De novo biosynthesis of the aggregation pheromone components ipsenol and ipsdienol by the pine bark beetles Ips paraconfusus Lanier and Ips pini (Say) (Coleoptera: Scolytidae).

The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ips pini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1-14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified radiolabeled monoterpene alcohols from-males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.

Induced oleoresin biosynthesis in grand fir as a defense against bark beetles.

Grand fir (Abies grandis) saplings and derived cell cultures are useful systems for studying the regulation of defensive oleoresinosis in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced production of monoterpene olefins (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. The pathways and enzymes involved in monoterpene and diterpene resin acid biosynthesis are described, as are the coinduction kinetics following stem injury as determined by resin analysis, enzyme activity measurements, and immunoblotting. The effects of seasonal development, light deprivation, and water stress on constitutive and wound-induced oleoresinosis are reported. Future efforts, including a PCR-based cloning strategy, to define signal transduction in the wound response and the resulting gene activation processes are delineated.

Irradiação de drogas vegetais: aspectos microbiológicos e químicos

Apesar da industrialização no setor farmacêutico, o emprego de drogas vegetais constitui desafio atual quando considerado alternativa terapêutica para as populações de baixa renda ou aquelas que apresentam tradição no uso dessas drogas. Além disso, tendências modernas valorizam a variedade de espécies com propriedades curativas, em particular as espécies brasileiras, desafiando os pesquisadores a intensificar investigações nessa área e induzindo à população um crescente consumo. Assim, questões relacionadas à qualidade dessas drogas apresentam fundamental importância. Devido à origem, a carga microbiana detectada nas mesmas é normalmente elevada, oferecendo riscos potenciais ao usuário. Desta forma, a avaliação de sua qualidade sanitária constitui etapa obrigatória no que se refere ao aspecto de segurança ao consumidor. Além disso, a eficácia terapêutica pode igualmente ser comprometida por decomposição de componentes, decorrente da ação de microrganismos. Com o objetivo de eliminar os efeitos decorrentes da biocarga presente nas drogas vegetais, agentes descontaminantes, de natureza física ou química, têm sido empregados. A utilização de tais procedimentos de descontaminação, prevista na legislação vigente, requer estudos relacionados à estabilidade dos princípios ativos após exposição ao agente selecionado. Dentre os agentes destaca-se a irradiação gama, amplamente utilizada em função de sua aplicabilidade na ausência de água e de temperatura elevada, além de apresentar alta penetrabilidade e reduzir, com eficácia, a carga microbiana viável. Os objetivos do presente trabalho foram avaliar os efeitos de diferentes doses de radiação ionizante sobre a carga microbiana de quatro espécies de drogas vegetais: alcachofra (Cynara scolymus L.), camomila (Matricaria recutita L.), ginkgo (Ginkgo biloba L.) e guaraná (Paullinia cupana H.B.K.), bem como detectar possíveis alterações provocadas pela radiação sobre os teores de seus princípios ativos. As análises microbiológicas e químicas foram realizadas antes e após irradiação com doses médias de 5,5 kGy, 11,4 kGy e 17,8 kGy. Os resultados obtidos anteriormente à irradiação revelaram elevados níveis de contaminação: média de 4,1 x106 para microrganismos aeróbicos totais e 3,3x105 para fungos. Após descontaminação, a dose média de 11,4 kGy, reduziu a carga de microrganismos aeróbicos totais a níveis menores ou iguais a 102 em todas as drogas, com exceção da camomila proveniente do fornecedor B (3,2x104). Para os fungos, a menor dose aplicada (5,5 kGy) foi suficiente para reduzir a contagem a níveis da ordem de 10. Com relação à determinação dos marcadores nas drogas vegetais, os resultados obtidos não revelaram alterações significativas nos teores de cafeína no guaraná e de glicosídeos flavonoídicos no ginkgo. Para a camomila, as amostras antes a após irradiação, apresentaram o mesmo teor de óleo volátil bem como ausência de diferenças significativas no teor de α-bisabolol. Em contraste, observou-se redução no teor de 7-glicosil apigenina após submissão à radiação ionizante, indicando degradação decorrente do processo. Com relação à alcachofra, permanece ainda desconhecida a influência da radiação devido à ausência de metodologias adequadas para extração e determinação da cinarina.

