1 are responsible for synthesis of MF and JH respectively. Our experiments did not show any involvement of SgFAMeT in JH biosynthesis in the desert locust. Effective and selective inhibitors of SgJHAMT and SgCYP15A1 would likely represent selective biorational locust control agents.     

The chemotaxonomic and medicinal significance of phenolic acids in Arctopus and Alepidea (Apiaceae subfamily Saniculoideae)

The occurrence of (R)-3′-O-β-d-glucopyranosylrosmarinic acid, rosmarinic acid and caffeic acid in two important South African medicinal plants is reported for the first time. (R)-3′-O-β-d-Glucopyranosylrosmarinic acid and rosmarinic acid were isolated and identified in several samples from three species of the genus Arctopus L. (sieketroos) and three species of the genus Alepidea F. Delaroche (ikhathazo), both recently shown to be members of the subfamily Saniculoideae of the family Apiaceae. The compounds occur in high concentrations (up to 15.3 mg of (R)-3′-O-β-d-glucopyranosylrosmarinic acid per g dry wt) in roots of Arctopus. Our results provide a rationale for the traditional uses of these plants, as the identified compounds are all known for their antioxidant activity, with rosmarinic acid further contributing to a wide range of biological activities. Furthermore, we confirm the idea that (R)-3′-O-β-d-glucopyranosylrosmarinic acid is a useful chemotaxonomic marker for the subfamily Saniculoideae.     

Characterization and evaluation of an arbitrary primed Polymerase Chain Reaction (PCR) product for the specific detection of Brucella species

Laboratory detection of Brucella is based largely on bacterial isolation and phenotypic characterization. These methods are lengthy and labor-intensive and have been associated with a heightened risk of laboratory-acquired infection. Antibody based indirect detection methods also suffer from limitations in proper diagnosis of the organism. To overcome these problems, nucleic acid amplification has been explored for rapid detection and confirmation of the presence of Brucella spp. PCR-based diagnostics is useful for screening large populations of livestock to identify infected individuals and confirms the presence of the pathogen. Random Amplification of Polymorphic DNA (RAPD) was performed and identified a 1.3 kb PCR fragment specifically amplifiable from DNA isolated from Brucella. A BLAST search revealed no significant homology with the reported sequences from species other than the members of Brucella. The isolated fragment seems to be a part of d-alanine–d-alanine ligase gene in Brucella sp. Translational BLAST revealed certain degree of homology of this sequence with orthologs of this gene reported from other microbial species at the deduced amino acid level. The sequence information was used to develop PCR based assays to detect Brucella sp. from various samples. The minimum detection limit of Brucella from blood and milk samples spiked with Brucella DNA was found to be 1 ng/ml and 10 ng/ml, respectively. In conclusion, we demonstrated that the PCR based detection protocol was successfully used for the detection of Brucella from various organs and spiked samples of diseased sheep. Diagnosis of Brucellosis by PCR based method reported in this study is relatively rapid, specific and simple.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 °C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

Drought stress increases the production of 5-hydroxynorvaline in two C4 grasses

Plants produce various compounds in response to water deficit. Here, the presence and identification of a drought-inducible non-protein amino acid in the leaves of two C₄ grasses is first reported. The soluble amino acids extracted from the leaves of three different species were measured by high-performance liquid chromatography of derivatives formed with o-phthaldialdehyde and β-mercaptoethanol. One amino acid that increased in amount with drought stress had a retention time not corresponding to any common amino acid. Its identity was determined by metabolite profiling, using ¹H NMR and GC-MS. This unusual amino acid was present in the dehydrated leaves of Cynodon dactylon (L.) Pers. and Zoysia japonica Steudel, but was absent from Paspalum dilatatum Poir. Its identity as 2-amino-5-hydroxypentanoic acid (5-hydroxynorvaline, 5-HNV) was confirmed by synthesis and co-chromatography of synthetic and naturally occurring compounds. The amount of 5-HNV in leaves of the more drought tolerant C₄ grasses, C. dactylon and Z. japonica, increased with increasing water deficit; therefore, any benefits from this unusual non-protein amino acid for drought resistance should be further explored.     

Dammarane triterpenes from Cabralea canjerana (Vell.) Mart. (Meliaceae): Their chemosystematic significance

The stem of Cabralea canjerana (Vell.) Mart. yielded three new dammarane triterpenes 20S,24S-epoxy-7β,25-dihydroxy-3,4-secodammar-4(28)-en-3-oic acid, 20S,24S-epoxy-7β,15α,25-trihydroxy-3,4-secodammar-4(28)-en-3-oic acid and 20S,24R-epoxy-7β,22ξ,25-trihydroxy-3,4-secodammar-4(28)-en-3-oic acid, which were identified on the basis of spectroscopic methods. The known dammarane triterpenes ocotillone, eichlerianic acid, shoreic acid and the sterols sitosterol, campesterol, stigmasterol, sitostenone and stigmast-5-en-3-one were also isolated and identified. The branches yielded the above three known dammaranes and eichlerialactone. The dammaranes in C. canjerana display strong similarities with Trichilieae tribe, which contains several dammaranes. The data reported herein thus provide firm support for placing Cabralea within the subfamily Melioideae, Trichilieae tribe.     