Sequencing and functional analysis of the genome of a nematode egg-parasitic fungus, Pochonia chlamydosporia

Pochonia chlamydosporia is a worldwide-distributed soil fungus with a great capacity to infect and destroy the eggs and kill females of plant-parasitic nematodes. Additionally, it has the ability to colonize endophytically roots of economically-important crop plants, thereby promoting their growth and eliciting plant defenses. This multitrophic behavior makes P. chlamydosporia a potentially useful tool for sustainable agriculture approaches. We sequenced and assembled ∼41 Mb of P. chlamydosporia genomic DNA and predicted 12,122 gene models, of which many were homologous to genes of fungal pathogens of invertebrates and fungal plant pathogens. Predicted genes (65%) were functionally annotated according to Gene Ontology, and 16% of them found to share homology with genes in the Pathogen Host Interactions (PHI) database. The genome of this fungus is highly enriched in genes encoding hydrolytic enzymes, such as proteases, glycoside hydrolases and carbohydrate esterases. We used RNA-Seq technology in order to identify the genes expressed during endophytic behavior of P. chlamydosporia when colonizing barley roots. Functional annotation of these genes showed that hydrolytic enzymes and transporters are expressed during endophytism. This structural and functional analysis of the P. chlamydosporia genome provides a starting point for understanding the molecular mechanisms involved in the multitrophic lifestyle of this fungus. The genomic information provided here should also prove useful for enhancing the capabilities of this fungus as a biocontrol agent of plant-parasitic nematodes and as a plant growth-promoting organism.

The 3-hydroxycineoles

Data concerning the 3-hydroxycineoles 1 and 2 are provided to enable the ready identification of these metabolites and to determine their enantiomeric excess in mixtures. An unusual S(N)2-type inversion at a tertiary center is observed during one synthetic approach.

Structural revision of shatavarins I and IV, the major components from the roots of Asparagus racemosus

The two major steroidal saponins from the roots of Asparagus racemosus were isolated by RP-HPLC and their structure determined by extensive NMR studies. Their structures did not match those reported previously for shatavarins. I and IV and were found to be 3-O-{[beta-D-glueopyranosy](1 -> 2)][alpha-L-rhamnopyranosyl(1 -> 4)]-beta-D-glucopyranosyl}-26-O-(P-D-glu(opyranosyl)-(25S)5 beta-furostan-3p,22 alpha,26-triol and 3-O-{[beta-D-glueopyranosyl(1 -> 2)][alpha-L-rhamnopyranosyl(1 -> 4)]-beta-D-glucopyrariosyl}-(25S)-5 beta-spirostan-3 beta-ol. (c) 2006 Elsevier Ltd. All rights reserved.

Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose

Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K-0.5 = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1 ',6 '-trichloro-4,1 ',6 '-trideoxygalactosucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K-i) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

Synthesis and characterization of Pd(II) and Pt(II) complexes with triazolopyrimidine derivatives: The crystal structure of [Pd2L2Cl4] where L=5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidine

The Pd(II) and Pt(II) complexes with triazolopyrimidine C-nucleosides L-1 (5,7-dimethyl-3-(2',3',5'-tri-O-benzoyl-beta-D-ribofuranosyl-s-triazolo)[4,3-a]pyrimidine), L-2 (5,7-dimethyl-3-beta-D-ribofuranosyl-s-triazolo [4,3-a]pyrimidine) and L-3 (5,7-dimethyl[1,5-a]-s-triazolopyrimidine), [Pd(en)(L-1)](NO3)(2), (Pd(bpy)(L-1)](NO3)(2), cis-Pd(L-3)(2)Cl-2, [Pd-2(L-3)(2)Cl-4]center dot H2O, cis-Pd(L-2)(2)Cl-2 and [Pt-3(L-1)(2)Cl-6] were synthesized and characterized by elemental analysis and NMR spectroscopy. The structure of the [Pd-2(L-3)(2)Cl-4]center dot H2O complex was established by Xray crystallography. The two L-3 ligands are found in a head to tail orientation, with a (PdPd)-Pd-... distance of 3.1254(17) angstrom.L-1 coordinates to Pd(II) through N8 and N1 forming polymeric structures. L-2 coordinates to Pd(II) through N8 in acidic solutions (0.1 M HCl) forming complexes of cis-geometry. The Pd(II) coordination to L-2 does not affect the sugar conformation probably due to the high stability of the C-C glycoside bond. (c) 2006 Elsevier B.V. All rights reserved.

Ecological genetics of Melaleuca quinquenervia (Myrtaceae): Population variation in Florida and its influence on performance of the biological control agent Oxyops vitiosa (Coleoptera: Curculionidae)

Melaleuca quinquenervia (Cav.) Blake (Myrtaceae) was imported into Florida from Australia over a century ago as a landscape plant. A favorable climate and periodic wildfires helped M. quinquenervia thrive; it now occupies about 200,000 hectares in southern Florida. A biological control (i.e., biocontrol) program against M. quinquenervia has been initiated, but not all biocontrol releases are successful. Some scientists have argued that poor biocontrol agent success may relate to genetic differences among populations of invasive weeds. I tested this premise by determining (1) the number and origins of M. quinquenervia introductions into Florida, (2) whether multiple introduction events resulted in the partitioning of Florida's M. quinquenervia populations into discrete biotypes, and (3) whether Oxyops vitiosa, an Australia snout beetle imported to control this weed, might discriminate among putative M. quinquenervia biotypes. Careful scrutiny of early horticultural catalogs and USDA plant introduction records suggested at least six distinct introduction events. Allozyme analyses indicated that the pattern of these introductions, and the subsequent redistribution of progeny, has resulted in geographic structuring of the populations in southern Florida. For example, trees on Florida's Gulf Coast had a greater effective number of alleles and exhibited greater heterozygosity than trees on the Atlantic Coast. Essential oil yields from M. quinquenervia leaves followed a similar trend; Gulf Coast trees yielded nearly twice as much oil as Atlantic Coast trees when both were grown in a common garden. These differences were partially explained by the predominance of a chemical phenotype (chemotype) very rich in the sesquiterpene (E)-nerolidol in M. quinquenervia trees from the Gulf Coast, but rich in a mixture of the monoterpene 1,8-cineole and the sesquiterpene viridiflorol in trees from the Atlantic Coast. Performance of O. vitiosa differed dramatically in laboratory studies depending on the chemotype of the foliage they were fed. Larval survivorship was four-fold greater on the (E)-nerolidol chemotype. Growth was also greater, with adult O. vitiosa gaining nearly 50% more biomass on the (E)-nerolidol plants than on the second chemotype. The results of this study thus confirmed the premise that plant genotype can affect the population dynamics of insects released as weed biocontrols. ^

Red mangroves emit hydrocarbons

The objective of this study is to investigate hydrocarbon species and amounts released by red mangrove foliage and determine if these quantities warrant future research on atmospheric chemical processing of these compounds. The field investigation took place during July 2001 at Key Largo, Florida Bay, Florida. Foliage still attached to plants was enclosed in cuvettes while air of known flow rates circulated around leaves to study, hydrocarbon emissions. Cuvette air samples underwent gas chromatographic analyses to determine species and amounts of hydrocarbons released by mangrove foliage. Red mangrove foliage emits isoprene and trace amounts of the monoterpenes of alpha-pinene, beta-pinene, camphene, and d-limonene. The mangrove flowers released these latter compounds in amounts ranging from 0.5 to 10 mg (monoterpene) per gram of dry biomass per hour. These fluxes are normalized to, the foliage temperature of 30 degreesC. When normalized to the foliage temperature of 30 degreesC and light levels of 1000 mumol m(-2) s(-1), isoprene emission rates as high as 0.092 +/- 0.109 mug (isoprene) per gram of dry biomass per hour were measured. Compared to terrestrial forest ecosystems, red mangroves are low isoprene emitters. During peak flowering periods in the summertime, however, red mangroves may emit sufficient amounts of monoterpenes to alter ground-level ozone concentrations and contribute to biogenic aerosol formation.

Secretome analysis of Trichoderma atroviride T17 biocontrol of Guignardia citricarpa

The fungal species Guignardia citricarpa is an important pathogen in citriculture. Members of the fungal genus Trichoderma are recognized as biocontrol agents but studies on the interactions between both fungi are scarce. This study aimed to identify extracellular proteins secreted by Trichoderma atroviride T17 that are related to the control of G. citricarpa. Two-dimensional gel electrophoresis (2D) was used to study the patterns of proteins secreted by T. atroviride T17 in medium containing glucose (control) and in medium containing G. citricarpa GC3 inactivated mycelium. We identified 59 of the 116 spots differentially expressed (50.86%) by LC–MS/MS. Of these, we highlight the presence of glycoside hydrolases (CAZy families 3, 43, 54, 76 and 93), chitinase, mutanase, a-1,3-glucanase, a-1,2-mannosidase, carboxylic hydrolase ester, carbohydrate-binding module family 13, glucan 1,3-b-glucosidase, a-galactosidase and Neutral protease 2. These proteins are related to mycoparasitism processes, stimuli and therefore to the biological control of pathogens. The results obtained are in agreement with reports describing an increase in the secretion of proteins related to mycoparasitism and biological control and a reduction in the secretion of proteins related to the metabolism of Trichoderma species grown in the presence of the pathogen. Moreover, these results are pioneer in understanding T. atroviride interaction with G. citricarpa. For the first time, we identified potential candidate proteins that may have a role in the antagonism mechanism of G. citricarpa by T. atroviride T17. Thus our results shed a light into the molecular mechanisms that T. atroviride use to control G. citricarpa.