Synthesis, aggregation behavior and cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid)

Synthesis, aggregation behavior and in vitro cholesterol solubilization studies of 16-epi-pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid, EPCA) are reported. The synthesis of this unnatural epimer of pythocholic acid (3α,12α,16α-trihydroxy-5β-cholan-24-oic acid, PCA) involves a series of simple and selective chemical transformations with an overall yield of 21% starting from readily available cholic acid (CA). The critical micellar concentration (CMC) of 16-epi-pythocholate in aqueous media was determined using pyrene as a fluorescent probe. In vitro cholesterol solubilization ability was evaluated using anhydrous cholesterol and results were compared with those of other natural di- and trihydroxy bile acids. These studies showed that 16-epi-pythocholic acid (16β-hydroxy-deoxycholic acid) behaves similar to cholic acid (CA) and avicholic acid (3α,7α,16α-trihydroxy-5β-cholan-24-oic acid, ACA) in its aggregation behavior and cholesterol dissolution properties.     

Metabolic alterations in the hamster co-infected with Schistosoma japonicum and Necator americanus

Co-infection with hookworm and schistosomes is a common phenomenon in sub-Saharan Africa, as well as in parts of South America and southeast Asia. As a first step towards understanding the metabolic response of a hookworm-schistosome co-infection in humans, we investigated the metabolic consequences of co-infection in an animal model, using a nuclear magnetic resonance (NMR)-based metabolic profiling technique, combined with multivariate statistical analysis. Urine and serum samples were obtained from hamsters experimentally infected with 250 Necator americanus infective L₃ and 100 Schistosoma japonicum cercariae simultaneously. In the co-infection model, similar worm burdens were observed as reported for single infection models, whereas metabolic profiles of co-infection represented a combination of the altered metabolite profiles induced by single infections with these two parasites. Consistent differences in metabolic profiles between the co-infected and non-infected control hamsters were observed from 4 weeks p.i. onwards. The predominant metabolic alterations in co-infected hamsters consisted of depletion of amino acids, tricarboxylic acid cycle intermediates (e.g. citrate and succinate) and glucose. Moreover, alterations of a series of gut microbial-related metabolites, such as decreased levels of hippurate, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid and trimethylamine-N-oxide, and increased concentrations of 4-cresol glucuronide and phenylacetylglycine were associated with co-infection. Our results provide a first step towards understanding the metabolic response of an animal host to multiple parasitic infections.     

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.     

Honeybees and beehives are rich sources for fructophilic lactic acid bacteria

Fructophilic lactic acid bacteria (FLAB) are a specific group of lactic acid bacteria (LAB) characterized and described only recently. They prefer fructose as growth substrate and inhabit only fructose-rich niches. Honeybees are high-fructose-consuming insects and important pollinators in nature, but reported to be decreasing in the wild. In the present study, we analyzed FLAB microbiota in honeybees, larvae, fresh honey and bee pollen. A total of 66 strains of LAB were isolated from samples using a selective isolation technique for FLAB. Surprisingly, all strains showed fructophilic characteristics. The 66 strains and ten FLAB strains isolated from flowers in a separate study were genotypically separated into six groups, four of which being identified as Lactobacillus kunkeei and two as Fructobacillus fructosus. One of the L. kunkeei isolates showed antibacterial activity against Melissococcus plutonius, a causative pathogen of European foulbrood, this protection being attributable to production of an antibacterial peptide or protein. Culture-independent analysis suggested that bee products and larvae contained simple Lactobacillus-group microbiota, dominated by L. kunkeei, although adult bees carried a more complex microbiota. The findings clearly demonstrate that honeybees and their products are rich sources of FLAB, and FLAB are potential candidates for future bee probiotics.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Enrichment and properties of urinary pre-S-phenylmercapturic acid (pre-SPMA)

In benzene metabolism, pre-S-phenylmercapturic acid (pre-SPMA) is the precursor to S-phenylmercapturic acid (SPMA). Urinary pre-SPMA/SPMA ratios are variable. For the determination of urinary SPMA as a biomarker of exposure to benzene it is essential to completely convert pre-SPMA to SPMA. We developed a procedure for the enrichment and determination of urinary pre-SPMA by LC–MS/MS which allowed us to trace the conversion of pre-SPMA to SPMA. Complete conversion was found upon treatment of urine with HCl (37%) at pH 1.1. Previously reported treatment of urine with concentrated H₂SO₄ was found to yield SPMA levels higher than after HCl treatment. The origin of that extra SPMA amount is unknown. In conclusion, our findings suggest that pre-treatment of urine with HCl to adjust the pH to 0.5–1 is essential for complete conversion of pre-SPMA to SPMA and should be applied prior to analysis of SPMA in urine.     

Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

麻疯树鲨烯合酶基因SQS的克隆及生物信息学分析

以麻疯树(Jatropha curcas L.)总RNA为模板,根据已报道的鲨烯合酶基因序列设计简并引物,用RACE方法克隆得到麻疯树鲨烯合酶基因全长cDNA,命名为JcSQS。JcSQS全长1609 bp,包含1个1242 bp的开放阅读框,预测麻疯树鲨烯合酶基因编码的蛋白含有413个氨基酸。JcSQS具有鲨烯合酶类的保守结构域,JcSQS 蛋白与蓖麻、柿、木榄等植物中SQS基因编码的氨基酸序列具有高度同源性。这为研究麻疯树萜烯类物质的生物合成和调控机制奠定了基础。     

Purification,properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.