HPLC-DAD-ESI/MS phenolic characterization and biological activity of Equisetum giganteum L.

Naturally-occurring phytochemicals have received a pivotal attention in the last years, due to the increasing evidences of biological activities. Equisetum giganteum L., commonly known as “giant horsetail”, is a native plant from Central and South America, being largely used in dietary supplements as diuretic, hemostatic, antiinflammatory and anti-rheumatic agents [1,2]. The aim of the present study was to evaluate the antioxidant (scavenging effects on 2,2-diphenyl-1-picrylhydrazyl radicals- RSA, reducing power- RP, β-carotene bleaching inhibition- CBI and lipid peroxidation inhibition- LPI), anti-inflammatory (inhibition of NO production in lipopolysaccharidestimulated RAW 264.7 macrophages) and cytotoxic (in a panel of four human tumor cell lines: MCF-7- breast adenocarcinoma, NCI-H460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma; and in non-tumor porcine liver primary cells- PLP2) properties of E. giganteum, providing a phytochemical characterization of its extract (ethanol/water, 80:20, v/v), by using highperformance liquid chromatography coupled to diode array detection and electrospray ionisation mass spectrometry (HPLC-DAD–ESI/MS). E. giganteum presented fourteen phenolic compounds, two phenolic acids and twelve flavonol glycoside derivatives, mainly kaempferol derivatives, accounting to 81% of the total phenolic content, being kaempferol-O-glucoside-O-rutinoside, the most abundant molecule (7.6 mg/g extract). The extract exhibited antioxidant (EC50 values = 123, 136, 202 and 57.4 μg/mL for RSA, RP, CBI and LPI, respectively), anti-inflammatory (EC50 value = 239 μg/mL) and cytotoxic (GI50 values = 250, 258, 268 and 239 μg/mL for MCF-7, NCI-H460, HeLa and HepG2, respectively) properties, which were positively correlated with its concentration in phenolic compounds. Furthermore, up to 400 μg/mL, it did not revealed toxicity in non-tumor liver cells. Thus, this study highlights the potential of E. giganteum extracts as rich sources of phenolic compounds that can be used in the food, pharmaceutical and cosmetic fields.

Anthrone and oxanthrone C,O-diglycosides from Picramnia teapensis

Two C,O-diglycosylated compounds, the anthrone picramnioside F, and the oxanthrone mayoside C, were isolated from the stem bark of Picramnia teapensis, along with the previously reported anthraquinones, 1-O-beta -D- and 8-O-beta -D-glucopyranosyl emodin. The compounds were separated by recycling-HPLC, and their structures were determined on the basis of spectroscopic analysis. CD measurements were used to establish the absolute configuration of the anthrone and oxanthrone. The antifungal activity of 1-O-beta -D- and 8-O--D-glucopyranosyl emodin against Leucoagaricus gongilophorus was shown to be similar to that of the lignan sesamin. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

Metabolites derived from the tropical seagrass Thalassia testudinum are bioactive against pathogenic Labyrinthula sp

Temperate and tropical seagrasses are susceptible to wasting disease outbreaks caused by pathogenic protists of the genus Labyrinthula. Even though there is an increasing awareness of the environmental conditions that influence the etiology of seagrass-. Labyrinthula disease dynamics, the biochemical basis of seagrass defense responses, in particular chemical defenses, is still vastly understudied. Using an in vitro bioassay, we provide evidence that previously characterized phenolic and potentially novel, undescribed non-phenolic metabolites derived from Thalassia testudinum Banks ex Konig exhibit anti-labyrinthulid activity. All phenolic compounds tested displayed dose-dependent behavior and selected combinations interacted synergistically. The flavone glycoside thalassiolin B was roughly 20-100 times more active than any phenolic acid tested. Based upon values reported in the literature, it was calculated that infected specimens of T. testudinum contain natural concentrations of phenolic acids that are consistently greater than what is required to inhibit Labyrinthula growth. This suggests that while there may be an ample supply of phenolic-based derivatives available to inhibit Labyrinthula growth, they may not be readily bio-accessible.Using a bioactivity-guided approach, a semi-purified chemical fraction from T. testudinum was found to contain anti-labyrinthulid activity. 1H NMR spectra for this fraction lacked aromatic hydrogen signals, suggesting that the bioactive compound was non-aromatic in nature. Furthermore, the LC-MS fragmentation patterns were suggestive of the presence of glycosylated natural products of an unknown structural class. This has the potential to provide a foundation for future chemical investigations.

Widespread convergence in toxin resistance by predictable molecular evolution

The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na(+)/K(+)-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na(+)/K(+)-ATPase H1-H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na(+)/K(+)-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.